Study On The Functions Of MiR-183 In Hcc And Construction Of Artificial MiRNA Cluster Expression Vector

Objective: (1) Study on the functions of miR-183 in Hepatocellular carcinoma (HCC). (2) Construct artificial miRNA cluster expression vector and analyse tumor cell cycle arrest induced by the artificial miRNA cluster. Moreover, a preliminary research of the relation between miRNA procession and the expression of the protein with miRNA host genes. Methods:(1)To study functions of miR-183 in HCC, we compared the miR-183 expression profile from HCC tumor tissues and adjacent normal liver tissues. We used bioinformatics methods to screen the potential target genes of miR-183. Luciferase reporter assay, western blot and qRT-PCR were conducted to confirm target association. Moreover, we further studied the role of miR-183 in hepatoma cell. DAPI analysis for morphological characteristics associated with apoptosis. (2) The recombinant plasmids (PGL-34a-424) were constructed by amplifing miR-34a and miR-424 precursors from a library of vectors expressing miRNAs and connecting them. The expression of artificial microRNA cluster was tested by RT-PCR. Then, the relation between miRNA procession and the expression of the protein with miRNA host genes was detected by Luciferase Reporter Assay after constructing mutant miRNA precursors. Moreover, the recombinant plasmids (pEGFP-34a-424) were constructed to analyze tumor cell cycle by flow cytometry. Results:(1) Expressions of miR-183 were examined further in an independent series of primary HCC tumors and adjacent nontumoral livers by using Real-time PCR method. The results showed that miR-183 was significantly up-regulated (2 to 300 fold) in 68% of tumors. We analyzed putative target genes by using the TargetScan bioinformaticss tools. The results of luciferase assays revealed that PDCD4 is a direct target gene of miR-183. Western blot and qRT-PCR results showed that miR-183 can downregulate PDCD4 mRNA and protein expression. PDCD4 is down-regulated in human HCC tissue compared to corresponding nontumoral liver tissue. Furthermore, PDCD4 is involved in TGF-β1 induced apoptotic signaling pathways in the HCC cell line Huh7. Both DAPI staining of cells and Annexin-V assays showed that the number of apoptotic cells induced by TGF-β1 treatment was reduced in the miR-183 transfectant. (2) Artificial miRNA cluster expression vector was constructed successfully and miRNA procession could suppress the expression of the protein with miRNA host genes by Luciferase Reporter Assay. Moreover, pEGFP-34a-424 induces tumor cell cycle G0/G1 phase arrest. Conclusions: (1) This study suggests that miR-183, up-regulated in HCC, represses the expression of the tumor-suppressor PDCD4 posttranscriptionally, and inhibits TGF-β1-induced apoptosis in human HCC cells. Therefore, miR-183 may play an important role in HCC development. (2) Artificial miRNA cluster can efficiently express mature miRNA, and miRNA procession could suppress the expression of the protein with miRNA host genes. Moreover, artificial miRNA cluster on cell cycle arrest was more effective than single miRNA.