The Expression Of MiR-224 In Human Hepatocellular Carcinoma Cells And Its Functional Analysis

Background and objectiveMicroRNAs (miRNAs) are a new class of non-protein-coding, endogenous, small RNAs and of about 20-24 nucleotides in length, which are cleaved from the 70~80 nt partially duplexed precursor by the RNase III Dice. Mature miRNAs are known to negatively regulate gene expression or destroy the stability of genes via incomplete or complete matching with the 3'UTR of their target genes at the post-transcriptional level. They are involved in processes such as the regulation of organismal development, nerve differentiation, cell proliferation, apoptosis and fat metabolism. By now, it is predicted that miRNAs may regulate the two-third whole human genes. Many miRNAs are has relatively conservative sequences among species.Though the foundation of these miRNAs are not clear, it is sure that they have different expressed patterns, which can regulate many physiology and pathology processes, and abnormal expression of miRNAs may result in some human diseases including tumors. miRNAs are recognized as an oncogene or anti-oncogene, they have the specific express profiles in different tumor tissues.Human hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. It has the strongest invasion, the fastest metastasis and the highest recurrence. Abnormal expression profiles of miRNAs in HCC tissues have been detected by many studies. In our study, we detected the different expression profiles of miRNAs in HepG2 and LO2 cell lines by miRNA microarray. To determine the validity of miRNA microarray, we verified miR-224 expression in HepG2 cells by qRT-PCR and Northern blot. Along with reports of the different expression patterns of miRNAs in HCC and non-tumor tissues, this allowed us to isolate miR-224 as a study target, since it is overexpressed in both HCC tissues and cells, and at the same time it has more higher expression was found in the placenta tissues than other normal tissues. Then the different experiments including MTT, FACS, Scratch wound, Transwell migration and Matrigel invasion assays were performed to decided whether miR-224 can influence the biological behaviors of HepG2 cells or not. The results showed miR-224 plays a very important role in maintenance of malignant phenotype of HepG2 cell, which is a new regulator in the cell migration and invasion of hepatocellular carcinoma cells.The key to study the function of miRNAs is to find out its target genes. Predicting the targets of miRNAs and investigating the interaction between miRNAs and the corresponding targets are becoming the study focus. By now, there are several bioinformatics ways to predicte the target genes of miRNAs, including MIRANDA, PICTAR, Target Scan and so on. We predicted the miR-224 target genes by different bioinformatics, and analyse the expression of PAK4 by Immunohistochemistry and Tissue chip in HCC tissues, which is one of its predicted genes. MiR-224 was significantly upregulated in HepG2 cells. Cell proliferation, migration and invasion, but not cell cycles, were altered after changing the expression of miR-224. Taking invasion and migration as a breakthrough, it shows miR-224 can increased expression of PAK4/ MMP-9 in HepG2 and LO2 cell by Western blot. So, miR-224 may indirectly regulate PAK4 expression in liver cancer. The results will not only help us to understand the role of miRNA during the hepatocarcinogenesis, but also provide a valid ways to look for a new target for the treatment of HCC.Methods1. MiRNA microarray was performed to detect the different expression profiles of miRNAs in HepG2 and LO2 cell lines. qRT-PCR and Northern blot were performed to determine the validity of miRNA microarray.2. MTT, FACS, Scratch wound, Transwell migration and Matrigel invasion assays were performed to investigate the miR-224 influences on the cell cycles, cell proliferation and apoposis, cell migration and invasion.3. The different bioinformatics ways to predicted the target genes of miR-224 which is overexpressed both in the HCC tissues and cells.4. Immunohistochemistry and Tissue chip showed the expression level of PAK4 in HCC and the relationship among the phosphorylation expression level of PAK4(p-PAK4) with the clinical pathology factor of HCC.5. Western blot was performed to decide the relationship among miR-224 with PAK4 and MMP-9. Results1. The result of miRNA microarray shows the pattern of miRNA expression in HepG2 cells was markedly different from LO2 cells. After normalization, the expression profiles of 143 miRNAs were determined between HepG2 and LO2 cells, revealing 66 upregulations and 77 downregulations miRNAs. MiR-224 was overexpressed both in HCC cells and tissues.2. Cell proliferation, apoptosis, migration and invasion but no cell cycles were altered after changing the expression of miR-224.3. MiR-224 positively regulating cell migration and invasion.4. There are hundreds of target gens were predicted by two bioinformatics ways, most of genes are involved in cell cycles, cell proliferation, differentiation and apoptosis, signal transduction, and so on. MiR-224 may indirectly regulate PAK4 expression in HCC.5. There is a functional linkage among the expression of miR-224 and PAK4 /MMP-9 which were two mediators of c tumor cell migration and invasion.6. The protein of PAK4 are higher expressed level in HCC tissues compare normal liver tissues, and p-PAK4 are higher expressed level than PAK4 in HCC tissues. p-PAK4 are located mainly in cell nucleus and the expressed positive rate of p-PAK4 has a close relation with the degree of cell differentiation,lymph node metastasis or TNM stage.Conclusions1. The overexpression of miR-224 is a very important event during the hepatocarcino- genesis.2. MiR-224 is involved in the regulation of cell proliferation and apoptosis but not cell cycles in HepG2 cell.3. MiR-224 is a new regulator in the cell migration and invasion of HepG2 cell.