微小分子miR-125b新靶基因的鉴定及其功能初步研究  

Identification and Functional Study of a New Target Gene of miR-125b

作　　者：王海洋[1,2] 曾泉[1,2] 张彪[1,2] 李思霆[1,2] 南雪[1,2] 何丽娟[1,2] 岳文[1,2] 周军年[1,2] 裴雪涛[1,2] 

机构地区：[1]军事医学科学院野战输血研究所干细胞与再生医学研究室,北京100850 [2]军事医学科学院华南干细胞与再生医学研究中心,广东广州510005

出　　处：《生物技术通讯》2016年第2期147-152,共6页Letters in Biotechnology

基　　金：国家自然科学基金青年科学基金(31301199);广州市健康医疗协同创新重大专项(201400000003-1)

摘　　要：目的:利用生物信息学方法预测miR-125b的新靶基因,在肝癌细胞系中进行验证和结合位点的鉴定,为阐明miR-125b在肝癌发生发展中的作用和机制提供新线索。方法:Western印迹分析在肝癌细胞中过表达miR-125b后上皮-间充质转化(EMT)相关蛋白的表达情况;对肝癌患者肿瘤组织及癌旁组织的miR-125b表达差异进行实时定量PCR检测,同时对SNAIL1的表达情况进行免疫组化检测;用生物信息学方法预测miR-125b的新靶基因;利用双萤光素酶报告系统验证miR-125b在预测的新靶基因mRNA的3'非翻译区是否有直接结合位点;构建新靶基因的真核过表达载体,检测新靶基因过表达后对miR-125b表达水平的影响。结果:在肝癌细胞中过表达miR-125b可抑制细胞EMT过程,下调α平滑肌肌动蛋白、神经钙黏素的表达;miR-125b在肝癌患者肿瘤组织中的表达远低于癌旁组织,而SNAIL1作为调控EMT的主要转录因子之一,其在肿瘤组织中的入核比例明显高于癌旁组织;用TargetScan预测到snail1是miR-125b的潜在靶点之一,但双萤光素酶实验表明snail1并非miR-125b的直接靶基因。此外,我们意外发现在肝癌细胞中过表达SNAIL1可上调miR-125b的表达。结论:miR-125b可抑制肝癌EMT过程,但并非直接通过靶向snail1发挥作用,两者在肝癌细胞中存在着复杂的间接调控关系。Objective： Predict and identify a new target gene of mi R-125 b, to provide new clues for exploringthe role and mechanism of mi R- 125 b in hepatocellular carcinogenesis. Methods： Using Western blot techniquesto test typical epithelial mesenchymal transition（EMT） related markers after mi R-125 b overexpression. The expres-sion level of mi R-125 b was detected in paired hepatocellular carcinoma（HCC） specimens by q RT-PCR, and theSNAIL1 expression pattern was found by immunohistochemistry（IHC）. Predicting binding sites of mi R-125 b by bio-informatic analysis. Performing luciferase reporter assay to confirm the direct combination of mi R- 125 b and thenew predicted target gene. Testing the effects of predicted target gene overexpression on mi R-125 b expression lev-el. Results： The expression of α-sooth muscle actin（α-SMA）, N-cadherin were significantly down-regulated afteroverexpression of mi R-125 b. The expression level of mi R-125 b was lower but the percentage of nuclear localizedSNAIL1 was higher in HCC tissues compared with corresponding adjacent nontumorous tissues. Bioinformatic analy-sis predicted snail1, a vital regulator of EMT, as a potential target of mi R-125 b. However, the luciferase reporterassay showed snail1 was not a direct target of mi R-125 b. Interestingly, we found that SNAIL1 overexpressing up-regulates mi R- 125 b expression level in HCC cells Conclusion： mi R- 125 b acts as an inhibitor of EMT. Theremay exist a complicated and indirect interaction between mi R-125 b and snail1.

关 键 词：miR-125b SNAIL1 肝细胞癌 

分 类 号：Q78[生物学—分子生物学] Q25