miRNA-101和miRNA-122功能研究

【作者】 李帅；

【导师】 孙志贤； 郑晓飞；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 microRNA(miRNA)是真核生物中一类长度约为22nt,由茎环结构前体加工而来的具有基因调控功能的小分子非编码RNA。miRNA通过与其靶基因mRNA的3′非翻译区(untranslated region, UTR)结合抑制靶基因翻译或导致靶基因mRNA的降解,从而在转录后水平调控真核生物的基因表达。miRNA广泛参与细胞增殖、凋亡、应激等重要生命过程的调控。大量研究表明,miRNA与多种肿瘤的发生有着极为密切的关系。我们首先研究了miRNA异常表达与肝细胞癌(Hepatocellular Carcinoma, HCC)发生发展之间的关系。利用miRNA芯片及本实验室建立的miRNA实时定量检测方法,我们检测了肝细胞癌组织及癌旁组织中miRNA的表达,发现miR-101、miR-148a、miR-182、miR-422b、miR-451在两种组织之间存在显著表达差异。运用生物信息学预测软件、荧光素酶报告基因实验以及Western Blot,我们发现miR-101能抑制fos癌基因的表达并抑制转录因子AP-1的活化。此外, Transwell细胞体外侵袭实验表明miR-101能够抑制fos的表达进而抑制肝癌细胞系的侵袭转移。本研究同时关注了肝脏中特异性高表达的miR-122。为了寻找miR-122的候选靶基因,我们对miR-122的成熟体进行了生物素(Biotin)标记,并将其转染入细胞,通过亲和纯化我们富集了miR-122的靶基因。此外,我们还发现miR-122能够抑制PRKRA的表达进而促进miRNA的加工成熟。取得的主要进展有:1.肝细胞癌中miRNA的异常表达。利用miRNA芯片及实时定量PCR检测了20例肝细胞癌病人癌组织和癌旁组织中miRNA的表达,发现在肝细胞癌组织中miR-101、miR-148a、miR-422b、miR-451表达显著升高,而miR-182表达显著降低。2. fos癌基因是miR-101的靶基因。荧光素酶报告基因实验证实miR-101通过作用于fos基因mRNA的3′非翻译区抑制其表达。在人胚肾细胞HEK 293中过表达miR-101可以显著抑制PMA对内源FOS蛋白的诱导表达。3. miR-101抑制转录因子AP-1的活化并抑制肝癌细胞系的侵袭转移。利用AP-1转录活性报告载体(pAP-1-Luc),表明miR-101能够抑制fos进而抑制由PMA诱导的转录因子AP-1的活化。利用Transwell细胞体外侵袭实验,发现miR-101能够抑制fos进而抑制由肝细胞生长因子(hepatocyte growth factor, HGF)诱导的肝癌细胞系的侵袭转移。4.运用实验方法寻找miR-122的候选靶基因。对miR-122成熟序列3′端进行生物素标记,将其转染入肝癌细胞系HepG2中,之后用温和方式裂解细胞并利用生物素与链亲和素之间的亲和性富集miR-122的靶基因。提取富集产物中的RNA,用实时定量PCR及基因芯片检测富集的基因,发现miR-122的多个已知靶基因得到了富集,并发现了一些新的候选靶基因。5. miR-122能够抑制PRKRA的表达进而促进miRNA的加工成熟。结合以上基因芯片结果和miRNA靶位点预测软件的预测结果,发现PRKRA是miR-122的候选靶基因。荧光素酶报告基因实验证实,miR-122可以直接作用于PRKRA的3′非翻译区。过表达miR-122可以显著抑制HepG2细胞中PRKRA的蛋白水平及其mRNA水平。miR-122和针对PRKRA的siRNA能够促进HeLa细胞中miRNA的加工成熟。综上,本研究揭示了肝细胞癌中异常表达的miR-101能够抑制fos癌基因的表达并抑制肝癌细胞系的侵袭转移。本研究还用亲和纯化方法寻找肝脏特异性miR-122的候选靶基因,发现miR-122可以抑制PRKRA进而促进miRNA的加工成熟。更多还原

【Abstract】 MicroRNAs (miRNAs) are small conserved RNA molecules of ~22 nucleotides which negatively modulate gene expression in animals and plants, primarily through base pairing to the 3′untranslated region (UTR) of target mRNAs, which leads to mRNA cleavage and/or translation repression. MiRNAs are involved in a variety of basic biological processes, such as cell proliferation, apoptosis and stress responses. Recent studies showed that miRNAs correlate with various cancers.First of all, tissue samples of 20 primary HCCs including cancerous and adjacent normal liver tissues were obtained from patients who underwent hepatectomy. Then we employed miRNA-microarray and miRNA-realtime-PCR (developed by our lab) to investigate the expression profile of miRNA in hepatocellular carcinoma (HCC). Results showed that miR-101, miR-148a, miR-182, miR-422b and miR-451 are differentially expressed in HCC. By using bioinformatic tool, dual luciferase reporter assay and Western blot analysis, we demonstrated that miR-101 repressed the expression of fos oncogene, a key component of the activator protein-1 (AP-1) transcription factor. Moreover, via a luciferase expression vector (pAP-1-Luc) driven by seven copies of an AP-1 cis-element, we observed that miR-101 expression inhibited PMA induced AP-1 activity. In in vitro Matrigel invasion and Transwell migration assays, enhanced miR-101 expression inhibited the invasion and migration of cultured HCC cells, respectively.Secondly, our study focused on hepato-specific miR-122. We employed 3′-end biotinylated synthetic miR-122 to identify its targets based on affinity purification. Quantitative RT-PCR analysis of the affinity purified RNA demonstrated a specific enrichment of several reported miR-122 targets. Using bioinformatic tool, dual luciferase reporter assay and Western blot analysis, we showed that miR-122 repressed the expression of PRKRA and the inhibition of PRKRA could facilitate the expression of miRNA in HeLa cells.The main progresses are listed:1. miRNA signature in HCC. By using miRNA-microarray and miRNA-realtime PCR we found miR-101, miR-148a, miR-422b and miR-451 were significantly down-regulated in HCC tissues compared with the adjacent normal liver tissues, while miR-182 was significantly up-regulated in HCC.2. fos is the target gene of miR-101. miRNA targets prediction software Targetscan shows that fos is a candidate target gene of miR-101. Using dual luciferase reporter assay we demonstrated that miR-101 could interact with the 3′UTR of fos directly. Moreover, the overexpression of miR-101 could inhibit the high expression of FOS protein induced by PMA in HEK293 cells.3. miR-101 inhibits both the activation of AP-1 and HGF-induced migration and invasion through the inhibition of fos gene. By using a luciferase expression vector (pAP-1-Luc) driven by seven copies of an AP-1 cis-element, we observed that miR-101 expression inhibited PMA induced AP-1 activity. In in vitro Matrigel invasion and Transwell migration assays, enhanced miR-101 expression inhibited the invasion and migration of cultured HCC cells, respectively.4. Isolation of the hepato-specific miR-122 targets using biotinylated synthetic miRNA. We employed 3′-end biotinylated synthetic miR-122 to identify its targets based on affinity purification. Quantitative RT-PCR analysis of the affinity purified RNA demonstrated a specific enrichment of several reported miR-122 targets. To find more targets of miR-122, the purified RNA was amplified and analyzed on microarray.5. miR-122 facilitates miRNA expression by inhibiting PRKRA. From above microarray data and the prediction of Targetscan, we found PRKRA is a candidate target of miR-122. Using dual luciferase reporter assay we showed that miR-122 could interact with the 3′UTR of PRKRA directly. The overexpresion of miR-122 in HepG2 cells inhibited the expression of PRKRA at both protein and mRNA level. Moreover, miR-122 and siRNA for PRKRA could facilitate the expression of miRNA in HeLa cell.On the whole, our investigation revealed that miR-101 was downregulated in HCC and invovled in the regulation of hepatoma carcinoma cell migration and invasion by regulating the expression of fos oncogene. The study on miR-122 found out many candidate target genes of miR-122. Moreover, miR-122 could facilitate the expression of miRNA in HeLa cell by inhibiting the expression of PRKRA.更多还原