miR-150靶向调控c-FLIP对结肠癌HT-29细胞自噬影响的实验研究

目的探讨上调结肠癌细胞miR-150表达对自噬的影响以及可能调控机制。方法体外培养HT-29细胞,转染miR-150 mimics或细胞型Fas相关死亡域样白介素-1β转换酶抑制蛋白(c-FLIP)基因,分为空白对照组、阴性对照组、miR-150 mimics组和miR-150 mimics+c-FLIP组,实时荧光定量PCR(q PCR)鉴定转染效果。q PCR检测各组miR-150和cFLIP mRNA表达;Western blotting检测各组自噬相关蛋白微管相关蛋白1轻链3(LC3-Ⅱ)、Beclin 1以及c-FLIP蛋白表达。采用细胞免疫荧光染色观察各组细胞自噬的情况。采用双荧光素酶报告实验验证miR-150与c-FLIP的靶向关系。结果 miR-150 mimics组和miR-150 mimics+c-FLIP组miR-150表达水平分别为2. 25±0. 13和2. 19±0. 16,均高于空白对照组的1. 00±0. 09和阴性对照组的1. 02±0. 13,差异有统计学意义(P<0. 05)。miR-150 mimics组c-FLIP mRNA和蛋白表达水平明显低于空白对照组和阴性对照组,差异有统计学意义(P<0. 05);而miR-150 mimics+c-FLIP组c-FLIP mRNA和蛋白表达水平均高于空白对照组、阴性对照组和miR-150 mimics组,差异有统计学意义(P<0. 05)。miR-150 mimics组细胞Beclin 1、LC3-Ⅱ蛋白表达量明显高于空白对照组和阴性对照组;miR-150 mimics+c-FLIP组细胞Beclin 1、LC3-Ⅱ蛋白表达量低于空白对照组、阴性对照组和miR-150 mimics组。miR-150 mimics组自噬细胞阳性率为(52. 92±6. 63)%,高于空白对照组的(33. 54±5. 40)%和阴性对照组的(31. 05±4. 97)%,差异有统计学意义(P<0. 05);miR-150 mimics+c-FLIP组自噬细胞阳性率为(22. 50±4. 38)%,低于空白对照组、阴性对照组、miR-150 mimics组。双荧光素酶报告基因实验证实c-FLIP是miR-150的下游靶基因。结论上调miR-150表达可通过负性调控c-FLIP提高结肠癌细胞HT-29的自噬活性。

Experimental study on the effect of miR-150 on autophagy in colon cancer HT-29 cells by targeting the regulation of c-FLIP

Objective To investigate the effect of upregulation of miR-150 expression in colon cancer cells on autophagy and its possible regulatory mechanisms.Methods HT-29 cells were cultured in vitro and transfected with miR-150 mimics or cell-type fas-associated death field-like interleukin-1(c-FLIP)genes.These cells were divided into blank control group,negative control group,miR-150 mimics group and miR-150 mimics+c-FLIP group.Real-time fluorescence quantitative PCR(qPCR)was used to identify the transfection effect.The mRNA expressions of miR-150 and c-FLIP in each group were detected by qPCR.Western blotting test groups of autophagy related protein microtubule associated protein 1 light chain 3(LC3-Ⅱ),Beclin 1 and c-FLIP protein expression.The autophagy of each group was observed by immunofluorescence staining.The dual luciferase reporting experiment was used to verify the targeting relationship between miR-150 and c-FLIP.Results The expression of miR-150 in the miR-150 mimics group and the miR-150 mimics+c-FLIP group were 2.25±0.13 and 2.19±0.16,respectively,which were both higher than those in the blank control group and the negative control group,and the differences were statistically significant(P<0.05).The mRNA and protein expression levels of c-FLIP in the miR-150 mimics group were significantly lower than those in the blank control group and the negative control group,and the difference was statistically significant(P<0.05).The mRNA and protein expression levels of c-FLIP in the miR-150 mimics+c-FLIP group were higher than those in the blank control group,the negative control group and the miR-150 mimics group,and the difference was statistically significant(P<0.05).The expression of Beclin 1,LC3-Ⅱproteins in miR-150 mimics group were higher than these in blank control group and NC group(P<0.05);the expression of Beclin 1,LC3-Ⅱproteins in miR-150 mimics+c-FLIP group was lower than these in blank control group,NC group and miR-150 mimics group.The positive rate of autophagy in the miR-150 mimics group was(52.92±6.63)%,higher than that in the blank control group and the negative control group,the difference was statistically significant(P<0.05).The positive rate of autophagy in the miR-150 mimics+c-FLIP group was lower than that in the blank control group,the negative control group and the miR-150 mimics group.The double luciferase reporter gene experiment confirmed that c-FLIP was the downstream target gene of miR-150.Conclusion Up-regulation of miR-150 expression negatively regulated the autophagy activity of HT-29 in colon cancer cells by c-FLIP.