miR-28-5p影响结肠癌细胞迁移活力的分子机制初探

A preliminary study of molecular mechanism:miR-28-5p affected migration activity of colon cancer cells

目的探讨miR-28-5p影响结肠癌细胞迁移活力的分子机制。方法选用结肠癌细胞(HCT116细胞)进行实验,实验分为两部分。第一部分实验:将HCT116细胞分为空白对照组(control组)和爱拉斯汀组(erastin组),control组不进行特殊处理,erastin组使用加有20μM erastin的培养基培养48 h。第二部分实验:将HCT116细胞分为拮抗剂阴性对照组(inhibitor-NC组)、miR-28-5p拮抗剂组(inhibitor组)、激动剂阴性对照组(mimics-NC组)、miR-28-5p激动剂组(mimics组),两组阴性对照分别转染拮抗剂阴性对照混合物和激动剂阴性对照混合物,miR-28-5p拮抗剂组转染miR-28-5p inhibitor以沉默miR-28-5p,miR-28-5p激动剂组转染miR-28-5p mimics以过表达miR-28-5p。用细胞划痕实验检测细胞迁移活力,实时荧光定量PCR(RT-PCR)检测HCT116细胞中miR-28-5p的相对表达水平,蛋白质免疫印迹(WB)检测VAT1L蛋白的相对表达量。比较48 h后erastin组与control组miR-28-5p、VAT1L表达水平及两组细胞划痕迁移率和转染完成后各组细胞miR-28-5p、VAT1L表达水平。结果第一部分:与control组相比,erastin组miR-28-5p的相对表达量、VAT1L蛋白的相对表达量均有降低(均P<0.05);与control组相比,erastin组的细胞迁移率降低(P<0.05)。第二部分:与inhibitor-NC组比较,miR-28-5p inhibitor组miR-28-5p的相对表达量降低(P<0.05);与mimics-NC组比较,miR-28-5p mimics组miR-28-5p的相对表达量增高(P<0.05)。与inhibitor-NC组比较,miR-28-5p inhibitor组VAT1L蛋白的相对表达量升高(P<0.05);与mimics-NC组比较,miR-28-5p mimics组VAT1L蛋白的相对表达量降低(P<0.05)。结论miR-28-5p可能通过抑制VAT1L的表达,以降低结肠癌细胞迁移活力。

Objective To explore molecular mechanism of miR-28-5p affecting migration viability of colon cancer cells.Methods Colon cancer cells(HCT116 cells)were selected for the experiment.The experiment was divided into two parts.Part I:HCT116 cells were divided into blank control group(control group)and erastin group(erastin group),the control group did not receive special treatment,and the erastin group was cultured for 48 hours with 20μM erastin.Part II:HCT116 cells were divided into inhibitor-NC group and miR-28-5p inhibitor group,mimics-NC group and miR-28-5p mimics group,and transfected with miR-28-5p antagonist and mimics,to silence and overexpress miR-28-5p.The cell migration activity was detected by cell scratch assay,the relative expression level of miR-28-5p in HCT116 cells was detected by real-time PCR(RT-PCR),and the relative expression level of VAT1L protein was detected by Western blot(WB).The expression levels of miR-28-5p and VAT1L in the erastin group and the control group after 48 hours,as well as the scratch migration rate of the two groups and the expression levels of miR-28-5p and VAT1L in each group after transfection were compared.Results Part I:Compared with the control group,the relative expression of miR-28-5p in the erastin group was decreased(P=0.035);compared with the control group,the relative expression of VAT1L protein in the erastin group was decreased(P=0.021);Compared with the control group,the cell migration rate in the erastin group was decreased(P=0.025).Part II:Compared with the inhibitor-NC group,the relative expression of miR-28-5p in the miR-28-5p inhibitor group was decreased(P=0.0001);the relative expression level increased(P=0.001).Compared with the inhibitor-NC group,the relative expression of VAT1L protein in the miR-28-5p inhibitor group increased(P=0.04);compared with the mimics-NC group,the relative expression of VAT1L protein in the miR-28-5p mimics group decreased(P=0.018).Conclusion miR-28-5p may reduce the migration activity of colon cancer cells via inhibiting VAT1L.