微小RNA-199b-3p靶向调控SOX6的表达及其对结肠癌细胞SW620增殖和凋亡的影响

目的 探讨微小RNA-199b-3p（miR-199b-3p）靶向调控SOX6的表达及其对结肠癌细胞SW620增殖及凋亡的影响。方法 采用Lipofectamine脂质体法将miR-199b-3p抑制剂及阴性对照（NC）转染至SW620细胞并分为抑制剂组和NC组，同时以未转染的SW620细胞作对照。采用实时荧光定量PCR（QPCR）法检测3组miR-199b-3p的表达以评价转染效果，MTT法和Annexin Ⅴ-FITC／PI流式细胞术检测转染后的增殖活性及凋亡率，QPCR和Western blotting检测各组凋亡相关基因（Bax和caspase-3）及SOX6的mRNA和蛋白水平，构建含有野生型及突变型SOX6-3’UTR的荧光报告基因质粒并采用双荧光素酶报告实验验证miR-199b-3p对SOX6的靶向调控作用。结果 QPCR检测显示，转染48 h后抑制剂组的miR-199b-3p的表达水平为0.526±0.034，低于未转染组的1.009±0.064和NC组的0.960±0.057，差异有统计学意义（P＜0.05）；与其余两组相比，抑制剂组转染24、48、72 h后的SW620细胞的增殖活性均降低（P＜0.05）；抑制剂组的凋亡率为（37.533±1.459）％，高于未转染组的（6.101±0.663）％和NC组的（8.753±1.061）％，差异均有统计学意义（P＜0.05）；抑制剂组SW620细胞的SOX6、Bax、caspase-3 mRNA和蛋白表达水平均高于未转染组和NC组，差异均有统计学意义（P＜0.05）。双荧光素酶报告基因实验表明 miR-199b-3p可显著抑制野生型 SOX6-3’UTR的荧光荧光素酶活性，而对突变型质粒转染细胞的荧光素酶活性并无影响。结论miR-199b-3p可靶向调控SOX6的表达，且下调miR-199b-3p表达可抑制SW620细胞的增殖并诱导凋亡，为结肠癌的治疗提供潜在的分子治疗靶点。

Effects of miRNA-199b-3p on the targeted regulation of SOX6 and proliferation and apoptosis of colon cancer cell line SW620

Objective To investigate the effects of microRNA-199b-3p (miR-199b-3p) on the expression of SOX6 and its effect on proliferation and apoptosis of colon cancer cell line SW620.Methods The miR-199b-3p inhibitor and negative control (NC) were transfected into SW620 cells by Lipofectamine liposome method and then divided into inhibitor group and NC group.The SW620 cells without transfection were used as control (non-transfection group).Real-time quantitative PCR (QPCR) was used to detect the expression of miR-199b-3p in three groups as to evaluate the transfection efficiency.MTT and Annexin Ⅴ-FITC/PI flow cytometry were used to detect the proliferative activity and apoptotic rate after transfection, respectively.The mRNA and protein levels of apoptosis related genes (Bax and caspase-3) and SOX6 were detected by QPCR and Western blotting.The luciferase reporter gene plasmids containing wild type and mutant SOX6-3'UTR were constructed.The effect of miR-199b-3p on the regulation of SOX6 was verified by double luciferase reporter assay.Results The QPCR results at 48 h after transfection showed that the level of miR-199b-3p in the inhibitor group was 0.526±0.034, lower than 1.009±0.064 of non-transfection group and 0.960±0.057 of NC group, and the difference was statistically significant (P<0.05).Compared with other two groups, the proliferative activity of the inhibitor group was decreased at 24, 48 and 72 h after transfection (P<0.05).The apoptotic rate of the inhibitor group was (37.533±1.459)%, higher than (6.101±0.663)% of non-transfection group and (8.753±1.061)% of NC group, and the difference was statistically significant (P<0.05).The mRNA and protein levels of SOX6, Bax and caspase-3 in inhibitor group were higher than those in non-transfection group and the NC group, and the difference was statistically significant (P<0.05).Dual luciferase reporter gene assay showed that miR-199b-3p could significantly inhibit the luciferase activity of wild-type SOX6-3'UTR, but had no effect on the luciferase activity of mutant plasmid transfected cells.Conclusion MiR-199b-3p can regulate the expression of SOX6, and inhibiting the expression of miR-199b-3p can inhibit the proliferation of SW620 cells and induce apoptosis, which provide a potential molecular therapeutic target for the treatment of colon cancer.