The Differential Expression Profile Of MicroRNAs Between Hepatocellular Carcinoma And Embryonic Hepatocyte In The Rats

Objective:To explore the miRNA(microRNA)differential expression profile between hepatocellular carcinoma and embryonic hepatocyte in the rats,and to provide the evidence that miRNAs are involved in the molecular pathogensis of HCC.Methods:1. 150 male inbred rats(75-100Kg)are divided into experimental group and control group randomly,the rat of liver cancer model is established by low dose DEN.2. Rat hepatocellular carcinoma tissues and embryonic hepatocyte tisseues are cultured by primary culture in vitro. The total RNAs are extracted by Trizol, then miRNAs are stained by Hy3.3. Hybridizations are made by applying the cell miRNAs to mammalian miRNA chip. Data analysis is proceeded by software of SAM version 3.0.Results: 1. The rat of liver cancer model is established by 0.15% DEN successfully. Gross dissection find that liver textures is hard and is full of carcinoma nodules. The result of HE staining and the change of carcinoma pathology is consentaneous,destroyed hepatic lobules,abnormal hepatocellular nodules, hyperplastic fibrous tissues.2.Primary inoculation 24 h after the cells can be seen from 3-5 months, composed of epithelioid cell clones,7 d after the discovery by a large number of cloned cells to form a stable mass,shape is polygon,the edge is clear. One-step extraction of cells using Trizol total RNA,is opropanol precipitation enrichment through RNA,quantitative UV spectrophotometer. According to RNA at A260/280 value≥1.7,as well as formaldehyde denaturing gel electrophoresis, 28s,18s rRNA bands clarity,brightness ratio near 2:1,to meet the requirements of miRNA chip experiments.3. Hybridizations are made by applying the cell miRNAs to mammalian miRNA chip. Data analysis is proceeded by software of SAM version 3.0. Total 78 differential expressed miRNAs are found ,including 68 over-expression and 10low-expression miRNAs. Data analysis reveals that some miRNAs are associated with some different tumors, especially ,the has-miR-21 may play a crucial role in HCC.Conclusion:From miRNA gene chip,we receive a total of 78 differentially expressed miRNAs.This is a sufficient proof that the occurrence of the miRNAs in the liver,the development of the timing of the expression of differences.Then with the subsequent in-depth study,it is likely to become early liver a very important cancer marker,making the early diagnosis of liver cancer,early treatment can be achieved. Rat hepatoma cells increase the number of miRNA down more and more (68:10),this is prompted by rat hepatoma cells in a significant reduction in the expression of tumor suppressor genes,oncogene expression,resulting in the occurrence of cancer cells.At the same time,we also make use of bioinformatics to predict miRNA in the level of transcription or translation of target genes and find that hsa-miR-21 and matrix metalloproteinases (MMPs) inhibitor RECK, TIMP3 expression is closely related. Hsa-miR-21 inhibits the expression of the high-RECK,TIMP3 expression inhibitor,allowing increased expression of MMPs,and a large number of MMPs expression in liver cancer growth, invasion and metastasis,such as the process plays a very important role.