Yang et al. Cancer Cell Int (2020) 20:343 https://doi.org/10.1186/s12935-020-01430-5 Cancer Cell International PRIMARY RESEARCH Open Access Long non-coding RNA CTBP1-AS2 enhances cervical cancer progression via up-regulation of ZNF217 through sponging miR-3163 Shanshan Yang1†, Feng Shi2†, Yuting Du1, Zhao Wang1, Yue Feng1, Jiayu Song1, Yunduo Liu3* and Min Xiao4* Abstract Background: Long non-coding RNAs (lncRNAs) play signifcant roles in tumorigenesis and can contribute to identifcation of novel therapeutic targets for cancers. This paper was aimed at exploring the role of CTBP1 divergent transcript (CTBP1-AS2) in cervical cancer (CC) progression. Methods: qRT-PCR and western blot assays were used to detect relevant RNA and protein expressions. In vitro func- tional assays, including CCK8, EdU, TUNEL and transwell assays were applied to explore the functions of CTBP1-AS2 in CC cell proliferation, apoptosis and migration. In vivo animal study was utilized to investigate the role of CTBP1-AS2 in tumor growth. Luciferase reporter, RNA pull down and RIP assays were performed to determine the specifc mechani- cal relationship between CTBP1-AS2, miR-3163 and ZNF217. Results: CTBP1-AS2 was signifcantly overexpressed in CC cell lines. Knockdown of CTBP1-AS2 curbed cell prolifera- tion, migration and invasion, while stimulated cell apoptosis in vitro. CTBP1-AS2 facilitated xenograft tumor growth in vivo. Cytoplasmic CTBP1-AS2 was found to be a miR-3163 sponge in CC cells. MiR-3163 inhibition abolished the anti-tumor efects of CTBP1-AS2 knockdown. Additionally, Zinc fnger protein 217 (ZNF217) was identifed as a direct target of miR-3163. CTBP1-AS2 acted as a miR-3163 sponge to elevate ZNF217 expression. ZNF217 up-regulation abrogated the tumor suppressing efects of CTBP1-AS2 knockdown. Conclusion: CTBP1-AS2 regulates CC progression via sponging miR-3163 to up-regulate ZNF217. Keywords: CTBP1-AS2, miR-3163, ZNF217, Cervical cancer Background approximately 300,000 patients die of cervical cancer Cervical cancer (CC) is the fourth most common diag- worldwide [2]. Human papilloma virus (HPV) is the nosed cancer and the fourth leading cause of cancer- major cause for the high risk of CC. Based on cancer related deaths in females globally [1]. Each year, more statistics in 2019, there were 13,170 estimated new than 500,000 cervical cancer cases are diagnosed and cases and 4250 estimated deaths in the United States [3]. Recently, an increasing trend of morbidity and mortality of CC has been discovered in China [4, 5]. *Correspondence: [email protected]; [email protected] Global strategies for the prevention and screening of † Shanshan Yang and Feng Shi are co-frst authors CC remain to be improved based on various geographic 3 Department of Gynecological Oncology, Harbin Medical University Cancer Hospital, No. 150 Haping Road, Nangang District, Harbin 150081, settings and health systems [6]. Preclinical models have Heilongjiang, China been used for the treatment of CC patients [7]. At pre- 4 Department of Breast Surgery, Harbin Medical University Cancer sent, radiotherapy chemotherapy and surgery remain Hospital, No. 150 Haping Road, Nangang District, Harbin 150081, Heilongjiang, China the main clinical therapeutic methods for patients Full list of author information is available at the end of the article with CC [8–10]. Terefore, it is essential to explore the © The Author(s) 2020. 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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 in a credit line to the data. Yang et al. Cancer Cell Int (2020) 20:343 Page 2 of 13 molecular mechanisms behind the initiation and devel- Materials and methods opment of CC. Tissue samples Long non-coding RNAs (lncRNAs) are a class of Tis study was executed between 2014 and 2019, with RNAs longer than 200 nucleotides but lack the pro- the ethical approval from the Ethics Committee of tein-coding potential. Recent fndings indicated that Harbin Medical University Cancer Hospital. Patients lncRNAs play vital roles in gene regulation at the tran- without Human papillomavirus (HPV) infection were scriptional level [11]. Dysregulation of lncRNAs is excluded from this study. All 72 participants had signed associated with a series of biological processes, such the written informed consent. Highly sensitive poly- as cell proliferation, apoptosis, invasion and migration merase chain reaction (PCR) techniques were used to [12–14]. Furthermore, lncRNAs have been identifed detect the HPV. Te number of patients infected with as novel biomarkers of many cancers [15, 16]. To date, HPV-18, HPV-11, HPV-45 and HPV-68 were separately the pathologic roles of most lncRNAs remain unknown, 25, 19, 15, 13. Te 72 CC samples and adjacent normal which indicates the extensive potential of lncRNAs in samples from CC patients were collected and instantly the prediction and treatment of various cancers. maintained in the liquid nitrogen at − 80 °C. In CC, some lncRNAs were discovered to be aber- rantly expressed and exerted important biological func- Cell lines tions. For instance, linc00511 is highly expressed in CC Human cervical cancer cell lines, including SiHa (HPV and knockdown of linc00511 dampens CC cell prolif- positive), HeLa (HPV positive), MS751 (HPV positive) eration and reduces drug resistance to paclitaxel [17]. and C33A (HPV negative) as well as the normal cervi- CTBP1 divergent transcript (CTBP1-AS2), as a newly cal epithelial cells (H8) were purchased from Shanghai identifed lncRNA, was limitedly reported in cancers. Institute of Cell Biology (Shanghai, China), cultured Te only report on the role of CTBP1-AS2 in cancer routinely in RPMI-1640 medium (Invitrogen, Carls- is that CTBP1-AS2 predicts unfavorable prognosis of bad, CA, USA) at 37 °C with 5% CO 2. 10% fetal bovine papillary thyroid cancer [18]. However, the biological serum (FBS; Termo Fisher Scientifc, Waltham, MA, role of CTBP1-AS2 in the carcinogenesis and develop- USA) and antibiotics were applied to supplement the ment of CC has not been studied yet. culture medium. MicroRNAs (miRNAs) are small ncRNAs with a size between 20 and 25 nt. Based on previous studies, miR- Extraction of total RNA and qRT‑PCR NAs can exert various functions in human cancers. Total RNA was extracted from HeLa and SiHa cells For instance, anti-miR-203 suppresses cell growth and using 1 mL of TRIzol (Invitrogen) and reversely tran- stemness in ER-positive breast cancer via targeting scribed into cDNA using PrimeScript RT reagent Kit SOCS3 [19]. MiR-17-5p and miR-20a-5p inhibit hepa- (Takara, Kyoto, Japan) or miRNA reverse transcrip- tocellular carcinoma metastasis [20]. MiR-17 acts as an tion PCR kit (Ribobio; Guangzhou, China). Te relative oncogene in hepatocellular carcinoma through down- gene expression level was measured by SYBR Green regulation of Smad3 [21]. It has been reported that PCR Master Mix (Invitrogen) or SYBR ® PrimeScript® miR-3163 targets ADAM-17 and inhibits the Notch miRNA RT-PCR Kit (Takara), and Step-One Plus Real- pathway to enhance the sensitivity of HCC cells to anti- Time PCR System (Applied Biosystems, Foster City, tumor agents in hepatocellular carcinoma [22]. MiR- CA, USA), and quantifed by the comparative 2−ΔΔCt 3163 promotes colorectal cancer cell growth in vivo method. GAPDH mRNA or U6 snRNA served as the [23]. Tis study was aimed to explore the interaction endogenous control. Te sequences of PCR primers between CTBP1-AS2 and miR-3163. were provided in Additional fle 1: Table S1. Zinc fnger protein 217 (ZNF217) is protein-cod- ing gene contributing to the tumorigenesis of various Cell transfection human cancers. Te coordination between ZNF217 and When the cell density was about 70%, cell transfec- LSD1 facilitates hepatocellular carcinoma progression tion was performed in 24-well plates with CO at 37 °C [24]. ZNF217 is targeted by miR-211-3p and reverses 2 for 48 h utilizing Lipofectamine 2000 (Invitrogen). the efects of miR-211-3p on proliferative and migra- Te duplicate short hairpin RNAs for CTBP1-AS2, tory potentials of non-small cell lung cancer cells [25]. ZNF217 (sh-CTBP1-AS2#1/2, sh-ZNF217#1/2), plas- To summarize, the current study focused on investi- mid pcDNA3.1/ZNF217 were designed by Genepharm gating the role of CTBP1-AS2/miR-3163/ZNF217 axis (Shanghai, China), as well as their relative negative con- in the biological behaviors of HPV-positive cells. trol RNA (sh-Ctrl, pcDNA3.1). MiR-3163 mimics and NC mimics, miR-3163 inhibitor and NC inhibitor were Yang et al. Cancer Cell Int (2020) 20:343 Page 3 of 13 also produced by Genepharm. Relevant sequences were HRP-tagged secondary antibodies (all from Abcam, provided in Additional fle 1: Table S1. Cambridge, MA, USA). GAPDH served as internal con- trol. Samples were analyzed by enhanced chemilumines- Cell counting kit‑8 (CCK‑8) cence reagent (Santa Cruz Biotechnology, Santa Cruz, HeLa or SiHa cells were planted into 96-well plates at CA, USA).