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Long non-coding RNA PVT1 predicts poor prognosis and induces radioresistance by regulating DNA repair and cell apoptosis in nasopharyngeal carcinoma

Long non-coding RNA PVT1 predicts poor prognosis and induces radioresistance by regulating DNA... The long non-coding RNA, plasmacytoma variant translocation 1 (PVT1), is highly expressed in a variety of tumors, and is believed to be a potential oncogene. However, the role and mechanism of action of PVT1 in the carcinogenesis and progression of nasopharyngeal carcinomas (NPCs) remains unclear. In this study, for the first time, we have discovered that PVT1 shows higher expression in NPCs than in normal nasopharyngeal epithelial tissue, and patients with NPCs who show higher expression of PVT1 have worse progression-free and overall survivals. Additionally, we observed that the proliferation of NPC cells decreased, and their rate of apoptosis increased; these results indicated that the knockdown of PVT1 expression in the NPC cells induced radiosensitivity. Further, we have shown that the knockdown of PVT1 expression can induce apoptosis in the NPC cells by influencing the DNA damage repair pathway after radiotherapy. In general, our study shows that PVT1 may be a novel biomarker for prognosis and a new target for the treatment of NPCs. Additionally, targeting PVT1 may be a potential strategy for the clinical management of NPC and for the improvement of the curative effect of radiation in NPCs. 4–8 Introduction gained a lot of attention . Radiotherapy is the principal 9,10 Nasopharyngeal carcinoma (NPC) is a malignant tumor and most effective treatment for NPCs . In recent years, 1–3 arising from nasopharyngeal epithelial (NPE) cells . studies have found that concurrent chemoradiotherapy NPC exhibits strong regional and ethnic differences; it is significantly improved the survival times of patients with one of the most common malignant tumors in Southeast locally advanced NPC, but radioresistance continued to Asia and Southern China, and every year the increasing occur in some patients after they received radiotherapy number of newly diagnosed cases and the mortalities have alone, neoadjuvant chemotherapy combined with radiotherapy, or concurrent chemoradiotherapy; these caused local and distant metastases of the tumor and led 11–13 to poor prognoses . Therefore, studying the potential Correspondence: Zhaoyang Zeng (zengzhaoyang@csu.edu.cn)or Guiyuan Li (lgy@csu.edu.cn) mechanisms of the occurrence of radioresistance in NPCs Hunan Key Laboratory of Translational Radiation Oncology, Hunan Cancer can provide more effective strategies for clinical treatment Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, and prolong the survival times of NPC patients. Central South University, Changsha, Hunan, China The Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Currently, many studies have investigated the mechan- Ministry of Education, Cancer Research Institute, Central South University, 14 isms of radioresistance, such as DNA damage , tumor Changsha, Hunan, China 15,16 17,18 stem cells , and autophagy . In particular, Full list of author information is available at the end of the article Edited by A Stephanou © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; He et al. Cell Death and Disease (2018) 9:235 Page 2 of 12 radioresistance due to DNA damage has been extensively related to poor relapse-free survival (RFS) and overall studied. Radiation can induce DNA damage in tumor survivals (OS) of these patients. Further studies found that cells, further leading to apoptosis and necrosis in these PVT1 may affect the prognosis by regulating radio- cells: this is an important method of clinical treatment for resistance of NPC. tumors. DNA damage is divided into single- or double- strand breaks in DNA and damages in bases. In tumor Results cells, the damaged DNA is identified by many proteins; PVT1 is highly expressed in NPC and is associated with some of these bind with the damaged DNA to mask these poor prognoses in patients with NPCs damaged areas, which consequently are not recognized by We analyzed normal nasopharyngeal epithelium and the repair system, and this process eventually leads to cell NPC tissue samples for differences in lncRNA expression. apoptosis. However, some proteins bind with the After obtaining gene expression data for our samples damaged DNA to initiate the repair pathway, and the (GSE64634) , we also downloaded another NPC gene enhancement of the ability to repair DNA damage often expression data from the Affymetrix Human Genome causes tumor cell radioresistance. Additionally, several U133 Plus 2.0 platform-based studies in the Gene studies have observed that the levels of some proteins that Expression Omnibus (GEO) database (GSE12452 and are related to radioresistance, including 14-3-3σ, Maspin, GSE53819) . Among the differentially expressed the heat shock protein GRP78, manganese-dependent lncRNAs, PVT1 was highly expressed in the NPC samples superoxide dismutase , and Raf kinase inhibitor protein in all three above-mentioned datasets (Fig. 1a–c). PVT1 (RKIP) , can predict the sensitivity of NPC cells to expression was detected in paraffin-embedded section radiotherapy. In addition to protein-encoding genes, long samples from patients with NPCs by in situ hybridization non-coding RNAs (lncRNAs) also play an important role (Fig. 1d). PVT1 shows significantly higher expression in in radioresistance. lncRNAs are about 200 nucleotides NPC tissues, compared to the controls with normal long and are located in the nucleus or cytoplasm of nasopharyngeal tissue (64%, 60/94 vs. 18% 6/33; P < 0.001; 20–24 eukaryotic cells . Studies have shown that lncRNAs Fig. 1e). The radiosensitivity of patients with high 25–27 are involved in DNA damage repair and can regulate expression of PVT1 was lower than that of patients with the differentiation of stem cells . The abnormal expres- low expression (88.6%, 39/44 vs. 39.6% 19/48; P < 0.001; sion and regulation of these lncRNAs is closely related to Fig. 1f). Patients with NPC who show high expression of the occurrence and development of malignant PVT1 have shorter RFS (P = 0.0028, Fig. 1g) and OS (P = 6,24,29–35 tumors . A few studies have shown that lncRNAs 0.0006, Fig. 1h). In conclusion, PVT1 is highly expressed 36–40 have effects on tumor radioresistance . These studies in patients with NPC, and this high expression is asso- suggest that lncRNA may be new potential molecular ciated with poor prognoses in these patients. targets for radiosensitivity. Plasmacytoma variant translocation 1 (PVT1) is a Knockdown of PVT1 enhances the radiosensitivity of NPC lncRNA gene, which was first discovered in mouse plas- cell lines macytoma in the mid 1980s, and in recent years, many As radiotherapy is the main treatment for NPC, we studies have found that PVT1 is involved in the occur- reasoned that the clinical outcomes of NPC are closely rence and development of various malignant tumors. related to the effects of radiotherapy. Since the high PVT1 shows very low expression in most normal human expression of PVT1 is associated with the poor prognoses tissues, but is highly expressed in many malignant tumors of patients, we explored whether PVT1 affects the and tumor cell lines. For example, PVT1 is highly radiosensitivity of NPCs. First, we reduced PVT1 41–43 expressed in tissues and cell lines of gastric cancer , expression by small interfering RNA (siRNA) treatment of 44–46 45 non-small-cell lung cancer , cervical cancer , and the NPC cell lines 5-8F and CNE2. Quantitative real-time colorectal cancer , and the increase in its expression polymerase chain reaction (qPCR) results confirmed that levels is significantly related to the degree of infiltration of the siRNA interfered with and reduced PVT1 expression the malignant tumor, the TNM (tumor–node–metastasis) effectively (Fig. 2a). Next, the cells of the control group staging, and the regional lymph node metastasis. The (transfected with scrambled RNA) and the PVT1 knock- above data indicate that PVT1 may be an important down group each received irradiation doses of 0, 2, 4, 6, cancer gene, and may be involved in the occurrence and and 8 Gy. At 12 days after exposure, the clone formation development of tumors through various mechanisms. test showed that the cell proliferation in the PVT1 However, the influence of PVT1 on the occurrence and interference and control groups showed no significant development of NPCs have not been reported yet, and the differences in the absence of radiotherapy; after receiving role of PVT1 in the process of NPC radioresistance has radiotherapy, the number and sizes of colonies in the not yet been studied. In our study, we have found that PVT1 knockdown group significantly reduced, and with PVT1 is highly expressed in patients with NPC and is the increase in radiation dose, the clone formation ability Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 3 of 12 P = 0.0006 P = 0.0028 Fig. 1 PVT1 is upregulated in NPC tissues and that is associated with poor prognosis. PVT1 was upregulated in the Gene Expression Profiling (GEP) datasets a GSE64634, b GSE12452, and c GSE53819, **P < 0.01, ***P < 0.001. d Representative images of the detection of the expression of PVT1 in NPC tissues and adjacent non-tumor NPE tissues by in situ hybridization. Upper panel: magnification = ×200, scale bars = 50 μm; lower panel: magnification = ×400, scale bars = 20 μm. e The expression of PVT1 in NPC tissues (n = 94) is higher than in non-tumor NPE tissues (n = 33). f The expression levels of PVT1 in the radioresistant and radiosensitive NPC tissues. Treatment data could not be obtained for two patients. Kaplan–Meier survival curves of NPC patients show that NPC patients with high expression of PVT1 (n = 60) have shorter g RFS and h OS than patients with low expression of PVT1 (n = 34) Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 4 of 12 Fig. 2 PVT1 knockdown enhances radiosensitivity in NPC cells. a Compared to a scrambled RNA sequence (used as negative control (NC)), the siRNA specifically targeting PVT1 (siPVT1) significantly inhibits PVT1 expression in the NPC cell lines CNE2 and 5-8F. b Clone formation assay shows that PVT1 knockdown induces radiosensitivity in NPC cells. CNE2 and 5-8F cells were transfected by scrambled RNA (NC) or siRNA targeting PVT1 (siPVT1), and irradiated with 2–8 Gy of radiation; 12 days later the cells were stained (left panel) and dose survival curves were constructed using linear quadratic model analysis (right panel). c Proliferation of NPC cells following radiotherapy. In the NPC cell lines 5-8F and CNE2, PVT1 knockdown via siRNA treatment and irradiation with a dose of 6 Gy radiation shows sustained growth inhibition, although PVT1 knockdown alone does not reduce the proliferation of these cells. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance. further decreased (Fig. 2b). Then, we measured the pro- abilities of the 5-8F and CNE2 cells that did not receive liferation ability of NPC cell lines with or without PVT1 radiation exposure. However, after exposure to a dose of knockdown after radiotherapy. The results showed that 6 Gy radiation, the proliferation ability of these cell lines PVT1 knockdown had no effect on the proliferation significantly decreased after PVT1 knockdown (Fig. 2c). Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 5 of 12 Fig. 3 PVT1 knockdown promotes apoptosis in NPC cells after radiotherapy. CNE2 and 5-8F cells were transfected by either scrambled RNA (NC) or siRNA targeting PVT1 (siPVT1), and irradiated with a dose of 6 Gy and 4Gy X-radiation, respectively. Apoptosis in NPC cells was detected by flow cytometry. In NPC cells, PVT1 knockdown promotes apoptosis induced by radiotherapy, although PVT1 knockdown alone did not significantly induce apoptosis. **P < 0.01 Finally, these results show that silencing PVT1 in NPC enhanced the radiosensitivity and reduced the prolifera- cells can significantly inhibit the clone formation and cell tion of NPC cells after radiotherapy, we investigated proliferation abilities after radiotherapy and may lead to whether the radiosensitivity enhanced by PVT1 knock- radiosensitivity. down occurs via the regulation of apoptosis in NPC cells. We reduced PVT1 expression using siRNAs in 5-8F and Knockdown of PVT1 could promote the apoptosis of NPC CNE2 cells and analyzed cell apoptosis before and after cells induced by radiotherapy radiotherapy by flow cytometry. As shown in Fig. 3, before The major effect of radiotherapy is the induction of radiotherapy, the proportion of apoptotic cells in both the apoptosis in cancer cells. Because PVT1 knockdown PVT1 knockdown and control groups showed no Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 6 of 12 Fig. 4 Knockdown of PVT1 can attenuate the ability of DNA repair in NPC cells after radiotherapy. NPC cell lines 5-8F and CNE2 were transfected by either scrambled RNA (NC) or siRNA that targeted Fig. 5 Knockdown of PVT1 activates caspases and PARP after PVT1 (siPVT1), and irradiated with a dose of 8 Gy X-radiation. The radiotherapy. NPC cell lines 5-8F and CNE2 were transfected by activation of key proteins, ATM, p53, and Chk2, in the DNA repair either scrambled RNA (NC) or siRNA that targeted PVT1 (siPVT1), and pathway was detected by western blot analysis by measuring their irradiated with a dose of 6 Gy X-radiation. The activation of caspase-7, phosphorylation levels (p-ATM, p-p53, p-Chk2). Knockdown of caspase-9, and PARP was detected by western blot analysis. PVT1 PVT1 significantly reduced the phosphorylation levels of these knockdown significantly increased the cleavage of caspase-7, caspase- proteins after radiotherapy. GAPDH was used as an internal control. 9, and PARP. GAPDH was used as an internal control. significant differences; however, after radiotherapy, the PVT1 in cell apoptosis after radiotherapy, we examined proportion of apoptotic cells of the PVT1 knockdown the activation of caspase-7, caspase-9, and poly (ADP- group increased significantly compared to those of the ribose) polymerase (PARP), which are the most important control group. These results showed that interfering with regulators of cell apoptosis. The NPC cell lines 5-8F and the expression of PVT1 can promote apoptosis induced CNE2 were transfected with either scrambled sequences by radiotherapy in NPC cells, and thus can increase the (as negative control (NC)) or siRNAs that target PVT1 radiosensitivity in these cells. (siPVT1) and exposed to 6 Gy radiation; 48 h later, the cells were collected to detect the expression levels of the PVT1 knockdown results in diminution of the DNA repair cleaved (activated) caspases and PARP . The results ability through the ATM–p53 (mutated in ataxia showed that the activation levels of caspase-7, caspase-9, telangiectasia–p53) pathway and PARP in the cells targeted with siPVT1 were higher In tumor cells, the rapid repair of DNA damage after than those in the cells targeted by the scrambled radiotherapy inhibits cell apoptosis, leading to radio- sequences. These results suggest that a decrease in PVT1 resistance. To investigate the effect of PVT1 knockdown expression leads to the activation of apoptotic pathway on the DNA repair pathway after radiotherapy, we proteins after radiotherapy, thereby enhancing the radio- examined the changes in key proteins of the DNA repair sensitivity (Fig. 5). pathway in NPC cells after radiotherapy. NPC cells were transfected with either scrambled RNA or siRNAs tar- Discussion geting PVT1; 24 h later, the cells were irradiated at a dose The PVT1 gene is located in the human chromosome of 8 Gy. After 8 h, the cells were collected to extract 8q24 region near the well-known proto-oncogene MYC. protein, and the expression of key proteins on DNA repair The c-Myc protein encoded by the MYC gene is the hub pathway, such as phosphorylated ATM kinase (p-ATM), of many important growth signal transduction path- p-p53, and phosphorylated checkpoint kinase 2 (p-Chk2), ways . PVT1 has been a hotspot in related studies on was detected by western blot analysis. Knockdown of lncRNA in recent years. Present studies have found that PVT1 significantly decreased the phosphorylation levels PVT1 is highly expressed in many tumors. It can inhibit 45,52 of ATM, p53, and Chk2 (Fig. 4). These results indicated the apoptosis of tumor cells , promote cell prolifera- 44,53,54 that PVT1 knockdown causes a decrease in the DNA tion by regulating the cell cycle in tumors , and repair ability of NPC cells after radiotherapy and enhances affect the invasion and metastasis of tumors , which their radiosensitivity. plays an important role in the occurrence and develop- ment of malignant tumors. However, its effects on NPCs Knockdown of PVT1 could promote apoptosis through have never been reported. caspases In this study, we first investigated the PVT1 expression Radiotherapy induces DNA damage and triggers apop- levels in clinical samples from patients with NPCs. We tosis; caspases, a family of cysteine proteases, are the observed that PVT1 is highly expressed in NPC tissues, central regulators of apoptosis. To elucidate the role of and patients with NPC who show high expression of Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 7 of 12 PVT1 have poor prognoses in terms of both RFS and OS. PVT1 expression following radiotherapy can activate the The residual radioresistance in patients with NPC after apoptosis cascade pathway, and significantly enhance the radiotherapy is the root of NPC recurrence and the activation levels of caspase-9, caspase-7, and PARP pro- leading cause of patient deaths, which implies that PVT1 tein. Specifically, caspase-9 activation increases caspase-7 may affect radioresistance and ultimately lead to poor activation, which increases PARP cleavage, leading to an prognosis of patients with NPC. Therefore, next, we stu- increased rate of apoptosis. died whether PVT1 affects the radiosensitivity of NPC. We have reported, for the first time, the effects of PVT1 Our results confirmed that PVT1 knockdown can inhibit on the radiosensitivity of tumor cells, and have pre- the proliferation ability and colony-forming ability of NPC liminarily explored the molecular mechanisms of the cells after radiotherapy, resulting from the increased cell influence of PVT1 on NPC radioresistance; however, the apoptosis. specific activation pathways need further elucidation. The biological effects of radiotherapy are mainly DNA PVT1 and Myc colocalize in the same chromosome band, strand breakage and cell apoptosis. The common repair and some studies have reported that Myc and PVT1 can pathways of cells include non-homologous end-joining synergistically promote tumorigenesis. Myc enhances its terminal attachment repair based on DNA-dependent own expression by interacting with the PVT1 promoter, protein kinase and the homologous recombination (HR) and the high expression of Myc may promote PVT1 repair pathway based on the ATM gene . In HR repair, expression, forming a positive feedback loop. Addition- the ATM gene is highly sensitive to radiation . When the ally, PVT1 can prevent protein phosphorylation, which DNA double-strand breaks, ATM is activated, damaged stabilizes the c-Myc protein and promotes tumorigen- 66,67 DNA complexes are collected to the breaking point, and esis . In addition, c-Myc can participate in the repair of the DNA repair and apoptosis pathways are mediated DNA double-strand breaks induced by radiotherapy by through the phosphorylation of downstream products regulating ATM phosphorylation . Further, PVT1 and c- such as Chk2 and p53. Chk2, an important signal trans- Myc may interact in NPCs, regulating the activation of duction protein in the DNA damage pathway, activates a ATM phosphorylation that causes radioresistance in variety of downstream DNA repair genes by phosphor- NPC. The possible interaction and common mechanisms ylation. Studies have shown that the increased activation of action between the DNA repair pathway proteins, of ATM/Chk2 leads to the enhancement of DNA repair, PVT1, and Myc, are shown in Fig. 6. Additionally, Myc is resulting in radioresistance . Activated ATM can closely related to the occurrence and development of increase the levels of activated p53 protein and the ability NPC, can affect the growth of NPC cells by regulating the for DNA repair. On the contrary, low expression of p- ATM can only partially activate the p53 protein, decrease the repair ability, and lead to cell apoptosis . Our results show that PVT1 knockdown in NPC cells resulted in decreased ATM/Chk2/p53 activation phosphorylation, weakened DNA repair ability, increased tumor apoptosis, and enhanced radiosensitivity. To investigate how PVT1 influences cell apoptosis during radiotherapy, we examined the activation of some apoptosis-associated proteins. The caspase family are key components in the apoptotic pathway. The activation of caspase factors can trigger the apoptotic pathway, resulting in a cascade effect. Caspase-9, which is in the upstream of the cascade reaction, can be self-activated by the participation of other proteins. Activated caspase-9 can activate caspase-7 downstream; activated caspase-7 combines with a specific substrate to cause cell apopto- 60,61 sis . PARP, a key cleavage substrate for caspase-7 binding in apoptotic pathways, can detect the signal of DNA strand breaks, and is regarded as a receptor for DNA damage . Following radiotherapy, the concentra- tion of phosphorylated p53 proteins decreases, and repair functions decline, which can increase the activation of Fig. 6 The mechanism of action of PVT1 in the resistance to PARP protein cleavage, promoting the apoptosis of tumor radiotherapy in nasopharyngeal carcinoma 63–65 cells . Our research shows that the knockdown of Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 8 of 12 69,70 expression of microRNA (miRNA) , and can partici- between the normal nasopharyngeal epithelium and NPC 22,32 pate in the occurrence and development of NPC by tissue samples in these three datasets . affecting the expression of downstream target genes . PVT1 can also regulate downstream target genes by Tissue samples encoding miRNA and can regulate the expression of Tissue samples of NPC and NPE for this study were miRNA by fusing with Myc . PVT1 can also encode a obtained from the Xiangya Hospital and the Affiliated series of miRNAs and regulate the expression of down- Cancer Hospital of Central South University (Changsha, 73,74 stream target genes . For instance, PVT1 fuses with China), including 94 NPC and 33 NPE paraffin-embedded Myc and enhances PVT1 transcription through the tissue samples for the confirmation of PVT1 expression above-mentioned feedback loop, resulting in high by in situ hybridization. This study was approved by the expression of PVT1-encoded miR-1204, regulation of Research Ethics Board of the Xiangya Hospital and the downstream target genes of miR-1204, and the promotion Affiliated Cancer Hospital of Central South University, of tumor cell proliferation . Further studies can elucidate and written informed consents were obtained from all whether PVT1 regulates miR-1204 in NPC and affects the patients for research purposes. Clinicopathological data related genes of radioresistance or directly encodes were collected from patient medical records (Supple- miRNA and influences the radiosensitivity of NPC. mental Table 1). All patients with NPC had been histo- In normal mammogenesis, PVT1 may function as a pathologically diagnosed with poorly differentiated competitive endogenous RNA (ceRNA) and interact with squamous cell carcinomas, and had received no previous the miR-200 family to regulate mRNA expression , but it treatment. Samples of NPE tissues from the patients with remains unclear whether it may function as a ceRNA and chronic inflammation of nasopharyngeal mucosa in the interact with miRNA to participate in the expression of same period were also collected and used for controls. downstream target genes to regulate NPC radio- Patients without distant metastasis received radical sensitivity. In addition, PVT1 can regulate the aberrant radiotherapy alone using a modified linear accelerator in methylation of p15/p16 gene promoter region by col- the Affiliated Cancer Hospital of Central South Uni- 42,76 79,80 lecting EZH2, downregulating p15/p16 , and upregu- versity . The total dose of administered radiotherapy lating the ability of promoting cell proliferation and anti- was 68 to 72 Gy (2 Gy/fraction, 5 days/week, followed by apoptosis of multidrug resistance protein (MDR1), rest for 2 days). Lymph node-negative patients received a mammalian target of rapamycin (mTOR), multidrug total dose of 60 Gy, and lymph node-positive patients resistance-associated proteins (MRP), and hypoxia/ received a total dose of 70 Gy. hypoxia-inducible factor-1α (HIF-1α) . In thyroid car- The clinical evaluation of radiosensitivity in patients is cinoma, PVT1 is involved in the formation of thyroid based on the first re-examination of computer tomo- carcinomas by collecting EZH2 and regulating the graphy scans after undergoing radiotherapy for a month, expression of thyrotropin receptor (TSHR) . Therefore, according to the following criteria: patients with radio- we hypothesized that in NPC, PVT1 might epigenetically resistant NPC were defined as those with persistent dis- regulate the related genes of radioresistance, thereby ease (incomplete regression of primary tumor and/or neck affecting the radiosensitivity of NPC. lymph nodes) at >3 months, or with local recurrent dis- The role of PVT1 in NPC radioresistance is summarized ease in the nasopharynx or neck lymph nodes or both in Fig. 6. Specifically, PVT1 can promote the radiotherapy within 12 months of completion of radiotherapy. Patients resistance of nasopharyngeal carcinoma. First, PVT1 can with radiosensitive NPC were defined as those who did promote DNA repair by phosphorylation of ATM/Chk2/ not experience either local residual lesions over 3 months p53 signaling pathway in NPC cells. PVT1 can also sig- (complete regression) or local recurrent disease for over nificantly downregulate cleaved caspase-9, cleaved cas- 12 months after completion of radiotherapy. The patients pase-7, and cleaved PARP, thereby inhibiting apoptosis were followed-up for 5–10 years. Disease-free survival was and ultimately causing radiation resistance. defined as the duration from the date of completion of primary radiotherapy to the date when recurrence or Materials and methods distant metastasis was confirmed by clinical evidence or Data mining and analysis pathological diagnosis. OS was defined as the period that To identify functional lncRNAs in NPC, we downloaded patients were alive from the date of first diagnosis to the three sets of gene expression profiling data for naso- date of death due to cancer progression or date of the end pharyngeal carcinomas, GSE64634, GSE12452, and of follow-up. GSE53819, from the Affymetrix Human Genome U133 Plus 2.0 platform-based studies in the GEO database. We In situ hybridization used the Significant Analysis of Microarray (SAM) soft- In situ hybridization was performed to detect PVT1 ware to analyze the differential expression of lncRNAs expression in NPC specimens using three different Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 9 of 12 nucleotide probes from different regions of PVT1; gly- U-3′; the sequences of non-target scrambled RNA con- ceraldehyde 3-phosphate dehydrogenase (GAPDH) was trols were provided by Invitrogen. For gene knockdown, used as positive control. The sequences of PVT1 probes cells were seeded overnight and transfected either with was as follows: Probe-1: 5′-GGT CGG ACT AGA AAA the siRNAs targeting LINC PVT1 or non-target scram- CCG GTC TTC CTC TAA TTT T-3′; Probe-2: 5′-GAG bled control siRNAs (Invitrogen) using Lipofectamine ACT GTA AAA ACT TCT CAG GTC TTA GGA-3′; RNAiMAX Reagent (Invitrogen) in OptiMEM medium Probe-3: 5′-CTC ATA AAA CTC TAA CCT CTT AAT (Invitrogen). TCT CGG TCA G-3′. The probes were synthesized and labeled with Digoxigenin-11-dUTP (DIG-dUTP) at the 3′ Cell proliferation assay and 5′ ends (Invitrogen, Shanghai, China). In situ hybri- The cells transfected with siRNA were seeded into 96- dization was performed as previously described . All well plates (500 cells per well), incubated for 24 h, and sections were independently scored by two pathologists irradiated with a dose of 6 Gy radiation. According to the who were blinded to the clinicopathological features and manufacturer’s instructions, a Cell Counting Kit-8 (CCK- the clinical data. 8) kit (Dojindo Laboratories, Kumamoto, Japan) was used to determine cell proliferation after irradiation. The In situ hybridization evaluation criteria absorbance of each well was monitored by a spectro- The intensity and the distribution range of the staining photometer at a wavelength of 450 nm. Each experiment were determined by semi-quantitative treatment . Two was performed at least three times in triplicate wells. pathologists scored the results according to the following criteria. (1) Staining intensity: no observed cell staining Cloning formation assay was scored as 0; cells with light-brown cell staining as 1; A cloning formation assay was used for accessing the cells stained brown with no background staining, or dark- radiosensitivity of the NPC cells after incubation. First, brown-stained cells with light-brown background staining the cells were seeded in 6-well plates (500, 1000, 2000, were recorded as moderately positive, as 2; dark-brown- 3000, and 4000 cells per well in triplicate) and incubated stained cells with no background staining were recorded overnight. Next, the cells were exposed to irradiation as strongly positive as 3. (2) Number of positive cells: no doses of 0, 2, 4, 6, and 8 Gy, respectively. Then, after positive cells were scored as 0; less than 25% of positive 12 days of culture, the surviving fractions (>50 cells) were cells as 1; between 25 and 50% positive cells as 2; positive stained with 0.5% crystal violet and counted under a cells over 50% recorded as strongly positive as 3. The final microscope. Finally, the data were fitted to a linear scores were obtained by multiplying the two scores. The quadratic model to create the cloning formation survival results were as follows: 0 was considered as negative curves to evaluate the radiosensitivity of these cells. expression, and the final score was 0; 1−2 as weakly positive, and the final score was 1; 3−4 as moderately Cell apoptosis assay positive, and the final score was 2; 6−9 as strong positive, First, the 5-8F and CNE2 cells were seeded in 6-well and the final score was 3. plates and transfected with siRNA for 24 h. Next, these All the target cells of each tissue were counted under cells were exposed to irradiation doses of 4 and 6 Gy, ×10 magnification, and the counting was repeated twice. respectively. After 48 h, the cells were harvested. The average values obtained from the counting was According to the protocols, an annexin V-fluorescein used for statistical analysis. In situ hybridization scores of isothiocyanate/propidium iodide apoptosis detection kit 2 and 3 indicated high expression of PVT1, and immu- (BD, San Jose, CA, USA) was used to stain cells and the nohistochemistry scores of 0 and 1 indicated low cell apoptosis was immediately detected by flow expression of PVT1. All the samples were scored inde- cytometry. pendently by two experienced pathologists who were double-blinded. Real-time PCR Cellular RNA was extracted using TRIzol reagent Cell line and siRNAs (Invitrogen, Carlsbad, CA, USA). For performing Real- Human NPC cell lines CNE2 and 5-8F were maintained Time Quantitative Reverse Transcription PCR (qRT- 47,49–53,81 in our laboratory . Cells were grown in RPMI- PCR), RNA was reverse transcribed to cDNA by using a 1640 medium (Life Technologies, Grand Island, NY) PrimeScript RT reagent Kit (Takara, Dalian, China). qRT- supplemented with 10% fetal bovine serum (Life Tech- PCR was performed using a SYBR_PremixExTaqII kit nologies), and 1% penicillin–streptomycin (Life Technol- (Takara, Dalian, China) in the CFX96 Real-Time PCR ogies) in a humidified incubator with 5% CO at 37 °C. Detection System (Bio-Rad, Hercules, CA, USA) to The sequence of the LINC PVT1 targeting siRNAs was as determine the relative expression levels of target genes. follows: 5′-CAC UAC UGA CCU UGC AGC UUA UUA The sequences of qRT-PCR primers are as follows: LINC Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 10 of 12 PVT1: forward primer 5′-TGG CTG AGA GGG TTG Acknowledgements This study was supported by grants from the National Natural Science AGA TC-3′ and reverse primer 5′-GCT GTA TGT GCC Foundation of China (81472531, 81572787, 81672683, 81672993, 81772928, AAG GTC AC-3′; β-actin: forward primer 5′-TCA CCA and 81702907), the Natural Science Foundation of Hunan Province ACT GGG ACG ACA TG-3′ and reverse primer 5′-GTC (2015JJ1022, 2016JC2035, and 2017SK2105), the Overseas Expertise Introduction Project for Discipline Innovation (111 Project, No. 111-2-12), and ACC GGA GTC CAT CAC GAT-3′; β-actin was used as the Fundamental Research Funds for the Central South University (2016zzts478 reference and normalization control. and 2014zzts066). Authors’ contributions Western blotting Y.H. wrote the manuscript and performed most assays. Y.J. and F.W. performed Lysis, electrophoresis, and target protein visualization the experiments related to radioresistance. Y.T., L.Y., and J.L. collected clinical were performed as described previously. Briefly, 50 μgof samples and performed qRT-PCR. P.Y., Q.N., and J.P. collected and analyzed the data. Q.L., F.X., C.G., B.X., X.L., M.Z., Y.L., and W.X. participated in the design of cell lysates were separated by 10% sodium dodecyl this study. Z.Z. and G.L. designed this study. All the authors have read and sulfate–polyacrylamide gel electrophoresis and transferred approved the final manuscript. onto a polyvinylidene fluoride membrane (Millipore, Billerica). To assess protein expression, the blots were Author details incubated overnight at 4 °C with the following primary Hunan Key Laboratory of Translational Radiation Oncology, Hunan Cancer antibodies: rabbit antibodies against caspase-7, caspase-9, Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, PARP, phospho-ATM (Ser1981), phospho-Chk2 Central South University, Changsha, Hunan, China. The Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, (a serine/threonine kinase) (Thr68), and mouse antibody Cancer Research Institute, Central South University, Changsha, Hunan, China. against phospho-p53 (Ser15) (Cell Signaling, Danvers, The Key Laboratory of Carcinogenesis of the Chinese Ministry of Health, MA). After washing with tris-buffered saline-Tween, Xiangya Hospital, Central South University, Changsha, Hunan, China. Hunan Key Laboratory of Nonresolving Inflammation and Cancer, Disease Genome the blots were incubated with horseradish peroxidase- Research Center, The Third Xiangya Hospital, Central South University, conjugated secondary antibodies (Cell Signaling Changsha, Hunan, China. Department of Cancer Biology, Lerner Research Technology) for 1 h at 37 °C. The signal was visualized Institute, Cleveland Clinic, Cleveland, OH, USA using enhanced chemiluminescence (EMD-Millipore, Conflict of interest Billerica, MA) and quantified by densitometry The authors declare that they have no conflict of interest. using ImageJ software (http://rsb.info.nih.gov/ij). GAPDH (Cell Signaling Technology) was used as a Publisher's note loading control. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Statistical analysis Data analyses were conducted by the GraphPad Prism 5 Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-018-0265-y). (GraphPad, La Jolla, CA). Survival curves were plotted using the Kaplan–Meier method, and compared using the Received: 4 October 2017 Revised: 9 December 2017 Accepted: 12 log-rank test. Student’s t-tests were used to evaluate December 2017 significant differences between any two groups of data; one-way analysis of variance was used if there were more than two groups. The dose survival curve was calculated by a linear quadratic model (Y = exp(−(a*x + b*(x^2)))). References 1. Thompson, L. Nasopharyngeal carcinoma. Ear. Nose. Throat J. 84,404–405 All data are represented as means ± standard (2005). deviations. P < 0.05 was considered statistically 2. Tu, C. et al. Genome-wide analysis of 18 Epstein-Barr viruses isolated from significant. primary nasopharyngeal carcinoma biopsy specimens. J. 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Long non-coding RNA PVT1 predicts poor prognosis and induces radioresistance by regulating DNA repair and cell apoptosis in nasopharyngeal carcinoma

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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Immunology; Cell Culture; Antibodies
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Abstract

The long non-coding RNA, plasmacytoma variant translocation 1 (PVT1), is highly expressed in a variety of tumors, and is believed to be a potential oncogene. However, the role and mechanism of action of PVT1 in the carcinogenesis and progression of nasopharyngeal carcinomas (NPCs) remains unclear. In this study, for the first time, we have discovered that PVT1 shows higher expression in NPCs than in normal nasopharyngeal epithelial tissue, and patients with NPCs who show higher expression of PVT1 have worse progression-free and overall survivals. Additionally, we observed that the proliferation of NPC cells decreased, and their rate of apoptosis increased; these results indicated that the knockdown of PVT1 expression in the NPC cells induced radiosensitivity. Further, we have shown that the knockdown of PVT1 expression can induce apoptosis in the NPC cells by influencing the DNA damage repair pathway after radiotherapy. In general, our study shows that PVT1 may be a novel biomarker for prognosis and a new target for the treatment of NPCs. Additionally, targeting PVT1 may be a potential strategy for the clinical management of NPC and for the improvement of the curative effect of radiation in NPCs. 4–8 Introduction gained a lot of attention . Radiotherapy is the principal 9,10 Nasopharyngeal carcinoma (NPC) is a malignant tumor and most effective treatment for NPCs . In recent years, 1–3 arising from nasopharyngeal epithelial (NPE) cells . studies have found that concurrent chemoradiotherapy NPC exhibits strong regional and ethnic differences; it is significantly improved the survival times of patients with one of the most common malignant tumors in Southeast locally advanced NPC, but radioresistance continued to Asia and Southern China, and every year the increasing occur in some patients after they received radiotherapy number of newly diagnosed cases and the mortalities have alone, neoadjuvant chemotherapy combined with radiotherapy, or concurrent chemoradiotherapy; these caused local and distant metastases of the tumor and led 11–13 to poor prognoses . Therefore, studying the potential Correspondence: Zhaoyang Zeng (zengzhaoyang@csu.edu.cn)or Guiyuan Li (lgy@csu.edu.cn) mechanisms of the occurrence of radioresistance in NPCs Hunan Key Laboratory of Translational Radiation Oncology, Hunan Cancer can provide more effective strategies for clinical treatment Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, and prolong the survival times of NPC patients. Central South University, Changsha, Hunan, China The Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Currently, many studies have investigated the mechan- Ministry of Education, Cancer Research Institute, Central South University, 14 isms of radioresistance, such as DNA damage , tumor Changsha, Hunan, China 15,16 17,18 stem cells , and autophagy . In particular, Full list of author information is available at the end of the article Edited by A Stephanou © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; He et al. Cell Death and Disease (2018) 9:235 Page 2 of 12 radioresistance due to DNA damage has been extensively related to poor relapse-free survival (RFS) and overall studied. Radiation can induce DNA damage in tumor survivals (OS) of these patients. Further studies found that cells, further leading to apoptosis and necrosis in these PVT1 may affect the prognosis by regulating radio- cells: this is an important method of clinical treatment for resistance of NPC. tumors. DNA damage is divided into single- or double- strand breaks in DNA and damages in bases. In tumor Results cells, the damaged DNA is identified by many proteins; PVT1 is highly expressed in NPC and is associated with some of these bind with the damaged DNA to mask these poor prognoses in patients with NPCs damaged areas, which consequently are not recognized by We analyzed normal nasopharyngeal epithelium and the repair system, and this process eventually leads to cell NPC tissue samples for differences in lncRNA expression. apoptosis. However, some proteins bind with the After obtaining gene expression data for our samples damaged DNA to initiate the repair pathway, and the (GSE64634) , we also downloaded another NPC gene enhancement of the ability to repair DNA damage often expression data from the Affymetrix Human Genome causes tumor cell radioresistance. Additionally, several U133 Plus 2.0 platform-based studies in the Gene studies have observed that the levels of some proteins that Expression Omnibus (GEO) database (GSE12452 and are related to radioresistance, including 14-3-3σ, Maspin, GSE53819) . Among the differentially expressed the heat shock protein GRP78, manganese-dependent lncRNAs, PVT1 was highly expressed in the NPC samples superoxide dismutase , and Raf kinase inhibitor protein in all three above-mentioned datasets (Fig. 1a–c). PVT1 (RKIP) , can predict the sensitivity of NPC cells to expression was detected in paraffin-embedded section radiotherapy. In addition to protein-encoding genes, long samples from patients with NPCs by in situ hybridization non-coding RNAs (lncRNAs) also play an important role (Fig. 1d). PVT1 shows significantly higher expression in in radioresistance. lncRNAs are about 200 nucleotides NPC tissues, compared to the controls with normal long and are located in the nucleus or cytoplasm of nasopharyngeal tissue (64%, 60/94 vs. 18% 6/33; P < 0.001; 20–24 eukaryotic cells . Studies have shown that lncRNAs Fig. 1e). The radiosensitivity of patients with high 25–27 are involved in DNA damage repair and can regulate expression of PVT1 was lower than that of patients with the differentiation of stem cells . The abnormal expres- low expression (88.6%, 39/44 vs. 39.6% 19/48; P < 0.001; sion and regulation of these lncRNAs is closely related to Fig. 1f). Patients with NPC who show high expression of the occurrence and development of malignant PVT1 have shorter RFS (P = 0.0028, Fig. 1g) and OS (P = 6,24,29–35 tumors . A few studies have shown that lncRNAs 0.0006, Fig. 1h). In conclusion, PVT1 is highly expressed 36–40 have effects on tumor radioresistance . These studies in patients with NPC, and this high expression is asso- suggest that lncRNA may be new potential molecular ciated with poor prognoses in these patients. targets for radiosensitivity. Plasmacytoma variant translocation 1 (PVT1) is a Knockdown of PVT1 enhances the radiosensitivity of NPC lncRNA gene, which was first discovered in mouse plas- cell lines macytoma in the mid 1980s, and in recent years, many As radiotherapy is the main treatment for NPC, we studies have found that PVT1 is involved in the occur- reasoned that the clinical outcomes of NPC are closely rence and development of various malignant tumors. related to the effects of radiotherapy. Since the high PVT1 shows very low expression in most normal human expression of PVT1 is associated with the poor prognoses tissues, but is highly expressed in many malignant tumors of patients, we explored whether PVT1 affects the and tumor cell lines. For example, PVT1 is highly radiosensitivity of NPCs. First, we reduced PVT1 41–43 expressed in tissues and cell lines of gastric cancer , expression by small interfering RNA (siRNA) treatment of 44–46 45 non-small-cell lung cancer , cervical cancer , and the NPC cell lines 5-8F and CNE2. Quantitative real-time colorectal cancer , and the increase in its expression polymerase chain reaction (qPCR) results confirmed that levels is significantly related to the degree of infiltration of the siRNA interfered with and reduced PVT1 expression the malignant tumor, the TNM (tumor–node–metastasis) effectively (Fig. 2a). Next, the cells of the control group staging, and the regional lymph node metastasis. The (transfected with scrambled RNA) and the PVT1 knock- above data indicate that PVT1 may be an important down group each received irradiation doses of 0, 2, 4, 6, cancer gene, and may be involved in the occurrence and and 8 Gy. At 12 days after exposure, the clone formation development of tumors through various mechanisms. test showed that the cell proliferation in the PVT1 However, the influence of PVT1 on the occurrence and interference and control groups showed no significant development of NPCs have not been reported yet, and the differences in the absence of radiotherapy; after receiving role of PVT1 in the process of NPC radioresistance has radiotherapy, the number and sizes of colonies in the not yet been studied. In our study, we have found that PVT1 knockdown group significantly reduced, and with PVT1 is highly expressed in patients with NPC and is the increase in radiation dose, the clone formation ability Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 3 of 12 P = 0.0006 P = 0.0028 Fig. 1 PVT1 is upregulated in NPC tissues and that is associated with poor prognosis. PVT1 was upregulated in the Gene Expression Profiling (GEP) datasets a GSE64634, b GSE12452, and c GSE53819, **P < 0.01, ***P < 0.001. d Representative images of the detection of the expression of PVT1 in NPC tissues and adjacent non-tumor NPE tissues by in situ hybridization. Upper panel: magnification = ×200, scale bars = 50 μm; lower panel: magnification = ×400, scale bars = 20 μm. e The expression of PVT1 in NPC tissues (n = 94) is higher than in non-tumor NPE tissues (n = 33). f The expression levels of PVT1 in the radioresistant and radiosensitive NPC tissues. Treatment data could not be obtained for two patients. Kaplan–Meier survival curves of NPC patients show that NPC patients with high expression of PVT1 (n = 60) have shorter g RFS and h OS than patients with low expression of PVT1 (n = 34) Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 4 of 12 Fig. 2 PVT1 knockdown enhances radiosensitivity in NPC cells. a Compared to a scrambled RNA sequence (used as negative control (NC)), the siRNA specifically targeting PVT1 (siPVT1) significantly inhibits PVT1 expression in the NPC cell lines CNE2 and 5-8F. b Clone formation assay shows that PVT1 knockdown induces radiosensitivity in NPC cells. CNE2 and 5-8F cells were transfected by scrambled RNA (NC) or siRNA targeting PVT1 (siPVT1), and irradiated with 2–8 Gy of radiation; 12 days later the cells were stained (left panel) and dose survival curves were constructed using linear quadratic model analysis (right panel). c Proliferation of NPC cells following radiotherapy. In the NPC cell lines 5-8F and CNE2, PVT1 knockdown via siRNA treatment and irradiation with a dose of 6 Gy radiation shows sustained growth inhibition, although PVT1 knockdown alone does not reduce the proliferation of these cells. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance. further decreased (Fig. 2b). Then, we measured the pro- abilities of the 5-8F and CNE2 cells that did not receive liferation ability of NPC cell lines with or without PVT1 radiation exposure. However, after exposure to a dose of knockdown after radiotherapy. The results showed that 6 Gy radiation, the proliferation ability of these cell lines PVT1 knockdown had no effect on the proliferation significantly decreased after PVT1 knockdown (Fig. 2c). Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 5 of 12 Fig. 3 PVT1 knockdown promotes apoptosis in NPC cells after radiotherapy. CNE2 and 5-8F cells were transfected by either scrambled RNA (NC) or siRNA targeting PVT1 (siPVT1), and irradiated with a dose of 6 Gy and 4Gy X-radiation, respectively. Apoptosis in NPC cells was detected by flow cytometry. In NPC cells, PVT1 knockdown promotes apoptosis induced by radiotherapy, although PVT1 knockdown alone did not significantly induce apoptosis. **P < 0.01 Finally, these results show that silencing PVT1 in NPC enhanced the radiosensitivity and reduced the prolifera- cells can significantly inhibit the clone formation and cell tion of NPC cells after radiotherapy, we investigated proliferation abilities after radiotherapy and may lead to whether the radiosensitivity enhanced by PVT1 knock- radiosensitivity. down occurs via the regulation of apoptosis in NPC cells. We reduced PVT1 expression using siRNAs in 5-8F and Knockdown of PVT1 could promote the apoptosis of NPC CNE2 cells and analyzed cell apoptosis before and after cells induced by radiotherapy radiotherapy by flow cytometry. As shown in Fig. 3, before The major effect of radiotherapy is the induction of radiotherapy, the proportion of apoptotic cells in both the apoptosis in cancer cells. Because PVT1 knockdown PVT1 knockdown and control groups showed no Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 6 of 12 Fig. 4 Knockdown of PVT1 can attenuate the ability of DNA repair in NPC cells after radiotherapy. NPC cell lines 5-8F and CNE2 were transfected by either scrambled RNA (NC) or siRNA that targeted Fig. 5 Knockdown of PVT1 activates caspases and PARP after PVT1 (siPVT1), and irradiated with a dose of 8 Gy X-radiation. The radiotherapy. NPC cell lines 5-8F and CNE2 were transfected by activation of key proteins, ATM, p53, and Chk2, in the DNA repair either scrambled RNA (NC) or siRNA that targeted PVT1 (siPVT1), and pathway was detected by western blot analysis by measuring their irradiated with a dose of 6 Gy X-radiation. The activation of caspase-7, phosphorylation levels (p-ATM, p-p53, p-Chk2). Knockdown of caspase-9, and PARP was detected by western blot analysis. PVT1 PVT1 significantly reduced the phosphorylation levels of these knockdown significantly increased the cleavage of caspase-7, caspase- proteins after radiotherapy. GAPDH was used as an internal control. 9, and PARP. GAPDH was used as an internal control. significant differences; however, after radiotherapy, the PVT1 in cell apoptosis after radiotherapy, we examined proportion of apoptotic cells of the PVT1 knockdown the activation of caspase-7, caspase-9, and poly (ADP- group increased significantly compared to those of the ribose) polymerase (PARP), which are the most important control group. These results showed that interfering with regulators of cell apoptosis. The NPC cell lines 5-8F and the expression of PVT1 can promote apoptosis induced CNE2 were transfected with either scrambled sequences by radiotherapy in NPC cells, and thus can increase the (as negative control (NC)) or siRNAs that target PVT1 radiosensitivity in these cells. (siPVT1) and exposed to 6 Gy radiation; 48 h later, the cells were collected to detect the expression levels of the PVT1 knockdown results in diminution of the DNA repair cleaved (activated) caspases and PARP . The results ability through the ATM–p53 (mutated in ataxia showed that the activation levels of caspase-7, caspase-9, telangiectasia–p53) pathway and PARP in the cells targeted with siPVT1 were higher In tumor cells, the rapid repair of DNA damage after than those in the cells targeted by the scrambled radiotherapy inhibits cell apoptosis, leading to radio- sequences. These results suggest that a decrease in PVT1 resistance. To investigate the effect of PVT1 knockdown expression leads to the activation of apoptotic pathway on the DNA repair pathway after radiotherapy, we proteins after radiotherapy, thereby enhancing the radio- examined the changes in key proteins of the DNA repair sensitivity (Fig. 5). pathway in NPC cells after radiotherapy. NPC cells were transfected with either scrambled RNA or siRNAs tar- Discussion geting PVT1; 24 h later, the cells were irradiated at a dose The PVT1 gene is located in the human chromosome of 8 Gy. After 8 h, the cells were collected to extract 8q24 region near the well-known proto-oncogene MYC. protein, and the expression of key proteins on DNA repair The c-Myc protein encoded by the MYC gene is the hub pathway, such as phosphorylated ATM kinase (p-ATM), of many important growth signal transduction path- p-p53, and phosphorylated checkpoint kinase 2 (p-Chk2), ways . PVT1 has been a hotspot in related studies on was detected by western blot analysis. Knockdown of lncRNA in recent years. Present studies have found that PVT1 significantly decreased the phosphorylation levels PVT1 is highly expressed in many tumors. It can inhibit 45,52 of ATM, p53, and Chk2 (Fig. 4). These results indicated the apoptosis of tumor cells , promote cell prolifera- 44,53,54 that PVT1 knockdown causes a decrease in the DNA tion by regulating the cell cycle in tumors , and repair ability of NPC cells after radiotherapy and enhances affect the invasion and metastasis of tumors , which their radiosensitivity. plays an important role in the occurrence and develop- ment of malignant tumors. However, its effects on NPCs Knockdown of PVT1 could promote apoptosis through have never been reported. caspases In this study, we first investigated the PVT1 expression Radiotherapy induces DNA damage and triggers apop- levels in clinical samples from patients with NPCs. We tosis; caspases, a family of cysteine proteases, are the observed that PVT1 is highly expressed in NPC tissues, central regulators of apoptosis. To elucidate the role of and patients with NPC who show high expression of Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 7 of 12 PVT1 have poor prognoses in terms of both RFS and OS. PVT1 expression following radiotherapy can activate the The residual radioresistance in patients with NPC after apoptosis cascade pathway, and significantly enhance the radiotherapy is the root of NPC recurrence and the activation levels of caspase-9, caspase-7, and PARP pro- leading cause of patient deaths, which implies that PVT1 tein. Specifically, caspase-9 activation increases caspase-7 may affect radioresistance and ultimately lead to poor activation, which increases PARP cleavage, leading to an prognosis of patients with NPC. Therefore, next, we stu- increased rate of apoptosis. died whether PVT1 affects the radiosensitivity of NPC. We have reported, for the first time, the effects of PVT1 Our results confirmed that PVT1 knockdown can inhibit on the radiosensitivity of tumor cells, and have pre- the proliferation ability and colony-forming ability of NPC liminarily explored the molecular mechanisms of the cells after radiotherapy, resulting from the increased cell influence of PVT1 on NPC radioresistance; however, the apoptosis. specific activation pathways need further elucidation. The biological effects of radiotherapy are mainly DNA PVT1 and Myc colocalize in the same chromosome band, strand breakage and cell apoptosis. The common repair and some studies have reported that Myc and PVT1 can pathways of cells include non-homologous end-joining synergistically promote tumorigenesis. Myc enhances its terminal attachment repair based on DNA-dependent own expression by interacting with the PVT1 promoter, protein kinase and the homologous recombination (HR) and the high expression of Myc may promote PVT1 repair pathway based on the ATM gene . In HR repair, expression, forming a positive feedback loop. Addition- the ATM gene is highly sensitive to radiation . When the ally, PVT1 can prevent protein phosphorylation, which DNA double-strand breaks, ATM is activated, damaged stabilizes the c-Myc protein and promotes tumorigen- 66,67 DNA complexes are collected to the breaking point, and esis . In addition, c-Myc can participate in the repair of the DNA repair and apoptosis pathways are mediated DNA double-strand breaks induced by radiotherapy by through the phosphorylation of downstream products regulating ATM phosphorylation . Further, PVT1 and c- such as Chk2 and p53. Chk2, an important signal trans- Myc may interact in NPCs, regulating the activation of duction protein in the DNA damage pathway, activates a ATM phosphorylation that causes radioresistance in variety of downstream DNA repair genes by phosphor- NPC. The possible interaction and common mechanisms ylation. Studies have shown that the increased activation of action between the DNA repair pathway proteins, of ATM/Chk2 leads to the enhancement of DNA repair, PVT1, and Myc, are shown in Fig. 6. Additionally, Myc is resulting in radioresistance . Activated ATM can closely related to the occurrence and development of increase the levels of activated p53 protein and the ability NPC, can affect the growth of NPC cells by regulating the for DNA repair. On the contrary, low expression of p- ATM can only partially activate the p53 protein, decrease the repair ability, and lead to cell apoptosis . Our results show that PVT1 knockdown in NPC cells resulted in decreased ATM/Chk2/p53 activation phosphorylation, weakened DNA repair ability, increased tumor apoptosis, and enhanced radiosensitivity. To investigate how PVT1 influences cell apoptosis during radiotherapy, we examined the activation of some apoptosis-associated proteins. The caspase family are key components in the apoptotic pathway. The activation of caspase factors can trigger the apoptotic pathway, resulting in a cascade effect. Caspase-9, which is in the upstream of the cascade reaction, can be self-activated by the participation of other proteins. Activated caspase-9 can activate caspase-7 downstream; activated caspase-7 combines with a specific substrate to cause cell apopto- 60,61 sis . PARP, a key cleavage substrate for caspase-7 binding in apoptotic pathways, can detect the signal of DNA strand breaks, and is regarded as a receptor for DNA damage . Following radiotherapy, the concentra- tion of phosphorylated p53 proteins decreases, and repair functions decline, which can increase the activation of Fig. 6 The mechanism of action of PVT1 in the resistance to PARP protein cleavage, promoting the apoptosis of tumor radiotherapy in nasopharyngeal carcinoma 63–65 cells . Our research shows that the knockdown of Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 8 of 12 69,70 expression of microRNA (miRNA) , and can partici- between the normal nasopharyngeal epithelium and NPC 22,32 pate in the occurrence and development of NPC by tissue samples in these three datasets . affecting the expression of downstream target genes . PVT1 can also regulate downstream target genes by Tissue samples encoding miRNA and can regulate the expression of Tissue samples of NPC and NPE for this study were miRNA by fusing with Myc . PVT1 can also encode a obtained from the Xiangya Hospital and the Affiliated series of miRNAs and regulate the expression of down- Cancer Hospital of Central South University (Changsha, 73,74 stream target genes . For instance, PVT1 fuses with China), including 94 NPC and 33 NPE paraffin-embedded Myc and enhances PVT1 transcription through the tissue samples for the confirmation of PVT1 expression above-mentioned feedback loop, resulting in high by in situ hybridization. This study was approved by the expression of PVT1-encoded miR-1204, regulation of Research Ethics Board of the Xiangya Hospital and the downstream target genes of miR-1204, and the promotion Affiliated Cancer Hospital of Central South University, of tumor cell proliferation . Further studies can elucidate and written informed consents were obtained from all whether PVT1 regulates miR-1204 in NPC and affects the patients for research purposes. Clinicopathological data related genes of radioresistance or directly encodes were collected from patient medical records (Supple- miRNA and influences the radiosensitivity of NPC. mental Table 1). All patients with NPC had been histo- In normal mammogenesis, PVT1 may function as a pathologically diagnosed with poorly differentiated competitive endogenous RNA (ceRNA) and interact with squamous cell carcinomas, and had received no previous the miR-200 family to regulate mRNA expression , but it treatment. Samples of NPE tissues from the patients with remains unclear whether it may function as a ceRNA and chronic inflammation of nasopharyngeal mucosa in the interact with miRNA to participate in the expression of same period were also collected and used for controls. downstream target genes to regulate NPC radio- Patients without distant metastasis received radical sensitivity. In addition, PVT1 can regulate the aberrant radiotherapy alone using a modified linear accelerator in methylation of p15/p16 gene promoter region by col- the Affiliated Cancer Hospital of Central South Uni- 42,76 79,80 lecting EZH2, downregulating p15/p16 , and upregu- versity . The total dose of administered radiotherapy lating the ability of promoting cell proliferation and anti- was 68 to 72 Gy (2 Gy/fraction, 5 days/week, followed by apoptosis of multidrug resistance protein (MDR1), rest for 2 days). Lymph node-negative patients received a mammalian target of rapamycin (mTOR), multidrug total dose of 60 Gy, and lymph node-positive patients resistance-associated proteins (MRP), and hypoxia/ received a total dose of 70 Gy. hypoxia-inducible factor-1α (HIF-1α) . In thyroid car- The clinical evaluation of radiosensitivity in patients is cinoma, PVT1 is involved in the formation of thyroid based on the first re-examination of computer tomo- carcinomas by collecting EZH2 and regulating the graphy scans after undergoing radiotherapy for a month, expression of thyrotropin receptor (TSHR) . Therefore, according to the following criteria: patients with radio- we hypothesized that in NPC, PVT1 might epigenetically resistant NPC were defined as those with persistent dis- regulate the related genes of radioresistance, thereby ease (incomplete regression of primary tumor and/or neck affecting the radiosensitivity of NPC. lymph nodes) at >3 months, or with local recurrent dis- The role of PVT1 in NPC radioresistance is summarized ease in the nasopharynx or neck lymph nodes or both in Fig. 6. Specifically, PVT1 can promote the radiotherapy within 12 months of completion of radiotherapy. Patients resistance of nasopharyngeal carcinoma. First, PVT1 can with radiosensitive NPC were defined as those who did promote DNA repair by phosphorylation of ATM/Chk2/ not experience either local residual lesions over 3 months p53 signaling pathway in NPC cells. PVT1 can also sig- (complete regression) or local recurrent disease for over nificantly downregulate cleaved caspase-9, cleaved cas- 12 months after completion of radiotherapy. The patients pase-7, and cleaved PARP, thereby inhibiting apoptosis were followed-up for 5–10 years. Disease-free survival was and ultimately causing radiation resistance. defined as the duration from the date of completion of primary radiotherapy to the date when recurrence or Materials and methods distant metastasis was confirmed by clinical evidence or Data mining and analysis pathological diagnosis. OS was defined as the period that To identify functional lncRNAs in NPC, we downloaded patients were alive from the date of first diagnosis to the three sets of gene expression profiling data for naso- date of death due to cancer progression or date of the end pharyngeal carcinomas, GSE64634, GSE12452, and of follow-up. GSE53819, from the Affymetrix Human Genome U133 Plus 2.0 platform-based studies in the GEO database. We In situ hybridization used the Significant Analysis of Microarray (SAM) soft- In situ hybridization was performed to detect PVT1 ware to analyze the differential expression of lncRNAs expression in NPC specimens using three different Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 9 of 12 nucleotide probes from different regions of PVT1; gly- U-3′; the sequences of non-target scrambled RNA con- ceraldehyde 3-phosphate dehydrogenase (GAPDH) was trols were provided by Invitrogen. For gene knockdown, used as positive control. The sequences of PVT1 probes cells were seeded overnight and transfected either with was as follows: Probe-1: 5′-GGT CGG ACT AGA AAA the siRNAs targeting LINC PVT1 or non-target scram- CCG GTC TTC CTC TAA TTT T-3′; Probe-2: 5′-GAG bled control siRNAs (Invitrogen) using Lipofectamine ACT GTA AAA ACT TCT CAG GTC TTA GGA-3′; RNAiMAX Reagent (Invitrogen) in OptiMEM medium Probe-3: 5′-CTC ATA AAA CTC TAA CCT CTT AAT (Invitrogen). TCT CGG TCA G-3′. The probes were synthesized and labeled with Digoxigenin-11-dUTP (DIG-dUTP) at the 3′ Cell proliferation assay and 5′ ends (Invitrogen, Shanghai, China). In situ hybri- The cells transfected with siRNA were seeded into 96- dization was performed as previously described . All well plates (500 cells per well), incubated for 24 h, and sections were independently scored by two pathologists irradiated with a dose of 6 Gy radiation. According to the who were blinded to the clinicopathological features and manufacturer’s instructions, a Cell Counting Kit-8 (CCK- the clinical data. 8) kit (Dojindo Laboratories, Kumamoto, Japan) was used to determine cell proliferation after irradiation. The In situ hybridization evaluation criteria absorbance of each well was monitored by a spectro- The intensity and the distribution range of the staining photometer at a wavelength of 450 nm. Each experiment were determined by semi-quantitative treatment . Two was performed at least three times in triplicate wells. pathologists scored the results according to the following criteria. (1) Staining intensity: no observed cell staining Cloning formation assay was scored as 0; cells with light-brown cell staining as 1; A cloning formation assay was used for accessing the cells stained brown with no background staining, or dark- radiosensitivity of the NPC cells after incubation. First, brown-stained cells with light-brown background staining the cells were seeded in 6-well plates (500, 1000, 2000, were recorded as moderately positive, as 2; dark-brown- 3000, and 4000 cells per well in triplicate) and incubated stained cells with no background staining were recorded overnight. Next, the cells were exposed to irradiation as strongly positive as 3. (2) Number of positive cells: no doses of 0, 2, 4, 6, and 8 Gy, respectively. Then, after positive cells were scored as 0; less than 25% of positive 12 days of culture, the surviving fractions (>50 cells) were cells as 1; between 25 and 50% positive cells as 2; positive stained with 0.5% crystal violet and counted under a cells over 50% recorded as strongly positive as 3. The final microscope. Finally, the data were fitted to a linear scores were obtained by multiplying the two scores. The quadratic model to create the cloning formation survival results were as follows: 0 was considered as negative curves to evaluate the radiosensitivity of these cells. expression, and the final score was 0; 1−2 as weakly positive, and the final score was 1; 3−4 as moderately Cell apoptosis assay positive, and the final score was 2; 6−9 as strong positive, First, the 5-8F and CNE2 cells were seeded in 6-well and the final score was 3. plates and transfected with siRNA for 24 h. Next, these All the target cells of each tissue were counted under cells were exposed to irradiation doses of 4 and 6 Gy, ×10 magnification, and the counting was repeated twice. respectively. After 48 h, the cells were harvested. The average values obtained from the counting was According to the protocols, an annexin V-fluorescein used for statistical analysis. In situ hybridization scores of isothiocyanate/propidium iodide apoptosis detection kit 2 and 3 indicated high expression of PVT1, and immu- (BD, San Jose, CA, USA) was used to stain cells and the nohistochemistry scores of 0 and 1 indicated low cell apoptosis was immediately detected by flow expression of PVT1. All the samples were scored inde- cytometry. pendently by two experienced pathologists who were double-blinded. Real-time PCR Cellular RNA was extracted using TRIzol reagent Cell line and siRNAs (Invitrogen, Carlsbad, CA, USA). For performing Real- Human NPC cell lines CNE2 and 5-8F were maintained Time Quantitative Reverse Transcription PCR (qRT- 47,49–53,81 in our laboratory . Cells were grown in RPMI- PCR), RNA was reverse transcribed to cDNA by using a 1640 medium (Life Technologies, Grand Island, NY) PrimeScript RT reagent Kit (Takara, Dalian, China). qRT- supplemented with 10% fetal bovine serum (Life Tech- PCR was performed using a SYBR_PremixExTaqII kit nologies), and 1% penicillin–streptomycin (Life Technol- (Takara, Dalian, China) in the CFX96 Real-Time PCR ogies) in a humidified incubator with 5% CO at 37 °C. Detection System (Bio-Rad, Hercules, CA, USA) to The sequence of the LINC PVT1 targeting siRNAs was as determine the relative expression levels of target genes. follows: 5′-CAC UAC UGA CCU UGC AGC UUA UUA The sequences of qRT-PCR primers are as follows: LINC Official journal of the Cell Death Differentiation Association He et al. Cell Death and Disease (2018) 9:235 Page 10 of 12 PVT1: forward primer 5′-TGG CTG AGA GGG TTG Acknowledgements This study was supported by grants from the National Natural Science AGA TC-3′ and reverse primer 5′-GCT GTA TGT GCC Foundation of China (81472531, 81572787, 81672683, 81672993, 81772928, AAG GTC AC-3′; β-actin: forward primer 5′-TCA CCA and 81702907), the Natural Science Foundation of Hunan Province ACT GGG ACG ACA TG-3′ and reverse primer 5′-GTC (2015JJ1022, 2016JC2035, and 2017SK2105), the Overseas Expertise Introduction Project for Discipline Innovation (111 Project, No. 111-2-12), and ACC GGA GTC CAT CAC GAT-3′; β-actin was used as the Fundamental Research Funds for the Central South University (2016zzts478 reference and normalization control. and 2014zzts066). Authors’ contributions Western blotting Y.H. wrote the manuscript and performed most assays. Y.J. and F.W. performed Lysis, electrophoresis, and target protein visualization the experiments related to radioresistance. Y.T., L.Y., and J.L. collected clinical were performed as described previously. Briefly, 50 μgof samples and performed qRT-PCR. P.Y., Q.N., and J.P. collected and analyzed the data. Q.L., F.X., C.G., B.X., X.L., M.Z., Y.L., and W.X. participated in the design of cell lysates were separated by 10% sodium dodecyl this study. Z.Z. and G.L. designed this study. All the authors have read and sulfate–polyacrylamide gel electrophoresis and transferred approved the final manuscript. onto a polyvinylidene fluoride membrane (Millipore, Billerica). To assess protein expression, the blots were Author details incubated overnight at 4 °C with the following primary Hunan Key Laboratory of Translational Radiation Oncology, Hunan Cancer antibodies: rabbit antibodies against caspase-7, caspase-9, Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, PARP, phospho-ATM (Ser1981), phospho-Chk2 Central South University, Changsha, Hunan, China. The Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, (a serine/threonine kinase) (Thr68), and mouse antibody Cancer Research Institute, Central South University, Changsha, Hunan, China. against phospho-p53 (Ser15) (Cell Signaling, Danvers, The Key Laboratory of Carcinogenesis of the Chinese Ministry of Health, MA). After washing with tris-buffered saline-Tween, Xiangya Hospital, Central South University, Changsha, Hunan, China. Hunan Key Laboratory of Nonresolving Inflammation and Cancer, Disease Genome the blots were incubated with horseradish peroxidase- Research Center, The Third Xiangya Hospital, Central South University, conjugated secondary antibodies (Cell Signaling Changsha, Hunan, China. Department of Cancer Biology, Lerner Research Technology) for 1 h at 37 °C. The signal was visualized Institute, Cleveland Clinic, Cleveland, OH, USA using enhanced chemiluminescence (EMD-Millipore, Conflict of interest Billerica, MA) and quantified by densitometry The authors declare that they have no conflict of interest. using ImageJ software (http://rsb.info.nih.gov/ij). GAPDH (Cell Signaling Technology) was used as a Publisher's note loading control. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Statistical analysis Data analyses were conducted by the GraphPad Prism 5 Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-018-0265-y). (GraphPad, La Jolla, CA). Survival curves were plotted using the Kaplan–Meier method, and compared using the Received: 4 October 2017 Revised: 9 December 2017 Accepted: 12 log-rank test. Student’s t-tests were used to evaluate December 2017 significant differences between any two groups of data; one-way analysis of variance was used if there were more than two groups. The dose survival curve was calculated by a linear quadratic model (Y = exp(−(a*x + b*(x^2)))). References 1. Thompson, L. Nasopharyngeal carcinoma. Ear. Nose. Throat J. 84,404–405 All data are represented as means ± standard (2005). deviations. P < 0.05 was considered statistically 2. Tu, C. et al. Genome-wide analysis of 18 Epstein-Barr viruses isolated from significant. primary nasopharyngeal carcinoma biopsy specimens. J. 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