Detection Of MiRNA And MRNA In The Plasma Of Patients With Liver Disease

MicroRNAs (miRNAs) are a class of small (19~24nt), non-coding endogenous RNAs, which are highly conserved during evolution. Most miRNAs regulate the translation of target gene by binding to 3'-untranslated region (3'-UTR) to affect the growth and development of organisms, they also participate in many diseases progresses. According to bioinformatics predictions, there are thousands of miRNA gene in the human genome regulating 1/3 gene expression of genome. Recent studies on the molecular mechanism of miRNA not only reveal the physiological function of miRNA, but also explain the abnormal expression patterns of many disease-related miRNAs.It has been confirmed that there are hundreds of miRNA in the plasma of healthy people. miRNAs surviving from endogenous RNase are stable and abundant in plasma, which indicates that they probably play an important role in tumor diagnosis. Since miRNAs are involved in almost every step of tumorigenesis, it has good prospects in early diagnosis, prewarning and treatment of cancer. This study is designed to find those abnormally expressed liver plasma miRNAs. The Poly (A) tailing technique (established by our laboratory) and miRNA quantitative real-time PCR were applied to detect the miRNAs profiles in healthy plasma and liver-disease plasma and to select the miRNAs with discrepant profiles. The aim was to figure out the hepatology-specific expressed miRNAs in plasma.Firstly, this study optimized the miRNA extraction procedure and system with TRI Reagent. Secondly, the expression profiles of miR-21, miR-221 and miR-224 in the plasma of healthy people and liver-disease patients were quantitatively detected. Thirdly, it has been reported that AFP mRNA, PKM2 mRNA and ALB mRNA exist in the plasma of the liver cancer patients, so the study also paid attention to their expression profiles in the plasma of the healthy people and liver-cirrhosis patients. Main progresses are listed as following:1. Optimize the miRNA extraction procedure. The proper ratio of plasma and TRI Reagent is 1:5. And the total RNA extrated from more than 800μl plasma is suitable for the poly-A-tailed RT-PCR analysis of plasma miRNA.2. It's been verified that the expression profiles of miR-21, miR-221 and miR-224 in liver-disease patients'plasma are higher than those in healthy people's plasma. After the clinical classification of liver-cirrhosis, the statistic analysis showed that the expression profiles of miR-21, miR-221 and miR-224 in different types of liver-cirrhosis patients'plasma are higher than those in healthy people's plasma.3. Analyze the relationship between clinical biochemical index and detected miRNAs. Most clinical biochemical index have no obvious relative risks with detected miR-21, miR-221 and miR-224.4. The expression profiles of AFP mRNA, PKM2 mRNA and ALB mRNA in both healthy people and liver-cirrhosis patients'plasma were detected and verified to be discrepant expressed.5. Analyze the correlation between clinical biochemical index and detected mRNA. It was verified by non-parametric test that there was no significant relative risks among the expression of AFP mRNA, PKM2 mRNA and clinical biochemical index. However, ALB mRNA expression has statistically significant relative risks withγ-glutamyl transpeptadase(GGT) and serum albumin(ALB)..In conclusion, real-time PCR was applied to detect the expression of some particular miRNAs and mRNAs in liver-disease patients'plasma in this study. Moreover, the study focused on the change progress of miRNA and mRNA in liver-disease patients'plasma, which lends support for the further research. It is possible to find the miRNA biomarkers for liver cancer early diagnosis if there are large enough samples and long-term follow-up study. This study will help people to know liver disease better.