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Long Non‑Coding RNA Plasmacytoma Variant Translocation 1 Gene Promotes the Development of Cervical Cancer Via the NF‑Κb Pathway

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Long Non‑Coding RNA Plasmacytoma Variant Translocation 1 Gene Promotes the Development of Cervical Cancer Via the NF‑Κb Pathway MOLECULAR MEDICINE REPORTS 20: 2433-2440, 2019 Long non‑coding RNA plasmacytoma variant translocation 1 gene promotes the development of cervical cancer via the NF‑κB pathway CHANG WANG1, HAO ZOU2, HONGJUAN YANG1, LEI WANG1, HUIJUN CHU1, JINWEN JIAO1, YANKUI WANG1 and AIPING CHEN1 1Department of Gynecology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266590; 2Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China Received November 16, 2018; Accepted May 20, 2019 DOI: 10.3892/mmr.2019.10479 Abstract. The long noncoding RNA plasmacytoma variant of cervical cancer cells, potentially via the NF‑κB pathway. translocation 1 gene (LncRNA PVT1) has an important role in The data from the present study will contribute to the current tumor occurrence and development, yet the role and underlying knowledge surrounding the theoretical basis of cervical cancer molecular mechanisms of this RNA in cervical cancer have and provide a new perspective for the treatment of cervical not yet been elucidated. In the present study, three cervical cancer. cancer cell lines (HeLa, Ca Ski and SiHa) were used to verify how LncRNA PVT1 mediates cervical cancer development, Introduction and the H8 cell line was used as a control. A LncRNA PVT1 overexpression vector or small interfering RNAs targeting There were ~500,000 people with cervical cancer worldwide LncRNA PVT1 were transfected into cervical cancer cells to in 2012, and in women, cervical cancer ranks second in cancer generate LncRNA PVT1 overexpression and silencing in these occurrence and 15th in cancer mortality (1-3). Currently, the cells. LncRNA PVT1 overexpression accelerated the growth of pathogenesis of cervical cancer is not fully clear, but human cervical cancer cells by advancing the cell cycle and inhibiting papillomavirus (HPV) infection is established to be the most cellular apoptosis; increases in Cyclin D1 (CCND1) mRNA closely associated risk factor for cervical cancer occurrence. and activated Bcl-2 protein expression levels also supported However, high-risk HPV infection alone is not enough to this finding. Furthermore, NF‑κB activation and expression induce tumor progression (4). was increased by LncRNA PVT1 overexpression. In addition, Previous studies have revealed that HPV activates the NF‑κB activation or inhibition induced changes in cell viability, NF‑κB pathway to promote expression of different anti-apop- accompanied by changes in CCND1 and Bcl-2 expression. tosis genes that induce cervical cancer development through Increases or decreases in microRNA‑16 (miR‑16) expres- inhibition of cellular apoptosis (5‑8). NF‑κB an essential sion (using miR mimics and inhibitors) also corresponded nuclear transcription factor composed of RelA/p65, p50, p52 to changes in LncRNA PVT1 expression, in vitro. miR‑16 and NF‑κB family members (9). NF‑κB pathway activation is mimics and inhibitor had opposite effects to those of NF‑κB associated with the development of various tumors and can activity, and miR‑16 was demonstrated to directly interact with promote tumor cell growth, proliferation and anti-apoptotic the NF‑κB gene as measured using the dual-luciferase assay. effects to promote malignant transformation and tumor cell In summary, LncRNA PVT1 inhibits the effect of miR‑16, metastases (10,11). NF‑κB pathway activation is a common promoting the cell cycle and inhibiting cellular apoptosis mechanism used to promote tumor survival; the activated NF‑κB induces expression of various anti-apoptotic genes, such as the cellular inhibitor of apoptosis protein (12), the X-linked inhibitor of apoptosis protein (13) and Bcl-2 family proteins (14). In addition, NF‑κB activation was shown to be Correspondence to: Professor Yankui Wang, Department of present in cervical cancer (15), suggesting that NF‑κB may Gynecology, The Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Shinan, Qingdao, Shandong 266590, P.R. China have a role in the development and progression of cervical E-mail: [email protected] cancer. MicroRNAs (miRs) serve as markers of cervical cancer Key words: cervical cancer cell, long non-coding RNA, plasmacytoma progression (16), and it was previously reported that miR‑16 has variant translocation 1 gene, microRNA‑16, NF‑κB, cell proliferation an important role in the regulation of cell apoptosis, proliferation, and apoptosis migration and invasion of cervical cancer (17-19). In addition, miR‑16 targets inhibitor of nuclear factor κB kinase subunit β (Iκ-Bα) in hepatocellular carcinoma (20), and decreases NF‑κB 2434 WANG et al: REGULATION OF CERVICAL CANCER gene expression; thus, reducing the levels of activated NF‑κB (21). gene were obtained via artificial nucleotide synthesis at Beijing Therefore, miR‑16 has the important role of regulating the Tianyi Huiyuan Bioscience & Technology Inc. and the expres- NF‑κB signaling pathway. On the other hand, long non-coding sion plasmid constructed. The siRNA (5'-GAG​CUG​CGA​GCA​ RNAs (lncRNAs) are involved in different tumor processes (22) AAG​AUG​U‑3') and control siRNA (si‑NC; 5'-GAU​CGU​ACU​ through regulation of target gene expression at the transcriptional, AUA​GCU​UGU​A‑3') of LncRNA PVT1 were purchased from post-transcriptional and epigenetic levels (23-25). LncRNAs Guangzhou RiboBio Co., Ltd. also at serve as precursors of small interfering RNAs (siRNAs), miRs and piwi-interacting RNAs (26,27), and can compete with Cell transfection. Fugene6 transfection reagent (Roche miR as competing endogenous RNAs (ceRNAs) to achieve Diagnostics) was used according to the manufacturer's inter‑communication and regulation (28‑33). Furthermore, the protocol. The overexpression vector (final concentration lncRNA‑plasmacytoma variant translocation 1 (LncRNA PVT1) 1 µg/ml) or siRNA of LncRNA PVT1 (final concentration gene was reported to be a biomarker of tumorigenesis and 50 nm) were transfected into HeLa, Ca Ski and SiHa cells associated with the development of cervical cancer (34-37) by in 96-well plates or 100 mm dishes with 1x105 cells/ml and competitively inhibiting miRs (38,39). cultured for 48 or 60 h, respectively, and the pEGFP‑N3 empty Although no evidence currently exists to support the vector or si‑NC were also transfected with the corresponding hypothesis that the interaction between LncRNA PVT1 and internal controls. Additionally, HeLa cells were respectively miR‑16 induces cervical cancer development, it is speculated treated with 5 µg/ml lipopolysaccharides (LPS; Beyotime that the LncRNA PVT1 gene promotes cervical cancer devel- Institute of Biotechnology) plus the lncRNA PVT1 siRNA, opment by inhibiting miR expression and activating the NF‑κB with pyrrolidinedithiocarbamate (PDTC; 50 µmol/l; Beyotime pathway. In the present study, three known cervical cancer Institute of Biotechnology) alone, or with PDTC (50 µmol/l) cell lines (HeLa, Ca Ski and SiHa cells) and the H8 cell line on the basis of the lncRNA PVT1 overexpression treatment for (as an external control of three cervical cancer cell lines) were 8 h to activate or inhibit the NF‑κB pathway, and then the cells selected as the experimental models for in vitro experiments to were continuously cultured for 60 h. In addition, using the investigate the effect and molecular mechanisms of LncRNA Fugene6 transfection reagent, HeLa cells with 60% confluency PVT1 on cervical cancer development. were transiently transfected with the miR‑16 mimics (final concentration 100 nm) or miR‑16 inhibitor (final concentration Materials and methods 50 nm). All cells were collected for the experiments below following 60 h treatment. Cell culture. As it involved the use of human materials, the present study was approved by the Ethics Committee of the MTT assay. Following culture of the cells from the four cell Affiliated Hospital of Qingdao University (Qingdao, China) lines in 96-well plates with different treatments or no treatment according to the experimental requirements of the school. for 60 h, cell numbers were determined using the MTT assay. HeLa, Ca Ski, SiHa and H8 cells were purchased from the The assay was conducted by adding 20 µl MTT (5 mg/ml) to National Infrastructure of Cell Line Resources of China. H8 each well of the plates and incubating the plates for 4 h at 37˚C. cell line (the human cervical epithelial immortalized cell Following removal of the supernatant, formazan crystals were line) was used as the external control of the three cervical dissolved in 200 µl dimethyl sulfoxide, and the absorbance cancer cell lines, HeLa, Ca Ski and SiHa. Each cell line was was measured at 490 nm by microplate reader (Thermo Fisher cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) Scientific, Inc.). There were eight replicate wells for each group containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in to ensure the accuracy of the experiments. 100 mm culture dishes (Corning Inc.) and incubated in a 5% CO2. incubator at 37˚C. The media was changed every 3 days, Flow cytometry (FCM). Following treatment with or without and the cells were passaged once they reached 80% conflu- the LncRNA‑PVT1 siRNA for 48 h, HeLa cells were ence. Additionally, 293T cells (purchased from the National collected to use for cell cycle or cell apoptosis analyses using Infrastructure of Cell Line Resources of China) were used in a FACSCanto II flow cytometer (Becton, Dickinson and co-transfection experiment and cultured with the same afore- Company). For the cell cycle analysis, cells were centrifuged mentioned treatment. To ensure cell activity, the cells from at 300 x g for 5 min at 4˚C and washed with ice‑cold PBS passage 2-4 were used for further experiments. following digestion with trypsin. The cells were then mixed with 0.25% Triton X-100 and 5 µl propidium iodide (PI) and Construction of LncRNA PVT1 and 3'‑untranslated region incubated for 30 min at room temperature in the dark. Cells (3'UTR) of NF‑κB overexpression vectors and siRNA of LncRNA were resuspended in 0.5 ml PBS and analyzed immediately.
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