MicroRNA-122介导Mex3a表达抑制膀胱癌细胞恶性生物学行为的机制研究

Mechanism underlying the role of microRNA-122 in inhibiting the malignant biological behavior of bladder cancer cells via regulating the expression of Mex3a

摘要:

目的 探究miR-122介导Mex3a表达抑制膀胱癌细胞增殖、迁移，促进细胞凋亡的发生机制。方法 通过实时荧光定量聚合酶链反应检测miR-122和Mex3a在正常人膀胱上皮细胞SVHUC-1和人膀胱癌细胞系T24和HT1376中的表达。构建过表达miR-122和低表达Mex3a的T24细胞，并通过CCK-8法、细胞划痕实验和流式细胞术评估miR-122和Mex3a对膀胱癌细胞增殖、迁移和凋亡的影响。双萤光素酶实验验证miR-122和Mex3a的靶向关系。在过表达miR-122的细胞中进一步过表达Mex3a，检测细胞增殖、迁移、凋亡和PI3K/Akt信号通路的变化。结果 SVHUC-1细胞miR-122相对表达量较T24、HT1376细胞高（P <0.05），Mex3a mRNA相对表达量较T24、HT1376细胞低（P <0.05）。miR-122 mimic组miR-122相对表达量较mimic NC组高（P <0.05）。mimic NC组与miR-122 mimic组在0、24、48、72 h的T24细胞吸光度值比较，经重复测量设计的方差分析，结果 ①不同时间点的细胞吸光度值有差异（P <0.05）；②mimic NC组与miR-122 mimic组的细胞吸光度值有差异（P <0.05），miR-122 mimic组细胞增殖能力较mimic NC组被抑制；③mimic NC组与miR-122 mimic组细胞吸光度值变化趋势有差异（P <0.05）。mimic NC组划痕愈合率较miR-122 mimic组高（P <0.05）。mimic NC组细胞凋亡率较miR-122 mimic组低（P <0.05）。mimic NC组WT-Mex3a相对表达量较miR-122 mimic组高（P <0.05）。mimic NC组Mex3a mRNA相对表达量较miR-122 mimic组高（P <0.05）。si-NC组Mex3a mRNA相对表达量较si-Mex3a组高（P <0.05）。si-NC组p-PI3K、p-Akt蛋白较si-Mex3a组高（P <0.05）。si-NC组与si-Mex3a组在0、24、48和72 h的T24细胞吸光度值比较，经重复测量设计的方差分析，结果 ①不同时间点间的细胞吸光度值有差异（P <0.05）；②si-NC组与si-Mex3a组细胞吸光度值有差异（P <0.05）；③si-NC组与si-Mex3a组细胞吸光度值变化趋势有差异（P <0.05）。si-NC组划痕愈合率较si-Mex3a组高（P <0.05）。si-NC组细胞凋亡率较si-Mex3a组低（P <0.05）。miR-122 mimic+ oe-Mex3a组Mex3a mRNA相对表达量较miR-122 mimic+ oe-N组高（P <0.05）。miR-122 mimic+ oe-NC组p-PI3K、p-AKT蛋白相对表达量较miR-122 mimic+ oe-Mex3a组低（P <0.05）。miR-122 mimic+ oe-NC组与miR-122 mimic+ oe-Mex3a组在0、24、48、72 h的T24细胞吸光度值比较，经重复测量设计的方差分析，结果 ①不同时间点的细胞吸光度值有差异（P <0.05）；②miR-122 mimic+ oe-NC组与miR-122 mimic+ oe-Mex3a组细胞吸光度值有差异（P <0.05），miR-122 mimic+ oe-Mex3a组较miR-122 mimic+ oe-NC组细胞增殖能力增强；③miR-122 mimic+ oe-NC组与miR-122 mimic+oe-Mex3a组细胞吸光度值变化趋势有差异（P <0.05）。miR-122 mimic+ oe-NC组划痕愈合率较miR-122 mimic+ oe-Mex3a组低（P <0.05）。miR-122 mimic+ oe-NC组细胞凋亡率较miR-122 mimic+ oe-Mex3a组高（P <0.05）。结论 miR-122可能通过抑制Mex3a的表达，抑制PI3K/Akt信号通路，从而抑制膀胱癌细胞的增殖、迁移，并促进凋亡。

Abstract:

Objective To investigate the mechanism whereby microRNA-122 (miR-122) inhibited the proliferation and migration yet promoted the apoptosis of bladder cancer cells via mediating the expression of Mex3a.Methods The qRT-PCR was applied to detect the expressions of miR-122 and Mex3a in normal human bladder epithelial cell line SVHUC-1 and human bladder cancer cell lines T24 and HT1376. T24 cell lines overexpressing miR-122 and downregulating Mex3a were constructed, and the effects of miR-122 and Mex3a expressions on the proliferation, migration and apoptosis of bladder cancer cells were assessed by CCK-8 assay, scratch assay and flow cytometry, respectively. The regulatory relationship between miR-122 and Mex3a was detected by luciferase reporter assay. Besides, Mex3a was further overexpressed in cells overexpressing miR-122 to verify the roles of miR-122 and Mex3a in cell proliferation, migration, and apoptosis as well as the regulation of the PI3K/Akt signaling pathway.Results The relative expression of miR-122 in SVHUC-1 cells was higher than that in T24 and HT1376 cells (P < 0.05), while the relative expression of Mex3a mRNA in SVHUC-1 cells was lower than that in T24 and HT1376 cells (P < 0.05). The relative expression of miR-122 in the miR-122 mimic group was higher than that in the mimic NC group (P < 0.05). The optical density of T24 cells in the mimic NC group and the miR-122 mimic group at 0, 24 h, 48 h and 72 h were compared via repeated measures ANOVA, which revealed that they were different among the time points (P < 0.05) and between the groups (P < 0.05). Specifically, the proliferation of cells in the miR-122 mimic group was inhibited relative to that in the mimic NC group. Besides, the change trend of the optical density of T24 cells was different between the mimic NC group and the miR-122 mimic group (P < 0.05). As suggested by the scratch assay, the wound healing rate in the mimic NC group was higher than that in the miR-122 mimic group (P < 0.05). The apoptosis rate in the mimic NC group was lower than that in the miR-122 mimic group (P < 0.05). The relative expression of WT-Mex3a in the mimic NC group was higher than that in the miR-122 mimic group (P < 0.05), and the relative expression of Mex3a mRNA in the mimic NC group was also higher than that in the miR-122 mimic group (P < 0.05). The relative expression of Mex3a mRNA in the si-NC group was higher than that in the si-Mex3a group (P < 0.05). The protein expressions of p-PI3K and p-Akt in the si-NC group were higher than those in the si-Mex3a group (P < 0.05). The optical density of T24 cells in the si-NC group and the si-Mex3a group at 0, 24 h, 48 h and 72 h were compared via repeated measures ANOVA, which revealed that they were different among the time points (P < 0.05) and between the groups (P < 0.05), and that the change trend of the optical density of T24 cells was different between the two groups (P < 0.05). The wound healing rate in the si-NC group was higher than that in the si-Mex3a group (P < 0.05), while the apoptosis rate in the si-NC group was lower than that in the si-Mex3a group (P < 0.05). The relative expression of Mex3a mRNA in the miR-122 mimic+ oe-Mex3a group was higher than that in the miR-122 mimic+ oe-NC group (P < 0.05). The protein expressions of p-PI3K and p-Akt in the miR-122 mimic+ oe-NC group were lower than those in the miR-122 mimic+ oe-Mex3a group (P < 0.05). The optical density of T24 cells in the miR-122 mimic+ oe-NC group and the miR-122 mimic+ oe-Mex3a group at 0, 24 h, 48 h and 72 h were compared via repeated measures ANOVA, which revealed that they were different among the time points (P < 0.05) and between the groups (P < 0.05). Specifically, the proliferation capability of cells in the miR-122 mimic+ oe-Mex3a group was higher than that in the miR-122 mimic+ oe-NC group (P < 0.05). In addition, the change trend of the optical density of T24 cells was different between the miR-122 mimic+ oe-NC group and the miR-122 mimic+ oe-Mex3a group (P < 0.05). The wound healing rate in the miR-122 mimic+ oe-NC group was lower than that in the miR-122 mimic+ oe-Mex3a group (P < 0.05), while the apoptosis rate in the miR-122 mimic+ oe-NC group was higher than that in the miR-122 mimic+ oe-Mex3a group (P < 0.05).Conclusions MiR-122 inhibits the PI3K/Akt signaling pathway by suppressing the expression of Mex3a, thus restraining the proliferation and metastasis of bladder cancer cells while promoting their apoptosis.