miR-325-3p通过靶向HE4抑制卵巢癌细胞SKOV3化疗耐药性的研究  

MiR-325-3p inhibits chemotherapy resistance of the ovarian cancer cell line SKOV3 by targeting HE4

作　　者：王恒[1] WANG Heng(Guizhou Aerospace Hospital,Guizhou,Zunyi 563000,China)

机构地区：[1]贵州航天医院,贵州省遵义市563000

出　　处：《河北医药》2024年第20期3051-3055,共5页Hebei Medical Journal

基　　金：贵州省科教青年英才培训工程(编号:黔省专合字(2019)334号)。

摘　　要：目的探讨miR-325-3p通过靶向人附睾蛋白4(HE4)基因对卵巢癌细胞SKOV3顺铂(CDDP)耐药的影响。方法采用浓度梯度递增法构建对CDDP耐药的卵巢癌细胞株SKOV3/DDP。CCK-8检测经不同浓度梯度的CDDP处理后亲代细胞SKOV3和耐药细胞SKOV3/DDP对CDDP的耐药性。随后,将SKOV3/DDP细胞随机分为空白对照组,阴性对照组和miR-325-3p组。其中,空白对照组不做任何处理,阴性对照组细胞转染阴性对照miR-NC,miR-325-3p组转染miR-325-3pmimic。采用CCK-8检测细胞半数抑制浓度(IC50)。流式细胞术检测细胞凋亡率。TargetScan数据库预测miR-325-3p与HE4 mRNA 3’UTR的结合位点。双荧光素酶报告基因实验验证miR-325-3p与HE4 mRNA 3’UTR的靶向性。实时荧光定量PCR(qRT-PCR)检测细胞中miR-325-3p和HE4 mRNA表达。Westernblotting实验检测HE4蛋白表达。结果亲代细胞SKOV3对CDDP的IC50为14.15μM,耐药细胞SKOV3/DDP对CDDP的IC50为45.29μM,且耐药倍数(RI)为3.20。与亲代细胞SKOV3比较,耐药细胞株SKOV3/DDP中miR-325-3p表达明显降低(P<0.05),而HE4 mRNA和蛋白表达明显升高(P<0.05)。另外,与空白对照组比较,阴性对照组中miR-325-3p表达、HE4 mRNA和蛋白表达、对CDDP的IC50以及细胞凋亡率均无明显差异(P>0.05)。与阴性对照组比较,miR-325-3p组中miR-325-3p表达、细胞凋亡率均显著升高(P<0.05),而对CDDP的IC50、HE4 mRNA和蛋白表达明显降低(P<0.05)。结论miR-325-3p通过靶向HE4抑制卵巢癌细胞SKOV3对CDDP的耐药性。Objective To investigate the effect of miR-325-3p on cisplatin(CDDP)resistance of the ovarian cancer cell line SKOV3 by targeting human epididymis protein 4(HE4).Methods The CDDP-resistant ovarian cancer cell line SKOV3/DDP was established by DDP induction with a gradient concentration.The cell counting kit-8(CCK-8)assay was performed to detect the CDDP resistance of SKOV3 cells and SKOV3/DDP cells treated with CDDP at varied concentrations.Subsequently,SKOV3/DDP cells were treated with blank control,or transfected with negative control and miR-325-3p mimic.The half maximal inhibitory concentration(IC50)was measured by CCK-8 assay.Cell apoptosis was examined by flow cytometry.The binding site between miR-325-3p and HE43’UTR was predicted in the TargetScan,and verified by dual-luciferase reporter assay.Quantitative real-time PCR(qRT-PCR)was used to detect the mRNA expression of miR-325-3p and HE4.Western blotting was performed to detect the protein expression of HE4.Results The IC50 of SKOV3 and SKOV3/DDP against CDDP was 14.15μmol/L,and 45.29μmol/L,respectively.The resistance index(RI)was 3.20.Compared with the parental cells,miR-325-3p was significantly downregulated in SKOV3/DDP cells,while the mRNA and protein levels of HE4 were significantly upregulated(all P<0.05).There were no significant differences in miR-325-3p expression,mRNA and protein expressions of HE4,IC50 and cell apoptotic rate in cells treated with blank control versus those transfected with negative control(P>0.05).Transfection of miR-322-3p mimic significantly upregulated miR-325-3p and increased apoptotic rate,and decreased IC50 and mRNA and protein levels of HE4(all P<0.05).Conclusion MiR-325-3p inhibits CDDP resistance of ovarian cancer cells by targeting HE4.

关 键 词：miR-325-3p HE4 卵巢癌细胞 CDDP 耐药性 

分 类 号：R737.31[医药卫生—肿瘤]