MiR-142-5p通过靶向IGF2BP3调控多柔比星诱导的肝癌细胞凋亡  

MiR-142-5p promotes hepatocellular carcinoma cell apoptosis induced by dexorubicin with targetting IGF2BP3

作　　者：胡烨[1] 许小青[1] 王冰晶[1] 徐贺楠[1] 侯晋[2] 罗晓玲[3] 梁安民[3] 曹雪涛[1,2] 

机构地区：[1]中国医学科学院北京协和医学院基础医学研究所,北京100006 [2]第二军医大学免疫学研究所暨医学免疫学国家重点实验室,上海200433 [3]广西医科大学附属肿瘤医院,广西南宁530021

出　　处：《中国肿瘤生物治疗杂志》2017年第6期581-587,共7页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.31390431);国家重点基础研究发展规划(973计划)资助项目(No.2013CB530503)~~

摘　　要：目的:探讨miR-142-5p对多柔比星诱导的原发性肝细胞癌(hepatocellular carcinoma,HCC)细胞凋亡的影响及其作用机制。方法:收集广西医科大学附属肿瘤医院88例HCC患者手术切除的癌组织及癌旁组织(距癌灶组织边缘2~5 cm)标本。采用实时荧光定量PCR检测人HCC组织和癌旁组织、人正常肝细胞以及HCC细胞系中miR-142-5p的表达量。向HCC细胞SMMC-7721中转染miR-142-5p mimics,流式细胞术检测过表达miR-142-5p后SMMC-7721细胞在多柔比星(doxorubicin)(1μg/ml)诱导下凋亡的变化;生物信息学方法预测miR-142-5p可靶向结合胰岛素样生长因子2 mRNA结合蛋白3(insulin-like growth factor 2 mRNA-binding protein 3,IGF2BP3)基因,并采用荧光素酶报告基因实验进行验证。采用实时荧光定量PCR及Western blotting检测过表达miR-142-5p的SMMC-7721细胞中IGF2BP3的mRNA及蛋白表达情况。结果:与癌旁组织和正常肝细胞相比,HCC组织(-6.91±2.61 vs-11.59±2.59,P<0.01)和多种HCC细胞系中miR-142-5p呈明显低表达(均P<0.01);过表达miR-142-5p可显著促进多柔比星诱导的HCC细胞SMMC-7721的凋亡[(49.40±3.47)%vs(19.50±1.74)%,P<0.01];过表达miR-142-5p可明显降低HCC细胞中IGF2BP3的mRNA及蛋白表达水平(P<0.01),敲减IGF2BP3表达可进一步促进多柔比星诱导的SMMC-7721细胞的凋亡(P<0.01)。荧光素酶报告基因实验结果显示,miR-142-5p能够抑制IGF2BP3的3'UTR荧光素酶报告基因的活性。结论:miR-142-5p在HCC组织标本和体外培养细胞系中的表达水平均显著降低,转染miR-142-5p mimics后能够促进多柔比星诱导的HCC细胞的凋亡,其机制可能与miR-142-5p靶向作用IGF2BP3从而促进HCC细胞凋亡有关。Objective：To investigate the function and molecular mechanisms of micro RNA 142 5p （ miR 142 5p） on the doxorubicin induced apoptosis of primary hepatocellular carcinoma （HCC）.Methods： Paired HCC tissues and adjacent non cancerous tissue specimens （2 5cm away from the lesion） were surgically collected from 88 patients who were diagnosed with primary HCC in Tumor Hospital Affiliated to Guangxi Medical University between October 2001 and July 2005. qRT PCR was used to detect the miR 142 5p expressions in those HCC tissues, adjacent non cancerous tissues, normal hepatocellular cell line and HCC cell lines. HCC SMMC 7721 cells were transfected with miR 142 5p mimics, and flow cytometry was used to determine the changes in doxorubicin （1 μg/ml） induced apoptosis of SMMC 7721 cells. Bioinformatics predicted that miR 142 5p could bind with insulin like growth factor 2 Mrna binding protein 3 （IGF2BP3）, which was then validated by luciferase reporter gene assay. qRT PCR and Western blotting were separately used to detect the mRNA and protein expressions of IGF2BP3 in SMMC 7721 cells that over express miR 142 5p. Results： Compared with the adjacent non tumor tissues, miR 142 5p was significantly lower in HCC tissues （-6.91±261 vs -11.59±259, P〈0.01） and this decrease was also found in HCC cell line compared with normal human liver cells （P〈0.01）; over expression of miR 142 5p significantly promoted the doxorubicin induced apoptosis of HCC SMMC 7721 cells （／[49.4±3.47／]% vs ／[19.50±1.74／]%, P〈0.01）. miR 142 5p over expression could obviously inhibit the mRNA and protein expressions of IGF2BP3 in SMMC 7721 cells. Luciferase reporter gene assay also showed that miR 142 5p over expression could inhibit the luciferase activity of 3′UTR of IGF2BP3. Conclusion： miR 142 5p expression significantly decreased in both HCC tissues and HCC cell lines. miR 142 5p mimics transfection promoted doxorubicin induced apoptosis of

关 键 词：肝细胞癌 miR-142-5p 多柔比星 凋亡 胰岛素样生长因子2 mRNA结合蛋白3基因 

分 类 号：R735.7[医药卫生—肿瘤] R730.54[医药卫生—临床医学]