摘要
目的:探讨miR-30a-5p靶向三结构域蛋白31(TRIM31)基因对结直肠癌(CRC)细胞5-氟尿嘧啶(5-FU)耐药的影响,并阐明其作用机制。方法:收集27例5-FU化疗敏感(5-FU/S组)CRC患者和23例5-FU化疗耐药(5-FU/R组)CRC癌患者的CRC组织,实时荧光定量PCR(RT-qPCR)法检测癌组织中miR-30a-5p和TRIM31 mRNA表达水平,Pearson相关分析法分析miR-30a-5p和TRIM31 mRNA表达的相关性。体外培养人CRC 5-FU耐药细胞HT-29/5-FU细胞及其亲本HT-29细胞,MTT法检测2种细胞增殖活性并计算半数抑制浓度(IC50)和耐药指数(RI),RT-qPCR法检测2种细胞中miR-30a-5p和TRIM31 mRNA表达水平,Western blotting法检测2种细胞中TRIM31蛋白表达水平。将HT-29/5-FU细胞分为空白对照组、mimics NC组(转染miR-30a-5p阴性对照mimics NC)、miR-30a-5p mimics组(转染miR-30a-5p mimics)、miR-30a-5p mimics+vector组(转染miR-30a-5p mimics和阴性对照空载体)和miR-30a-5p mimics+TRIM31组(转染miR-30a-5p mimics和TRIM31过表达载体)。RT-qPCR法检测各组HT-29/5-FU细胞中miR-30a-5p和RTIM31 mRNA表达水平,Western blotting法检测各组HT-29/5-FU细胞中TRIM31蛋白表达水平,MTT法检测各组HT-29/5-FU细胞增殖活性,流式细胞术检测各组HT-29/5-FU细胞凋亡率,双荧光素酶报告基因系统验证miR-30a-5p与TRIM31的靶向关系。结果:与5-FU/S组比较,5-FU/R组患者CRC组织中miR-30a-5p表达水平降低(P<0.01),TRIM31 mRNA表达水平升高(P<0.01),miR-30a-5p和TRIM31 mRNA表达水平呈负相关关系(R2=0.885,P<0.01)。与HT-29细胞比较,HT-29/5-FU细胞中miR-30a-5p表达水平降低(P<0.01),TRIM31 mRNA和蛋白表达水平升高(P<0.01)。HT-29/5-FU细胞和HT-29细胞的IC50值分别为(104.41±0.22)和(9.82±0.31)mg·L^(-1),RI值为12.42。与空白对照组和mimics NC组比较,miR-30a-5p mimics组HT-29/5-FU细胞中miR-30a-5p表达水平升高(P<0.01),TRIM31蛋白表达水平降低(P<0.01),HT-29/5-FU细胞增殖活性明显降低(P<0.01),HT-29/5-FU细胞凋亡率明显升高(P<0.01)。与miR-30a-5p mimics组比较,miR-30a-5p mimics+TRIM31组HT-29/5-FU细胞中TRIM31蛋白表达水平升高(P<0.01),HT-29/5-FU细胞增殖活性明显升高(P<0.01),HT-29/5-FU细胞凋亡率明显降低(P<0.01)。双荧光素酶报告基因系统证实TRIM31是miR-30a-5p的靶基因。结论:miR-30a-5p通过靶向TRIM31基因表达增强CRC耐药细胞对5-FU的敏感性。
Objective:To investigate the effect of miR-30a-5p on 5-fluorouracil(5-FU)resistant of colorectal cancer(CRC)cells by targeting tripartite motif-containing protein 31(TRIM31)gene,and to clarify its mechanism.Methods:The CRC tissue of 27 patients with 5-FU chemotherapy-sensitive colorectal cancer(5-FU/S group)and 23 patients with 5-FU chemotherapy-resistant colorectal cancer(5-FU/R group)were collected.The expression levels of miR-30a-5p and TRIM31 mRNA in CRC tissue of the patients were detected by Real-time fluorescence quantitative PCR(RT-qPCR)method;the correlation between miR-30a-5p and TRIM31 mRNA expressions was analyzed by Pearson correlation analysis.The human CRC 5-FU resistant cells HT-29/5-FU cells and its parent HT-29 cells were cultured in vitro.The proliferation activities of two kinds of cells were detected by MTT assay,and the median inhibitory concentration(IC50)and drug resistance index(RI)were calculated;the expression levels of miR-30a-5p and TRIM31 mRNA in two kinds of cells were detected by RT-qPCR method;the expression levels of TRIM31 protein in two kinds of cells were detected by Western blotting method.The HT-29/5-FU cells were further divided into blank control group,mimics NC group,miR-30a-5p mimics group,miR-30a-5p mimics+vector group and miR-30a-5p mimics+TRIM31 group.The cells in blank control group were not transfected,the cells in miR-30a-5p mimics group were transfected with miR-30a-5p mimics,the cells in miR-30a-5p mimics+vector group were transfected with miR-30a-5p mimics and negative control of vector,and the cells in miR-30a-5p mimics+TRIM31 group were transfected with miR-30a-5p mimics and TRIM31 over-expression vector.The expression levels of miR-30a-5p and TRIM31mRNA in the HT-29/5-FU cells in various groups were detected by RT-qPCR method;the expression levels of TRIM31 protein in the HT-29/5-FU cells in various groups were detected by Western blotting method;the proliferation activities of the HT-29/5-FU cells in various groups were detected by MTT method;the apoptotic rates of the HT-29/5-FU cells in various groups were detected by flow cytometry;the targeted relationship between miR-30a-5p and TRIM31 was verified by dual-luciferase reporter gene system.Results:Compared with 5-FU/S group,the expression level of miR-30a-5p in cancer tissue of the patients in 5-FU/R group was decreased(P<0.01),and the expression level of TRIM31 mRNA was increased(P<0.01).The expression levels of miR-30a-5p and TRIM31 mRNA were negatively correlated(R2=0.885,P<0.01).Compared with HT-29 cells,the expression level of miR-30a-5p in the HT-29/5-FU cells was decreased(P<0.01),and the expression levels of TRIM31 mRNA and protein were increased(P<0.01).The IC50values of HT-29/5-FU and HT-29 cells were(104.41±0.22)and(9.82±0.31)mg·L^(-1),respectively,and the RI value was 12.42.Compared with blank control group or mimics NC group,the expression level of miR-30a-5p in the HT-29/5-FU cells in miR-30a-5p mimics group was increased(P<0.01),the expression level of TRIM31 protein was decreased(P<0.01),the proliferation activity of HT-29/5-FU cells was significantly decreased(P<0.01),and the apoptotic rate of HT-29/5-FU cells was significantly increased(P<0.01).Compared with miR-30a-5p mimics group,the expression level of TRIM31 protein in the HT-29/5-FU cells in imiR-30a-5p mimics+TRIM31 group was increased(P<0.01),the proliferation activity of HT-29/5-FU cells was significantly increased(P<0.01),and the apoptotic rate of HT-29/5-FU cells was significantly decreased(P<0.01).The dual-luciferase reporter system results confirmed that TRIM31 was the target gene of miR-30a-5p.Conclusion:Over-expression of miR-30a-5p enhances the sensitivity of the CRC drug-resistance cells to 5-FU by targeting the TRIM31 gene expression.
作者
卢瑞云
谷敬锋
张建
张新
徐菲
LU Ruiyun;GU Jingfeng;ZHANG Jian;ZHANG Xin;XU Fei(Department of Gastrointestinal Surgery,First Hospital,Hebei Medical University,Shijiazhuang 050039,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2021年第3期714-723,共10页
Journal of Jilin University：Medicine Edition
基金
河北省中医药管理局科研计划项目(2017179)。
