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Original Article Long Noncoding RNA TOB1-AS1, an Epigenetically Silenced Gene, Functioned As a Novel Tumor Suppressor by Sponging Mir-27B in Cervical Cancer

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Original Article Long Noncoding RNA TOB1-AS1, an Epigenetically Silenced Gene, Functioned As a Novel Tumor Suppressor by Sponging Mir-27B in Cervical Cancer Am J Cancer Res 2018;8(8):1483-1498 www.ajcr.us /ISSN:2156-6976/ajcr0079106 Original Article Long noncoding RNA TOB1-AS1, an epigenetically silenced gene, functioned as a novel tumor suppressor by sponging miR-27b in cervical cancer Jihang Yao1, Zhenghong Li1, Ziwei Yang2, Hui Xue1, Hua Chang1, Xue Zhang1, Tianren Li1, Kejun Guo1 Departments of 1Gynecology, 2Clinical Laboratory, The First Hospital of China Medical University, Shenyang 110001, Liaoning, China Received April 21, 2018; Accepted July 9, 2018; Epub August 1, 2018; Published August 15, 2018 Abstract: Cervical cancer is one of the most common cancers in females, accounting for a majority of cancer- related deaths in worldwide. Long non-coding RNAs (lncRNAs) have been identified as critical regulators in many tumor-related biological processes. Thus, investigation into the function and mechanism of lncRNAs in the develop- ment of cervical cancer is very necessary. In this study, we found that the expression of TOB1-AS1 was significantly decreased in cervical cancer tissues compared with the adjacent normal tissues. The methylation status of TOB1- AS1-related CpG island was analyzed using methylation specific PCR and bisulfite sequencing analysis, revealing that the aberrant hypermethylation of TOB1-AS1-related CpG island was frequently observed in primary tumors and cervical cancer cells. The expression of TOB1-AS1 in cervical cancer cells could be reversed by demethylation agent treatment. Functionally, overexpression of TOB1-AS1 significantly inhibited cell proliferation, cell cycle progression, invasion and induced apoptosis, while knockdown of TOB1-AS1 exhibited the opposite effect. Furthermore, it was determined that TOB1-AS1 was able to bind and degrade the expression of miR-27b. Upregulation of miR-27b promoted cell growth, cell cycle transition from G1 phase to S phase, and invasion and reduced apoptosis, phe- nomenon could be reversed by TOB1-AS1. Inhibition of miR-27b attenuated the promotive effect of si-TOB1-AS1 on cellular processes. Upregulation of TOB1-AS1 also suppressed tumor growth in vivo. Clinically, methylation of TOB1- AS1 and low expression of TOB1-AS1 was significantly correlated with tumor stage and tumor size, respectively. Univariate and multivariate analyses confirmed that low level of TOB1-AS1 was an independent risk factor for death. In conclusion, we suggested that the epigenetically silenced TOB1-AS1 was unable to restrain miR-27b, which con- tributed to cervical cancer progression. Keywords: TOB1-AS1, methylation, miR-27b, proliferation, apoptosis, invasion, cervical cancer Introduction RNAs), a group of ncRNAs (more than 200 nucleotides), have attracting more and more Cervical cancer is the second leading cause of attentions in the field of cancer biology. In- cancer-related death in women worldwide, with creasing studies has revealed a close relation an estimated incidence of 500,000 cases and between lncRNAs and human malignancy [4, 5]. approximately 233,000 deaths per year [1, 2]. Several lncRNAs have been verified to be asso- Cervical cancer is a multi-step process involv- ciated with various tumor related processes, ing the deregulation of multiple genes; there- such as proliferation, apoptosis, migration, and fore, studies on the potential mechanisms un- invasion [6, 7]. For example, PVT1 was upregu- derlying the initiation and progression of cervi- lated in cervical cancer cells and knockdown of cal cancer are absolutely necessary. PVT1 led to an inhibition of cell viability and motility [8]. Overexpression of HOXA11-AS con- It is well known that 98% of DNA sequence in tributed to cell proliferation, migration, and human genome may be transcribed to non-cod- invasion in vitro, while silencing of HOXA11-AS ing RNAs (ncRNAs) that lack the capacity to exhibited the opposite effect on cellular pro- encode proteins [3]. Long non-coding RNAs (lnc- cesses [9]. Additionally, many differentially TOB1-AS1/miR-27b axis regulates cervical cancer progression expressed lncRNAs, such as MEG3 and therapy before surgery. The histological type MALAT1, have potential application in clinical and tumor stage were identified according to diagnosis and prognosis for cancer patients the International Federation of Gynecology and [10, 11]. Obstetrics (FIGO) classification system. All the tissue samples were snap-frozen in liquid nitro- MicroRNAs (miRNAs) are a class of ncRNAs gen immediately following surgical resection, with a length of 18-22 nucleotides that have and stored in -80°C until use. been reported to be oncogenes or tumor sup- pressors [12]. Accumulating studies have The human cervical cancer cell lines (SiHa, reported that lncRNAs may exert their roles in HeLa, CaSki, and C33A) human immortalized carcinogenesis by competitively binding to tu- keratinocytes (HaCaT) cells were purchased mor-related miRNAs [13, 14]. For example, from the Cell Bank of the Chinese Academy of MEG3 decreased the expression level of miR- Sciences (Shanghai, China) and cultured in 21-5p, followed by inducing cell growth inhibi- DMEM (Gibco, Waltham, MA, USA) medium sup- tion and apoptosis in cervical cancer cells [15]. plemented with 10% fetal bovine serum (FBS, CCAT1 antagonized the effect of miR-410, Gibco), 100 g/ml streptomycin sulfate and 100 which could promote cell proliferation and re- U/ml penicillin sodium. All cells were main- duce apoptosis by suppressing ITPKB expres- tained at 37°C in a humidified atmosphere with sion in colon cancer [16]. HOTAIR knockdown 5% CO2. led to the inhibition of migration and invasion via decreasing of miR-206 in cervical cancer Plasmid construction and oligonucleotide cells [17]. transfection Human transducer of ERBB2.1 (TOB1), a mem- Small interfering RNA (siRNA) and non-specific ber of the TOB/BTG family, was reported as an control siRNA (si-ctrl) were synthesized by anti-proliferative protein in various cancers. GenePharma Co., Ltd. (Shanghai, China) and Decreased expression of TOB1 has been transfected into cells by using Lipofectamine observed in many cancers including breast, 3000 (Invitrogen, Carlsbad, CA, USA). The sequ- lung, and skin carcinoma [18-20]. Besides, ences of si-TOB1-AS1 were as follows: si-RNA1, upregulation of TOB1 promoted cell migration 5’-GCACCCGATTAATTGAATA-3’; si-RNA2, 5’-GC- and invasion and induced apoptosis in gastric GACTCGGATCCGTTTAT-3’. To construct TOB1- cancer cells [21, 22]. TOB1 antisense RNA 1 AS1 overexpression plasmid, the complemen- (TOB1-AS1), a novel lncRNA, is originated from tary DNA of TOB1-AS1 was amplified and cloned the TOB1 gene cluster located on chromosome into pcDNA3.1 vector (Invitrogen) and named 17q21.33 in an antisense manner. However, as pcTOB1-AS1. The empty vector was used as the function and molecular mechanism of the negative control (pcDNA3.1). MiR-27b mi- TOB1-AS1 have not been investigated. In this mic, miR-27b inhibitor, mimic negative control study, we focused on the function and me- (mim con), and inhibitor negative control (inh chanim of TOB1-AS1 in the development of cer- con) were synthesized by GenePharma Co., Ltd. vical cancer. (Shanghai, China). The miRNAs, siRNAs or plas- mids were transfected/co-transfected by using Materials and methods Lipofectamine 3000 reagent (Invitrogen) ac- cording to the manufacturer’s instructions. At Clinical samples and cells 48 h after transfection/co-transfection, cells were subjected to further experimentation. Cervical cancer tissues (n=50) and adjacent non-tumor tissues (n=50) were obtained from Quantitative real-time PCR (qRT-PCR) patients who were diagnosed at the Department of Gynecology, the First Hospital of China Total RNA was extracted from cells and tissues Medical University between Feb 2011 and Dec using TRIzol reagent (Invitrogen). The Prime- 2012. This study was approved by the Ethics Script™ Reverse Transcription Kit (TakaRa, Approval Committee of the First Hospital of Japan) was used to generated complementary China Medical University and all written in- DNA using in an ABI 7500 System (ABI, Foster formed consents were signed by patients. None City, CA, USA). MiRNA was isolated using the of the patients had received chemo- or radio- mir-Vana miRNA Isolation Kit (Ambion, Austin, 1484 Am J Cancer Res 2018;8(8):1483-1498 TOB1-AS1/miR-27b axis regulates cervical cancer progression TX, USA) and cDNA was synthesized using the 20 μM) of 5-aza-2-deoxycytidine (5-aza) (Sigma, SuperScript II Reverse kit (Invitrogen). The St. Louis, MO, USA). The fresh medium contain- SYBR Green Real-time PCR Master Mix (Takara) ing 5-aza was changed every 24 h for 3 days was used in qRT-PCR reaction and the quantita- and cells were obtained at 72 h. tive data was calculated using the 2-ΔΔCt meth- od. GAPDH and U6 were used as internal con- Cell proliferation assay trol. Primers were as follows: TOB1-AS1, 5’-GC- 3 CAGGCCTAGAAGCTTTTG-3’ (forward), 5’-TCTT- A total of 2×10 cells (per well) were seeded in CCCACCCCTTCTCCTA-3’ (reverse); GAPDH, 5’- 96-well plates in triplicate. At 24, 48, 72, and CGACTTATACATGGCCTTA-3’ (forward), 5’-TTCC- 96 h after transfection, the cells were incubat- GATCACTGTTGGAAT-3’ (reverse); U6, 5’-CTCG- ed with MTT (5 mg/mL; Sigma) for 4 h at 37°C. CTTCGGCAGCACA-3’ (forward), 5’-AACGCTTCA- DMSO was added to each well to stop the reac- CGAATTTGCGT-3’ (reverse). All samples were tion and the optical density (OD) of each well run in triplicates. was measured at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Methylation specific PCR (MSP) and bisulphite sequencing analysis Cell cycle distribution and apoptosis analysis DNA was isolated from cells and tissues using The transfected cells were trypsinized, washed, DNeasy Blood & Tissue Kit (Qiagen, Düsseldorf, fixed with ice-cold 70% ethanol, and stored at Germany) and then subjected to bisulfite treat- 4°C overnight. Then, the cells were washed ment using the EpiTect Bisulfite kit (Qiagen). For with PBS and stained with propidium iodide (PI, MSP assay, the primer pairs for both the meth- Sigma)/RNAase for 30 min at room tempera- ylated and unmethylated sequences was used ture.
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