miR-3126-5p靶向 LASP1抑制结直肠癌细胞的增殖和迁移的机制探讨

目的探讨微小RNA(miRNA)-3126-5p抑制靶基因LIM和SH3蛋白1(LIM and SH3 protein 1.LASP1)对结直肠癌细胞增殖和迁移能力的影响。方法采用实时定量聚合酶链式反应(qRT-PCR)法检测结直肠癌细胞株(HT-29、HCT116、LoVo、SW480)和正常肠黏膜上皮细胞(HIEC)中miR-3126-5p的表达水平。选择表达水平最低的细胞株作为实验对象,实验分为2组:阴性对照组(转染miR-NC)和miR-3126-5p组(转染miR-3126-5p),转染48 h后收集各组细胞。qRT-PCR法检测各组细胞miR-3126-5p的表达水平。采用MTS法和划痕愈合实验分别检测各组细胞的增殖水平和迁移能力。采用生物信息学软件microRNA.org和双荧光素酶报告基因实验分别预测和验证miR-3126-5p的靶基因。采用qRT-PCR和Western blot检测各组细胞靶基因的表达水平。计量资料以均数土标准差(Mean±SO)表示,两两组间比较采用t检验,多组间比较采用单因素方差分析结果与正常肠黏膜上皮细胞(HIEC)相比,结直肠癌细胞株miR-3126-5p的表达水平显著降低(P<0.05),表达水平最低的细胞株是HCT116细胞(P<0.01)。阴性对照组和miR-3126-5P组HCT116细胞中miR-3126-5p的表达分别为(1.05±0.16)和(7.91±1.26),差异具有统计学意义(t=5.40,P<0.01)。与阴性对照组相比,miR-3126-5p组HCT116细胞的增殖能力显著降低(P<0.05),迁移能力显著下降(t=4.52,F<0.01)。microRNA.org显示miR-3126-5p与LIM和SH3蛋白1(LASP1)基因mRNA存在互补结合位点。miR-3126-5p和L4SP/mRNA能够靶向结合(P<0.01)。与阴性对照组相比,miR-3126-5p组H CT116细胞中L4SP/基因表达显著降低(t=4.56,P<0.01)。结论结直肠癌细胞株中miR-3126-5p呈低表达,miR-3126-5p能够通过抑制靶基因LASP1降低结直肠癌HCT116细胞的增殖和迁移能力。

Mechanism of miR-3126-5p targeting LASP1 to inhibit the proliferation and migration of colorectal cancer cells

Objective To explore the effect of microRNA(miRNA)-3126-5p on the proliferation and migration of colorectal cancer cells by inhibiting the expression of LIM and SH3 protein 1(LASP1).Methods Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-3126-5p in colorectal cancer cell lines(HT-29,HCT116,LoVo,SW480)and normal intestinal mucosal epithelial cells(HIEC).The cell line with the lowest expression level was selected as the experimental object.The experiment was divided into 2 groups:the negative control group(transfected with miR-NC)and the miR-3126-5p group(transfected with miR-3126-5p).Cells of each group were collected 48h after transfection.qRT-PCR method was used to detect the expression level of miR-3126-5p in each group.The MTS method and the scratch healing experiment were used to detect the proliferation level and migration ability of the cells in each group.The hioinfomiatirs software microRNA.org and the dual-luciferase reporter gene experiment were used to predict and verify the target genes of miR-3126-5p,respectively.qRT-PCR and Western blot were used to detect the expression levels of target genes in each group of cells.Measurement data were expressed as mean±standard deviation(Mean±SD),t test was used for comparison between two groups,and one-way analysis of variance was used for comparison between multiple groups.Results Compared with normal intestinal mucosal epithelial cells(HIEC),the expression level of colorectal cancer cell line miR-3126-5p was significantly reduced(P<0.05),and the cell line with the lowest expression level was HCT116 cells(P<0.01).The expression of miR-3126-5p in HCT116 cells in the negative control group and miR-3126-5p group were(1.05±0.16)and(7.91±1.26)respectively,and the difference was statistically significant(t=5.40,P<0.01).Compared with the negative control group,the proliferation ability of HCT116 cells in the miR-3126-5p group was significantly reduced(t=4.52,P<0.05),and the niigiation ability was significantly reduced(P<0.01).microRNA.org shows that miR-3126-5p has complementary binding sites with LIM and SH3 protein 1(LASP1)gene mRNA.miR-3126-5p can target LASP1 mRNA(P<0.01).Compared with the negative control group,the expression of LASP1 gene in HCT116 cells of the miR-3126-5p group was significantly reduced(t=4.56,P<0.01).Conclusion The expression of miR-3126-5p in colorectal cancer cell lines is low,and miR-3126-5p can reduce the proliferation and migration ability of colorectal cancer HCT116 cells by inhibiting the expression of the target gene LASP1.