柚皮苷中药单体调节miR-216a基因对结直肠癌细胞增殖和凋亡的影响研究

Effects of naringin on the proliferation and apoptosis of colorectal cancer cells by regulating miR-216a gene in vitro

目的探讨柚皮苷中药单体靶向调节miR-216a基因对结直肠癌细胞增殖、凋亡的影响及机制。方法qRT-PCR检测三株结直肠癌细胞(SW620、LOVO和HCT116)miR-216a和KIAA1199基因mRNA表达,Western blotting检测KIAA1199蛋白表达。双荧光素酶报告基因实验检测KIAA1199-WT/MUT与miR-216a mimics共转染后的荧光素酶活性,qRT-PCR及Western blotting检测miR-216a mimics/inhibitor转染SW620细胞后KIAA1199基因mRNA及蛋白表达。MTT、流式细胞术分别检测miR-216a、靶向KIAA1199及柚皮苷(0.4 mmol/L、0.8 mmol/L和1.6 mmol/L)对SW620细胞活力、凋亡的影响。qRT-PCR检测柚皮苷对miR-216a基因和KIAA1199基因表达的影响,Western blotting检测柚皮苷对KIAA1199蛋白表达的影响。结果三株结直肠癌细胞miR-216a基因表达明显降低,KIAA1199基因mRNA及蛋白表达均明显升高(P<0.05)。选择SW620细胞作为研究对象。过表达miR-216a可明显抑制SW620细胞活力,促进细胞凋亡(P<0.05),同时过表达miR-216a和KIAA1199可减弱过表达miR-216a对SW620细胞活力和凋亡的影响。不同浓度柚皮苷均可抑制SW620细胞活力,促进细胞凋亡,上调miR-216a基因表达,下调KIAA1199基因表达,呈剂量依赖性(P<0.05)。结论柚皮苷中药单体可引起miR-216a表达水平改变进而调控KIAA1199表达,从而影响结直肠癌细胞增殖和凋亡。

Objective To investigate the effects of naringin on the proliferation and apoptosis of colorectal cancer cells by regulating miR-216a gene in vitro,and ti explore its action mechanism.Methods The expression levels of mRNA expression levels of miR-216a and KIAA1199 in three colorectal cancer cells(SW620,LoVo and HCT116)were detected by qRT-PCR,and the expression levels of KIAA1199 protein were detected by Western Blot.Double luciferase reporter gene assay was used to detect luciferase activity after CO transfection of KIAA1199-WT/MUT and miR-216a mimics.And qRT-PCR and Western blotting were used to detect the expression levels of KIAA1199 gene mRNA and protein after miR-216a mimics/inhibitor were transfected into SW620 cells.MTT and flow cytometry were used to detect the effects of miR-216a targeting KIAA1199 and naringin on the activity and apoptosis of SW620 cells.Moreover qRT-PCR was used to detect the effects of naringin on miR-216a gene and KIAA1199 gene expression,and Western Blot was used to detect the effects of naringin on KIAA1199 protein expression.Results The expression levels of miR-216a gene in three colorectal cancer cells were significantly decreased,however,the expression levels of KIAA1199 gene mRNA and protein were significantly increased(P<0.05).The overexpression of miR-216a could significantly inhibit the activity of SW620 cells and promote cell apoptosis(P<0.05),while overexpression of miR-216a and KIAA1199 could decrease the effects of overexpression of miR-216a on the activity and apoptosis of SW620 cells.In addition different concentrations of naringin could inhibit the activity of SW620 cells,promote cell apoptosis,up regulate miR-216a gene expression,down regulate KIAA1199 gene expression in a dose-dependent manner(P<0.05).Conclusion Naringin can change the expression levels of miR-216a and regulate the expression of KIAA1199,thus affecting the proliferation and apoptosis of colorectal cancer cells.