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Long noncoding RNA PVT1 modulates hepatocellular carcinoma cell proliferation and apoptosis by recruiting EZH2

Long noncoding RNA PVT1 modulates hepatocellular carcinoma cell proliferation and apoptosis by... Background: We aimed to figure out the molecular network of PVT1 and EZH2 on hepatocellular carcinoma (HCC) cells growth. We also explored the interaction between PVT1, EZH2, MDM2 and P53. Methods: Microarray analysis was performed to screen for abnormally expressed genes in HCC tissues and PVT1 was identified as one gene significantly upregulated in HCC. CCK-8 assay, colony formation assay, and flow cytom- etry detected cell vitality, proliferation and apoptosis, respectively. RIP and RNA pull-down assays were employed to examine the connection between PVT1 and EZH2. The effect of PVT1 on the stability of EZH2 protein and the impact of EZH2 on MDM2 were detected by ELISA. Co-immunoprecipitation assay was used to evaluate the relationship between MDM2 and EZH2. Western blot detected the expression of EZH2, MDM2 and P53. Results: Up-regulated PVT1 was detected in HCC. Knockdown of PVT1 inhibited HCC cell propagation and promoted apoptotic cells. PVT1 could improve EZH2 protein stability by binding to EZH2 protein but have no significant impact on EZH2 mRNA expression. EZH2 protein stabilized MDM2 protein expression by binding to MDM2 protein. PVT1 enhanced the protein expression of EZH2 and MDM2 as well as inhibited P53 protein expression. Conclusions: PVT1 promoted HCC cell propagation and inhibited apoptotic cells by recruiting EZH2, stabilizing MDM2 protein expression and restraining P53 expression. Keywords: Hepatocellular carcinoma, PVT1, EZH2, MDM2, P53 Background Long non-coding RNAs (lncRNAs) with more than As an aggressive malignancies, hepatocellular carcinoma 200 nucleotides are short of protein coding potential [4]. (HCC) results in high mortality of patients [1]. Despite LncRNAs have been demonstrated to play a critical bio- great advances in uncovering molecular mechanisms logical role in carcinogenesis by regulating gene expres- underlying HCC over the past decades, HCC still men- sion [5]. Some publications reported that besides cancer aces patients’ life with high rate of tumor recurrence and diagnosis and prognosis, lncRNAs can serve as potential metastasis [2]. Therapy such as surgery, radiotherapy target of tumors treatment [6]. In HCC, some differen - and chemotherapy still has limited efficacy because most tially expressed lncRNAs have been characterized as patients are diagnosed at advantaged stages [3]. Thus, tumor promoter or suppressor. For example, linc00052 it’s urgent to characterize the pathogenic mechanisms of was down-regulated in HCC, and the upregulation of HCC in order to identify novel targets for HCC. linc00052 inhibited HCC cells migration and invasion [7]. In contrast, lncRNA PCAT-1 expression was aberrantly up-regulated in HCC, which induced HCC cell invasion and migration [8]. These studies revealed the diversity of *Correspondence: youngstrar_sf@163.com lncRNAs impacts on HCC progression. Scientific Research Office, Zibo Central Hospital, No. 54 West The plasmacytoma variant translocation 1 gene (PVT1) Gongqingtuan Road, Zhangdian District, Zibo 255000, Shandong, China is a copy number amplification-associated lncRNA [ 9]. Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Guo et al. Cancer Cell Int (2018) 18:98 Page 2 of 10 PVT1 functions as an oncogene, which contributes to the Microarray analysis phenotype of multiple cancers [10]. For example, PVT1 The gene microarray hybridization and sample analysis promoted the growth of non-small cell lung cancer [11]. were performed by Ribobio (Guangzhou, China), using PVT1 was up-regulated in thyroid tissues and cells, and the Human 8 × 60  k LncRNA Expression Microarray silenced PVT1 significantly restrained cell propagation V3.0 (AS-LNC-M-V3.0, Arraystar Inc., Rockville, USA). and arrested cell cycle at G0/G1 phase [12]. PVT1 has Total RNA was quantified with NanoDrop 2000 (Thermo been discovered to be associated with HCC progression Fisher Scientific Inc., USA). After synthesized with total in many literatures. Wang et  al. elucidated that PVT1 RNA, cDNA was labeled according to Nimblegen Gene promoted proliferation and cell cycling in HCC cells [13]. Expression Analysis protocol (Nimblegen Systems, Inc., Zhang et  al. found that PVT1 had high diagnostic value WI, USA). Data analysis was conducted by NimbleScan in HCC [14]. Therefore, the functions of PVT1 in HCC (Nim-blegen, USA). need to be further investigated. The enhancer of zeste homolog 2 (EZH2) is a subu - Cell culture nit of the multi-enzyme complex polycomb repressive Three Human HCC cells (HepG2, Huh7, SK-HEP-1, complex 2 and is involved in chromatin compaction and and BEL-7404) and a normal liver cell (HL-7702) were gene repression [15]. EZH2 was verified to contribute to purchased from the BeNa Culture Collection (Beijing, the aggressiveness of various human cancers [15]. For China). HepG2 and SK-HEP-1 in minimum essential example, EZH2 promotes cell proliferation in laryngeal medium (MEM, GIBCO BRL, USA) were cultured with carcinoma [16], inducing cell metastasis in oral cancer 10% fetal bovine serum (FBS, GIBCO BRL, USA); Huh7 [17], increasing cell invasion in endometrial cancer [18] was cultured in Dulbecco’s modified Eagle’s medium Dysregulation of EZH2 in HCC has been found in some (DMEM) with 10% FBS (GIBCO BRL); BEL-7404 studies. For instance, up-regulated EZH2 was measured and HL-7702 were maintained in RPMI1640 medium in HCC tissues, which was positively correlated with (GIBCO BRL, USA) supplemented with 10% FBS tumor grade and clinical stage [19]. Cheng et  al. found (GIBCO BRL). that downregulation of EZH2 inhibited HCC cell growth through inhibition of β-catenin signaling [20]. Some QRT‑PCR studies reported the mechanism of PVT1 and EZH2. TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and For example, PVT1 was verified to induce the increase NanoDrop 2000 (Thermo Fisher Scientific Inc., USA) of EZH2 in gastric cancer [10], thyroid cancer [12] and were utilized to isolate and quantify total RNA. RNA glioma [21]. However, the connection of PVT1 and EZH2 was reversely transcribed to cDNA by using ReverTra in HCC remains unclear. Ace qPCR RT Kit (Toyobo, Japan). QRT-PCR was con- In conclusion, our study demonstrated the effects of ducted by THUNDERBIRD SYBR qPCR Mix (Toyobo, −∆∆Ct PVT1 and EZH2 in HCC. In current study, we measured Japan). Internal controls were U6 and GADPH. 2 PVT1 expression in HCC, and examined the effects of method determined comparative quantification. Primer PVT1 on HCC cell activities. In addition, we performed sequences were exhibited at Table 1. experiments to confirm the connection between PVT1 and EZH2 as well as MDM2 in HCC cells. Our study showed that PVT1 was highly expressed in HCC tissues Table 1 Primer sequence and these results highlighted the crucial role of PVT1 in HCC, which may function as a therapies target for HCC. Gene Sequence PVT1-F 5′-ATA GAT CCT GCC CTG TTT GC-3′ PVT1-R 5′-CAT TTC CTG CTG CCG TTT TC-3 Methods GAPDH-F 5′-GGA GCG AGA TCC CTC CAA AAT-3′ Tissue specimens GAPDH-R 5′-GGC TGT TGT CAT ACT TCT CATGG-3′ A total of 121 HCC tissue samples and matched non- EZH2-F 5′-TTG TTG GCG GAA GCGTG-3′ tumor normal tissue samples were obtained from EZH2-R 5′-TCC CTA GTC CCG CGC AAT GTGC-3 patients who underwent surgical resection without pcDNA3.1-PVT1-F 5′-GGG GTA CCC TCC GGG CAG AGC GCG TGTG-3′ any form of preoperative chemotherapy and/or radia- pcDNA3.1-PVT1-R 5′-CGG GAT CCT AGA CAC GAG GCC GGC CACGC-3′ tion therapy at Zibo Central Hospital. All tissues were si-EZH2 5′-AUC AGC UCG UCU GAA CCU CUU-3′ immediately stored at − 80  °C in liquid nitrogen. Writ- si-NC 5′- GGG CCA GAC TGG GAA GAA A -3′ ten informed consent was taken from all participants and si-MDM2 5′-GTG TGT AAT AAG GGA GAT A-3′ this study was approved by the ethics committee of Zibo si-PVT1 5′-CAG CCA TCA TGA TGG TAC T-3′ Central Hospital. Guo et al. Cancer Cell Int (2018) 18:98 Page 3 of 10 Cell transfection USA) according to the manual. The supernatants of cell Si-PVT1, PVT1-pcDNA3.1 and pcDNA3.1 were syn- extracts were incubated with treated-beads for 6 h. We thesized by GenePharma (Shanghai, China). Cells used the RIP wash buffer to wash the beads for 6 times. were seeded into 6-well plates (1 × 10 ). Transfection The purified RNA was used for qRT-PCR analysis. was performed by Lipofectamine 2000 (Life Technolo- gies, USA). Cells transfected with pcDNA3.1 served RNA pull‑down assay as NC group; cells transfected with PVT1-pcDNA3.1 PVT1 and the antisense RNA were transcribed by was regarded as PVT1 group; cells transfected with si- mMESSAGE mMACHINE T7 Kit (Ambion, USA) and PVT1 and pcDNA3.1 served as si-PVT1 group. RNeasy Mini Kit (Qiagen, Valencia, CA) in vitro, biotin- labeled using Pierce RNA 3′ End Desthiobiotinylation CCK‑8 assay Kit (Thermo Scientific, USA). 1  mg total protein from 8 × 10 HepG2 and Huh 7 cells per well transfected with HepG2 to Huh7 cell extracts were mixed with 50 pmol of si-NC or si-PVT1 were incubated into 96 well and cell biotinylated PVT1 for 1 h, and then added with 60 µL of vitality was assessed by Cell Counting Kit-8 (Biotech- Streptavidin Beads (Invitrogen) for 1 h. The proteins were well, Shanghai, China) at 24, 48, 72 and 96 h according resolved by 10% SDS-PAGE, detected by conventional to the manual. Absorbance was recorded at 490 nm. western blot analysis. Colony information assay ELISA Cells transfected with si-NC or si-PVT1 were collected After transfected with si-NC, si-PVT1 or si-EZH2, at logarithmic growth phase. Afterwards, cells were HepG2 and Huh7 cells in 24-well plates (2 × 10 /well) placed in a 6-well plate (1 × 10 /well) for 2 weeks. After were added with cyclohexane (CHX, 100  μg/mL, Sigma, discarding the medium, we used 4% paraformaldehyde USA). Total protein was extracted from cell culture to fix cells for 15  min, and added Giemsa solution to media using RIPA buffer (Beyotime, Shanghai, China), stain for 5 min. followed by blocking with 5% non-fat PBS for 1  h. After participation of primary antibody for 30  min, cells were added with second antibody for another 30 min and then Flow cytometry added with TMB for 15  min. 2  mol/L H SO was added Cell apoptosis was assessed by Annexin V-FITC (Key- 2 4 for 5  min and the optical density was read at 450  nm gen, China). Cells were re-suspended in 500 μL binding using a VICTOR3 Microplate Reader (Perkin Elmer; buffer containing 5  μL propidium iodide (PI) and 5  μL Waltham, MA). annexin V-FITC. Cells were analyzed by BD Accuri C6 flow cytometer (BD, USA). Co‑immunoprecipitation (Co‑IP) assay For co-immunoprecipitation, HepG2 and Huh7 cells at Western blot 48-hour post-transfection were washed twice with PBS Protein samples were harvested from cells to were lysed and lysed in CytoBuster Protein Extraction Reagent (Mil- in RIPA buffer (Beyotime, Shanghai, China). The con - lipore). Cell lysates were purified with Protein G Plus- centration of protein was determined using Pierce BCA Agarose beads (Santa Cruz) and incubated with prepared Protein Assay Kit (Pierce, Rockford, IL, USA). After sepa- antibody-conjugated beads overnight. The beads were rated by 10% SDS-polyacrylamide gel electrophoresis dissolved in 30  μL SDS-PAGE loading buffer for protein (SDS-PAGE), proteins were transferred to polyvinylidene analysis with the indicated antibodies. difluoride membranes (PVDF, Millipore, USA), blocked with TBST including 5% non-fat skimmed milk. Primary Statistical analysis antibodies were added (anti-P53, ab1431, 0.5  µg/mL; GraphPad Prism 6.0 software (GraphPad Software Inc., anti-GAPDH, ab181603, 1:10,000; anti-EZH2, ab191080, USA) analyzed all statistics. The measured parameters 1:500; anti-MDM2, ab38618, 1:1000, Abcam, Cambridge, presented as mean ± SD. Statistical tests for data analysis MA, USA). After washed three times with TBST, second were two-tailed t test and Chi square tests. A probability anti-body was added (IgG-HRP, ab7090, 1:2000, Abcam, level of 0.05 was chosen for statistical significance. Cambridge, MA, USA) for 1.5  h. Signal detection was carried out with an ECL system (Life Technology, USA). Results PVT1 increased in HCC tissues and correlated RNA immunoprecipitation (RIP) assay with clinicopathologic characters of HCC RIP assay was conducted by the Magna RIP RNA-Bind- Microarray analysis elucidated that PVT1 was up-reg- ing Protein Immunoprecipitation Kit (Millipore, MA, ulated in HCC tissues (Fig.  1a, fold change > 2, P < 0.05). Guo et al. Cancer Cell Int (2018) 18:98 Page 4 of 10 Fig. 1 PVT1 was up-regulated in HCC tissues. a Heatmap indicated that PVT1 was up-regulated in tumors and down-regulated in normal tissues. b QRT-PCR revealed that PVT1 expression in tumors was much higher than that in normal tissues. **P < 0.01, compared with normal tissues The results of qRT-PCR confirmed overexpression of Table 2 Clinicopathological correlation of  lncRNA PVT1 expression in human HCCs PVT1 in HCC tissues (Fig.  1b). Furthermore, through comparing the clinical information of 121 patients, we a Variables No. of casesPVT1 expression P value* found that PVT1 expression was linked to tumor size, Low High number, grade and stage (Table 2). Age (years) 0.99 ≥ 60 47 28 19 Knockdown of PVT1 inhibited HCC cell propagation < 60 74 44 30 and promoted apoptotic cells Gender 0.801 QRT-PCR evaluated PVT1 expression in different HCC Female 65 38 27 cell lines. As shown in Fig.  2a, four cell lines (HepG2, Male 56 34 22 Huh7, SK-HEP-1 and BEL-7404) expressed higher lev- AFP level (ng/mL) 0.082 els of PVT1 compared with normal cell line (HL-7702). ≥ 400 60 31 29 PVT1 in HepG2 and Huh7 cell lines exhibited the high- < 400 61 41 20 est level among four cell line and were selected for further Tumor size (cm) 0.003 experiments. After knockdown of PVT1 in HepG2 and ≥ 5 62 29 33 Huh7 cells, PVT1 expression decreased compared with < 5 59 43 16 control group (Fig. 2b, P < 0.05). CCK-8 and colony forma- Tumor number 0.003 tion assays elucidated that cell vitality and proliferation in Single 54 40 14 si-PVT1 group were attenuated compared with control Multiple 67 32 35 group (Fig.  2c, d, all P < 0.05). Furthermore, knockdown Tumor grade 0.001 of PVT1 induced apoptosis in HepG2 and Huh7 cells G1 59 46 13 through flow cytometry analysis (Fig. 2 e, P < 0.05). G2 + G3 62 26 36 Tumor stage 0.010 PVT1 improved EZH2 protein stability I + II 67 52 15 RIP experiments were conducted in HepG2 and Huh7 III 54 20 34 cells, which revealed PVT1 enrichment in EZH2-RNA PVT1 plasmacytoma variant translocation 1, AFP alphafetoprotein participation compared with si-NC group (Fig.  3a, * For analysis of correlation between PVT1 and clinical features, Pearson’s Chi P < 0.05). In addition, knockdown of PVT1 had no impact square tests were used. Results were considered statistically significant at on EZH2 mRNA expression (Fig. 3b) and RNA pull-down P < 0.05 with italics font assays in HepG2 and Huh7 cells revealed that EZH2 The average expression level was used as the cutoff. Low expression of PVT1 in 72 patients was classified as values below the fold change of 5.44 while high interacted with PVT1 (Fig.  3c, P < 0.05). However, PVT1 PVT1 expression in 49 patients was classified as values above the level was not directly bound to MDM2 or P53 (Additional Guo et al. Cancer Cell Int (2018) 18:98 Page 5 of 10 Fig. 2 Knockdown of PVT1 inhibited HCC cell proliferation and promoted cell apoptosis. a PVT1 was highly expressed in four HCC cell lines (HepG2, Huh7, SK-HEP-1 and BEL-7404) compared with normal cell line (HL-7702). **P < 0.01, *P < 0.05, compared with HL-7702 cell line. b PVT1 expression in HepG2 and Huh7 cells transfected with si-PVT1 was lower than that in cells transfected with si-NC. *P < 0.05, compared with si-NC group. c Cell vitality of HepG2 and Huh7 cells transfected with si-PVT1 was lower than that in cell s transfected with si-NC detected by CCK-8 assay. *P < 0.01, **P < 0.05, compared with si-NC group. d Colony formation assay indicated that the number of colony cells in si-PVT1 group was lower than that in si-NC group. *P < 0.05, compared with si-NC group. e Flow cytometry showed that apoptosis rate in si-PVT1 group was higher than that in si-NC group. *P < 0.05, compared with si-NC group Guo et al. Cancer Cell Int (2018) 18:98 Page 6 of 10 Fig. 3 PVT1 could bind to EZH2. a RIP assay indicated that PVT1 levels of EZH2 RIP vs IgG RIP in si-PVT1 group was higher than that in si-NC group. **P < 0.01, compared with si-NC group. b QRT-PCR indicated that EZH2 mRNA expression in si-PVT1 group had no significant difference between that in si-NC group. c RNA pull-down assay indicated that PVT1 bound to EZH2. d Western blot revealed that EZH2 protein expression in si-PVT1 group was lower than that si-NC group. *P < 0.05, compared with si-NC group. e Western blot indicated that after added with 100 μg/mL CHX, si-PVT1 had greater inhibitory effect on EZH2 protein expression with the increase of time. f ELISA assay revealed that EZH2 protein expression in si-PVT1 group was lower than that in si-NC group with the increase of time after added with 100 μg/mL CHX. *P < 0.05, compared with si-NC group Guo et al. Cancer Cell Int (2018) 18:98 Page 7 of 10 file  1: Figure S1). In contrast, si-PVT1 inhibited EZH2 knockdown significantly inhibited prostate cancer protein expression (Fig.  3d, P < 0.05). Western blot and growth in vivo and in vitro and promoted cell apoptosis ELISA assays demonstrated that si-PVT1 decreased [26]. Zhuang et al. found that PVT1 silencing inhibited the EZH2 protein stability (Fig.  3e, f, P < 0.05). Taken bladder cancer cell growth and induced apoptosis [27]. together, PVT1 could improve EZH2 protein stability but Based on previous studies, we speculated that PVT1 have no significant impact on EZH2 mRNA expression. might have impacts on HCC cell process. In cur- rent study, PVT1 in HCC was aberrantly higher. After knockdown of PVT1, inhibited HCC cell proliferation EZH2 protein stabilized MDM2 protein expression was inhibited while cell apoptosis was enhanced. Con- In Co-IP assay, we found that EZH2 could bind to MDM2 sistently, Gou et al. also found that PVT1 was increased (Fig.  4a). To further study the interaction between in HCC, which promoted cell proliferation and inva- EZH2 and MDM2, we successfully transfected HepG2 sion in HCC [6]. Lan et  al. demonstrated that PVT1 and Huh7 cells with si-EZH2 and si-MDM2 (Fig.  4b, promoted proliferation, invasion and migration in HCC P < 0.05). Measured by western blot, knockdown of EZH2 cells by regulating miR-186-5p [28]. decreased MDM2 protein expression, whereas knock- EZH2 has been thought to contribute to malignant down of MDM2 had no significant impact on EZH2 pro - transformation due to its role in regulating funda- tein expression (Fig.  4c). Meanwhile, the MDM2 protein mental cellular processes [29]. Previously, some stud- half-life decreased after knockdown of EZH2 (Fig.  4d, e, ies have reported the correlation between PVT1 and P < 0.05). Therefore, EZH2 protein improved the stability EZH2. For example, PVT1 induced lung adenocarci- of MDM2 protein. noma progression through LATS2/MDM2/P53 path- way suppressed by EZH2 [30]. Kong et al. revealed that PVT1 enhanced EZH2 and MDM2 expression as well PVT1 recruited EZH2 and contributed to gastric can- as inhibited P53 protein expression cer growth [10]. In our study, we performed RIP, RNA After transfected with pcDNA3.1-PVT1 and si-PVT1 in pull-down and ELISA assays to examine the relation- HepG2 and Huh7 cells, western blot was employed to ship between PVT1 and EZH2. All experiments dem- determine the protein expression of EZH2, MDM2 and onstrated that PVT1 improved EZH2 protein stability P53. The results elucidated that overexpression of PVT1 by binding to EZH2. Furthermore, we found that EZH2 improved the protein expression of EZH2 and MDM2 protein stabilized MDM2 protein expression by binding as well as inhibited P53 protein expression. In contrast, to MDM2 detected by co-IP assay. Previous study has knockdown of PVT1 had the opposite effects (Fig. 5). claimed that as a key negative regulator of the tumor suppressor p53, MDM2 is recruited to target gene pro- Discussion moters by EZH2 [31]. We also employed western blot Mounting evidence has highlighted the critical roles of to measure the effects of PVT1 on EZH2, MDM2 and lncRNAs in HCC cellular process [22]. We performed P53. The results indicated that PVT1 enhanced the series experiments to investigate the function of PVT1 on protein expressions of EZH2 and MDM2 as well as HCC. First, we confirmed PVT1 overexpression in HCC, inhibited P53 protein expression. In this study, we first and inhibition of PVT1 suppressed HCC cell propaga- clearly elucidated the molecular mechanism underlying tion and promoted apoptotic cells. Second, we employed PVT1, EZH2, MDM2 and P53 in HCC. RIP, RNA pull-down and ELISA assays to examine the However, some concerns still existed in our study. In connection of PVT1 and EZH2, finding that PVT1 could current study, we only investigated the effects of PVT1 improve EZH2 protein stability by binding to EZH2. on P53 expression, while the molecular network between Meanwhile, the results of co-IP assay revealed that EZH2 EZH2, MDM2 and P53 need to be further explored. On protein stabilized MDM2 protein expression. Addition- the other hand, the pathway of PVT1, EZH2 and P53 in ally, PVT1 enhanced the protein expression of EZH2 and HCC should be taken into consideration. MDM2 as well as inhibited P53 protein expression in HCC cells. LncRNAs are involved in regulating biological func- Conclusions tions and gene expression in physiological and patho- In summary, our findings suggested that PVT1 had posi - logical contexts, such as cancer [23]. Growing evidence tive effects on HCC cell growth. Additionally, PVT1 sta - has clarified the functions of different lncRNAs in bilized the protein expression of EZH2 and MADM2. HCC, including lncRNA-PVT1 [24]. PVT1 functioned PVT1 might be a potential therapies target of HCC treat- as an oncogenic lncRNA in multiple types of cancers ment. Our experiments may improve the diagnostic abil- [25]. PVT1 overexpression has been identified as pre - ity of biomarkers for HCC in clinical practice. dictor for many carcinomas [9]. For example, PVT1 Guo et al. Cancer Cell Int (2018) 18:98 Page 8 of 10 Fig. 4 EZH2 protein stabilized MDM2 protein expression. a Co-IP assay indicated that EZH2 bound to MDM2. b QRT-PCR indicated that EZH2 mRNA in si-EZH2 group was lower than that in si-NC group. **P < 0.01, compared with si-NC group. c Western blot showed that MDM2 protein expression in si-EZH2 group was lower than that in si-NC group, while EZH2 protein expression in si-MDM2 group had no significant difference with that in si-NC group. d Western blot indicated that after added with 100 μg/mL CHX, si-EZH2 had greater inhibitory effect on MDM2 protein expression with the increase of time. f ELISA assay revealed that MDM2 protein expression in si-EZH2 group was lower than that in si-NC group with the increase of time after added with 100 μg/mL CHX. *P < 0.05, compared with si-NC group Guo et al. Cancer Cell Int (2018) 18:98 Page 9 of 10 Fig. 5 PVT1 enhanced the protein expression of EZH2 and MDM2 as well as inhibited P53 protein expression. a, b Western blot demonstrated that overexpression of PVT1 improved the protein expression level of EZH2 and MDM2 as well as inhibited P53 protein expression in HepG2 and Huh7 cells. In contrast, knockdown of PVT1 inhibited the protein expression level of EZH2 and MDM2 as well as promoted P53 protein expression Competing interests Additional file The authors declare that they have no competing interests. Availability of data and materials Additional file 1: Figure S1. PVT1 could not directly bind to MDM2 or Please contact the corresponding author for data. P53. (A) RNA pull-down assay indicated that PVT1 could not directly bind to MDM2 or P53. Consent for publication Obtained. Ethics approval and consent to participate Abbreviations This study was approved by the ethics committee of Zibo Central Hospital. Co-IP: co-immunoprecipitation; CHX: cyclohexane; EZH2: enhancer of zeste ( The related Ethics Committee Approval Form has been achieved.) homolog 2; HCC: hepatocellular carcinoma; lncRNAs: long non-coding RNAs; PI: propidium iodide; PVT1: plasmacytoma variant translocation 1 gene; RIP: Funding RNA immunoprecipitation; SDS-PAGE: SDS-polyacrylamide gel electrophoresis. Not applicable. Authors’ contributions JG made substantial contributions to conception and design. LL made acqui- Publisher’s Note sition of data. CH and CW made analysis and interpretation of data. JG have Springer Nature remains neutral with regard to jurisdictional claims in pub- been involved in drafting the manuscript. LL, CH and CW have been involved lished maps and institutional affiliations. in revising it critically for important intellectual content. All authors agreed to be accountable for all aspects of the work. All authors read and approved the Received: 20 April 2018 Accepted: 12 June 2018 final manuscript. 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Long noncoding RNA PVT1 modulates hepatocellular carcinoma cell proliferation and apoptosis by recruiting EZH2

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Publisher
Springer Journals
Copyright
2018 The Author(s)
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1475-2867
DOI
10.1186/s12935-018-0582-3
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Abstract

Background: We aimed to figure out the molecular network of PVT1 and EZH2 on hepatocellular carcinoma (HCC) cells growth. We also explored the interaction between PVT1, EZH2, MDM2 and P53. Methods: Microarray analysis was performed to screen for abnormally expressed genes in HCC tissues and PVT1 was identified as one gene significantly upregulated in HCC. CCK-8 assay, colony formation assay, and flow cytom- etry detected cell vitality, proliferation and apoptosis, respectively. RIP and RNA pull-down assays were employed to examine the connection between PVT1 and EZH2. The effect of PVT1 on the stability of EZH2 protein and the impact of EZH2 on MDM2 were detected by ELISA. Co-immunoprecipitation assay was used to evaluate the relationship between MDM2 and EZH2. Western blot detected the expression of EZH2, MDM2 and P53. Results: Up-regulated PVT1 was detected in HCC. Knockdown of PVT1 inhibited HCC cell propagation and promoted apoptotic cells. PVT1 could improve EZH2 protein stability by binding to EZH2 protein but have no significant impact on EZH2 mRNA expression. EZH2 protein stabilized MDM2 protein expression by binding to MDM2 protein. PVT1 enhanced the protein expression of EZH2 and MDM2 as well as inhibited P53 protein expression. Conclusions: PVT1 promoted HCC cell propagation and inhibited apoptotic cells by recruiting EZH2, stabilizing MDM2 protein expression and restraining P53 expression. Keywords: Hepatocellular carcinoma, PVT1, EZH2, MDM2, P53 Background Long non-coding RNAs (lncRNAs) with more than As an aggressive malignancies, hepatocellular carcinoma 200 nucleotides are short of protein coding potential [4]. (HCC) results in high mortality of patients [1]. Despite LncRNAs have been demonstrated to play a critical bio- great advances in uncovering molecular mechanisms logical role in carcinogenesis by regulating gene expres- underlying HCC over the past decades, HCC still men- sion [5]. Some publications reported that besides cancer aces patients’ life with high rate of tumor recurrence and diagnosis and prognosis, lncRNAs can serve as potential metastasis [2]. Therapy such as surgery, radiotherapy target of tumors treatment [6]. In HCC, some differen - and chemotherapy still has limited efficacy because most tially expressed lncRNAs have been characterized as patients are diagnosed at advantaged stages [3]. Thus, tumor promoter or suppressor. For example, linc00052 it’s urgent to characterize the pathogenic mechanisms of was down-regulated in HCC, and the upregulation of HCC in order to identify novel targets for HCC. linc00052 inhibited HCC cells migration and invasion [7]. In contrast, lncRNA PCAT-1 expression was aberrantly up-regulated in HCC, which induced HCC cell invasion and migration [8]. These studies revealed the diversity of *Correspondence: youngstrar_sf@163.com lncRNAs impacts on HCC progression. Scientific Research Office, Zibo Central Hospital, No. 54 West The plasmacytoma variant translocation 1 gene (PVT1) Gongqingtuan Road, Zhangdian District, Zibo 255000, Shandong, China is a copy number amplification-associated lncRNA [ 9]. Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Guo et al. Cancer Cell Int (2018) 18:98 Page 2 of 10 PVT1 functions as an oncogene, which contributes to the Microarray analysis phenotype of multiple cancers [10]. For example, PVT1 The gene microarray hybridization and sample analysis promoted the growth of non-small cell lung cancer [11]. were performed by Ribobio (Guangzhou, China), using PVT1 was up-regulated in thyroid tissues and cells, and the Human 8 × 60  k LncRNA Expression Microarray silenced PVT1 significantly restrained cell propagation V3.0 (AS-LNC-M-V3.0, Arraystar Inc., Rockville, USA). and arrested cell cycle at G0/G1 phase [12]. PVT1 has Total RNA was quantified with NanoDrop 2000 (Thermo been discovered to be associated with HCC progression Fisher Scientific Inc., USA). After synthesized with total in many literatures. Wang et  al. elucidated that PVT1 RNA, cDNA was labeled according to Nimblegen Gene promoted proliferation and cell cycling in HCC cells [13]. Expression Analysis protocol (Nimblegen Systems, Inc., Zhang et  al. found that PVT1 had high diagnostic value WI, USA). Data analysis was conducted by NimbleScan in HCC [14]. Therefore, the functions of PVT1 in HCC (Nim-blegen, USA). need to be further investigated. The enhancer of zeste homolog 2 (EZH2) is a subu - Cell culture nit of the multi-enzyme complex polycomb repressive Three Human HCC cells (HepG2, Huh7, SK-HEP-1, complex 2 and is involved in chromatin compaction and and BEL-7404) and a normal liver cell (HL-7702) were gene repression [15]. EZH2 was verified to contribute to purchased from the BeNa Culture Collection (Beijing, the aggressiveness of various human cancers [15]. For China). HepG2 and SK-HEP-1 in minimum essential example, EZH2 promotes cell proliferation in laryngeal medium (MEM, GIBCO BRL, USA) were cultured with carcinoma [16], inducing cell metastasis in oral cancer 10% fetal bovine serum (FBS, GIBCO BRL, USA); Huh7 [17], increasing cell invasion in endometrial cancer [18] was cultured in Dulbecco’s modified Eagle’s medium Dysregulation of EZH2 in HCC has been found in some (DMEM) with 10% FBS (GIBCO BRL); BEL-7404 studies. For instance, up-regulated EZH2 was measured and HL-7702 were maintained in RPMI1640 medium in HCC tissues, which was positively correlated with (GIBCO BRL, USA) supplemented with 10% FBS tumor grade and clinical stage [19]. Cheng et  al. found (GIBCO BRL). that downregulation of EZH2 inhibited HCC cell growth through inhibition of β-catenin signaling [20]. Some QRT‑PCR studies reported the mechanism of PVT1 and EZH2. TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and For example, PVT1 was verified to induce the increase NanoDrop 2000 (Thermo Fisher Scientific Inc., USA) of EZH2 in gastric cancer [10], thyroid cancer [12] and were utilized to isolate and quantify total RNA. RNA glioma [21]. However, the connection of PVT1 and EZH2 was reversely transcribed to cDNA by using ReverTra in HCC remains unclear. Ace qPCR RT Kit (Toyobo, Japan). QRT-PCR was con- In conclusion, our study demonstrated the effects of ducted by THUNDERBIRD SYBR qPCR Mix (Toyobo, −∆∆Ct PVT1 and EZH2 in HCC. In current study, we measured Japan). Internal controls were U6 and GADPH. 2 PVT1 expression in HCC, and examined the effects of method determined comparative quantification. Primer PVT1 on HCC cell activities. In addition, we performed sequences were exhibited at Table 1. experiments to confirm the connection between PVT1 and EZH2 as well as MDM2 in HCC cells. Our study showed that PVT1 was highly expressed in HCC tissues Table 1 Primer sequence and these results highlighted the crucial role of PVT1 in HCC, which may function as a therapies target for HCC. Gene Sequence PVT1-F 5′-ATA GAT CCT GCC CTG TTT GC-3′ PVT1-R 5′-CAT TTC CTG CTG CCG TTT TC-3 Methods GAPDH-F 5′-GGA GCG AGA TCC CTC CAA AAT-3′ Tissue specimens GAPDH-R 5′-GGC TGT TGT CAT ACT TCT CATGG-3′ A total of 121 HCC tissue samples and matched non- EZH2-F 5′-TTG TTG GCG GAA GCGTG-3′ tumor normal tissue samples were obtained from EZH2-R 5′-TCC CTA GTC CCG CGC AAT GTGC-3 patients who underwent surgical resection without pcDNA3.1-PVT1-F 5′-GGG GTA CCC TCC GGG CAG AGC GCG TGTG-3′ any form of preoperative chemotherapy and/or radia- pcDNA3.1-PVT1-R 5′-CGG GAT CCT AGA CAC GAG GCC GGC CACGC-3′ tion therapy at Zibo Central Hospital. All tissues were si-EZH2 5′-AUC AGC UCG UCU GAA CCU CUU-3′ immediately stored at − 80  °C in liquid nitrogen. Writ- si-NC 5′- GGG CCA GAC TGG GAA GAA A -3′ ten informed consent was taken from all participants and si-MDM2 5′-GTG TGT AAT AAG GGA GAT A-3′ this study was approved by the ethics committee of Zibo si-PVT1 5′-CAG CCA TCA TGA TGG TAC T-3′ Central Hospital. Guo et al. Cancer Cell Int (2018) 18:98 Page 3 of 10 Cell transfection USA) according to the manual. The supernatants of cell Si-PVT1, PVT1-pcDNA3.1 and pcDNA3.1 were syn- extracts were incubated with treated-beads for 6 h. We thesized by GenePharma (Shanghai, China). Cells used the RIP wash buffer to wash the beads for 6 times. were seeded into 6-well plates (1 × 10 ). Transfection The purified RNA was used for qRT-PCR analysis. was performed by Lipofectamine 2000 (Life Technolo- gies, USA). Cells transfected with pcDNA3.1 served RNA pull‑down assay as NC group; cells transfected with PVT1-pcDNA3.1 PVT1 and the antisense RNA were transcribed by was regarded as PVT1 group; cells transfected with si- mMESSAGE mMACHINE T7 Kit (Ambion, USA) and PVT1 and pcDNA3.1 served as si-PVT1 group. RNeasy Mini Kit (Qiagen, Valencia, CA) in vitro, biotin- labeled using Pierce RNA 3′ End Desthiobiotinylation CCK‑8 assay Kit (Thermo Scientific, USA). 1  mg total protein from 8 × 10 HepG2 and Huh 7 cells per well transfected with HepG2 to Huh7 cell extracts were mixed with 50 pmol of si-NC or si-PVT1 were incubated into 96 well and cell biotinylated PVT1 for 1 h, and then added with 60 µL of vitality was assessed by Cell Counting Kit-8 (Biotech- Streptavidin Beads (Invitrogen) for 1 h. The proteins were well, Shanghai, China) at 24, 48, 72 and 96 h according resolved by 10% SDS-PAGE, detected by conventional to the manual. Absorbance was recorded at 490 nm. western blot analysis. Colony information assay ELISA Cells transfected with si-NC or si-PVT1 were collected After transfected with si-NC, si-PVT1 or si-EZH2, at logarithmic growth phase. Afterwards, cells were HepG2 and Huh7 cells in 24-well plates (2 × 10 /well) placed in a 6-well plate (1 × 10 /well) for 2 weeks. After were added with cyclohexane (CHX, 100  μg/mL, Sigma, discarding the medium, we used 4% paraformaldehyde USA). Total protein was extracted from cell culture to fix cells for 15  min, and added Giemsa solution to media using RIPA buffer (Beyotime, Shanghai, China), stain for 5 min. followed by blocking with 5% non-fat PBS for 1  h. After participation of primary antibody for 30  min, cells were added with second antibody for another 30 min and then Flow cytometry added with TMB for 15  min. 2  mol/L H SO was added Cell apoptosis was assessed by Annexin V-FITC (Key- 2 4 for 5  min and the optical density was read at 450  nm gen, China). Cells were re-suspended in 500 μL binding using a VICTOR3 Microplate Reader (Perkin Elmer; buffer containing 5  μL propidium iodide (PI) and 5  μL Waltham, MA). annexin V-FITC. Cells were analyzed by BD Accuri C6 flow cytometer (BD, USA). Co‑immunoprecipitation (Co‑IP) assay For co-immunoprecipitation, HepG2 and Huh7 cells at Western blot 48-hour post-transfection were washed twice with PBS Protein samples were harvested from cells to were lysed and lysed in CytoBuster Protein Extraction Reagent (Mil- in RIPA buffer (Beyotime, Shanghai, China). The con - lipore). Cell lysates were purified with Protein G Plus- centration of protein was determined using Pierce BCA Agarose beads (Santa Cruz) and incubated with prepared Protein Assay Kit (Pierce, Rockford, IL, USA). After sepa- antibody-conjugated beads overnight. The beads were rated by 10% SDS-polyacrylamide gel electrophoresis dissolved in 30  μL SDS-PAGE loading buffer for protein (SDS-PAGE), proteins were transferred to polyvinylidene analysis with the indicated antibodies. difluoride membranes (PVDF, Millipore, USA), blocked with TBST including 5% non-fat skimmed milk. Primary Statistical analysis antibodies were added (anti-P53, ab1431, 0.5  µg/mL; GraphPad Prism 6.0 software (GraphPad Software Inc., anti-GAPDH, ab181603, 1:10,000; anti-EZH2, ab191080, USA) analyzed all statistics. The measured parameters 1:500; anti-MDM2, ab38618, 1:1000, Abcam, Cambridge, presented as mean ± SD. Statistical tests for data analysis MA, USA). After washed three times with TBST, second were two-tailed t test and Chi square tests. A probability anti-body was added (IgG-HRP, ab7090, 1:2000, Abcam, level of 0.05 was chosen for statistical significance. Cambridge, MA, USA) for 1.5  h. Signal detection was carried out with an ECL system (Life Technology, USA). Results PVT1 increased in HCC tissues and correlated RNA immunoprecipitation (RIP) assay with clinicopathologic characters of HCC RIP assay was conducted by the Magna RIP RNA-Bind- Microarray analysis elucidated that PVT1 was up-reg- ing Protein Immunoprecipitation Kit (Millipore, MA, ulated in HCC tissues (Fig.  1a, fold change > 2, P < 0.05). Guo et al. Cancer Cell Int (2018) 18:98 Page 4 of 10 Fig. 1 PVT1 was up-regulated in HCC tissues. a Heatmap indicated that PVT1 was up-regulated in tumors and down-regulated in normal tissues. b QRT-PCR revealed that PVT1 expression in tumors was much higher than that in normal tissues. **P < 0.01, compared with normal tissues The results of qRT-PCR confirmed overexpression of Table 2 Clinicopathological correlation of  lncRNA PVT1 expression in human HCCs PVT1 in HCC tissues (Fig.  1b). Furthermore, through comparing the clinical information of 121 patients, we a Variables No. of casesPVT1 expression P value* found that PVT1 expression was linked to tumor size, Low High number, grade and stage (Table 2). Age (years) 0.99 ≥ 60 47 28 19 Knockdown of PVT1 inhibited HCC cell propagation < 60 74 44 30 and promoted apoptotic cells Gender 0.801 QRT-PCR evaluated PVT1 expression in different HCC Female 65 38 27 cell lines. As shown in Fig.  2a, four cell lines (HepG2, Male 56 34 22 Huh7, SK-HEP-1 and BEL-7404) expressed higher lev- AFP level (ng/mL) 0.082 els of PVT1 compared with normal cell line (HL-7702). ≥ 400 60 31 29 PVT1 in HepG2 and Huh7 cell lines exhibited the high- < 400 61 41 20 est level among four cell line and were selected for further Tumor size (cm) 0.003 experiments. After knockdown of PVT1 in HepG2 and ≥ 5 62 29 33 Huh7 cells, PVT1 expression decreased compared with < 5 59 43 16 control group (Fig. 2b, P < 0.05). CCK-8 and colony forma- Tumor number 0.003 tion assays elucidated that cell vitality and proliferation in Single 54 40 14 si-PVT1 group were attenuated compared with control Multiple 67 32 35 group (Fig.  2c, d, all P < 0.05). Furthermore, knockdown Tumor grade 0.001 of PVT1 induced apoptosis in HepG2 and Huh7 cells G1 59 46 13 through flow cytometry analysis (Fig. 2 e, P < 0.05). G2 + G3 62 26 36 Tumor stage 0.010 PVT1 improved EZH2 protein stability I + II 67 52 15 RIP experiments were conducted in HepG2 and Huh7 III 54 20 34 cells, which revealed PVT1 enrichment in EZH2-RNA PVT1 plasmacytoma variant translocation 1, AFP alphafetoprotein participation compared with si-NC group (Fig.  3a, * For analysis of correlation between PVT1 and clinical features, Pearson’s Chi P < 0.05). In addition, knockdown of PVT1 had no impact square tests were used. Results were considered statistically significant at on EZH2 mRNA expression (Fig. 3b) and RNA pull-down P < 0.05 with italics font assays in HepG2 and Huh7 cells revealed that EZH2 The average expression level was used as the cutoff. Low expression of PVT1 in 72 patients was classified as values below the fold change of 5.44 while high interacted with PVT1 (Fig.  3c, P < 0.05). However, PVT1 PVT1 expression in 49 patients was classified as values above the level was not directly bound to MDM2 or P53 (Additional Guo et al. Cancer Cell Int (2018) 18:98 Page 5 of 10 Fig. 2 Knockdown of PVT1 inhibited HCC cell proliferation and promoted cell apoptosis. a PVT1 was highly expressed in four HCC cell lines (HepG2, Huh7, SK-HEP-1 and BEL-7404) compared with normal cell line (HL-7702). **P < 0.01, *P < 0.05, compared with HL-7702 cell line. b PVT1 expression in HepG2 and Huh7 cells transfected with si-PVT1 was lower than that in cells transfected with si-NC. *P < 0.05, compared with si-NC group. c Cell vitality of HepG2 and Huh7 cells transfected with si-PVT1 was lower than that in cell s transfected with si-NC detected by CCK-8 assay. *P < 0.01, **P < 0.05, compared with si-NC group. d Colony formation assay indicated that the number of colony cells in si-PVT1 group was lower than that in si-NC group. *P < 0.05, compared with si-NC group. e Flow cytometry showed that apoptosis rate in si-PVT1 group was higher than that in si-NC group. *P < 0.05, compared with si-NC group Guo et al. Cancer Cell Int (2018) 18:98 Page 6 of 10 Fig. 3 PVT1 could bind to EZH2. a RIP assay indicated that PVT1 levels of EZH2 RIP vs IgG RIP in si-PVT1 group was higher than that in si-NC group. **P < 0.01, compared with si-NC group. b QRT-PCR indicated that EZH2 mRNA expression in si-PVT1 group had no significant difference between that in si-NC group. c RNA pull-down assay indicated that PVT1 bound to EZH2. d Western blot revealed that EZH2 protein expression in si-PVT1 group was lower than that si-NC group. *P < 0.05, compared with si-NC group. e Western blot indicated that after added with 100 μg/mL CHX, si-PVT1 had greater inhibitory effect on EZH2 protein expression with the increase of time. f ELISA assay revealed that EZH2 protein expression in si-PVT1 group was lower than that in si-NC group with the increase of time after added with 100 μg/mL CHX. *P < 0.05, compared with si-NC group Guo et al. Cancer Cell Int (2018) 18:98 Page 7 of 10 file  1: Figure S1). In contrast, si-PVT1 inhibited EZH2 knockdown significantly inhibited prostate cancer protein expression (Fig.  3d, P < 0.05). Western blot and growth in vivo and in vitro and promoted cell apoptosis ELISA assays demonstrated that si-PVT1 decreased [26]. Zhuang et al. found that PVT1 silencing inhibited the EZH2 protein stability (Fig.  3e, f, P < 0.05). Taken bladder cancer cell growth and induced apoptosis [27]. together, PVT1 could improve EZH2 protein stability but Based on previous studies, we speculated that PVT1 have no significant impact on EZH2 mRNA expression. might have impacts on HCC cell process. In cur- rent study, PVT1 in HCC was aberrantly higher. After knockdown of PVT1, inhibited HCC cell proliferation EZH2 protein stabilized MDM2 protein expression was inhibited while cell apoptosis was enhanced. Con- In Co-IP assay, we found that EZH2 could bind to MDM2 sistently, Gou et al. also found that PVT1 was increased (Fig.  4a). To further study the interaction between in HCC, which promoted cell proliferation and inva- EZH2 and MDM2, we successfully transfected HepG2 sion in HCC [6]. Lan et  al. demonstrated that PVT1 and Huh7 cells with si-EZH2 and si-MDM2 (Fig.  4b, promoted proliferation, invasion and migration in HCC P < 0.05). Measured by western blot, knockdown of EZH2 cells by regulating miR-186-5p [28]. decreased MDM2 protein expression, whereas knock- EZH2 has been thought to contribute to malignant down of MDM2 had no significant impact on EZH2 pro - transformation due to its role in regulating funda- tein expression (Fig.  4c). Meanwhile, the MDM2 protein mental cellular processes [29]. Previously, some stud- half-life decreased after knockdown of EZH2 (Fig.  4d, e, ies have reported the correlation between PVT1 and P < 0.05). Therefore, EZH2 protein improved the stability EZH2. For example, PVT1 induced lung adenocarci- of MDM2 protein. noma progression through LATS2/MDM2/P53 path- way suppressed by EZH2 [30]. Kong et al. revealed that PVT1 enhanced EZH2 and MDM2 expression as well PVT1 recruited EZH2 and contributed to gastric can- as inhibited P53 protein expression cer growth [10]. In our study, we performed RIP, RNA After transfected with pcDNA3.1-PVT1 and si-PVT1 in pull-down and ELISA assays to examine the relation- HepG2 and Huh7 cells, western blot was employed to ship between PVT1 and EZH2. All experiments dem- determine the protein expression of EZH2, MDM2 and onstrated that PVT1 improved EZH2 protein stability P53. The results elucidated that overexpression of PVT1 by binding to EZH2. Furthermore, we found that EZH2 improved the protein expression of EZH2 and MDM2 protein stabilized MDM2 protein expression by binding as well as inhibited P53 protein expression. In contrast, to MDM2 detected by co-IP assay. Previous study has knockdown of PVT1 had the opposite effects (Fig. 5). claimed that as a key negative regulator of the tumor suppressor p53, MDM2 is recruited to target gene pro- Discussion moters by EZH2 [31]. We also employed western blot Mounting evidence has highlighted the critical roles of to measure the effects of PVT1 on EZH2, MDM2 and lncRNAs in HCC cellular process [22]. We performed P53. The results indicated that PVT1 enhanced the series experiments to investigate the function of PVT1 on protein expressions of EZH2 and MDM2 as well as HCC. First, we confirmed PVT1 overexpression in HCC, inhibited P53 protein expression. In this study, we first and inhibition of PVT1 suppressed HCC cell propaga- clearly elucidated the molecular mechanism underlying tion and promoted apoptotic cells. Second, we employed PVT1, EZH2, MDM2 and P53 in HCC. RIP, RNA pull-down and ELISA assays to examine the However, some concerns still existed in our study. In connection of PVT1 and EZH2, finding that PVT1 could current study, we only investigated the effects of PVT1 improve EZH2 protein stability by binding to EZH2. on P53 expression, while the molecular network between Meanwhile, the results of co-IP assay revealed that EZH2 EZH2, MDM2 and P53 need to be further explored. On protein stabilized MDM2 protein expression. Addition- the other hand, the pathway of PVT1, EZH2 and P53 in ally, PVT1 enhanced the protein expression of EZH2 and HCC should be taken into consideration. MDM2 as well as inhibited P53 protein expression in HCC cells. LncRNAs are involved in regulating biological func- Conclusions tions and gene expression in physiological and patho- In summary, our findings suggested that PVT1 had posi - logical contexts, such as cancer [23]. Growing evidence tive effects on HCC cell growth. Additionally, PVT1 sta - has clarified the functions of different lncRNAs in bilized the protein expression of EZH2 and MADM2. HCC, including lncRNA-PVT1 [24]. PVT1 functioned PVT1 might be a potential therapies target of HCC treat- as an oncogenic lncRNA in multiple types of cancers ment. Our experiments may improve the diagnostic abil- [25]. PVT1 overexpression has been identified as pre - ity of biomarkers for HCC in clinical practice. dictor for many carcinomas [9]. For example, PVT1 Guo et al. Cancer Cell Int (2018) 18:98 Page 8 of 10 Fig. 4 EZH2 protein stabilized MDM2 protein expression. a Co-IP assay indicated that EZH2 bound to MDM2. b QRT-PCR indicated that EZH2 mRNA in si-EZH2 group was lower than that in si-NC group. **P < 0.01, compared with si-NC group. c Western blot showed that MDM2 protein expression in si-EZH2 group was lower than that in si-NC group, while EZH2 protein expression in si-MDM2 group had no significant difference with that in si-NC group. d Western blot indicated that after added with 100 μg/mL CHX, si-EZH2 had greater inhibitory effect on MDM2 protein expression with the increase of time. f ELISA assay revealed that MDM2 protein expression in si-EZH2 group was lower than that in si-NC group with the increase of time after added with 100 μg/mL CHX. *P < 0.05, compared with si-NC group Guo et al. Cancer Cell Int (2018) 18:98 Page 9 of 10 Fig. 5 PVT1 enhanced the protein expression of EZH2 and MDM2 as well as inhibited P53 protein expression. a, b Western blot demonstrated that overexpression of PVT1 improved the protein expression level of EZH2 and MDM2 as well as inhibited P53 protein expression in HepG2 and Huh7 cells. In contrast, knockdown of PVT1 inhibited the protein expression level of EZH2 and MDM2 as well as promoted P53 protein expression Competing interests Additional file The authors declare that they have no competing interests. Availability of data and materials Additional file 1: Figure S1. PVT1 could not directly bind to MDM2 or Please contact the corresponding author for data. P53. (A) RNA pull-down assay indicated that PVT1 could not directly bind to MDM2 or P53. Consent for publication Obtained. Ethics approval and consent to participate Abbreviations This study was approved by the ethics committee of Zibo Central Hospital. Co-IP: co-immunoprecipitation; CHX: cyclohexane; EZH2: enhancer of zeste ( The related Ethics Committee Approval Form has been achieved.) homolog 2; HCC: hepatocellular carcinoma; lncRNAs: long non-coding RNAs; PI: propidium iodide; PVT1: plasmacytoma variant translocation 1 gene; RIP: Funding RNA immunoprecipitation; SDS-PAGE: SDS-polyacrylamide gel electrophoresis. Not applicable. Authors’ contributions JG made substantial contributions to conception and design. LL made acqui- Publisher’s Note sition of data. CH and CW made analysis and interpretation of data. JG have Springer Nature remains neutral with regard to jurisdictional claims in pub- been involved in drafting the manuscript. LL, CH and CW have been involved lished maps and institutional affiliations. in revising it critically for important intellectual content. All authors agreed to be accountable for all aspects of the work. All authors read and approved the Received: 20 April 2018 Accepted: 12 June 2018 final manuscript. 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Journal

Cancer Cell InternationalSpringer Journals

Published: Dec 1, 2018

Keywords: cancer research; cell biology

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