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LncRNA PVT1 promotes the growth of HPV positive and negative cervical squamous cell carcinoma by inhibiting TGF-β1

LncRNA PVT1 promotes the growth of HPV positive and negative cervical squamous cell carcinoma by... Background: Our study aimed to investigate the role of lncRNA PVT1 in cervical squamous cell carcinoma. Materials and methods: A total of 156 patients with cervical squamous cell carcinoma were enrolled in this study and human papillomavirus (HPV ) infection was detected by highly sensitive PCR techniques. Serum levels of PVT1 in patients infected with different HPVs and healthy controls was detected by qRT-PCR and compared. Serum levels of PVT1 were also compared among patients with different sizes of tumor. ROC curve analysis was performed to evalu- ate the diagnostic values of serum for cervical squamous cell carcinoma. Survival curves were plotted by Kaplan– Meier method and compared to evaluate the prognostic values of serum PVT1 for this disease. Eec ff ts of PVT1 siRNA silencing and overexpression on proliferation of cervical squamous cell carcinoma cells were explored by CCK-8 assay. Western blot was performed to detect the expression of TGF-β1 after PVT1 siRNA silencing and overexpression. Results: No significant differences in serum levels of PVT1 were detected among patients infected with different HPVs and HPV-negative patients. However, serum levels of PVT1 were significantly higher in all patient groups than in healthy control group. Serum level of PVT1 increased with the increased sizes of primary tumor. Serum PVT1 accu- rately predicted the disease and its prognosis. PVT1 siRNA silencing inhibited the proliferation of cancer cells and reduced the expression of TGF-β1, while PVT1 overexpression played an opposite role. Conclusion: LncRNA PVT1 promotes the growth HPV positive and negative cervical squamous cell carcinoma by inhibiting TGF-β1. Keywords: Cervical squamous cell carcinoma, lncRNA PVT1, TGF-β1 Background the incidence of cervical cancer and certain genotypes of At present, cervical cancer is considered to be the fourth HPV have been well established [5]. With the application most common type of cancer that leads to unacceptable of HPV infection screening and increased HPV vaccina- high modality rate [1]. Cervical adenocarcinoma and tion rate, incidence of cervical squamous cell carcinoma cervical squamous cell carcinoma are two major sub- has been reduced during 20th century, while incidence of types of cervical cancer, and the latter one accounts for this disease showed no further decrease during last sev- more than 80% of all cases [2]. Human papillomaviruses eral decades because of the existing of other risk factors (HPV) infection is the most common cause of occur- other than HPV infection in the pathogenesis of cervical rence of cervical cancer [3, 4], and correlations between squamous cell carcinoma [6]. In addition, the prognosis of HPV-negative cervical cancer is usually poor [7]. Long non-coding RNA is a group of non-coding RNAs *Correspondence: zuur718b@yeah.net contain more than 200 nucleotides [8]. Studies in last Xiuli Wang and Guichan Wang contributed equally to this work 1 several decades have shown that different lncRNAs have Department of Gynecology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai 264000, Shandong, People’s Republic different functions in most normal biological as well as of China pathological processes in the human body [9]. HPV 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. Wang et al. Cancer Cell Int (2018) 18:70 Page 2 of 8 infection is related to the altered expression of certain cell lines Ect1/E6E7 (HPV positive) and HCvEpC (HPV lncRNAs [10], while differentially expression lncRNAs in negative) were used as control group. All cell lines were cervical cancers are not reported. LncRNA PVT1 plays purchased from ATCC (USA). All cells were culture a role as oncogene in the development of various types under the conditions recommended by ATCC. Cells were of malignant tumors, such as non-small cell lung cancer harvest during logarithmic growth phase for following [11], gastric cancer [12] and so on. However, the roles of experiments. PVT1 in cervical squamous cell carcinoma still haven’t been well studied. Establishment of PVT1 siRNA silencing and overexpression In this study, expression of PVT1 in serum of patients cell lines with cervical squamous cell carcinoma was detected, and PVT1 siRNA (Catalog No. AV16708, Thermo Fisher Sci - serum levels of PVT1 were compared among patients entific, USA) was used to establish PVT1 RNA silencing infected with different HPVs and in different stages of cell lines, and Silencer Select Negative Control No. 1 primary tumors. In addition, the diagnostic and prognos- siRNA (Catalog No. 4390843, Thermo Fisher Scientific, tic values of serum PVT1 for cervical squamous cell car- USA) was used as a negative control. PVT1 overexpres- cinoma were analyzed. Effects of PVT1 siRNA silencing sion vector was established by inserting an EcoRI–EcoRI and overexpression on proliferation of cervical squamous fragment containing PVT1 cDNA into pIRSE2-EGFP cell carcinoma cells and expression of TGF-β1 were also vector (Clontech, Palo Alto, CA, USA). Cells were cul- explored. tured over night to reach 80–90% confluent, and trans - fection was performed by incubating with Lipofectamine Patients 2000 reagent (11668-019, Invitrogen, Carlsbad, USA) at A total of 156 patients with cervical squamous cell carci- 37 °C for 4 h. noma were enrolled from January 2009 to January 2012 in Affiliated Yantai Yuhuangding Hospital of Qingdao University. All patients were diagnosed by pathological Cell proliferation assay tests and imaging examinations. Human papillomavirus Cells were inoculated onto 96-well plates with 5  ×  10 (HPV) infection was detected by highly sensitive poly- cells per well. Cells were cultured at 37  °C for 3–5  h to merase chain reaction (PCR) techniques. Among them, reach cell adhesion, and 100  μl of DMEM medium was 21 patients were HPV negative, 47 patients were infected added. Cells were cultured at cultured at 37 °C, and 10 μl with HPV-16, 45 patients were infected with HPV-18, of CCK-8 was added at 24, 48, 72 and 96  h later. After 15 patients were infected with HPV-11, 17 patients were incubation for another 3  h, OD values at 450  nm were infected with HPV-45, and 11 patients were infected with measured using a microplate reader. HPV-68. All patients received surgical resection, and tumor tissues and adjacent healthy tissues were collected during surgical operation. Tumor size was measured and Real‑time quantitative reverse transcription PCR patients were divided into for groups according to the Trizol reagent (Invitrogen, USA) was used to extract long diameter: I (0–1 cm), II (1–3 cm), III (3–5 cm) and total RNA from tumor tissues, normal tissues, serum IV (> 5 cm). There were 23 patients in stage I, 34 in stage and in  vitro cultured cells according the instructions. II, 54 in stage III and 45 in stage IV. At the same time, After that, cDNA was then synthesized using total RNA 39 normal healthy females with the same age distribution as template and SuperScript III Reverse Transcriptase were also included to serve as control group. This study system (Thermo Fisher Scientific, USA). PCR reaction has been approved by the ethics committee of our hospi- system was prepared using cDNA and SYBR Green tal, and participants signed informed consent. Real-Time PCR Master Mixes (Thermo Fisher Scientific, USA). The following primers were used: 5′-TGA GAA Blood collection and serum preparation CTG TCC TTA CGT GACC-3′ (forward) and 5′-AGA GCA Fasting blood (20  ml) was collected from each partici- CCA AGA CTG GCT CT-3′(reverse) for PVT1; 5′-GAC pant on the day of admission. Blood samples were kept at CTC TAT GCC AAC ACA GT-3′ (forward) and 5′-AGT room temperature for 1  h, followed by centrifugation at ACT TGC GCT CAG GAG GA-3′ (reverse) for β-actin. 1500 rpm for 15 min to collect serum samples. PCR reaction was carried out on CFX96 Touch Real- Time PCR Detection System (Bio-Rad, USA). PCR reac- Cell lines and cell culture tion conditions were: 95 °C for 40 s, followed by 40 cycles In this study, human cervical squamous cell carcinoma of 95  °C for 12  s and 60  °C for 37  s. Ct values were pro- −ΔΔCT cell lines SiHa (HPV positive) and C33A (HPV negative) cessed using 2 method, and relative expression level were included. At the same time, human normal cervical of PVT1 was normalized to endogenous control β-actin. Wang et al. Cancer Cell Int (2018) 18:70 Page 3 of 8 Western‑blot in serum levels of PVT1 were detected among patients Total protein were extracted from cells using Cell lysis infected with different HPVs and HPV-negative patients. solutions (Thermo Fisher Scientific, USA) and quanti - However, serum levels of PVT1 were significantly higher fied by BCA assay. Gel electrophoresis (10% SDS-PAGE) in all patient groups than in healthy control group. Those was performed with 30 µg protein from each sample, fol- data suggest that upregulation of PVT1 may participate lowed by transmembrane to PVDF membranes. Block- in the pathogenesis of cervical squamous cell carcinoma ing was performed with 5% skimmed milk at room through a HPV-independent pathway. temperature for 2 h. After that membranes were washed and incubated with primary antibodies including rab- Comparison of serum levels of PVT1 among patients bit anti-TGF-β1 antibody (1:2000, ab92486, Abcam) and with different sizes of tumor anti-GAPDH antibody (1:1000, ab9485, Abcam) over- Our data have showed that serum levels of PVT1 were night at 4 °C. The next day, membranes were washed and abnormally increased in patients with cervical squamous further incubated with anti-rabbit IgG-HRP secondary cell carcinoma. Therefore, serum levels of PVT1 may also antibody (1:1000, MBS435036, MyBioSource) for 2  h at be different in patients with different sizes of tumor. As room temperature. Signal detection was performed using shown in Fig.  2, serum levels of PVT1 were significantly ECL (Sigma-Aldrich, USA) method. Relative expression increased with the increased sizes of cervical squamous level of TGF-β1 was normalized to endogenous control cell carcinoma. GAPDH using Image J software. Diagnostic and prognostic values of serum PVT1 Statistical analysis for cervical squamous cell carcinoma SPSS19.0 (SPSS Inc., USA) statistical software was used. ROC curve analysis was performed to evaluate the diag- Measurement data were expressed as ( x¯ ± s ), and t test nostic values of serum PVT1 for cervical squamous cell was used for the comparisons between two groups, and carcinoma. As shown in Fig. 3a, the area under the curve comparisons among multiple groups were performed by of serum PVT1 in the diagnosis was 0.9030 with 95% one way analysis of variance, followed by LSD test. Com- confident interval of 0.8567–0.9472 (p < 0.0001), indicat- parisons of count data were performed using Chi square ing that serum PVT1 is an accurate biomarker for cervi- test. p < 0.05 was considered to be statistically significant. cal squamous cell carcinoma. According to the median serum level of serum PVT1 patients were divided into Results high level group and low level group. Kaplan–Meier Serum levels of PVT1 in different patient groups method was used to draw survival curves for both and control group groups. Log-rank test was used to compare two survival Serum levels of PVT1 was detected by qRT-PCR and curves. As shown in Fig.  3b, overall survival of cervical compared among different patient groups and control squamous cell carcinoma patient with high serum level of group. In this study, five HPV strains, including HPV-16, PVT1 was significantly shorter that that of patients with HPV-18, HPV-11, HPV-45 and HPV-68 were detected low serum level of PVT1 (p < 0.05). Those data suggest in tumor tissues of patients with cervical squamous cell that serum level of PVT1 may serve as prognostic marker carcinoma. As shown in Fig.  1, no significant differences for patients with gastric cancer. Fig. 2 Comparison of serum levels of PVT1 among patients in Fig. 1 Serum levels of PVT1 in different patient groups and control different stages of primary tumor. *Compared with one stage before, group p < 0.05 Wang et al. Cancer Cell Int (2018) 18:70 Page 4 of 8 Fig. 3 Diagnostic and prognostic values of serum PVT1 for cervical squamous cell carcinoma. a ROC curve analysis for the diagnostic values of serum PVT1 for cervical squamous cell carcinoma; b comparison of survival curves of patients with high and low serum level of PVT1 Eec ff ts of PVT1 siRNA silencing and overexpression Eec ff ts of PVT1 siRNA silencing and overexpression on proliferation of cervical squamous cell carcinoma on expression of TGF‑β1 Expression of PVT1 in human cervical squamous cell To further investigated the interactions between TGF- carcinoma cell lines SiHa (HPV positive) and C33A β1 and PVT1, eeff cts of PVT1 siRNA silencing and (HPV negative) and normal cervical cell lines Ect1/ overexpression on expression of TGF-β1 in two cervi- E6E7 (HPV positive) and HCvEpC (HPV negative) was cal squamous cell carcinoma cell lines were explored. detected by qRT-PCR. As shown in Fig.  4a, expression As shown in Fig.  5a, PVT1 siRNA silencing significantly level of PVT1 was significantly higher in two cervical increased the expression level of TGF-β1 in both cell squamous cell carcinoma cell lines than in two normal lines (p < 0.05). In contrast, PVT1 overexpression signifi - cells. However, no significant differences in expression cantly reduced the expression of TGF-β1 in two cell lines level of PVT1 were found between SiHa and C33A or (p < 0.05). Especially in C33A cell line, expression level Ect1/E6E7 and HCvEpC, indicating that HPV infection of PVT1 reduced by more than eight times after PVT1 has no significant effects on the expression of PVT1 overexpression. Those data suggest that PVT1 can nega - in those cell lines. PVT1 siRNA silencing and overex - tively regulate the expression of TGF-β1 to participate in pression cell lines were established to investigate the the growth of cervical squamous cell carcinoma. role of PVT1 in the proliferation of cervical squamous cell carcinoma cells. As shown in Fig. 4b, PVT1 siRNA Discussion silencing significantly inhibited cell proliferation of Most cases of cervical squamous cell carcinoma are two cervical squamous cell carcinoma cell lines. In caused by HPV infection [3]. HPV infection is usually contrast, PVT1 overexpression significantly promoted harmless but long-term infection of HPV may increase cell proliferation of two cervical squamous cell carci - the risk of the occurrence of malignant tumors in vari- noma cell lines (Fig.  4c). TGF-β1 has anti-proliferative ous sites, such as cervix, vagina, penis, anus and oro- functions [13], and PVT1 can regulate the expression pharynx [3]. Different genotypes of HPV causes different of TGF-β1 [14]. In this study, treatment with TGF-β1 incidences of cervical squamous cell carcinoma, and two (10  ng/ml, Sigma-Aldrich, USA) for 1  h not signifi- genotypes of HPV, including HPV-16 and -18 causes cantly inhibited cell proliferation of two cervical squa- about 70% cases of HPV-positive cervical squamous cell carcinoma [3]. Consistent with previous studies, in mous cell carcinoma cell lines, but also significantly this study, 135 patients with cervical squamous cell car reduce the enhancing effects of PVT1 overexpression - on cell proliferation. Those data suggest that expres- cinoma were proved to be HPV-positive, accounting sion level of PVT1 is a positive regulator of prolifera- for 86.5% of all the cases. In addition, 47 patients were tion of cervical squamous cell carcinoma cells, and this infected with HPV-16 and 45 patients were infected with function is very likely to be achieved through interac- HPV-18, accounting for 68.4% of all the cases. HPV infec- tions with TGF-β1. tion is a major but not the only cause of this disease. In Wang et al. Cancer Cell Int (2018) 18:70 Page 5 of 8 Fig. 4 Eec ff ts of PVT1 siRNA silencing and overexpression on proliferation of cervical squamous cell carcinoma. a Expression of PVT1 in different cell lines; b Eec ff ts of PVT1 siRNA silencing on proliferation of cervical squamous cell carcinoma; c Eec ff ts of PVT1 siRNA overexpression on proliferation of cervical squamous cell carcinoma. *p < 0.05; C, control cells without transfection; NC, negative control cells transfected with negative control siRNA or empty vector; siRNA, cells transfected with PVT1 siRNA; Over, cells transfected with PVT1 expression vector Wang et al. Cancer Cell Int (2018) 18:70 Page 6 of 8 Fig. 5 Eec ff ts of PVT1 siRNA silencing and overexpression on expression of TGF-β1. a Eec ff ts of PVT1 siRNA silencing on expression of TGF-β1; b effects of PVT1 overexpression on expression of TGF-β1. *p < 0.05; C, control cells without transfection; NC, negative control cells transfected with negative control siRNA or empty vector; siRNA, cells transfected with PVT1 siRNA; Over, cells transfected with PVT1 expression vector this study, 21 patients were proved to be HPV-negative, that expression level of the lncRNA PVT1 was sig- accounting for 13.5% of all the cases. nificantly increased in cancer patients than in normal LncRNA PVT1 plays a role as oncogene in the healthy controls, and the upregulated expression level development of various types of malignant tumors of PVT1 was significantly correlated with poor prog- and shows abnormally upregulated expression. In nosis of those patients [15]. In another study, expres- the study of pancreatic cancer, Huang et  al. found sion level of PVT1 was also found to be abnormally Wang et al. Cancer Cell Int (2018) 18:70 Page 7 of 8 increased in gastric cancer patients, and abnormal Conclusion expression of PVT1 showed promising diagnostic and In conclusion, expression of PVT1 in patients with prognostic values for this disease [16]. Previous stud- cervical squamous cell carcinoma was not affected ies also confirmed that PVT1 is upregulated in cervical by HPV infection. Serum levels of PVT1 were sig- cancer and promote the progression of disease [17– nificantly higher in cervical squamous cell carcinoma 19]. Consistent with previous studies, in this study, patients than in healthy controls. Serum level of PVT1 expression level of PVT1 was found to be significantly increased with the increased size of primary tumor. higher in cervical squamous cell carcinoma patients Serum PVT1 can serve as a promising diagnostic and than in normal controls. In addition, expression level prognostic biomarker for cervical squamous cell carci- of PVT1 was increased with the increased stage of noma PVT1 siRNA silencing inhibited the proliferation primary tumor. HPV infection can induce the abnor- of cancer cells and reduced the expression of TGF-β1, mal expression of certain lncRNAs [10]. In our study, while PVT1 overexpression played an opposite role. no significant differences in serum levels of PVT1 So we conclude that lncRNA PVT1 can promote the were detected among patients infected with different growth HPV positive and negative cervical squamous HPVs and HPV-negative patients. Those data suggest cell carcinoma by inhibiting TGF-β1. Our study is lim- that upregulated expression of PVT1 may participate ited by the small sample size. Future studies with bigger in the pathogenesis of cervical squamous cell carci- sample size are needed to further confirm the conclu - noma through a HPV infection-independent pathway. sions in the present study. Besides that, serum PVT1 also showed promising Authors’ contributions diagnostic and prognostic values for cervical squa- CL conceived and supervised the study. XW, GW, LZ, JC, and JH conducted the mous cell carcinoma. Therefore, PVT1 may serve as a experiments and performed the analysis. XW, GW, and CL wrote the paper. All authors read and approved the final manuscript. target to improve the treatment outcomes and prog- nosis of those patients. It is worth to note that several Author details lncRNAs, such as GAS5 [20] and HOTAIR [21] show Department of Gynecology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai 264000, Shandong, People’s Republic of China. clinical values for the diagnosis and prognosis of cervi- Department of Obstetrics and Gynecology, The Affiliated Yantai Yuhuang- cal cancers. Combination of multiple biomarkers may ding Hospital of Qingdao University, Yantai 264000, Shandong, People’s improve the diagnosis. Republic of China. Previous studies have shown that PVT1 can promote Acknowledgements the proliferation of various types of cancer cells, such Not applicable. as hepatocellular carcinoma cells [22], thyroid can- Competing interests cer cells [23] and so on. PVT1 siRNA silencing inhib- The authors declare that they have no competing interests. ited the proliferation of two cervical squamous cell carcinoma cell lines, while PVT1 overexpression pro- Availability of data and materials Not applicable. moted the proliferation of cancer cells, indicating that PVT1 expression may promote the growth of cervical Consent for publication squamous cell carcinoma by promoting the prolifera- Not applicable. tion of cancer cells. TGF-β1 plays different roles in the Ethics approval and consent to participate development and progression of tumors. TGF-β1 not The ethics committee of Affiliated Yantai Yuhuangding Hospital of Qingdao only inhibits the growth of tumors through its anti- University has approved this study, and participants signed informed consent. proliferative functions [13], but also promotes tumor Funding metastasis by inducing epithelial–mesenchymal transi- Not applicable. tion [24]. Expression of TGF-β1 was regulated by PVT1 [14]. Therefore, it will be reasonable to hypothesize that Publisher’s Note PVT1 may regulate TGF-β1 to participate in cervical Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. squamous cell carcinoma. In this study, PVT1 siRNA silencing significantly increased the expression level Received: 5 April 2018 Accepted: 27 April 2018 of TGF-β1. In contrast, PVT1 overexpression signifi - cantly reduced the expression of TGF-β1. Those data suggest that PVT1 can negatively regulate the expres- sion of TGF-β1 to participate in the growth of cervi- References 1. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality cal squamous cell carcinoma. However, the molecular worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int mechanism of the regulation of TGF-β1 by PVT1 is still J Cancer. 2015;136(5):E359–86. unknown. Therefore, further studies are still needed. Wang et al. Cancer Cell Int (2018) 18:70 Page 8 of 8 2. Yang PM, Chou CJ, Tseng SH, et al. Bioinformatics and in vitro experi- 15. Huang C, Yu W, Wang Q, et al. Increased expression of the lncRNA PVT1 mental analyses identify the selective therapeutic potential of interferon is associated with poor prognosis in pancreatic cancer patients. Minerva gamma and apigenin against cervical squamous cell carcinoma and Med. 2015;106(3):143–9. adenocarcinoma. Oncotarget. 2017;8:46145–62. 16. Yuan CL, Li H, Zhu L, et al. Aberrant expression of long noncoding RNA 3. Zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical PVT1 and its diagnostic and prognostic significance in patients with application. Nat Rev Cancer. 2002;2(5):342–50. gastric cancer. Neoplasma. 2016;63(3):442–9. 4. Crosbie EJ, Einstein MH, Franceschi S, et al. Human papillomavirus and 17. Yang JP, et al. Long noncoding RNA PVT1 as a novel serum bio- cervical cancer. Lancet. 2013;382(9895):889–99. marker for detection of cervical cancer. Eur Rev Med Pharmacol Sci. 5. Burd EM. Human papillomavirus and cervical cancer. Clin Microbiol Rev. 2016;20(19):3980–6. 2003;16(1):1–17. 18. Iden M, et al. The lncRNA PVT1 contributes to the cervical cancer 6. Hildesheim A, Gonzalez P, Kreimer AR, et al. Impact of human papil- phenotype and associates with poor patient prognosis. PLoS ONE. lomavirus (HPV ) 16 and 18 vaccination on prevalent infections and 2016;11(5):e0156274. rates of cervical lesions after excisional treatment. Am J Obstet Gynecol. 19. Gao YL, et al. Long noncoding RNA PVT1 facilitates cervical can- 2016;215(2):212. e1–15. cer progression via negative regulating of miR-424. Oncol Res. 7. Rodríguez-Carunchio L, Soveral I, Steenbergen RDM, et al. HPV-negative 2017;25(8):1391–8. carcinoma of the uterine cervix: a distinct type of cervical cancer with 20. Cao S, Liu W, Li F, et al. Decreased expression of lncRNA GAS5 predicts a poor prognosis. BJOG Int J Obstet Gynaecol. 2015;122(1):119–27. poor prognosis in cervical cancer. Int J Clin Exp Pathol. 2014;7(10):6776. 8. Perkel JM. Visiting “noncodarnia”. Biotechniques. 2013;54(6):301, 303–4. 21. Kim H, Lee DW, YIm GW, NAm EJ, Kim S, KIm SW, KIm Y T, et al. Long non- 9. Esteller M. Non-coding RNAs in human disease. Nat Rev Genet. coding RNA HOTAIR is associated with human cervical cancer progres- 2011;12(12):861–74. sion. Int J Oncol. 2015;46(2):521–30. 10. Nohata N, Abba MC, Gutkind JS. Unraveling the oral cancer lncRNAome: 22. Wang F, Yuan JH, Wang SB, et al. Oncofetal long noncoding RNA PVT1 identification of novel lncRNAs associated with malignant progression promotes proliferation and stem cell-like property of hepatocellular and HPV infection. Oral Oncol. 2016;59:58–66. carcinoma cells by stabilizing NOP2. Hepatology. 2014;60(4):1278–90. 11. Wan L, Sun M, Liu GJ, et al. Long noncoding RNA PVT1 promotes non- 23. Zhou Q, Chen J, Feng J, et al. Long noncoding RNA PVT1 modulates thy- small cell lung cancer cell proliferation through epigenetically regulating roid cancer cell proliferation by recruiting EZH2 and regulating thyroid- LATS2 expression. Mol Cancer Ther. 2016;15(5):1082–94. stimulating hormone receptor ( TSHR). Tumor Biol. 2016;37(3):3105–13. 12. Zhang X, Bu P, Liu L, et al. Overexpression of long non-coding RNA PVT1 24. Pang MF, Georgoudaki AM, Lambut L, et al. TGF-β1-induced EMT pro- in gastric cancer cells promotes the development of multidrug resist- motes targeted migration of breast cancer cells through the lymphatic ance. Biochem Biophys Res Commun. 2015;462(3):227–32. system by the activation of CCR7/CCL21-mediated chemotaxis. Onco- 13. Li J, Ballim D, Rodriguez M, et al. The anti-proliferative function of the TGF- gene. 2016;35(6):748–60. β1 signaling pathway involves the repression of the oncogenic TBX2 by its homologue TBX3. J Biol Chem. 2014;289(51):35633–43. 14. Liu E, Liu Z, Zhou Y, et al. Overexpression of long non-coding RNA PVT1 in ovarian cancer cells promotes cisplatin resistance by regulating apoptotic pathways. Int J Clin Exp Med. 2015;8(11):20565. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Cell International Springer Journals

LncRNA PVT1 promotes the growth of HPV positive and negative cervical squamous cell carcinoma by inhibiting TGF-β1

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Springer Journals
Copyright
Copyright © 2018 by The Author(s)
Subject
Biomedicine; Cancer Research; Cell Biology
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1475-2867
DOI
10.1186/s12935-018-0567-2
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Abstract

Background: Our study aimed to investigate the role of lncRNA PVT1 in cervical squamous cell carcinoma. Materials and methods: A total of 156 patients with cervical squamous cell carcinoma were enrolled in this study and human papillomavirus (HPV ) infection was detected by highly sensitive PCR techniques. Serum levels of PVT1 in patients infected with different HPVs and healthy controls was detected by qRT-PCR and compared. Serum levels of PVT1 were also compared among patients with different sizes of tumor. ROC curve analysis was performed to evalu- ate the diagnostic values of serum for cervical squamous cell carcinoma. Survival curves were plotted by Kaplan– Meier method and compared to evaluate the prognostic values of serum PVT1 for this disease. Eec ff ts of PVT1 siRNA silencing and overexpression on proliferation of cervical squamous cell carcinoma cells were explored by CCK-8 assay. Western blot was performed to detect the expression of TGF-β1 after PVT1 siRNA silencing and overexpression. Results: No significant differences in serum levels of PVT1 were detected among patients infected with different HPVs and HPV-negative patients. However, serum levels of PVT1 were significantly higher in all patient groups than in healthy control group. Serum level of PVT1 increased with the increased sizes of primary tumor. Serum PVT1 accu- rately predicted the disease and its prognosis. PVT1 siRNA silencing inhibited the proliferation of cancer cells and reduced the expression of TGF-β1, while PVT1 overexpression played an opposite role. Conclusion: LncRNA PVT1 promotes the growth HPV positive and negative cervical squamous cell carcinoma by inhibiting TGF-β1. Keywords: Cervical squamous cell carcinoma, lncRNA PVT1, TGF-β1 Background the incidence of cervical cancer and certain genotypes of At present, cervical cancer is considered to be the fourth HPV have been well established [5]. With the application most common type of cancer that leads to unacceptable of HPV infection screening and increased HPV vaccina- high modality rate [1]. Cervical adenocarcinoma and tion rate, incidence of cervical squamous cell carcinoma cervical squamous cell carcinoma are two major sub- has been reduced during 20th century, while incidence of types of cervical cancer, and the latter one accounts for this disease showed no further decrease during last sev- more than 80% of all cases [2]. Human papillomaviruses eral decades because of the existing of other risk factors (HPV) infection is the most common cause of occur- other than HPV infection in the pathogenesis of cervical rence of cervical cancer [3, 4], and correlations between squamous cell carcinoma [6]. In addition, the prognosis of HPV-negative cervical cancer is usually poor [7]. Long non-coding RNA is a group of non-coding RNAs *Correspondence: zuur718b@yeah.net contain more than 200 nucleotides [8]. Studies in last Xiuli Wang and Guichan Wang contributed equally to this work 1 several decades have shown that different lncRNAs have Department of Gynecology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai 264000, Shandong, People’s Republic different functions in most normal biological as well as of China pathological processes in the human body [9]. HPV 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. Wang et al. Cancer Cell Int (2018) 18:70 Page 2 of 8 infection is related to the altered expression of certain cell lines Ect1/E6E7 (HPV positive) and HCvEpC (HPV lncRNAs [10], while differentially expression lncRNAs in negative) were used as control group. All cell lines were cervical cancers are not reported. LncRNA PVT1 plays purchased from ATCC (USA). All cells were culture a role as oncogene in the development of various types under the conditions recommended by ATCC. Cells were of malignant tumors, such as non-small cell lung cancer harvest during logarithmic growth phase for following [11], gastric cancer [12] and so on. However, the roles of experiments. PVT1 in cervical squamous cell carcinoma still haven’t been well studied. Establishment of PVT1 siRNA silencing and overexpression In this study, expression of PVT1 in serum of patients cell lines with cervical squamous cell carcinoma was detected, and PVT1 siRNA (Catalog No. AV16708, Thermo Fisher Sci - serum levels of PVT1 were compared among patients entific, USA) was used to establish PVT1 RNA silencing infected with different HPVs and in different stages of cell lines, and Silencer Select Negative Control No. 1 primary tumors. In addition, the diagnostic and prognos- siRNA (Catalog No. 4390843, Thermo Fisher Scientific, tic values of serum PVT1 for cervical squamous cell car- USA) was used as a negative control. PVT1 overexpres- cinoma were analyzed. Effects of PVT1 siRNA silencing sion vector was established by inserting an EcoRI–EcoRI and overexpression on proliferation of cervical squamous fragment containing PVT1 cDNA into pIRSE2-EGFP cell carcinoma cells and expression of TGF-β1 were also vector (Clontech, Palo Alto, CA, USA). Cells were cul- explored. tured over night to reach 80–90% confluent, and trans - fection was performed by incubating with Lipofectamine Patients 2000 reagent (11668-019, Invitrogen, Carlsbad, USA) at A total of 156 patients with cervical squamous cell carci- 37 °C for 4 h. noma were enrolled from January 2009 to January 2012 in Affiliated Yantai Yuhuangding Hospital of Qingdao University. All patients were diagnosed by pathological Cell proliferation assay tests and imaging examinations. Human papillomavirus Cells were inoculated onto 96-well plates with 5  ×  10 (HPV) infection was detected by highly sensitive poly- cells per well. Cells were cultured at 37  °C for 3–5  h to merase chain reaction (PCR) techniques. Among them, reach cell adhesion, and 100  μl of DMEM medium was 21 patients were HPV negative, 47 patients were infected added. Cells were cultured at cultured at 37 °C, and 10 μl with HPV-16, 45 patients were infected with HPV-18, of CCK-8 was added at 24, 48, 72 and 96  h later. After 15 patients were infected with HPV-11, 17 patients were incubation for another 3  h, OD values at 450  nm were infected with HPV-45, and 11 patients were infected with measured using a microplate reader. HPV-68. All patients received surgical resection, and tumor tissues and adjacent healthy tissues were collected during surgical operation. Tumor size was measured and Real‑time quantitative reverse transcription PCR patients were divided into for groups according to the Trizol reagent (Invitrogen, USA) was used to extract long diameter: I (0–1 cm), II (1–3 cm), III (3–5 cm) and total RNA from tumor tissues, normal tissues, serum IV (> 5 cm). There were 23 patients in stage I, 34 in stage and in  vitro cultured cells according the instructions. II, 54 in stage III and 45 in stage IV. At the same time, After that, cDNA was then synthesized using total RNA 39 normal healthy females with the same age distribution as template and SuperScript III Reverse Transcriptase were also included to serve as control group. This study system (Thermo Fisher Scientific, USA). PCR reaction has been approved by the ethics committee of our hospi- system was prepared using cDNA and SYBR Green tal, and participants signed informed consent. Real-Time PCR Master Mixes (Thermo Fisher Scientific, USA). The following primers were used: 5′-TGA GAA Blood collection and serum preparation CTG TCC TTA CGT GACC-3′ (forward) and 5′-AGA GCA Fasting blood (20  ml) was collected from each partici- CCA AGA CTG GCT CT-3′(reverse) for PVT1; 5′-GAC pant on the day of admission. Blood samples were kept at CTC TAT GCC AAC ACA GT-3′ (forward) and 5′-AGT room temperature for 1  h, followed by centrifugation at ACT TGC GCT CAG GAG GA-3′ (reverse) for β-actin. 1500 rpm for 15 min to collect serum samples. PCR reaction was carried out on CFX96 Touch Real- Time PCR Detection System (Bio-Rad, USA). PCR reac- Cell lines and cell culture tion conditions were: 95 °C for 40 s, followed by 40 cycles In this study, human cervical squamous cell carcinoma of 95  °C for 12  s and 60  °C for 37  s. Ct values were pro- −ΔΔCT cell lines SiHa (HPV positive) and C33A (HPV negative) cessed using 2 method, and relative expression level were included. At the same time, human normal cervical of PVT1 was normalized to endogenous control β-actin. Wang et al. Cancer Cell Int (2018) 18:70 Page 3 of 8 Western‑blot in serum levels of PVT1 were detected among patients Total protein were extracted from cells using Cell lysis infected with different HPVs and HPV-negative patients. solutions (Thermo Fisher Scientific, USA) and quanti - However, serum levels of PVT1 were significantly higher fied by BCA assay. Gel electrophoresis (10% SDS-PAGE) in all patient groups than in healthy control group. Those was performed with 30 µg protein from each sample, fol- data suggest that upregulation of PVT1 may participate lowed by transmembrane to PVDF membranes. Block- in the pathogenesis of cervical squamous cell carcinoma ing was performed with 5% skimmed milk at room through a HPV-independent pathway. temperature for 2 h. After that membranes were washed and incubated with primary antibodies including rab- Comparison of serum levels of PVT1 among patients bit anti-TGF-β1 antibody (1:2000, ab92486, Abcam) and with different sizes of tumor anti-GAPDH antibody (1:1000, ab9485, Abcam) over- Our data have showed that serum levels of PVT1 were night at 4 °C. The next day, membranes were washed and abnormally increased in patients with cervical squamous further incubated with anti-rabbit IgG-HRP secondary cell carcinoma. Therefore, serum levels of PVT1 may also antibody (1:1000, MBS435036, MyBioSource) for 2  h at be different in patients with different sizes of tumor. As room temperature. Signal detection was performed using shown in Fig.  2, serum levels of PVT1 were significantly ECL (Sigma-Aldrich, USA) method. Relative expression increased with the increased sizes of cervical squamous level of TGF-β1 was normalized to endogenous control cell carcinoma. GAPDH using Image J software. Diagnostic and prognostic values of serum PVT1 Statistical analysis for cervical squamous cell carcinoma SPSS19.0 (SPSS Inc., USA) statistical software was used. ROC curve analysis was performed to evaluate the diag- Measurement data were expressed as ( x¯ ± s ), and t test nostic values of serum PVT1 for cervical squamous cell was used for the comparisons between two groups, and carcinoma. As shown in Fig. 3a, the area under the curve comparisons among multiple groups were performed by of serum PVT1 in the diagnosis was 0.9030 with 95% one way analysis of variance, followed by LSD test. Com- confident interval of 0.8567–0.9472 (p < 0.0001), indicat- parisons of count data were performed using Chi square ing that serum PVT1 is an accurate biomarker for cervi- test. p < 0.05 was considered to be statistically significant. cal squamous cell carcinoma. According to the median serum level of serum PVT1 patients were divided into Results high level group and low level group. Kaplan–Meier Serum levels of PVT1 in different patient groups method was used to draw survival curves for both and control group groups. Log-rank test was used to compare two survival Serum levels of PVT1 was detected by qRT-PCR and curves. As shown in Fig.  3b, overall survival of cervical compared among different patient groups and control squamous cell carcinoma patient with high serum level of group. In this study, five HPV strains, including HPV-16, PVT1 was significantly shorter that that of patients with HPV-18, HPV-11, HPV-45 and HPV-68 were detected low serum level of PVT1 (p < 0.05). Those data suggest in tumor tissues of patients with cervical squamous cell that serum level of PVT1 may serve as prognostic marker carcinoma. As shown in Fig.  1, no significant differences for patients with gastric cancer. Fig. 2 Comparison of serum levels of PVT1 among patients in Fig. 1 Serum levels of PVT1 in different patient groups and control different stages of primary tumor. *Compared with one stage before, group p < 0.05 Wang et al. Cancer Cell Int (2018) 18:70 Page 4 of 8 Fig. 3 Diagnostic and prognostic values of serum PVT1 for cervical squamous cell carcinoma. a ROC curve analysis for the diagnostic values of serum PVT1 for cervical squamous cell carcinoma; b comparison of survival curves of patients with high and low serum level of PVT1 Eec ff ts of PVT1 siRNA silencing and overexpression Eec ff ts of PVT1 siRNA silencing and overexpression on proliferation of cervical squamous cell carcinoma on expression of TGF‑β1 Expression of PVT1 in human cervical squamous cell To further investigated the interactions between TGF- carcinoma cell lines SiHa (HPV positive) and C33A β1 and PVT1, eeff cts of PVT1 siRNA silencing and (HPV negative) and normal cervical cell lines Ect1/ overexpression on expression of TGF-β1 in two cervi- E6E7 (HPV positive) and HCvEpC (HPV negative) was cal squamous cell carcinoma cell lines were explored. detected by qRT-PCR. As shown in Fig.  4a, expression As shown in Fig.  5a, PVT1 siRNA silencing significantly level of PVT1 was significantly higher in two cervical increased the expression level of TGF-β1 in both cell squamous cell carcinoma cell lines than in two normal lines (p < 0.05). In contrast, PVT1 overexpression signifi - cells. However, no significant differences in expression cantly reduced the expression of TGF-β1 in two cell lines level of PVT1 were found between SiHa and C33A or (p < 0.05). Especially in C33A cell line, expression level Ect1/E6E7 and HCvEpC, indicating that HPV infection of PVT1 reduced by more than eight times after PVT1 has no significant effects on the expression of PVT1 overexpression. Those data suggest that PVT1 can nega - in those cell lines. PVT1 siRNA silencing and overex - tively regulate the expression of TGF-β1 to participate in pression cell lines were established to investigate the the growth of cervical squamous cell carcinoma. role of PVT1 in the proliferation of cervical squamous cell carcinoma cells. As shown in Fig. 4b, PVT1 siRNA Discussion silencing significantly inhibited cell proliferation of Most cases of cervical squamous cell carcinoma are two cervical squamous cell carcinoma cell lines. In caused by HPV infection [3]. HPV infection is usually contrast, PVT1 overexpression significantly promoted harmless but long-term infection of HPV may increase cell proliferation of two cervical squamous cell carci - the risk of the occurrence of malignant tumors in vari- noma cell lines (Fig.  4c). TGF-β1 has anti-proliferative ous sites, such as cervix, vagina, penis, anus and oro- functions [13], and PVT1 can regulate the expression pharynx [3]. Different genotypes of HPV causes different of TGF-β1 [14]. In this study, treatment with TGF-β1 incidences of cervical squamous cell carcinoma, and two (10  ng/ml, Sigma-Aldrich, USA) for 1  h not signifi- genotypes of HPV, including HPV-16 and -18 causes cantly inhibited cell proliferation of two cervical squa- about 70% cases of HPV-positive cervical squamous cell carcinoma [3]. Consistent with previous studies, in mous cell carcinoma cell lines, but also significantly this study, 135 patients with cervical squamous cell car reduce the enhancing effects of PVT1 overexpression - on cell proliferation. Those data suggest that expres- cinoma were proved to be HPV-positive, accounting sion level of PVT1 is a positive regulator of prolifera- for 86.5% of all the cases. In addition, 47 patients were tion of cervical squamous cell carcinoma cells, and this infected with HPV-16 and 45 patients were infected with function is very likely to be achieved through interac- HPV-18, accounting for 68.4% of all the cases. HPV infec- tions with TGF-β1. tion is a major but not the only cause of this disease. In Wang et al. Cancer Cell Int (2018) 18:70 Page 5 of 8 Fig. 4 Eec ff ts of PVT1 siRNA silencing and overexpression on proliferation of cervical squamous cell carcinoma. a Expression of PVT1 in different cell lines; b Eec ff ts of PVT1 siRNA silencing on proliferation of cervical squamous cell carcinoma; c Eec ff ts of PVT1 siRNA overexpression on proliferation of cervical squamous cell carcinoma. *p < 0.05; C, control cells without transfection; NC, negative control cells transfected with negative control siRNA or empty vector; siRNA, cells transfected with PVT1 siRNA; Over, cells transfected with PVT1 expression vector Wang et al. Cancer Cell Int (2018) 18:70 Page 6 of 8 Fig. 5 Eec ff ts of PVT1 siRNA silencing and overexpression on expression of TGF-β1. a Eec ff ts of PVT1 siRNA silencing on expression of TGF-β1; b effects of PVT1 overexpression on expression of TGF-β1. *p < 0.05; C, control cells without transfection; NC, negative control cells transfected with negative control siRNA or empty vector; siRNA, cells transfected with PVT1 siRNA; Over, cells transfected with PVT1 expression vector this study, 21 patients were proved to be HPV-negative, that expression level of the lncRNA PVT1 was sig- accounting for 13.5% of all the cases. nificantly increased in cancer patients than in normal LncRNA PVT1 plays a role as oncogene in the healthy controls, and the upregulated expression level development of various types of malignant tumors of PVT1 was significantly correlated with poor prog- and shows abnormally upregulated expression. In nosis of those patients [15]. In another study, expres- the study of pancreatic cancer, Huang et  al. found sion level of PVT1 was also found to be abnormally Wang et al. Cancer Cell Int (2018) 18:70 Page 7 of 8 increased in gastric cancer patients, and abnormal Conclusion expression of PVT1 showed promising diagnostic and In conclusion, expression of PVT1 in patients with prognostic values for this disease [16]. Previous stud- cervical squamous cell carcinoma was not affected ies also confirmed that PVT1 is upregulated in cervical by HPV infection. Serum levels of PVT1 were sig- cancer and promote the progression of disease [17– nificantly higher in cervical squamous cell carcinoma 19]. Consistent with previous studies, in this study, patients than in healthy controls. Serum level of PVT1 expression level of PVT1 was found to be significantly increased with the increased size of primary tumor. higher in cervical squamous cell carcinoma patients Serum PVT1 can serve as a promising diagnostic and than in normal controls. In addition, expression level prognostic biomarker for cervical squamous cell carci- of PVT1 was increased with the increased stage of noma PVT1 siRNA silencing inhibited the proliferation primary tumor. HPV infection can induce the abnor- of cancer cells and reduced the expression of TGF-β1, mal expression of certain lncRNAs [10]. In our study, while PVT1 overexpression played an opposite role. no significant differences in serum levels of PVT1 So we conclude that lncRNA PVT1 can promote the were detected among patients infected with different growth HPV positive and negative cervical squamous HPVs and HPV-negative patients. Those data suggest cell carcinoma by inhibiting TGF-β1. Our study is lim- that upregulated expression of PVT1 may participate ited by the small sample size. Future studies with bigger in the pathogenesis of cervical squamous cell carci- sample size are needed to further confirm the conclu - noma through a HPV infection-independent pathway. sions in the present study. Besides that, serum PVT1 also showed promising Authors’ contributions diagnostic and prognostic values for cervical squa- CL conceived and supervised the study. XW, GW, LZ, JC, and JH conducted the mous cell carcinoma. Therefore, PVT1 may serve as a experiments and performed the analysis. XW, GW, and CL wrote the paper. All authors read and approved the final manuscript. target to improve the treatment outcomes and prog- nosis of those patients. It is worth to note that several Author details lncRNAs, such as GAS5 [20] and HOTAIR [21] show Department of Gynecology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai 264000, Shandong, People’s Republic of China. clinical values for the diagnosis and prognosis of cervi- Department of Obstetrics and Gynecology, The Affiliated Yantai Yuhuang- cal cancers. Combination of multiple biomarkers may ding Hospital of Qingdao University, Yantai 264000, Shandong, People’s improve the diagnosis. Republic of China. Previous studies have shown that PVT1 can promote Acknowledgements the proliferation of various types of cancer cells, such Not applicable. as hepatocellular carcinoma cells [22], thyroid can- Competing interests cer cells [23] and so on. PVT1 siRNA silencing inhib- The authors declare that they have no competing interests. ited the proliferation of two cervical squamous cell carcinoma cell lines, while PVT1 overexpression pro- Availability of data and materials Not applicable. moted the proliferation of cancer cells, indicating that PVT1 expression may promote the growth of cervical Consent for publication squamous cell carcinoma by promoting the prolifera- Not applicable. tion of cancer cells. TGF-β1 plays different roles in the Ethics approval and consent to participate development and progression of tumors. TGF-β1 not The ethics committee of Affiliated Yantai Yuhuangding Hospital of Qingdao only inhibits the growth of tumors through its anti- University has approved this study, and participants signed informed consent. proliferative functions [13], but also promotes tumor Funding metastasis by inducing epithelial–mesenchymal transi- Not applicable. tion [24]. Expression of TGF-β1 was regulated by PVT1 [14]. Therefore, it will be reasonable to hypothesize that Publisher’s Note PVT1 may regulate TGF-β1 to participate in cervical Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. squamous cell carcinoma. In this study, PVT1 siRNA silencing significantly increased the expression level Received: 5 April 2018 Accepted: 27 April 2018 of TGF-β1. In contrast, PVT1 overexpression signifi - cantly reduced the expression of TGF-β1. Those data suggest that PVT1 can negatively regulate the expres- sion of TGF-β1 to participate in the growth of cervi- References 1. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality cal squamous cell carcinoma. However, the molecular worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int mechanism of the regulation of TGF-β1 by PVT1 is still J Cancer. 2015;136(5):E359–86. unknown. Therefore, further studies are still needed. Wang et al. 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Overexpression of long non-coding RNA PVT1 in ovarian cancer cells promotes cisplatin resistance by regulating apoptotic pathways. Int J Clin Exp Med. 2015;8(11):20565. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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Cancer Cell InternationalSpringer Journals

Published: May 8, 2018

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