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  本文關(guān)鍵詞： 長鏈非編碼RNA NEAT1 hnRNP A2 RNA結(jié)合蛋白 人肝細(xì)胞癌　出處：《昆明醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：[目的]在人肝細(xì)胞癌(Hepatocellular carcinoma,HCC)標(biāo)本中檢測癌和癌旁長鏈非編碼 RNA nuclear enriched abundant transcript 1,NEAT1 的表達(dá)差異。應(yīng)用化學(xué)合成小干擾RNA(small interfering RNA,si-RNA)抑制兩組人肝癌細(xì)胞系(HepG2,SMMC-7721)NEAT1的轉(zhuǎn)錄,進(jìn)一步研究NEAT1轉(zhuǎn)錄降低后對HCC細(xì)胞的表達(dá)譜、侵襲和增殖功能的影響。通過RNA免疫共沉淀尋找長鏈非編碼RNANEAT1相關(guān)的RNA結(jié)合蛋白,并研究這些RNA結(jié)合蛋白參與NEAT1調(diào)節(jié)肝癌相關(guān)基因 heterogeneous nuclear ribonucleoprotein A2(hnRNPA2)表達(dá)的分子機(jī)制,闡述NEAT1調(diào)節(jié)hnRNPA2的表達(dá)對人肝細(xì)胞癌的進(jìn)展的影響,為NEAT1作為分子靶點(diǎn)應(yīng)用于人肝細(xì)胞癌的診治提供實(shí)驗依據(jù)。[方法]利用Trizol-氯仿提取HCC組織標(biāo)本癌和癌旁組織,細(xì)胞系(HepG2,SMMC-7721)中的 RNA,RT-qPCR 檢測各組 NEAT1 表達(dá)差異(GAPDH作為內(nèi)參),使用轉(zhuǎn)染試劑Lipofectamine 2000和siRNANEATl對HepG2和SMMC-7721細(xì)胞進(jìn)行NEAT1敲低,RT-qPCR檢測NEAT1敲低效果,并對NEAT1敲低組和對照組細(xì)胞進(jìn)行表達(dá)譜測序,檢測NEAT1敲低后對HCC細(xì)胞各腫瘤相關(guān)基因表達(dá)的影響,對NEAT1敲低組和對照組細(xì)胞進(jìn)行CFSE標(biāo)記的細(xì)胞增殖檢測、CCK8染色的細(xì)胞增殖檢測、Transwell細(xì)胞侵襲移動能力檢測來探究NEAT1對HCC細(xì)胞增殖和侵襲功能的影響。查詢Starbase 2.0數(shù)據(jù),尋找與NEAT1相互作用密切的RNA.結(jié)合蛋白,通過RNA免疫共沉淀明確能同時與NEAT1和NEAT1所調(diào)控基因mRNA相互作用的RNA結(jié)合蛋白,RNA pull-down明確RNA結(jié)合蛋白與NEAT1作用靶點(diǎn)片段,使用轉(zhuǎn)染試劑Lipofectamine 2000和 siRNANEAT1 對 HepG2 細(xì)胞進(jìn)行 NEAT1 敲低,Western-Blotting,免疫組化驗證NEAT1對hnRNP A2蛋白的調(diào)節(jié),使用逆轉(zhuǎn)錄病毒retroviral vectors作為載體建立hnRNPA2過表達(dá)模型并應(yīng)用于NEAT1低表達(dá)的HepG2細(xì)胞模型中,CCK8增殖實(shí)驗和Transwell小室研究NEAT1低表達(dá)的HepG2細(xì)胞過表達(dá)hnRNPA2后與對照組的增殖、侵襲功能。[結(jié)果]長鏈非編碼RNANEAT1在HCC細(xì)胞和癌組織中表達(dá)量高于癌旁組織(p0.001)。敲低HCC細(xì)胞系NEAT1表達(dá)導(dǎo)致很多參與腫瘤發(fā)生、發(fā)展通路的基因表達(dá)量改變(229個基因表達(dá)上調(diào),148個基因表達(dá)下調(diào),表達(dá)量改變超過2倍且p0.05),敲低HCC細(xì)胞系NEAT1表達(dá)降低了 HCC細(xì)胞體外增殖(p0.01)、侵襲(p0.001)的能力。U2AF65蛋白能夠與NEAT1和hnRNP A2 mRNA相互作用(富集度分別為IgG的82.659倍和53.527倍),NEAT1敲低降低了 hnRNPA2mRNA轉(zhuǎn)錄和蛋白表達(dá)(p0.001),這種調(diào)控作用可能與NEAT1-U2AF65復(fù)合物相關(guān),hnRNPA2過表達(dá)可以逆轉(zhuǎn)NEAT1低表達(dá)引起的HepG2細(xì)胞增殖和侵襲抑制作用(p0.01)。[結(jié)論]NEAT1通過調(diào)節(jié)hnRNPA2蛋白影響了 HCC細(xì)胞的增殖和侵襲能力,這一調(diào)節(jié)機(jī)制可能是人肝細(xì)胞癌診斷和治療的新靶點(diǎn)。
[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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.7

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本文編號：1494308