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MiRNA-101 And MiRNA-122 Function

Posted on:2011-12-16Degree:DoctorType:Dissertation
Country:ChinaCandidate:S LiFull Text:PDF
GTID:1110360308974934Subject:Biochemistry and Molecular Biology
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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.
Keywords/Search Tags:miR-101, miR-122, fos, AP-1, PRKRA
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