miR-199对结直肠癌细胞增殖、侵袭的影响及机制

Effects and mechanism of microRNA 199 on proliferation and invasion of colorectal cancer cells

目的探讨微小RNA-199(miR-199)对结直肠癌细胞增殖、侵袭的影响及其作用机制。方法常规培养人正常结肠上皮细胞系(HCoEpiC细胞)、结直肠癌细胞系(LOVE、SW620、SW480、HT29、DLD-1细胞),用实时定量PCR法检测细胞中miR-199表达。将SW620细胞随机分为miR-199过表达组、阴性对照组及miR-199+转铁蛋白受体1(TFR1)共表达组(miR-199mimics+TFR1组),分别将miR-199mimics、mimic-NC、miR-199mimics+TFR1过表达质粒转染至细胞内;另取部分未转染细胞作为空白对照组。实时荧光定量PCR法检测TFR1 mRNA表达,MTT实验检测细胞增殖能力,Transwell侵袭实验检测细胞侵袭能力,Western blotting法检测TFR1蛋白表达,生物学软件TargetScan及双荧光素酶报告基因实验分析miR-199的靶基因。结果人结直肠癌细胞系中miR-199相对表达量均低于人结肠上皮细胞系(P均<0.05),且SW620细胞中miR-199相对表达量最低,故选SW620细胞为实验细胞。miR-199过表达组miR-199相对表达量高于阴性对照组、空白对照组;转染48、72 h,miR-199过表达组A_(490)值低于阴性对照组、空白对照组(P均<0.05);miR-199过表达组侵袭细胞数少于阴性对照组、空白对照组(P均<0.05)。TFR1为miR-199潜在的预测靶基因,TFR1存在与miR-199结合的位点,TFR1 mRNA的3'UTR中存在与miR-199互补的序列。空白对照组、miR-199过表达组野生型3'UTR的TFR1萤光素酶活性比较差异有统计学意义(P<0.05),突变型3'UTR的TFR1萤光素酶活性比较差异无统计学意义(P>0.05)。miR-199过表达组TFR1 mRNA和蛋白表达均低于空白对照组、阴性对照组(P均<0.05)。miR-199过表达组A_(490)值低于阴性对照组、空白对照组、miR-199mimics+TFR1组(P均<0.05),侵袭细胞数少于阴性对照组、空白对照组、miR-199mimics+TFR1组(P均<0.05)。结论miR-199可抑制结直肠癌细胞增殖、侵袭,其机制可能与靶向下调TFR1表达有关。

Objective To explore the effects of microRNA-199(miR-199)on the proliferation and invasion of colorectal cancer cells,and to explore the mechanism of action.Methods Human normal colon epithelial cell line HCo EpiC and colorectal cancer cell lines(LOVE,SW620,SW480,HT29,and DLD-1)were routinely cultured.The miR-199 expression in cells was detected by using real-time quantitative PCR.SW620 cells were selected as the experimental cells.Cells were randomly divided into the miR-199 over-expression group,negative control group,and miR-199+TFR1 co-expression group(miR-199mimics+TFR1 group).The miR-199 mimics,mimic-NC,and miR-199mimics+TFR1 over-expression plasmids were transfected into the cells in the above groups by using LipofectamineTM 3000 transfection reagent.Some non-transfected cells were taken as the blank control groups.Real-time fluorescence quantitative PCR was used to detect TFR1 mRNA expression.MTT assay was used to detect the cell proliferation ability.Transwell invasion assay was used to detect cell invasion ability.Western blotting was used to detect TFR1 protein expression.Biological software TargetScan and dual luciferase reporter gene assay were used to analyze the target genes of miR-199.Results The relative expression level of miR-199 in human colorectal cancer cell lines was lower than that in human colon epithelial cell lines(P<0.05),and the relative expression level of miR-199 was the lowest in SW620 cells.Therefore,SW620 cells were selected as the experimental cells.The relative expression level of miR-199 was higher in the miR-199 over-expres sion group than in the negative control group and blank control group.After 48 and 72 h of transfection,the A_(490) values of the miR-199 over-expression group were lower than those in the negative control group and blank control group(all P<0.05).The number of invasive cells was smaller in the miR-199 over-expression group than in the negative control group and blank control group(both P<0.05).TFR1 was a potential predictive target gene for miR-199.TFR1 existed at the binding site of miR-199,and there were complementary sequences with miR-199 in the 3'UTR of TFR1 mRNA.There was a statistically significant difference in TFR1 luciferase activity between the blank control group and the miR-199 over-expression group in wild-type 3'UTR(P<0.05),while there was no statistically significant difference in TFR1 luciferase activity of mutant 3'UTR(P>0.05).The expression levels of TFR1 mRNA and protein in the miR-199 over-expression group were lower than those in the blank control group and negative control group(all P<0.05).The A_(490) value was lower in the miR-199 over-expression group than in the negative control group,blank control group,and miR-199 mimics+TFR1 group(all P<0.05).And the number of invasive cells was smaller than those of the negative control group,blank control group,and miR-199 mimics+TFR1 group(all P<0.05).Conclusion MiR-199 inhibits the proliferation and invasion of colorectal cancer cells,and the mechanism may be related to the targeted down-regulation of TFR1.