南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (7): 1022-1029.doi: 10.12122/j.issn.1673-4254.2021.07.08

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miR-let-7c-5p通过靶向HMGA2抑制膀胱癌细胞侵袭和迁移

姚 越，张 冲，熊言骏，韩 兵，高幸福，汪 盛   

  • 出版日期:2021-07-20 发布日期:2021-07-19

miR-let-7c-5p inhibits invasion and migration of bladder cancer cells by targeting HMGA2

  • Online:2021-07-20 Published:2021-07-19

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摘要： 目的 探讨miR-let-7c-5p能否调控HMGA2抑制膀胱癌细胞侵袭和迁移。方法 生物信息学方法确定miR-let-7c-5p的关键基因；RT-qPCR和Western blot检测膀胱癌和癌旁组织中miR-let-7c-5p mRNA和HMGA2 蛋白相对表达量。以人正常膀胱细胞SV-HUC-1作为对照，RT-qPCR和Western blot检测膀胱癌细胞系T24，UM-UC-3，5637细胞中miR-let-7c-5p mRNA和HMGA2蛋白的相对表达量。对UM-UC-3细胞中miR-let-7c-5p分别进行上调和下调，上调实验设模拟物阴性对照组（mimic-NC）、miR-let-7c-5p模拟物组（mimicmiR-let-7c-5p）、下调实验设阴性对照组（inhibitor NC）及miR-let-7c-5p抑制剂组（si-miR-let-7c-5p），双荧光素酶实验、RT-qPCR和Western blot法共同验证miR-let-7c-5p和HMGA2的靶向关系；Transwell小室检测单独上调或下调miR-let-7c-5p表达及同时下调miR-let-7c-5p和HMGA2表达UM-UC-3细胞侵袭和迁移的变化；Western blot检测上皮间质转化（EMT）相关蛋白（E-cadherin, N-cadherin, vimentin, Snail）相对表达量。结果 HMGA2为miR-let-7c-5p的靶基因之一；与癌旁组织相比，膀胱癌组织中miR-let-7c-5pmRNA相对表达量降低（P<0.05），HMGA2蛋白相对表达量增加（P< 0.05）；与SV-HUC-1细胞相比，UM-UC-3细胞中miR-let-7c-5p相对表达量显著降低，HMGA2显著增加（P=0.01）。上调miR-let-7c-5p表达，UM-UC-3细胞侵袭和迁移能力均显著下降（P<0.05）；下调miR-let-7c-5p表达，UM-UC-3细胞侵袭和迁移能力均显著增加（P<0.05）；当同时下调miR-let-7c-5p和HMGA2表达UM-UC-3细胞侵袭和迁移能力显著下降（P<0.05）。Western blot结果显示，上调miR-let-7c-5p表达，E-cadherin表达增加，N-cadherin，vimentin，Snail蛋白表达下降；下调miR-let-7c-5p表达，E-cadherin表达下降，N-cadherin，vimentin，Snail蛋白表达增加；同时下调miR-let-7c-5p和HMGA2表达能够抑制UM-UC-3细胞EMT（P<0.05）。结论 miR-let-7c-5p通过靶向HMGA2抑制EMT进而抑制UM-UC-3细胞侵袭和迁移。

关键词: 膀胱癌；miR-let-7c-5p；HMGA2；上皮间质转化；侵袭迁移

Abstract: Objective To investigate whether miR-let-7c-5p inhibits invasion and migration of bladder cancer cells by regulating HMGA2. Methods We used bioinformatics methods to determine the key genes of miR- let- 7c-5p. RT-qPCR and Western blotting were used to detect the expressions of miR- let-7c-5p mRNA and HMGA2 protein in bladder cancer and adjacent tissues. With human normal bladder SV-HUC-1 cells as the control, we detected the expression levels of miR-let-7c-5p mRNA and HMGA2 protein in bladder cancer cell lines T24, UM-UC-3 and 5637 with RT-qPCR and Western blotting. We observed the effects of miR-let-7c-5p upregulation (by transfection with a miR-let-7c-5p mimic), miR-let-7c-5p downregulation (using a miR-let-7c-5p inhibitor), and knockdown of both HMGA2 and miR-let-7c-5p on invasion, migration and expressions of epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin, vimentin, and Snail) in UM-UC-3 cells. Dual luciferase assay, RT-qPCR and Western blotting were used to verify the targeting relationship between miR-let-7c-5p and HMGA2. Results HMGA2 was identified as one of the target genes of miR- let-7c- 5p. Compared with the adjacent tissues, bladder cancer tissues showed a significantly decreased expression of miR-let-7c-5p and an increased expression of HMGA2 protein (P<0.05). In UM-UC-3 cells, the expression of miR- let-7c-5p was significantly reduced and that of HMGA2 was significantly increased as compared with those in SV-HUC-1 cells (P=0.001). Up-regulating miR-let-7c-5p expression significantly lowered the invasion and migration abilities of UM-UC-3 cells, and down- regulating miR-let-7c-5p expression obviously promoted the invasion and migration of UM-UC-3 cells (P<0.05). Knockdown of both miR-let-7c-5p and HMGA2 expression significantly lowered the invasion and migration (P<0.05) and inhibited the expressions of EMT-related proteins of UM-UC-3 cells (P<0.05). Conclusion miR-let-7c-5p inhibits EMT of bladder cancer UM-UC-3 cells by targeting HMGA2, thereby inhibiting the cell invasion and migration.

Key words: bladder cancer; miR-let-7c-5p; HMGA2; epithelial-mesenchymal transition; invasion and migration