| 樊 丽,许常娟,申茹萌,宁艳辉,杨 丽.miR-93-5p通过靶向CDKN1A促进多囊卵巢综合征颗粒样瘤细胞KGN的生长增殖[J].,2021,(5):840-845 |
| miR-93-5p通过靶向CDKN1A促进多囊卵巢综合征颗粒样瘤细胞KGN的生长增殖 |
| miR-93-5p Promotes the Growth and Proliferation of KGN in Granulomatous Tumor Cells of Polycystic Ovary Syndromeby Targeting CDKN1A |
| 投稿时间：2020-06-27 修订日期：2020-07-23 |
| DOI：10.13241/j.cnki.pmb.2021.05.008 |
| 中文关键词: 多囊卵巢综合征 miRNA-93-5p CDKN1A 增殖 |
| 英文关键词: Polycystic ovary syndrome miRNA-93-5p CDKN1A Proliferation |
| 基金项目:军队后勤科研项目（CWH16C0417） |
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| 中文摘要: |
| 摘要 目的：验证CDKN1A是miR-93-5p直接调控的靶基因，阐明miR-93-5p可通过靶向CDKN1A促进人卵巢颗粒样肿瘤细胞系KGN的生长增殖。方法：选取我院2016年6月-2019年6月期间确诊的40例多囊卵巢综合征（PCOS）患者作为研究对象，qRT-PCR检测PCOS病变卵巢组织和病灶旁正常卵巢组织（对照）中miR-93-5p和CDKN1A的表达水平。TargetScan软件用以预测miR-93-5p靶基因，并使CDKN1A所含3'-UTR克隆至目的基因下游（CDKN1A-wt或CDKN1A-mut），并分别与miR-93-5p模拟物（miR-93-5p mimics）以及其无关对照寡核苷酸序列（scramble）共转染，荧光素酶报告基因实验验证所预测的靶基因，qRT-PCR和Western blot分别检测转染miR-93-5p mimics及其对照scramble-1、miR-93-5p抑制剂（miR-93-5p inhibitor）及其对照scramble-2后的mRNA和蛋白表达水平。MTT实验验证分别转染miR-93-5p mimics、scramble、CDKN1A 质粒（pcDNA3.1-CDKN1A）、空载体vector （pcDNA3.1）、miR-93-5p+CDKN1A质粒、scramble+vector质粒到KGN细胞中后细胞的生长增殖活性。结果：miR-93-5p在PCOS病变卵巢组织中的表达水平显著高于正常卵巢组织，CDKN1A在PCOS患者卵巢组织中的表达水平显著低于正常卵巢组织（均P<0.05）。在共转染miR-93-5p mimics和CDKN1A-wt、scramble和CDKN1A-wt的两组中，与共转染scramble和CDKN1A-wt组相比，共转染miR-93-5p和CDKN1A-wt组的荧光素酶活性强度降低了约40.9% (P<0.05)。转染miR-93-5p mimics后，CDKN1A的mRNA和蛋白表达水平均显著下调（P<0.05）；转染miR-93-5p inhibitor后，CDKN1A的mRNA和蛋白表达水平均显著上调（P<0.05）。在细胞增殖实验中，转染miR-93-5p mimics后，KGN细胞的生长速度显著高于scramble组（P<0.05）；与vector组比，转染CDKN1A可显著抑制KGN细胞的生长（P<0.05）；同时转染miR-93-5p mimics和CDKN1A后，miR-93-5p对细胞增殖的促进作用降低（P<0.05）。结论：miR-93-5p通过靶向调控CDKN1A表达而促进人卵巢颗粒样肿瘤细胞系KGN的生长增殖。 |
| 英文摘要: |
| ABSTRACT Objective: To verify that CDKN1A is a target gene directly regulated by mir-93-5p, and to clarify that mir-93-5p can promote the growth and proliferation of human ovarian granulosa tumor cell line kgn by targeting CDKN1A. Methods: 40 patients with polycystic ovary syndrome (PCOS) diagnosed by our hospital from June 2016 to June 2019 were selected as the research objects, qRT-PCR was used to detect the expression of miR-93-5p and CDKN1A in diseased and normal ovarian tissues(control). the target gene of miR-93-5p was predicted by Targetscan software, and the 3 '- UTR contained in CDKN1A was cloned to the downstream of the target gene (CDKN1A-wt or CDKN1A-mut), and co transfected with miR-93-5p mimic (miR-93-5p mimics) and its unrelated control oligonucleotide sequence (scrabble) respectively. Luciferase reporter gene experiment verifies the predicted target gene, were verified by the fluorescent enzyme reporter gene experiment. The expression level of mRNA and protein of the target gene was detected by qRT PCR and Western blot methods. MTT assay was used to verify the growth and proliferation of kgn cells transfected with miR-93-5p mimics, scrabble, CDKN1A plasmid (pcDNA3.1-CDKN1A), vector（pcDNA3.1）, miR-93-5p + CDKN1A and scrabble + vector plasmids. Results: The expression level of miR-93-5p in diseased ovarian tissue was significantly higher than that of normal ovarian tissue, and the expression level of CDKN1A in ovarian tissue of PCOS patients was significantly lower than that of normal ovarian tissue(all P＜0.05). In the co transfection of miR-93-5p mimics and CDKN1A-wt, scrabble and CDKN1A-wt, compared with the co transfection of scrabble and CDKN1A-wt, the activity of luciferase in the co transfection of miR-93-5p and CDKN1A-wt decreased by 40.9% (P<0.05). After transfected by miR-93-5p mimics, the mRNA and protein expression of CDKN1A were significantly down regulated(P<0.05); after transfected by miR-93-5p inhibitor, the mRNA and protein expression of CDKN1A were significantly up regulated (P<0.05). In the experiment of cell proliferation, the growth rate of KGN cells was significantly higher than that of scrabble group (P<0.05) after transfected by miR-93-5p mimics; compared with vector group, CDKN1A could significantly inhibit the growth of KGN cells (P<0.05); After simultaneous transfected by miR-93-5p mimics and CDKN1A, the promoting effect of miR-93-5p on cell proliferation was decreased(P<0.05). Conclusion: miR-93-5p can promote the growth and proliferation of KGN by targeting CDKN1A expression. |
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