miR-424和miR-503对人直肠癌细胞系增殖的影响

Effects of miR-424 and miR-503 on the proliferation of rectal cancer cell lines

目的探讨miR-424和miR-503对人直肠癌细胞株SW620和SW480细胞增殖的影响,并筛选其靶基因。方法直肠癌细胞株SW620和SW480分为SW480组、SW480-miR-424组、SW480-miR-503组、SW620组、SW620-miR-424组、SW620-miR-503组;SW480组和SW620组细胞正常培养,SW480-miR-424组、SW480-miR-503组、SW620-miR-424组和SW620-miR-503组细胞分别采用慢病毒载体pSLIK-Zeo构建miR-424和miR-503直肠癌细胞系;SW620细胞系各组于不同培养时间进行细胞计数并绘制细胞生长曲线;采用荧光定量PCR法检测SW480组、SW620组细胞中miR-424和miR-503表达水平;采用RNA-seq法筛选miR-424和miR-503的靶基因,并采用Western blot对靶基因进行验证。结果miR-424和miR-503在SW480细胞中呈高表达,在SW620细胞中呈低表达;SW480组miR-424-3p(9.572±2.171)、miR-424-5p(13.891±1.124)和miR-503(6.792±1.412)表达水平高于SW620组(1.026±2.874、1.103±0.411、0.984±0.847),差异有统计学意义(P<0.01);各时间点SW620-miR-424组和SW620-miR-503组细胞数量均少于SW620组(P<0.01),SW620-miR-503组少于SW620-miR-424组(P<0.01);RNA-seq测序分析结果显示,SW620和SW480细胞共有1 413个差异表达基因,其中有50个肿瘤标志物,WEE1蛋白为极高表达者,miR-424和miR-503均可与WEE1的3UTR序列结合,且miR-424的结合能力高于miR-503;SW620组细胞WEE1蛋白表达水平(8.69±3.24)高于SW480组(5.94±2.37)(P<0.01),SW480-miR-424组和SW480-miR-503组细胞WEE1蛋白表达水平(2.19±1.72、3.45±2.16)明显低于SW480组(P<0.01),SW620-miR-424组和SW620-miR-503组细胞WEE1蛋白表达水平(4.31±2.63、5.37±3.25)明显低于SW620组(P<0.01)。结论miR-424和miR-503抑制细胞周期检查点激酶WEE1的表达,进而调节细胞的增殖能力,参与肿瘤发生、发展过程。

Objective To explore the effects of miR-424 and miR-503 on the proliferation of rectal cancer cell lines and screen the target genes. Methods Rectal cancer cell lines SW620 and SW480 were divided into SW480 group,SW480-miR-424 group,SW480-miR-503 group,SW620 group,SW620-miR-424 group and SW620-miR-503 group.SW480 group and SW620 group were cultured normally. SW480-miR-424 group, SW480-miR-503 group,SW620-miR-424 group and SW620-miR-503 group were constructed the miR-424 and miR-503 rectal cancer cell lines by lentiviral vector pSLIK-Zeo.The cell number was counted and the growth curve was drawn at different culture time in rectal cancer cell lines SW620.The levels of miR-424 and miR-503 were detected by fluorescence quantitative PCR in SW480 group and SW620 group.The target genes of miR-424 and miR-503 were screened by RNA-seq and verified by Western blot.Results MiR-424 and miR-503 were highly expressed in SW480 cells and lowly expressed in SW620 cells.The levels of miR-424-3 p(9.572±2.171),miR-424-5 p(13.891±1.124)and miR-503(6.792±1.412)in SW480 group were significantly higher than those in SW620 group(1.026±2.874,1.103±0.411,0.984±0.847)(P<0.01).The cell number was significantly lower in SW620-miR-424 group and SW620-miR-503 group than that in SW620 group(P<0.01),and in SW620-miR-503 group than that in SW620-miR-424 group(P<0.01).The results of RNA-seq sequencing showed that there were 1 413 differentially expressed genes in SW620 and SW480 cells,among which there were 50 tumor markers and WEE1 protein was highly expressed.Both miR-424 and miR-503 were bound with WEE1 3'UTR,and the binding ability of miR-424 was significantly higher than miR-503.The level of WEE1 protein was significantly higher in SW620 group(8.69±3.24)than that in SW480 group(5.94±2.37)(P<0.01),was significantly lower in SW480-miR-424 group(2.19±1.72)and SW480-miR-503 group(3.45±2.16)than that in SW480 group(P<0.01),and in SW620-miR-424 group(4.31±2.63)and SW620-miR-503 group(5.37±3.25)than that in SW620 group(P<0.01).Conclusion MiR-424 and miR-503 inhibit the expression of cell cycle checkpoint kinase WEE1,regulate cell proliferation and participates in tumorigenesis and progression.