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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AnnexinⅤ-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。
Objective To investigate the effect of microRNA-199b-3p (miR-199b-3p) on the expression of SOX6 and its effect on the 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 method and divided into inhibitor group and NC group. Meanwhile, untransfected SW620 cells were used as control. The expression of miR-199b-3p was detected by real-time fluorescence quantitative PCR (QPCR) to evaluate the transfection efficiency. The proliferation activity and apoptosis rate were detected by MTT assay and Annexin Ⅴ-FITC / PI flow cytometry. QPCR And Western blotting were used to detect the mRNA and protein levels of apoptosis-related genes (Bax and caspase-3) and SOX6 in each group. Fluorescent reporter plasmids containing wild-type and mutant SOX6-3’UTR were constructed and used luciferase reporter assay To verify the regulatory effect of miR-199b-3p on SOX6. Results The expression of miR-199b-3p in the inhibitor group was 0.526 ± 0.034 at 48 h after transfection, which was lower than that of the untransfected group (1.009 ± 0.064) and 0.960 ± 0.057 (NC group), and the difference was statistically significant (P <0.05). Compared with the other two groups, the proliferation activity of SW620 cells in the inhibitor group was significantly lower at 24, 48 and 72 h after transfection (P <0.05), and the apoptosis rate in the inhibitor group was (37.533 ± 1.459)%, higher than that of untransfected group (6.101 ± 0.663)% and NC group (8.753 ± 1.061)% respectively, the difference was statistically significant (P <0.05); inhibitor group of SW620 cells SOX6, Bax, The mRNA and protein expressions of caspase-3 were significantly higher than those of untransfected and NC groups (P <0.05). Dual luciferase reporter assay showed that miR-199b-3p significantly inhibited the luciferase activity of wild-type SOX6-3’UTR, but had no effect on the luciferase activity of the mutant plasmid transfected cells. Conclusion miR-199b-3p can regulate the expression of SOX6, and down-regulating the expression of miR-199b-3p can inhibit the proliferation and induce the apoptosis of SW620 cells, providing a potential target of molecular therapy for the treatment of colon cancer.