miRNA-125b靶向p16促进套细胞淋巴瘤细胞增殖和侵袭的机制探讨

The mechanical study of miRNA-125b targeting p16 to promote the proliferation and invasion of mantle cell lymphoma cells

目的:探讨miRNA-125b抑制靶基因p16对套细胞淋巴瘤细胞增殖和侵袭能力的影响。方法:选择人源套细胞淋巴瘤细胞系JeKo-1作为实验对象。实验共分为五组:空白对照组(不转染)、miRNA-125b阴性对照组(转染miRNA-125b inhibitor-NC)、miRNA-125b抑制剂组(转染miRNA-125b inhibitor)、p16 siRNA阴性对照组(转染p16 siRNA-NC)、p16 siRNA组(转染p16 siRNA)。于转染后48 h收集细胞。采用RT-PCR法检测各组JeKo-1细胞miRNA-125b的表达水平;采用双荧光素酶靶标实验验证miRNA-125b与p16基因的靶向关系;采用Western Blot和RT-PCR法检测各组细胞p16基因的表达水平;采用MTS法检测各组细胞增殖水平;采用流式细胞术检测各组细胞周期分布;采用Transwell侵袭实验检测各组细胞侵袭能力。结果:与空白对照组、miRNA-125b inhibitor-NC组相比,miRNA-125b inhibitor组miRNA-125b表达水平显著降低,而p16表达水平显著升高,细胞增殖受到抑制、Je Ko-1细胞穿膜细胞数显著减少,细胞G0/G1期比例显著增加,而S期比例逐渐减少(P <0.05);与p16 siRNA-NC组相比,p16 siRNA组p16表达水平明显被抑制,细胞增殖能力及细胞侵袭能力均显著升高,细胞G0/G1期比例显著降低,而S期、G2/M期比例升高,差异均具有统计学意义(P <0.05)。双荧光素酶报告基因实验分析结果显示,miRNA-125b与p16基因3’UTR端存在互补结合位点,miRNA-125b和p16能够靶向结合,p16基因是miRNA-125b的靶基因。结论:miRNA-125b能够通过抑制靶基因p16促进套细胞淋巴瘤细胞的增殖和侵袭能力,可通过抑制miRNA-125b,促进p16基因的表达,使套细胞淋巴瘤细胞阻滞于G0/G1期,抑制其增殖转化。

Objective:To investigate the effect of miRNA-125b on the proliferation and invasion of mantle cell lymphoma cells by inhibiting target gene p16.Methods:Human nested cell lymphoma cell line JeKo-1 was selected as the experimental object.The experiment was divided into five groups:Blank control group(non-transfection),miRNA-125b negative control group(transfection of miRNA-125b inhibitor-NC),miRNA-125b inhibitor group(transfection of miRNA-125b inhibitor),p16 siRNA-NC group(transfection of p16 siRNA negative control),p16 siRNA group(transfection of p16 siRNA).Cells were collected 48 hours after transfection.The expression of miRNA-125b in JeKo-1 cells was detected by RT-PCR.Targeting relationship between miRNA-125b and p16 gene was validated by double luciferase target assay.Western Blot and RT-PCR were used to detect the expression level of p16 gene in cells of each group.MTS assay was used to detect cell proliferation in each group.Cell cycle distribution was detected by flow cytometry.Transwell invasion assay was used to detect the invasive ability of cells in each group.Results:Compared with the blank control group and the miRNA-125b inhibitor-NC group,the expression level of miRNA-125b in the miRNA-125b inhibitor group decreased significantly while the expression level of p16 increased significantly.Cell proliferation was inhibited.The number of JeKo-1 cell penetrating membrane cells decreased significantly.The proportion of G0/G1 phase increased significantly,while the proportion of S phase decreased gradually(P<0.05).Compared with the p16 siRNA-NC group,the expression level of p16 in the p16 siRNA group was significantly inhibited.The cell proliferation and invasion ability were significantly increased.The ratio of G0/G1 phase was significantly decreased,while the ratio of S phase and G2/M phase was increased.The difference was statistically significant(P<0.05).The results of double luciferase reporter gene assay showed that there were complementary binding sites between miRNA-125b and 3'UTR of p16 gene.miRNA-125b and p16 could bind targeting.p16 gene was the target gene of miRNA-125b.Conclusion:miRNA-125b can promote the proliferation and invasiveness of mantle cell lymphoma cells by inhibiting target gene p16,and promote the expression of p16 gene by inhibiting miRNA-125b,so that mantle cell lymphoma cells can be blocked at G0/G1 phase and inhibit their proliferation and transformation.