MiR-152在膀胱癌中的表达以及对ERBB3/AKT2信号通路的调节作用

Expression of MiR-152 in Bladder Cancer and Its Effect on ERBB3/Akt Signal Pathway

[目的]探讨miR-152对ERBB3/Akt信号通路的靶向调控作用以及对膀胱癌细胞UM-UC-3增殖、侵袭和迁移活性的影响。[方法]收集2016年7月至2018年6月接受手术治疗的56例膀胱癌患者的肿瘤组织及相应的癌旁组织,提取总RNA,检测miR-152表达水平。体外培养UM-UC-3细胞,转染miR-152 mimics(模拟物组),同时设置空白对照组(CTL)和空转染组(NC),采用实时荧光定量聚合酶链式反应(qRT-PCR)检测miR-152的表达。采用MTT法、划痕实验和transwell小室侵袭实验观察CTL组、NC组和模拟物组细胞增殖、迁移和侵袭能力。采用双荧光素酶报告基因方法验证ERBB3与miR-152的关系。采用qRT-PCR和蛋白免疫印迹(Western blot)法检测UM-UC-3细胞中Snail、ERBB3、Akt2、p-Akt、c-myc表达量。[结果]Mi R-152在肿瘤组织中的表达量明显低于癌旁组织(P<0.05)。与膀胱上皮永生化细胞株SVHUC-1相比,miR-152在膀胱癌细胞T24、UM-UC-3和J82中的表达量明显降低(P<0.05),其中在UM-UC-3细胞中表达量最低。经qRT-PCR法检测,模拟物组UM-UC-3细胞miR-152的表达量明显低于CTL组和NC组细胞(P<0.05)。模拟物组UM-UC-3细胞的增殖率、迁移活性以及划痕愈合率均明显低于CTL组和NC组细胞(P<0.05)。经双荧光素酶基因报告分析,ERBB3是miR-152的下游靶基因。模拟物组UM-UC-3细胞Snail、ERBB3、Akt2、p-Akt、c-myc mRNA和蛋白表达水平均低于CTL组和NC组细胞(P<0.05)。且miR-152表达量与ERBB3呈负相关性(r=-0.875,P<0.05)。[结论]上调miR-152可抑制ERBB3/Akt/c-myc和ERBB3/Akt/Snail信号通路的异常激活,逆转膀胱癌UM-UC-3细胞上皮—间质转化,从而降低膀胱癌生长、侵袭和迁移的风险。

[Purpose] To investigate the expression of miR-152 in bladder cancer and its effect on proliferation,invasion and migration of bladder cancer cells. [Methods] The cancer and cancer adjacent tissue samples of 56 patients with bladder cancer were collected from July 2016 to June2018. Quantitative real-time PCR (qRT-PCR) was used to detect miR-152 in bladder cancer tissues and cancer adjacent tissues. Bladder cancer UM-UC-3 cells were cultured in vitro and transfected with miR-152 mimics (mimics group). The proliferation,migration and invasion of UM-UC-3 cells were detected by MTT,Transwell assay and scratch test,respectively. The expression of Snail,ERBB3,Akt2,p-Akt,c-myc mRNA and proteins in UM-UC-3 cells were detected by qRT-PCR and Western blot,respectively. [Results] The relative expression of miR-152 in cancer tissues was lower than that in cancer adjacent tissues (P<0.05). And the miR-152 levels in bladder cancer cell lines T24,UM-UC-3 and J82 were all down-regulated compared to the human bladder epithelial SV-HUC-1 cells (P<0.05),and the miR-152 level of UM-UC-3 cells was lowest. The miR-152 expression in UM-UC-3 cells in mimics group was higher than blank control (CTL) group and normal control (NC) group (P<0.05). The proliferation rate,migration cell number and wound healing rate of UM-UC-3 cells in mimics group were lower than those in CTL group and NC group (P<0.05). The ERBB3 was verified as a target gene of miR-152 by the dual luciferase reporter gene system. The expression of Snail,ERBB3,Akt2,p-Akt,c-myc mRNA and proteins in UM-UC-3 cells of mimics group were all lower than those in CTL group and NC group (P<0.05). And the miR-152 level in UM-UC-3 cells was negatively related with ERBB3 mRNA(r=-0.875,P<0.05). [Conclusion] The study shows that up-regulation of miR-152 could inhibit the proliferation,migration and invasion of UM-UC-3 cells by inhibition of ERBB3/Akt/c-myc and ERBB3/Akt/Snail signaling pathways.