miR-454-3p靶向TCF-4对人肾癌细胞GRC-1增殖、凋亡和迁移的影响

Effects of miR-454-3p on proliferation, apoptosis and migration of human kidney cancer cell GRC-1 via targeting TCF-4

目的探讨miR-454-3p与TCF-4的靶向关系,以及对人肾癌细胞GRC-1增殖、凋亡和迁移的影响。方法miR-454-3p mimic和(或)pLV-TCF-4转染GRC-1细胞后,采用qRT-PCR法检测miR-454-3p和TCF-4 mRNA水平;Western blot法检测TCF-4蛋白的表达;双荧光素酶报告实验验证miR-454-3p与TCF-4的靶向关系;MTT法检测细胞增殖情况;Hoechst染色检测细胞凋亡;划痕实验检测细胞迁移能力;体外侵袭实验检测细胞侵袭能力。结果miR-454-3p mimic抑制TCF-4 mRNA和蛋白的表达;pLV-TCF-4缓解miR-454-3p对TCF-4蛋白表达的抑制作用。miR-454-3p mimic与TCF-4野生型报告载体共转,荧光素酶活性降低;与TCF-4突变型报告载体共转后,荧光素酶活性无明显变化。与GRC-1组相比,miR-454-3p mimic转染到GRC-1细胞后,细胞增殖倍数明显降低(P<0.01),细胞凋亡率由(5.08±1.21)%增加到(23.12±1.82)%,伤口愈合率由(58.43±6.32)%降低到(32.15±5.16)%,侵袭细胞数目由113.54±9.27减少至51.21±6.03;共转染miR-454-3p mimic和pLV-TCF-4后,能够逆转上述改变。结论miR-454-3p可通过靶向TCF-4抑制人肾癌细胞GRC-1的增殖和迁移,诱导细胞凋亡。

Purpose To investigate the targeting relationship between miRNA454-3p and TCF-4,and the effects of miRNA454-3p on proliferation,apoptosis and migration of human renal cell carcinoma GRC-1.Methods miR-454-3p mimic and/or pLV-TCF4 transfected GRC-1 cells,the mRNA levels of miR-454-3p and TCF-4 were detected by qRT-PCR.The expression of TCF-4 protein was detected by Western blotting.The targeting relationship between miR-454-3p and TCF-4 was verified by dual luciferase reporter assay.Cell proliferation was detected by MTT assay.Apoptosis was detected by Hoechst.Cell migration ability was measured by a scratch test.Cell invasion ability was examined by Transwell.Results miR-454-3p mimic inhibited the expression of TCF-4 mRNA and protein.pLV-TCF-4 attenuated the inhibitory effect of miR-454-3p on TCF-4 protein expression.The luciferase activity was decreased after miR-454-3p mimic co-transformed with the TCF-4 wild-type reporter vector,however,there was no significant change in luciferase activity after co-transfection with the TCF-4 mutant reporter vector.Compared with the GRC-1 group,miR-454-3p mimic transfected into GRC-1 cells,the cell proliferation fold was significantly decreased(P<0.01),and the apoptotic rate was increased from(5.08±1.21)%to(23.12±1.82)%,the wound healing rate decreased from(58.43±6.32)%to(32.15±5.16)%,and the number of invasive cells decreased from 113.54±9.27 to 51.21±6.03.However,after co-transfection of miR-454-3p mimic and pLV-TCF-4,the above changes were reversed.Conclusion miR-454-3p could inhibit the proliferation,migration and induce apoptosis of GRC-1 cells by targeting TCF-4.