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E7 stimulated upregulation of MALAT1 gene in cervical cancer HeLa cells
Virus Research 281 (2020) 197907
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IL-6/STAT3 mediates the HPV18 E6/E7 stimulated upregulation of MALAT1 gene in cervical cancer HeLa cells
T
Yunpeng Haoa, Zhinan Yana, Aowei Zhanga, Shiyue Hua, Nan Wanga, Xue-Gang Luoa, Wenjian Maa, Tong-Cun Zhanga,b, Hongpeng Hea,* a
Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science and Technology, 300457, Tianjin, PR China b College of Life Sciences, Wuhan University of Science and Technology, 430081, Wuhan, PR China
ARTICLE INFO
ABSTRACT
Keywords: HPV18 E6/E7 IL-6/STAT3 MALAT1 Cervical cancer
MALAT1, a long non-coding RNA, is highly expressed in cervical cancer cells and plays an important role in the development of cervical cancer. However, the mechanism for the excessive expression of MALAT1 in cervical cancer remains unclear. High-risk HPVs are causative agents of cervical cancer and the IL-6/STAT3 signaling is closely correlated with the development of various cancers including cervical cancer. In this study, the roles of HPV18 E6/E7 and IL-6/STAT3 in the regulation of MALAT1 transcription in cervical cancer cells were investigated. It was found that HPV18 E6/E7 activated the IL-6/STAT3 signaling and, in reciprocal, IL-6/STAT3 strengthened HPV18 E6/E7 expression in HeLa cells. Both HPV18 E6/E7 and IL-6/STAT3 were involved in MALAT1 expression and they worked synergistically in the upregulation of MALAT1 gene. With luciferase reporter assays, a STAT3-binding sequence in the enhancer region of MALAT1 gene was demonstrated to be crucial for the IL-6- or STAT3-induced MALAT1 promoter activation. Taken together, our data suggest that IL-6/STAT3 mediates the HPV18 E6/E7 stimulated upregulation of MALAT1 gene in cervical cancer HeLa cells.
1. Introduction Cervical cancer is a malignant tumor accounting for a large proportion of cancer-related deaths among women worldwide. Globally, there are about 500,000 new cases and about 270,000 people die of cervical cancer every year (Eiben et al., 2003; Haie-Meder et al., 2010). One causative factor of cervical cancer is the high-risk human papillomavirus (HPV) mainly including HPV 16 and HPV 18 (Hengstermann et al., 2001; Walboomers et al., 1999; zur Hausen, 2009). High-risk HPV E6 and E7 are two viral oncoproteins that maintain the phenotype of cervical cancer by targeting many cellular proteins, such as p53, Rb, hTERT, HDACs and KDMs, to regulate multiple pathways contributing to cell proliferation, immortalization, genomic instability and immune evasion, etc (Chen et al., 2018; Hengstermann et al., 2001; McLaughlin-Drubin et al., 2012; Moody and Laimins, 2010; zur Hausen, 2009). The IL-6/STAT3 signaling pathway plays crucial roles in both inflammatory diseases and cancers (Hodge et al., 2005). In cervicovaginal secretions, the local production of IL-6 was related to the severity of cervical neoplasia (Tjiong et al., 1999). It has been reported that STAT3
is constitutively activated in HPV-positive cervical cancer cells to enhance EMT (Sobti et al., 2009). Therefore, both high-risk HPV and the IL-6/STAT3 inflammatory signaling are closely correlated with cervical cancer. Long non-coding RNAs (LncRNAs) have limited protein coding ability but play an important regulatory role in cell proliferation, differentiation, invasion, and self-renewal of tumor cells (Ernst and Morton, 2013; Hu et al., 2018; Morlando and Fatica, 2018). Many LncRNAs are abnormally expressed in cervical cancer cells, including MALAT1 which is one of the most extensively studied lncRNAs (Cui et al., 2017; Jiang et al., 2014; Yang et al., 2015; Zhang et al., 2015). MALAT1 is believed to promote cell proliferation and invasive growth of cervical cancer cells in various ways (Jiang et al., 2014; Wang et al., 2018a; Yang et al., 2015; Zhang et al., 2015). However, the molecular mechanism by which MALAT1 is excessively expressed in cervical cancer cells is still unclear. The purpose of this study was to investigate the effects of IL-6/ STAT3 and HPV18 E6/E7 on the expression of LncRNA MALAT1 in cervical cancer cells.
⁎ Corresponding author at: College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th. Avenue, Tianjin Economic and Technological Development Area (TEDA), 300457, Tianjin, PR China. E-mail address: [email protected] (H. He).
https://doi.org/10.1016/j.virusres.2020.197907 Received 5 October 2019; Received in revised form 23 January 2020; Accepted 26 February 2020 Available online 27 February 2020 0168-1702/ © 2020 Elsevier B.V. All rights reserved.
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Fig. 1. HPV18 E6/E7 upregulated MALAT1 and activated the IL-6/STAT3 signaling. (A) Overexpression of HPV18 E6/E7 upregulated MALAT1 expression. (B) HPV18 E6/E7 overexpression augmented IL-6 expression and STAT3 phosphorylation. (C) MALAT1 expression was decreased in HPV18 E6/E7 depleting HeLa cells. (D) Depletion of HPV18 E6/E7 with HPV18 E6/E7 specific siRNA. (E) HPV18 E6/E7 upregulated MALAT1 expression in C33A cells. (F) HPV18 E6/E7 upregulated MALAT1 expression in HT-29 cells.
2. Materials and methods
synthesized using reverse transcription kits purchased from Promega, USA. Quantitative real-time RCR was carried out using the SYBR Green Supermix purchased from DBI China and detected by ABI step one system. The 2−ΔΔCT method was used to calculate relative transcription levels.
2.1. Cell culture and transfection Cervical cancer HeLa cells (HPV18 positive), cervical cancer C33A cells (HPV-negative) and colon cancer HT-29 cells (HPV-negative) were cultured in DMEM-Low glucose (Gibico, USA) containing 10 % FBS (Kangyuan, China), 100 μg/ml streptomycin and 100 U/ml penicillin (Solarbio, China). Recombinant Human Interleukin-6 was purchased from Prime Gene, Shanghai, China and diluted in sterile PBS. AG-490 was purchased from Selleck, USA and diluted in DMSO. Plasmids pCMV-Tag2B-HPV18 E6E7 and pcDNA3.1-STAT3 were previously described (He et al., 2019; Liao et al., 2015) and transiently transfected into HeLa cells with TurboFect (Thermo, USA). following the manufacturer's instruction. All plasmids used in this study were re-sequenced to confirm their identities before transfection. The STAT3 interfering siRNA duplexes and HPV18 E6E7 interfering siRNA duplexes, designed and synthesized by RiboBio, Guangzhou, China, were introduced into cells with RiboFect transfection agent (RiboBio, Guangzhou, China).
2.3. Western-blot Antibodies against HPV18/16 E6/E7, STAT3 were from Santa Cruz Biotechnology, USA. Antibodies against GAPDH was from Beyotime Biotechnology, China. Anti-phospho-STAT3 was from Bioss, Beijing, China. Secondary antibodies were IRDye-conjugated donkey antimouse or anti-rabbit IgG (Licor Biosciences, USA). Protein samples were prepared with SDS lysis buffer. Whole cell lysates were separated in an SDS-PAGE and transferred to a nitrocellulose membrane. Protein bands were visualized using Odyssey Imaging system with GAPDH serving as the loading control. The optical band density and relative quantification of protein bands were analyzed with ImageJ. 2.4. Luciferase reporter assay
2.2. Extraction of total RNA and RT-PCR
pGL3-MALAT1-Luciferase reporter plasmids carrying MALAT1 promoter and enhancer sequences were constructed with pGL3 backbone
Total RNA was isolated with Trizol reagent (Sigma). cDNA was 2
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Fig. 2. IL-6/STAT3 signaling promoted MALAT1 expression in HeLa cells. (A) IL-6 stimulated the phosphorylation of STAT3. (B) IL-6 stimulated MALAT1 expression. (C) AG490 inhibited the phosphorylation of STAT3. (D) AG490 inhibited MALAT1 expression. (E) Overexpression of STAT3 in HeLa cells. (F) Knockdown of STAT3 in HeLa cells. (G) MALAT1 RNA level was increased with STAT3 overexpression. (H) MALAT1 RNA level was decreased in STAT3-depelting cells.
The data presented are results of three to six independent experiments and the GraphPad Prism 6 was used for statistical analysis. The data are presented as mean ± standard deviation. Two tailed p value < 0.05 or < 0.01 was indicated by * or **, respectively.
relative MALAT1 RNA level was increased by about 7-fold in C33A cells and by about 2-fold in HT-29 cells (Fig. 1E and F). These results further confirm the important role of HPV18 E6/E7 in MALAT1 expression. HPV16 and HPV18 were previously shown to induce the activation of STAT3 in cervical cancer cells (Morgan and Macdonald, 2019). To confirm this effect, IL-6 and STAT3 were examined in HeLa cells overexpressing HPV18 E6/E7. As shown in Fig. 1B, over-expression of HPV18 E6/E7 promoted the expression of IL-6 in HeLa cells meanwhile there were more phosphorylated-STAT3 in HPV18 E6/E7 overexpressing HeLa cells although the total STAT3 was not altered. These results agree with the previous reports, confirming that HPV proteins stimulate the IL6/STAT3 signaling pathway.
3. Results
3.2. IL-6/STAT3 enhanced the transcription MALAT1 gene
3.1. HPV18 E6/E7 upregulated the expression of lncRNA MALAT1and activated the IL-6/STAT3 signaling in HeLa cells
HPV18 E6/E7 upregulated MALAT1 expression and activated the IL6/STAT3 signaling in HeLa cells. Given that STAT3 is a well-defined transcription activator, it is possible that IL-6/STAT3 affects the expression of MALAT1. To test this possibility, the RNA level of MALAT1 was measured when STAT3 was either activated by IL-6 treatment or inhibited by AG490 treatment. As shown in Fig. 2A, in response to IL-6 treatment, more STAT3 was phosphorylated indicating the activation of STAT3. Meanwhile, the RNA level of MALAT1 was increased significantly (Fig. 2B). By contrast, in AG-490-treated cells, the phosphorylation of STAT3 was inhibited (Fig. 2C). Under this condition, the expression of MALAT1 was attenuated (Fig. 2D), indicating the involvement of IL-6/STAT3 signaling pathway in MALAT1 gene regulation. To confirm the involvement of STAT3 in MALAT1 expression, STAT3 was either overexpressed (Fig. 2E) or depleted (Fig. 2F) in HeLa cells. Results of RT-qPCR showed that the lncRNA level of MALAT1 increased following STAT3 overexpression (Fig. 2G) but diminished with STAT3-knockdown (Fig. 2H). These results confirm that STAT3 is required for the excessive expression of MALAT1 in HeLa cells. STAT3 is a transcription factor that directly interacts with regulatory sequences of target genes. In order to verify whether STAT3 directly regulates MALAT1 transcription, the sequences of MALAT1 gene were analyzed using online biology software GeneCards and a STAT3 binding site was identified downstream of the transcription start site of MALAT1 gene, which suggests that STAT3 may regulate MALAT1
using MluI and SmaI sites. For luciferase activity assays, Hela cell were transfected with pcDNA3.1-STAT3 for 24 h before the transfection of luciferase reporter plasmids for another 24 h. Then luciferase activity was measured with Luciferase Assay System (Promega) in a Synergy™ 4 luminometer (Bioteck, USA). 2.5. Statistical analyses
HPV is a causative agent in the development of cervical cancer. LncRNA MALAT1 was previously identified to be highly expressed and be correlated with the metastasis of cervical cancer (Yang et al., 2015; Zhang et al., 2015). To explore the relationship between HPV and MALAT1, HPV18 E6/E7 encoding plasmids were transfected into HeLa cells. The efficiency of HPV18 E6/E7 overexpression was tested with RT-qPCR and Western-blot (Fig. 1A, B). With the overexpression of HPV18 E6 and E7, the RNA level of MALAT1 was increased by about 2 folds (Fig. 1A), suggesting a positive role of HPV18 E6/E7 in the expression of MALAT1. To further demonstrate the role of HPV 18 E6 and E7 in MALAT1 expression, siRNA was applied to knockdown HPV18 E6/E7 (Fig. 1C and D). In these HPV18 E6/E7 depleting HeLa cells, the RNA level of MALAT1 was significantly downregulated (Fig. 1C). Together, these results support that HPV18 E6/E7 contribute to the highly expression of MALAT1 in cervical cancer cells. To test the effect of HPV18 E6/E7 on MALAT1 expression in HPVnegative cells, HPV18 E6/E7 plasmids were introduced into C33A, an HPV-negative cervical cancer cell line, and HT29, an HPV-negative colon cancer cell line. In these two cell lines, there is no endogenous HPV genes, thus the relative E6 and E7 mRNA levels seem to be increased very dramatically which is about 6 thousand folds. Meanwhile, 3
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Fig. 3. STAT3-binding sequence was critical for IL-6/STAT3 to stimulate MALAT1 promoter activation. (A) Construction of luciferase reporter plasmids for MALAT1 promoter activity with or without STAT3-binding enhancer sequence. (B) STAT3 overexpression enhanced the activity MALAT1 promoter with a STAT3binding sequence. (C) IL-6 stimulated the activity MALAT1 promoter with a STAT3-binding sequence.
transcription through this enhancer sequence. To examine the importance of the enhancer sequence, luciferase reporter plasmids of MALAT1 promoter containing enhancer sequence or not were constructed (Fig. 3A). Results of luciferase assays showed that overexpression of STAT3 elevated MALAT1 promoter activity with the STAT3-binding enhancer but had little effect on MALAT1 promoter without enhancer (Fig. 3B). Consistently, IL-6 failed to stimulate MALAT1 promoter without STAT3-binding sequence whereas significantly augmented the activity of MALAT1 promoter with enhancer (Fig. 3C). These data demonstrated that STAT3-binding sequence plays an important role in the transcriptional regulation of MALAT1 gene, suggesting a direct involvement of STAT3 in MALAT1 regulation.
with the upregulation of HPV18 E6/E7 mRNA level, results of westernblot showed that the protein levels of HPV18 E6 and E7 were increased in IL-6 treated HeLa cells (Fig. 4B). The above results suggest that IL-6/ STAT3 signaling upregulated the expression of HPV18 E6/E7 in HeLa cells. To further confirm the stimulatory effect of IL-6/STAT3 signaling on HPV18 E6/E7 expression, HeLa cells were treated with AG490, a blocker of IL-6/STAT3 signaling pathway. The RNA levels of HPV18 E6 and E7 were obviously decreased following AG490 treatment (Fig. 4C). Results of western-blot showed that, under the same condition, the protein levels of HPV18 E6 and E7 were downregulated (Fig. 4D). These results support that IL-6/STAT3 signaling contributes to the constitutive expression of HPV18 E6/E7 in HeLa cell. To determine the involvement of STAT3 in the regulation of HPV18 genes, STAT3 was overexpressed in HeLa cells. As shown in Fig. 4E, the RNA levels of HPV18 E6 and E7 increased significantly. Results of western-blot showed the successful overexpression of STAT3 and the augmented expression of HPV18 E6 and E7 (Fig. 4F), supporting the involvement of STAT3 in the regulation of HPV18 E6/E7 genes. To further confirm the importance of STAT3 in HPV18 E6/E7 expression, siSTAT3 was used to knockdown endogenous STAT3. In STAT3-deficient cells, the expression of HPV18 E6/E7 was decreased by
3.3. HPV18 E6/E7 and IL-6/Stat3 worked synergistically to upregulate MALAT1gene As shown in Fig. 1B, HPV18 E6/E7 promoted IL-6 expression and STAT3 activation. To ask whether IL-6/STAT3 signaling has any influence on the expression of HPV E6 and E7, cervical cancer HeLa cells were firstly treated with IL-6. The RNA levels of HPV18 E6 and E7 were measured with RT-qPCR and the results showed that the expression of HPV18 E6 and E7 was upregulated by around 2 folds (Fig. 4A). In line 4
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Fig. 4. IL-6/STAT3 signaling strengthened HPV18 E6/E7 expression in HeLa cells. (A) HPV18 E6/E7 mRNA levels and (B) HPV18 E6/E7 protein levels were increased in IL-6-treated cells. (C) HPV18 E6/E7 mRNA levels and (D) HPV18 E6/E7 protein levels were reduced in AG490-treated cells. (E) HPV18 E6/E7 mRNA levels and (F) HPV18 E6/E7 protein levels were upregulated by STAT3 overexpression. (G) HPV18 E6/E7 mRNA levels and (H) HPV18 E6/E7 protein levels were downregulated in STAT3-depleting cells.
about 40 % (Fig. 4G and H), suggesting that STAT3 is required for the efficient expression of HPV18 E6/E7. Taken together, the above results demonstrate that the IL-6/STAT3 signaling activated the expression of HPV18 E6/E7 in HeLa cells. Thus, HPV18 E6/E7 induces the constitutive activation of IL-6/STAT3 signaling and IL-6/STAT3 strengthens the expression of HPV18 E6/E7, thereby forming a positive feedback loop, in HeLa cells. HPV18 E6/E7 and IL-6/STAT3 reciprocally strengthen their expression or activation in in HeLa cells. To test the effects of HPV18 E6/ E7 and IL-6/STAT3 on MALAT1 gene regulation, HeLa cells were
transfected with both HPV18 E6/E7- and STAT3-encoding plasmids. Results of RT-qPCR showed that MALAT1 RNA level was further increased to about 3-folds with the co-overexpression (Fig. 5A). In HeLa cells co-treated with IL-6 and HPV18 E6/E7, MALAT1 expression was similarly upregulated (Fig. 5B). These results demonstrated that HPV18 E6/E7 and IL-6/STAT3 work synergistically in MALAT1 gene upregulation and IL-6/STAT3 mediates the HPV18 E6/E7 induced MALAT1 expression in HeLa cells (Fig. 5C).
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Fig. 5. HPV18 E6/E7 and IL-6/STAT3 synergistically promoted the expression of MALAT1. (A) MALAT1 RNA level was further increased following the cooverexpression of HPV18 E6/E7 and STAT3. (B) MALAT1 RNA level was further increased in cells overexpressing of HPV18 E6/E7 and co-treated with IL-6. (C) A model describing the roles of HPV18 E6/E7 and IL-6/STAT3 in the regulation of MALAT1 gene in cervical cancer HeLa cells.
4. Discussion
administration, Methodology. Wenjian Ma: Software, Writing - review & editing. Tong-Cun Zhang: Resources. Hongpeng He: Conceptualization, Supervision, Writing - original draft, Funding acquisition, Resources.
LncRNA MALAT1 was previously reported to be highly expressed in cervical cancer cells and was correlated with the metastasis of cervical cancer (Jiang et al., 2014; Yang et al., 2015; Zhang et al., 2015). Give the previously observed correlation between MALAT1 and cervical cancer development, MALAT1 was proposed to be a potential diagnostic marker (Cui et al., 2017). However, the mechanism by which MALAT1 is highly expressed in cervical cancer cells remains unclear. In this paper, our results revealed that HPV E6/E7 and IL-6/STAT3 signaling pathway work synergistically to upregulate the transcription of MALAT1 in cervical cancer HeLa cells, suggesting a cooperation of virus oncoproteins with cellular inflammatory signaling in cervical cancer development. The IL-6/STAT3 signaling pathway has been identified to be constitutively activated in a variety of cancer cells including cervical cancer to promote the survival and invasive growth of cancer cells (Shukla et al., 2013). This constitutive activation was proposed to be correlated with HPV16 E6 which upregulates IL-6/STAT3 expression or activation (Morgan et al., 2018; Ren et al., 2013). Herein, in HeLa cells, we observed that IL-6 expression was increased and STAT3 was phosphorylated following HPV18 E6/E7 overexpression which is in line with previous observations and further confirming the regulatory effects of HPV E6/E7 on IL-6/STAT3 signaling. Moreover, we found that IL-6/ STAT3 signaling reciprocally regulated HPV18 E6/E7 expression in Hela cells. STAT3 was previously shown to be required for HPV16 E6/ E7 in HPV16-positive cervical cancer cells (Shukla et al., 2013) and was critical for the IL-10 induced HPV16 E6/E7 expression (Arany et al., 2002). Thereby, our results, together with results from other studies, suggest a positive feedback between HPV E6/E7 and the STAT3 signaling. Based on this positive feedback loop, HPV18 E6/E7 and IL-6/ STAT3 function synergistically in the upregulation of MALAT1 gene in cervical cancer cells. Previously, paracrine IL-8 was shown to activate STAT3 which enhanced MALAT1 expression by binding to MALAT1 promoter in prostate cancer cells (Zheng et al., 2018). In head and neck squamous cell carcinoma, TGF-beta was shown to induce STAT3 expression and STAT3 in turn promoted MALAT1 transcription (Wang et al., 2018b). Herein, we demonstrated the up-regulation of MALAT1 by IL-6/STAT3 signaling in cervical cancer cells. These findings further demonstrate the correlation between the activated STAT3 signaling and the upregulated MALAT1 expression in cancer cells, supporting the potential therapeutic value of STAT3 in cancer treatment.
Declaration of Competing Interest All of the authors declare no conflict of interests. Acknowledgements This work was supported by National Natural Science Foundation of China (31301073), the Natural Science Foundation of Tianjin (No.18JCYBJC91500, No. 17JCZDJC33600) and the Innovative Research Team of Tianjin Municipal Education Commission (TD135015). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.virusres.2020.197907. References Arany, I., Grattendick, K.G., Tyring, S.K., 2002. Interleukin-10 induces transcription of the early promoter of human papillomavirus type 16 (HPV16) through the 5’-segment of the upstream regulatory region (URR). Antiviral Res. 55 (2), 331–339. Chen, X., Loo, J.X., Shi, X., Xiong, W., Guo, Y., Ke, H., Yang, M., Jiang, Y., Xia, S., Zhao, M., Zhong, S., He, C., Fu, L., Li, F., 2018. E6 protein expressed by high-risk HPV activates super-enhancers of the EGFR and c-MET oncogenes by destabilizing the histone demethylase KDM5C. Cancer Res. 78 (6), 1418–1430. Cui, X., Jing, X., Wu, X., 2017. The prognostic value of long non coding RNAs in cervical cancer: a meta-analysis. Oncotarget 8 (37), 62470–62477. Eiben, G.L., da Silva, D.M., Fausch, S.C., Le Poole, I.C., Nishimura, M.I., Kast, W.M., 2003. Cervical cancer vaccines: recent advances in HPV research. Viral Immunol. 16 (2), 111–121. Ernst, C., Morton, C.C., 2013. Identification and function of long non-coding RNA. Front. Cell. Neurosci. 7, 168. Haie-Meder, C., Morice, P., Castiglione, M., Group, E.G.W., 2010. Cervical cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 21 (Suppl 5), v37–40. He, H., Liu, X., Liu, Y., Zhang, M., Lai, Y., Hao, Y., Wang, Q., Shi, D., Wang, N., Luo, X.G., Ma, W., Zhang, T.C., 2019. Human papillomavirus E6/E7 and long noncoding RNA TMPOP2 mutually upregulated gene expression in cervical Cancer cells. J. Virol. 93 (8). Hengstermann, A., Linares, L.K., Ciechanover, A., Whitaker, N.J., Scheffner, M., 2001. Complete switch from Mdm2 to human papillomavirus E6-mediated degradation of p53 in cervical cancer cells. Proc. Natl. Acad. Sci. U.S.A. 98 (3), 1218–1223. Hodge, D.R., Hurt, E.M., Farrar, W.L., 2005. The role of IL-6 and STAT3 in inflammation and cancer. Eur. J. Cancer 41 (16), 2502–2512. Hu, G., Niu, F., Humburg, B.A., Liao, K., Bendi, S., Callen, S., Fox, H.S., Buch, S., 2018. Molecular mechanisms of long noncoding RNAs and their role in disease pathogenesis. Oncotarget 9 (26), 18648–18663. Jiang, Y., Li, Y., Fang, S., Jiang, B., Qin, C., Xie, P., Zhou, G., Li, G., 2014. The role of MALAT1 correlates with HPV in cervical cancer. Oncol. Lett. 7 (6), 2135–2141. Liao, X.H., Zheng, L., He, H.P., Zheng, D.L., Wei, Z.Q., Wang, N., Dong, J., Ma, W.J., Zhang, T.C., 2015. STAT3 regulated ATR via microRNA-383 to control DNA damage to affect apoptosis in A431 cells. Cell. Signal. 27 (11), 2285–2295. McLaughlin-Drubin, M.E., Meyers, J., Munger, K., 2012. Cancer associated human papillomaviruses. Curr. Opin. Virol. 2 (4), 459–466.
CRediT authorship contribution statement Yunpeng Hao: Investigation, Methodology, Data curation, Visualization, Writing - original draft. Zhinan Yan: Investigation, Methodology, Visualization, Data curation. Aowei Zhang: Investigation, Methodology. Shiyue Hu: Validation, Methodology. Nan Wang: Funding acquisition, Resources. Xue-Gang Luo: Project 6
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IL-6/STAT3 mediates the HPV18 E6/E7 stimulated upregulation of MALAT1 gene in cervical cancer HeLa cells
T
Yunpeng Haoa, Zhinan Yana, Aowei Zhanga, Shiyue Hua, Nan Wanga, Xue-Gang Luoa, Wenjian Maa, Tong-Cun Zhanga,b, Hongpeng Hea,* a
Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science and Technology, 300457, Tianjin, PR China b College of Life Sciences, Wuhan University of Science and Technology, 430081, Wuhan, PR China
ARTICLE INFO
ABSTRACT
Keywords: HPV18 E6/E7 IL-6/STAT3 MALAT1 Cervical cancer
MALAT1, a long non-coding RNA, is highly expressed in cervical cancer cells and plays an important role in the development of cervical cancer. However, the mechanism for the excessive expression of MALAT1 in cervical cancer remains unclear. High-risk HPVs are causative agents of cervical cancer and the IL-6/STAT3 signaling is closely correlated with the development of various cancers including cervical cancer. In this study, the roles of HPV18 E6/E7 and IL-6/STAT3 in the regulation of MALAT1 transcription in cervical cancer cells were investigated. It was found that HPV18 E6/E7 activated the IL-6/STAT3 signaling and, in reciprocal, IL-6/STAT3 strengthened HPV18 E6/E7 expression in HeLa cells. Both HPV18 E6/E7 and IL-6/STAT3 were involved in MALAT1 expression and they worked synergistically in the upregulation of MALAT1 gene. With luciferase reporter assays, a STAT3-binding sequence in the enhancer region of MALAT1 gene was demonstrated to be crucial for the IL-6- or STAT3-induced MALAT1 promoter activation. Taken together, our data suggest that IL-6/STAT3 mediates the HPV18 E6/E7 stimulated upregulation of MALAT1 gene in cervical cancer HeLa cells.
1. Introduction Cervical cancer is a malignant tumor accounting for a large proportion of cancer-related deaths among women worldwide. Globally, there are about 500,000 new cases and about 270,000 people die of cervical cancer every year (Eiben et al., 2003; Haie-Meder et al., 2010). One causative factor of cervical cancer is the high-risk human papillomavirus (HPV) mainly including HPV 16 and HPV 18 (Hengstermann et al., 2001; Walboomers et al., 1999; zur Hausen, 2009). High-risk HPV E6 and E7 are two viral oncoproteins that maintain the phenotype of cervical cancer by targeting many cellular proteins, such as p53, Rb, hTERT, HDACs and KDMs, to regulate multiple pathways contributing to cell proliferation, immortalization, genomic instability and immune evasion, etc (Chen et al., 2018; Hengstermann et al., 2001; McLaughlin-Drubin et al., 2012; Moody and Laimins, 2010; zur Hausen, 2009). The IL-6/STAT3 signaling pathway plays crucial roles in both inflammatory diseases and cancers (Hodge et al., 2005). In cervicovaginal secretions, the local production of IL-6 was related to the severity of cervical neoplasia (Tjiong et al., 1999). It has been reported that STAT3
is constitutively activated in HPV-positive cervical cancer cells to enhance EMT (Sobti et al., 2009). Therefore, both high-risk HPV and the IL-6/STAT3 inflammatory signaling are closely correlated with cervical cancer. Long non-coding RNAs (LncRNAs) have limited protein coding ability but play an important regulatory role in cell proliferation, differentiation, invasion, and self-renewal of tumor cells (Ernst and Morton, 2013; Hu et al., 2018; Morlando and Fatica, 2018). Many LncRNAs are abnormally expressed in cervical cancer cells, including MALAT1 which is one of the most extensively studied lncRNAs (Cui et al., 2017; Jiang et al., 2014; Yang et al., 2015; Zhang et al., 2015). MALAT1 is believed to promote cell proliferation and invasive growth of cervical cancer cells in various ways (Jiang et al., 2014; Wang et al., 2018a; Yang et al., 2015; Zhang et al., 2015). However, the molecular mechanism by which MALAT1 is excessively expressed in cervical cancer cells is still unclear. The purpose of this study was to investigate the effects of IL-6/ STAT3 and HPV18 E6/E7 on the expression of LncRNA MALAT1 in cervical cancer cells.
⁎ Corresponding author at: College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th. Avenue, Tianjin Economic and Technological Development Area (TEDA), 300457, Tianjin, PR China. E-mail address: [email protected] (H. He).
https://doi.org/10.1016/j.virusres.2020.197907 Received 5 October 2019; Received in revised form 23 January 2020; Accepted 26 February 2020 Available online 27 February 2020 0168-1702/ © 2020 Elsevier B.V. All rights reserved.
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Fig. 1. HPV18 E6/E7 upregulated MALAT1 and activated the IL-6/STAT3 signaling. (A) Overexpression of HPV18 E6/E7 upregulated MALAT1 expression. (B) HPV18 E6/E7 overexpression augmented IL-6 expression and STAT3 phosphorylation. (C) MALAT1 expression was decreased in HPV18 E6/E7 depleting HeLa cells. (D) Depletion of HPV18 E6/E7 with HPV18 E6/E7 specific siRNA. (E) HPV18 E6/E7 upregulated MALAT1 expression in C33A cells. (F) HPV18 E6/E7 upregulated MALAT1 expression in HT-29 cells.
2. Materials and methods
synthesized using reverse transcription kits purchased from Promega, USA. Quantitative real-time RCR was carried out using the SYBR Green Supermix purchased from DBI China and detected by ABI step one system. The 2−ΔΔCT method was used to calculate relative transcription levels.
2.1. Cell culture and transfection Cervical cancer HeLa cells (HPV18 positive), cervical cancer C33A cells (HPV-negative) and colon cancer HT-29 cells (HPV-negative) were cultured in DMEM-Low glucose (Gibico, USA) containing 10 % FBS (Kangyuan, China), 100 μg/ml streptomycin and 100 U/ml penicillin (Solarbio, China). Recombinant Human Interleukin-6 was purchased from Prime Gene, Shanghai, China and diluted in sterile PBS. AG-490 was purchased from Selleck, USA and diluted in DMSO. Plasmids pCMV-Tag2B-HPV18 E6E7 and pcDNA3.1-STAT3 were previously described (He et al., 2019; Liao et al., 2015) and transiently transfected into HeLa cells with TurboFect (Thermo, USA). following the manufacturer's instruction. All plasmids used in this study were re-sequenced to confirm their identities before transfection. The STAT3 interfering siRNA duplexes and HPV18 E6E7 interfering siRNA duplexes, designed and synthesized by RiboBio, Guangzhou, China, were introduced into cells with RiboFect transfection agent (RiboBio, Guangzhou, China).
2.3. Western-blot Antibodies against HPV18/16 E6/E7, STAT3 were from Santa Cruz Biotechnology, USA. Antibodies against GAPDH was from Beyotime Biotechnology, China. Anti-phospho-STAT3 was from Bioss, Beijing, China. Secondary antibodies were IRDye-conjugated donkey antimouse or anti-rabbit IgG (Licor Biosciences, USA). Protein samples were prepared with SDS lysis buffer. Whole cell lysates were separated in an SDS-PAGE and transferred to a nitrocellulose membrane. Protein bands were visualized using Odyssey Imaging system with GAPDH serving as the loading control. The optical band density and relative quantification of protein bands were analyzed with ImageJ. 2.4. Luciferase reporter assay
2.2. Extraction of total RNA and RT-PCR
pGL3-MALAT1-Luciferase reporter plasmids carrying MALAT1 promoter and enhancer sequences were constructed with pGL3 backbone
Total RNA was isolated with Trizol reagent (Sigma). cDNA was 2
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Fig. 2. IL-6/STAT3 signaling promoted MALAT1 expression in HeLa cells. (A) IL-6 stimulated the phosphorylation of STAT3. (B) IL-6 stimulated MALAT1 expression. (C) AG490 inhibited the phosphorylation of STAT3. (D) AG490 inhibited MALAT1 expression. (E) Overexpression of STAT3 in HeLa cells. (F) Knockdown of STAT3 in HeLa cells. (G) MALAT1 RNA level was increased with STAT3 overexpression. (H) MALAT1 RNA level was decreased in STAT3-depelting cells.
The data presented are results of three to six independent experiments and the GraphPad Prism 6 was used for statistical analysis. The data are presented as mean ± standard deviation. Two tailed p value < 0.05 or < 0.01 was indicated by * or **, respectively.
relative MALAT1 RNA level was increased by about 7-fold in C33A cells and by about 2-fold in HT-29 cells (Fig. 1E and F). These results further confirm the important role of HPV18 E6/E7 in MALAT1 expression. HPV16 and HPV18 were previously shown to induce the activation of STAT3 in cervical cancer cells (Morgan and Macdonald, 2019). To confirm this effect, IL-6 and STAT3 were examined in HeLa cells overexpressing HPV18 E6/E7. As shown in Fig. 1B, over-expression of HPV18 E6/E7 promoted the expression of IL-6 in HeLa cells meanwhile there were more phosphorylated-STAT3 in HPV18 E6/E7 overexpressing HeLa cells although the total STAT3 was not altered. These results agree with the previous reports, confirming that HPV proteins stimulate the IL6/STAT3 signaling pathway.
3. Results
3.2. IL-6/STAT3 enhanced the transcription MALAT1 gene
3.1. HPV18 E6/E7 upregulated the expression of lncRNA MALAT1and activated the IL-6/STAT3 signaling in HeLa cells
HPV18 E6/E7 upregulated MALAT1 expression and activated the IL6/STAT3 signaling in HeLa cells. Given that STAT3 is a well-defined transcription activator, it is possible that IL-6/STAT3 affects the expression of MALAT1. To test this possibility, the RNA level of MALAT1 was measured when STAT3 was either activated by IL-6 treatment or inhibited by AG490 treatment. As shown in Fig. 2A, in response to IL-6 treatment, more STAT3 was phosphorylated indicating the activation of STAT3. Meanwhile, the RNA level of MALAT1 was increased significantly (Fig. 2B). By contrast, in AG-490-treated cells, the phosphorylation of STAT3 was inhibited (Fig. 2C). Under this condition, the expression of MALAT1 was attenuated (Fig. 2D), indicating the involvement of IL-6/STAT3 signaling pathway in MALAT1 gene regulation. To confirm the involvement of STAT3 in MALAT1 expression, STAT3 was either overexpressed (Fig. 2E) or depleted (Fig. 2F) in HeLa cells. Results of RT-qPCR showed that the lncRNA level of MALAT1 increased following STAT3 overexpression (Fig. 2G) but diminished with STAT3-knockdown (Fig. 2H). These results confirm that STAT3 is required for the excessive expression of MALAT1 in HeLa cells. STAT3 is a transcription factor that directly interacts with regulatory sequences of target genes. In order to verify whether STAT3 directly regulates MALAT1 transcription, the sequences of MALAT1 gene were analyzed using online biology software GeneCards and a STAT3 binding site was identified downstream of the transcription start site of MALAT1 gene, which suggests that STAT3 may regulate MALAT1
using MluI and SmaI sites. For luciferase activity assays, Hela cell were transfected with pcDNA3.1-STAT3 for 24 h before the transfection of luciferase reporter plasmids for another 24 h. Then luciferase activity was measured with Luciferase Assay System (Promega) in a Synergy™ 4 luminometer (Bioteck, USA). 2.5. Statistical analyses
HPV is a causative agent in the development of cervical cancer. LncRNA MALAT1 was previously identified to be highly expressed and be correlated with the metastasis of cervical cancer (Yang et al., 2015; Zhang et al., 2015). To explore the relationship between HPV and MALAT1, HPV18 E6/E7 encoding plasmids were transfected into HeLa cells. The efficiency of HPV18 E6/E7 overexpression was tested with RT-qPCR and Western-blot (Fig. 1A, B). With the overexpression of HPV18 E6 and E7, the RNA level of MALAT1 was increased by about 2 folds (Fig. 1A), suggesting a positive role of HPV18 E6/E7 in the expression of MALAT1. To further demonstrate the role of HPV 18 E6 and E7 in MALAT1 expression, siRNA was applied to knockdown HPV18 E6/E7 (Fig. 1C and D). In these HPV18 E6/E7 depleting HeLa cells, the RNA level of MALAT1 was significantly downregulated (Fig. 1C). Together, these results support that HPV18 E6/E7 contribute to the highly expression of MALAT1 in cervical cancer cells. To test the effect of HPV18 E6/E7 on MALAT1 expression in HPVnegative cells, HPV18 E6/E7 plasmids were introduced into C33A, an HPV-negative cervical cancer cell line, and HT29, an HPV-negative colon cancer cell line. In these two cell lines, there is no endogenous HPV genes, thus the relative E6 and E7 mRNA levels seem to be increased very dramatically which is about 6 thousand folds. Meanwhile, 3
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Fig. 3. STAT3-binding sequence was critical for IL-6/STAT3 to stimulate MALAT1 promoter activation. (A) Construction of luciferase reporter plasmids for MALAT1 promoter activity with or without STAT3-binding enhancer sequence. (B) STAT3 overexpression enhanced the activity MALAT1 promoter with a STAT3binding sequence. (C) IL-6 stimulated the activity MALAT1 promoter with a STAT3-binding sequence.
transcription through this enhancer sequence. To examine the importance of the enhancer sequence, luciferase reporter plasmids of MALAT1 promoter containing enhancer sequence or not were constructed (Fig. 3A). Results of luciferase assays showed that overexpression of STAT3 elevated MALAT1 promoter activity with the STAT3-binding enhancer but had little effect on MALAT1 promoter without enhancer (Fig. 3B). Consistently, IL-6 failed to stimulate MALAT1 promoter without STAT3-binding sequence whereas significantly augmented the activity of MALAT1 promoter with enhancer (Fig. 3C). These data demonstrated that STAT3-binding sequence plays an important role in the transcriptional regulation of MALAT1 gene, suggesting a direct involvement of STAT3 in MALAT1 regulation.
with the upregulation of HPV18 E6/E7 mRNA level, results of westernblot showed that the protein levels of HPV18 E6 and E7 were increased in IL-6 treated HeLa cells (Fig. 4B). The above results suggest that IL-6/ STAT3 signaling upregulated the expression of HPV18 E6/E7 in HeLa cells. To further confirm the stimulatory effect of IL-6/STAT3 signaling on HPV18 E6/E7 expression, HeLa cells were treated with AG490, a blocker of IL-6/STAT3 signaling pathway. The RNA levels of HPV18 E6 and E7 were obviously decreased following AG490 treatment (Fig. 4C). Results of western-blot showed that, under the same condition, the protein levels of HPV18 E6 and E7 were downregulated (Fig. 4D). These results support that IL-6/STAT3 signaling contributes to the constitutive expression of HPV18 E6/E7 in HeLa cell. To determine the involvement of STAT3 in the regulation of HPV18 genes, STAT3 was overexpressed in HeLa cells. As shown in Fig. 4E, the RNA levels of HPV18 E6 and E7 increased significantly. Results of western-blot showed the successful overexpression of STAT3 and the augmented expression of HPV18 E6 and E7 (Fig. 4F), supporting the involvement of STAT3 in the regulation of HPV18 E6/E7 genes. To further confirm the importance of STAT3 in HPV18 E6/E7 expression, siSTAT3 was used to knockdown endogenous STAT3. In STAT3-deficient cells, the expression of HPV18 E6/E7 was decreased by
3.3. HPV18 E6/E7 and IL-6/Stat3 worked synergistically to upregulate MALAT1gene As shown in Fig. 1B, HPV18 E6/E7 promoted IL-6 expression and STAT3 activation. To ask whether IL-6/STAT3 signaling has any influence on the expression of HPV E6 and E7, cervical cancer HeLa cells were firstly treated with IL-6. The RNA levels of HPV18 E6 and E7 were measured with RT-qPCR and the results showed that the expression of HPV18 E6 and E7 was upregulated by around 2 folds (Fig. 4A). In line 4
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Fig. 4. IL-6/STAT3 signaling strengthened HPV18 E6/E7 expression in HeLa cells. (A) HPV18 E6/E7 mRNA levels and (B) HPV18 E6/E7 protein levels were increased in IL-6-treated cells. (C) HPV18 E6/E7 mRNA levels and (D) HPV18 E6/E7 protein levels were reduced in AG490-treated cells. (E) HPV18 E6/E7 mRNA levels and (F) HPV18 E6/E7 protein levels were upregulated by STAT3 overexpression. (G) HPV18 E6/E7 mRNA levels and (H) HPV18 E6/E7 protein levels were downregulated in STAT3-depleting cells.
about 40 % (Fig. 4G and H), suggesting that STAT3 is required for the efficient expression of HPV18 E6/E7. Taken together, the above results demonstrate that the IL-6/STAT3 signaling activated the expression of HPV18 E6/E7 in HeLa cells. Thus, HPV18 E6/E7 induces the constitutive activation of IL-6/STAT3 signaling and IL-6/STAT3 strengthens the expression of HPV18 E6/E7, thereby forming a positive feedback loop, in HeLa cells. HPV18 E6/E7 and IL-6/STAT3 reciprocally strengthen their expression or activation in in HeLa cells. To test the effects of HPV18 E6/ E7 and IL-6/STAT3 on MALAT1 gene regulation, HeLa cells were
transfected with both HPV18 E6/E7- and STAT3-encoding plasmids. Results of RT-qPCR showed that MALAT1 RNA level was further increased to about 3-folds with the co-overexpression (Fig. 5A). In HeLa cells co-treated with IL-6 and HPV18 E6/E7, MALAT1 expression was similarly upregulated (Fig. 5B). These results demonstrated that HPV18 E6/E7 and IL-6/STAT3 work synergistically in MALAT1 gene upregulation and IL-6/STAT3 mediates the HPV18 E6/E7 induced MALAT1 expression in HeLa cells (Fig. 5C).
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Fig. 5. HPV18 E6/E7 and IL-6/STAT3 synergistically promoted the expression of MALAT1. (A) MALAT1 RNA level was further increased following the cooverexpression of HPV18 E6/E7 and STAT3. (B) MALAT1 RNA level was further increased in cells overexpressing of HPV18 E6/E7 and co-treated with IL-6. (C) A model describing the roles of HPV18 E6/E7 and IL-6/STAT3 in the regulation of MALAT1 gene in cervical cancer HeLa cells.
4. Discussion
administration, Methodology. Wenjian Ma: Software, Writing - review & editing. Tong-Cun Zhang: Resources. Hongpeng He: Conceptualization, Supervision, Writing - original draft, Funding acquisition, Resources.
LncRNA MALAT1 was previously reported to be highly expressed in cervical cancer cells and was correlated with the metastasis of cervical cancer (Jiang et al., 2014; Yang et al., 2015; Zhang et al., 2015). Give the previously observed correlation between MALAT1 and cervical cancer development, MALAT1 was proposed to be a potential diagnostic marker (Cui et al., 2017). However, the mechanism by which MALAT1 is highly expressed in cervical cancer cells remains unclear. In this paper, our results revealed that HPV E6/E7 and IL-6/STAT3 signaling pathway work synergistically to upregulate the transcription of MALAT1 in cervical cancer HeLa cells, suggesting a cooperation of virus oncoproteins with cellular inflammatory signaling in cervical cancer development. The IL-6/STAT3 signaling pathway has been identified to be constitutively activated in a variety of cancer cells including cervical cancer to promote the survival and invasive growth of cancer cells (Shukla et al., 2013). This constitutive activation was proposed to be correlated with HPV16 E6 which upregulates IL-6/STAT3 expression or activation (Morgan et al., 2018; Ren et al., 2013). Herein, in HeLa cells, we observed that IL-6 expression was increased and STAT3 was phosphorylated following HPV18 E6/E7 overexpression which is in line with previous observations and further confirming the regulatory effects of HPV E6/E7 on IL-6/STAT3 signaling. Moreover, we found that IL-6/ STAT3 signaling reciprocally regulated HPV18 E6/E7 expression in Hela cells. STAT3 was previously shown to be required for HPV16 E6/ E7 in HPV16-positive cervical cancer cells (Shukla et al., 2013) and was critical for the IL-10 induced HPV16 E6/E7 expression (Arany et al., 2002). Thereby, our results, together with results from other studies, suggest a positive feedback between HPV E6/E7 and the STAT3 signaling. Based on this positive feedback loop, HPV18 E6/E7 and IL-6/ STAT3 function synergistically in the upregulation of MALAT1 gene in cervical cancer cells. Previously, paracrine IL-8 was shown to activate STAT3 which enhanced MALAT1 expression by binding to MALAT1 promoter in prostate cancer cells (Zheng et al., 2018). In head and neck squamous cell carcinoma, TGF-beta was shown to induce STAT3 expression and STAT3 in turn promoted MALAT1 transcription (Wang et al., 2018b). Herein, we demonstrated the up-regulation of MALAT1 by IL-6/STAT3 signaling in cervical cancer cells. These findings further demonstrate the correlation between the activated STAT3 signaling and the upregulated MALAT1 expression in cancer cells, supporting the potential therapeutic value of STAT3 in cancer treatment.
Declaration of Competing Interest All of the authors declare no conflict of interests. Acknowledgements This work was supported by National Natural Science Foundation of China (31301073), the Natural Science Foundation of Tianjin (No.18JCYBJC91500, No. 17JCZDJC33600) and the Innovative Research Team of Tianjin Municipal Education Commission (TD135015). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.virusres.2020.197907. References Arany, I., Grattendick, K.G., Tyring, S.K., 2002. Interleukin-10 induces transcription of the early promoter of human papillomavirus type 16 (HPV16) through the 5’-segment of the upstream regulatory region (URR). Antiviral Res. 55 (2), 331–339. Chen, X., Loo, J.X., Shi, X., Xiong, W., Guo, Y., Ke, H., Yang, M., Jiang, Y., Xia, S., Zhao, M., Zhong, S., He, C., Fu, L., Li, F., 2018. E6 protein expressed by high-risk HPV activates super-enhancers of the EGFR and c-MET oncogenes by destabilizing the histone demethylase KDM5C. Cancer Res. 78 (6), 1418–1430. Cui, X., Jing, X., Wu, X., 2017. The prognostic value of long non coding RNAs in cervical cancer: a meta-analysis. Oncotarget 8 (37), 62470–62477. Eiben, G.L., da Silva, D.M., Fausch, S.C., Le Poole, I.C., Nishimura, M.I., Kast, W.M., 2003. Cervical cancer vaccines: recent advances in HPV research. Viral Immunol. 16 (2), 111–121. Ernst, C., Morton, C.C., 2013. Identification and function of long non-coding RNA. Front. Cell. Neurosci. 7, 168. Haie-Meder, C., Morice, P., Castiglione, M., Group, E.G.W., 2010. Cervical cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 21 (Suppl 5), v37–40. He, H., Liu, X., Liu, Y., Zhang, M., Lai, Y., Hao, Y., Wang, Q., Shi, D., Wang, N., Luo, X.G., Ma, W., Zhang, T.C., 2019. Human papillomavirus E6/E7 and long noncoding RNA TMPOP2 mutually upregulated gene expression in cervical Cancer cells. J. Virol. 93 (8). Hengstermann, A., Linares, L.K., Ciechanover, A., Whitaker, N.J., Scheffner, M., 2001. Complete switch from Mdm2 to human papillomavirus E6-mediated degradation of p53 in cervical cancer cells. Proc. Natl. Acad. Sci. U.S.A. 98 (3), 1218–1223. Hodge, D.R., Hurt, E.M., Farrar, W.L., 2005. The role of IL-6 and STAT3 in inflammation and cancer. Eur. J. Cancer 41 (16), 2502–2512. Hu, G., Niu, F., Humburg, B.A., Liao, K., Bendi, S., Callen, S., Fox, H.S., Buch, S., 2018. Molecular mechanisms of long noncoding RNAs and their role in disease pathogenesis. Oncotarget 9 (26), 18648–18663. Jiang, Y., Li, Y., Fang, S., Jiang, B., Qin, C., Xie, P., Zhou, G., Li, G., 2014. The role of MALAT1 correlates with HPV in cervical cancer. Oncol. Lett. 7 (6), 2135–2141. Liao, X.H., Zheng, L., He, H.P., Zheng, D.L., Wei, Z.Q., Wang, N., Dong, J., Ma, W.J., Zhang, T.C., 2015. STAT3 regulated ATR via microRNA-383 to control DNA damage to affect apoptosis in A431 cells. Cell. Signal. 27 (11), 2285–2295. McLaughlin-Drubin, M.E., Meyers, J., Munger, K., 2012. Cancer associated human papillomaviruses. Curr. Opin. Virol. 2 (4), 459–466.
CRediT authorship contribution statement Yunpeng Hao: Investigation, Methodology, Data curation, Visualization, Writing - original draft. Zhinan Yan: Investigation, Methodology, Visualization, Data curation. Aowei Zhang: Investigation, Methodology. Shiyue Hu: Validation, Methodology. Nan Wang: Funding acquisition, Resources. Xue-Gang Luo: Project 6
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