长链非编码RNA NEAT1通过调节hnRNP A2蛋白表达促进人肝癌细胞增殖和侵袭的研究

发布时间：2018-02-07 11:49

本文关键词： 长链非编码RNA NEAT1 hnRNP A2 RNA结合蛋白人肝细胞癌　出处：《昆明医科大学》2017年博士论文　论文类型：学位论文

【摘要】：[目的]在人肝细胞癌(Hepatocellular carcinoma,HCC)标本中检测癌和癌旁长链非编码 RNA nuclear enriched abundant transcript 1,NEAT1 的表达差异。应用化学合成小干扰RNA(small interfering RNA,si-RNA)抑制两组人肝癌细胞系(HepG2,SMMC-7721)NEAT1的转录,进一步研究NEAT1转录降低后对HCC细胞的表达谱、侵袭和增殖功能的影响。通过RNA免疫共沉淀寻找长链非编码RNANEAT1相关的RNA结合蛋白,并研究这些RNA结合蛋白参与NEAT1调节肝癌相关基因 heterogeneous nuclear ribonucleoprotein A2(hnRNPA2)表达的分子机制,阐述NEAT1调节hnRNPA2的表达对人肝细胞癌的进展的影响,为NEAT1作为分子靶点应用于人肝细胞癌的诊治提供实验依据。[方法]利用Trizol-氯仿提取HCC组织标本癌和癌旁组织,细胞系(HepG2,SMMC-7721)中的 RNA,RT-qPCR 检测各组 NEAT1 表达差异(GAPDH作为内参),使用转染试剂Lipofectamine 2000和siRNANEATl对HepG2和SMMC-7721细胞进行NEAT1敲低,RT-qPCR检测NEAT1敲低效果,并对NEAT1敲低组和对照组细胞进行表达谱测序,检测NEAT1敲低后对HCC细胞各肿瘤相关基因表达的影响,对NEAT1敲低组和对照组细胞进行CFSE标记的细胞增殖检测、CCK8染色的细胞增殖检测、Transwell细胞侵袭移动能力检测来探究NEAT1对HCC细胞增殖和侵袭功能的影响。查询Starbase 2.0数据,寻找与NEAT1相互作用密切的RNA.结合蛋白,通过RNA免疫共沉淀明确能同时与NEAT1和NEAT1所调控基因mRNA相互作用的RNA结合蛋白,RNA pull-down明确RNA结合蛋白与NEAT1作用靶点片段,使用转染试剂Lipofectamine 2000和 siRNANEAT1 对 HepG2 细胞进行 NEAT1 敲低,Western-Blotting,免疫组化验证NEAT1对hnRNP A2蛋白的调节,使用逆转录病毒retroviral vectors作为载体建立hnRNPA2过表达模型并应用于NEAT1低表达的HepG2细胞模型中,CCK8增殖实验和Transwell小室研究NEAT1低表达的HepG2细胞过表达hnRNPA2后与对照组的增殖、侵袭功能。[结果]长链非编码RNANEAT1在HCC细胞和癌组织中表达量高于癌旁组织(p0.001)。敲低HCC细胞系NEAT1表达导致很多参与肿瘤发生、发展通路的基因表达量改变(229个基因表达上调,148个基因表达下调,表达量改变超过2倍且p0.05),敲低HCC细胞系NEAT1表达降低了 HCC细胞体外增殖(p0.01)、侵袭(p0.001)的能力。U2AF65蛋白能够与NEAT1和hnRNP A2 mRNA相互作用(富集度分别为IgG的82.659倍和53.527倍),NEAT1敲低降低了 hnRNPA2mRNA转录和蛋白表达(p0.001),这种调控作用可能与NEAT1-U2AF65复合物相关,hnRNPA2过表达可以逆转NEAT1低表达引起的HepG2细胞增殖和侵袭抑制作用(p0.01)。[结论]NEAT1通过调节hnRNPA2蛋白影响了 HCC细胞的增殖和侵袭能力,这一调节机制可能是人肝细胞癌诊断和治疗的新靶点。
[Abstract]:[objective] to detect the difference of the expression of RNA nuclear enriched abundant transcript 1n (NEAT1) between human hepatocellular carcinoma (HCC) and human hepatocellular carcinoma (HCC) specimens. The chemically synthesized small interfering RNA(small interfering RNAsi-RNA) was used to inhibit the transcription of human hepatocellular carcinoma cell line HepG2SMMC-7721NEAT1. To further study the effect of NEAT1 transcription reduction on the expression profile, invasion and proliferation of HCC cells. Long chain RNA binding proteins associated with RNANEAT1 were identified by RNA immunoprecipitation. To study the molecular mechanism of these RNA binding proteins involved in NEAT1 regulating the expression of heterogeneous nuclear ribonucleoprotein A2hnRNPA2, and to elucidate the effect of NEAT1 on the progression of human hepatocellular carcinoma. To provide experimental basis for the application of NEAT1 as a molecular target in the diagnosis and treatment of human hepatocellular carcinoma. [methods] Trizol-chloroform was used to extract HCC tissue samples from cancer and adjacent tissues. NEAT1 expression difference was detected by RT-qPCR in HepG2SMMC-7721 cell line. Lipofectamine 2000 and siRNANEATl were used to detect the NEAT1 knockdown effect on HepG2 and SMMC-7721 cells by NEAT1 knockdown qPCR, and the expression profiles of NEAT1 knockout group and control group were sequenced. To detect the effect of NEAT1 knockout on the expression of tumor-related genes in HCC cells. The cell proliferation of NEAT1 knockout group and control group was detected by CFSE labeled cell proliferation assay with CCK8 staining to explore the effect of NEAT1 on the proliferation and invasion function of HCC cells, and to inquire about the data of Starbase 2.0, so as to explore the effect of NEAT1 on the proliferation and invasion function of HCC cells. NEAT1 binding proteins were identified by RNA immunoprecipitation. RNA binding proteins, which interact with mRNA genes regulated by NEAT1 and NEAT1 at the same time, were identified to identify RNA binding protein and NEAT1 binding target fragments by RNA immunoprecipitation. NEAT1 knockout Western-Blottingwas performed on HepG2 cells using transfection reagents Lipofectamine 2000 and siRNANEAT1. Immunohistochemistry was used to verify the regulation of NEAT1 on hnRNP A2 protein. HnRNPA2 overexpression model was established by using retrovirus retroviral vectors as vector, and was used in HepG2 cell model with low expression of NEAT1 and Transwell chamber to study the proliferation of HepG2 cells with low NEAT1 expression after over-expression of hnRNPA2 and control group. Invasive function. [results] the expression of long chain noncoding RNANEAT1 in HCC cells and cancer tissues was higher than that in adjacent tissues. Low expression of NEAT1 in HCC cells resulted in many tumorigenesis. The amount of gene expression in the development pathway was changed (229 genes were up-regulated, 148 genes were down-regulated. The expression of U2AF65 protein could interact with NEAT1 and hnRNP A2 mRNA (enrichment was 82.659 times of IgG and 53.527 times of IgG). HnRNPA2mRNA transcription and protein expression were reduced, which may be related to the overexpression of NEAT1-U2AF65 complex, which could reverse the proliferation and invasion inhibition of HepG2 cells induced by low expression of NEAT1. [conclusion] NEAT1 may influence the expression of hnRNPA2 by regulating hnRNPA2 protein. Proliferation and invasion of HCC cells, This regulatory mechanism may be a new target for the diagnosis and treatment of human hepatocellular carcinoma.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

【参考文献】