摘要
目的:探讨miR-769-5p在上皮性卵巢癌(EOC)血清外泌体中的表达情况,及其在EOC细胞增殖、迁移和侵袭的影响的机制。方法:采用反转录-荧光定量聚合酶链式反应(RT-qPCR)验证miR-769-5p在恶性组、良性组和健康组血清外泌体样本中的表达水平。采用Lipofectamine 3000转染构建过表达和敲减细胞株,分组为:miR-769-5p mimics组、mimics-NC组、miR-769-5p inhibitor组和inhibitor-NC组,通过CCK-8实验、划痕愈合实验和Transwell小室实验验证miR-769-5p对EOC细胞增殖、迁移和侵袭的影响。对过表达miR-769-5p的EOC细胞进行转录组测序,并结合公共数据库的预测结果,确定miR-769-5p的潜在靶基因。双荧光素酶报告基因实验验证miR-769-5p和靶基因的关系。RT-qPCR和蛋白质印迹法进一步验证miR-769-5p对靶基因的调控作用。结果:恶性组血清外泌体中miR-769-5p的表达水平较健康组和良性组更低(P<0.05)。与mimics-NC组比较,miR-769-5p mimics组的SKOV3和OVCAR3两种EOC细胞OD450值、迁移率、侵袭个数更低(P<0.05)。与inhibitor-NC组比较,miR-769-5p inhibitor组的两种EOC细胞在OD450值、迁移率、侵袭个数更高(P<0.05)。双荧光素酶报告基因实验结果显示,共转染miR-769-5p mimics与pEZX-FR02-TFAM-WT后,与mimics-NC组,TFAM-WT型组转染后荧光值显著下降(P<0.01),共转染miR-769-5p mimics与pEZX-FR02-report-TFAM-MUT后,与mimics-NC组比较,TFAM-MUT型组转染后荧光值差异无统计学意义(P>0.05)。实验表明,miR-769-5p能够结合TFAM的3'-UTR区域。与mimics-NC组比较,miR-769-5p mimics组EOC细胞TFAM mRNA和TFAM蛋白表达量更低(P<0.05)。结论:miR-769-5p在EOC血清外泌体中显著低表达,具有成为肿瘤早期诊断生物标志物的潜力。同时,miR-769-5p可能通过靶向TFAM抑制EOC细胞的增殖、迁移和侵袭。
Objective:To investigate the expression of miR-769-5p in serum exosomes of epithelial ovarian cancer(EOC)and its mechanism of influence on EOC cell proliferation,migration,and invasion.Methods:The expression level of miR-769-5p in serum exosomes from the malignant group,benign ovarian disease group,and healthy control group was detected using reverse transcription quantitative polymerase chain reaction(RT-qPCR).Lipofectamine 3000 transfection was used to construct overexpression and knockdown cell lines,which were divided into four groups:miR-769-5p mimics group,mimics-NC group,miR-769-5p inhibitor group,and inhibitor-NC group.The effects of miR-769-5p on cell proliferation,migration,and invasion were assessed using CCK-8,wound healing,and Transwell assays.Transcriptome sequencing was conducted on EOC cells overexpressing miR-769-5p,and potential target genes were predicted using public databases.The interaction between miR-769-5p and its target gene was validated by dual-luciferase reporter assay.RT-qPCR and Western blotting were used to further confirm the regulatory effect of miR-769-5p on the target gene.Results:The expression level of miR-769-5p in serum exosomes from the malignant group was significantly lower than that in the healthy control and benign ovarian disease groups(P<0.05).Compared with the mimics-NC group,the miR-769-5p mimics group exhitied lower OD 450 values,migration rates,and numbers of invading cells in both SKOV3 and OVCAR3 EOC cell lines(P<0.05).Compared with the inhibitor-NC group,cells in the miR-769-5p inhibitor group exhibited increased OD 450 values,migration rates,and invasion counts(P<0.05).The dual-luciferase reporter assay showed that co-transfection of miR-769-5p mimics with pEZX-FR02-TFAM-WT significantly reduced the luciferase activity compared to the mimics-NC group(P<0.01),whereas co-transfection of miR-769-5p mimics with pEZX-FR02-TFAM-MUT showed no significant difference in luciferase activity compared to the control(P>0.05).These findings indicate that miR-769-5p can directly bind to the 3′-UTR region of TFAM.Furthermore,compared with the mimics-NC group,EOC cells transfected with miR-769-5p mimics showed significantly decreased TFAM mRNA and protein expression levels(P<0.05).Conclusion:miR-769-5p is significantly downregulated in EOC serum exosomes and may serve as a promising biomarker for early tumor diagnosis.Moreover,miR-769-5p may inhibit the proliferation,migration,and invasion of ovarian cancer cells by targeting TFAM.
作者
金卓婷
王丹
葛佳
郑茜文
张勇
JIN Zhuoting;WANG Dan;GE Jia(Department of Obstetrics and Gynecology,Mianyang Central Hospital,School of Medicine,University of Electronic Science and Technology of China,Mianyang Sichuan 621000,China;Department of Obstetrics and Gynecology,The Affiliated Hospital of Southwest Medical University,Lvzhou Sichuan 646000,China)
出处
《实用妇产科杂志》
北大核心
2025年第6期520-525,共6页
Journal of Practical Obstetrics and Gynecology
