Plasma MicroRNA, Potential Biomarkers For HBV-related Hepatocelluar Carcinoma And Chronic Liver Damage

An estimated400million hepatitis B virus (HBV) infected individuals worldwide, among about1/3people are in China where HBV is the leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC)."HBV-cirrhosis-HCC" was described as the three paces of HBV carriers.HCC represents the major histological type of liver cancer and likely accounts for70%-85%of all cases of liver cancer. It is one of the most common and rapidly fatal human malignancies worldwide and the second cancer to cause death in China. The prognosis of patients depends on the stage when the disease is diagnosed. Most patients were diagnosed with HCC at their late stage in China. The5-year survival in total HCC patients is7%. This illustrates the early cancer detection is critical for the treatment and the patient survival. Alpha fetal protein (AFP) is the most common clinical indicator for HCC diagnosis, but the high level of AFP also can be detected in the serum of germ cell tumor, gastroenteric tumor and cirrhosis patients. Besides, the serum level of AFP in30%HCC patients can be normal. Therefore, discovery of new biomarkers for early diagnosis of HCC is very important and imperative.Liver damage caused by chronic HBV infection was the archcriminal of liver cirrhosis and HCC. Monitoring the dynamic change of liver damage and timely intervention is very important for management of the progression of liver cirrhosis and HCC. At present, liver biopsy and pathology examination is the golden standard to diagnosis liver damage. Pathomorphology examination can intuitively reveal the degree of hepatocyte injury, inflammation and fibrosis. However, the invasive test leads to major complications like hemorrhoea or bile leakage. This is unacceptable by many patients. Serum alanine aminotransferase (ALT) is often used as liver damage biomarker in clinical circumstance. Nevertheless, ALT often be charged for its low sensitivity and specificity. The serum activity of ALT has little relationship with tissue morphology. Therefore, to find new biomarkers for HBV-related liver damage which can reflect the degree of liver damage in some extent and superior to ALT is in urgent need now.One approach to address this issue might be based on small regulatory RNA molecules, in particular on microRNA which was first reported in1993, and becoming a new star in molecular biology research field. MicroRNA was an evolutionary conserved class of endogenously expressed small RNAs of20-25nucleotides in size. It is non-coding RNA and can target mRNA by bonding to the3’UTR. MicroRNA was discovered with critical functions in cellular development, differentiation, proliferation and apoptosis. It expresses in a tissue-and time-specific way. Researchers found in different cancer, microRNA shows relatively specific expression profile and has potential to serve as a new kind of biomarker for cancers. Actually, microRNA is found with very stable form in circulating because of the specific structure which can keep microRNA from RNA enzyme. Circulating microRNA is considered can serve as new biomarkers for diseases. High-throughput microRNA quantification technologies, such as microRNA microarray, TaqMan-based real-time PCR (qRT-PCR) microRNA assays, have provided powerful tools to study the global microRNA profile in all kinds of disease.To discovery blood-based microRNA biomarkers for early HCC detection, microarrays were used to screen the expression of plasma microRNA HCC patients. Candidate microRNA was validated by qRT-PCR. The data was statistical analysed and a logistic model was established. Meanwhile, we found microRNA showed an unusual high level in plasma of CHB patients during the studying. Therefore, we recruited more CHB plasma and selected corresponding liver biopsy tissue to do further study with the same technology platform. MicroRNA dynamic changes were revealed with the severity of HBV related chronic liver damage. PART ONE Plasma microRNA, potential biomarkers for HBV-related Hepatocellular CarcinomaPurpose:More than60%of patients with hepatocellular carcinoma (HCC) do not receive curative therapy as a result of late clinical presentation and diagnosis. We aimed to identify plasma microRNA for diagnosing hepatitis B virus (HBV)-related HCC.Method:Plasma microRNA expression was investigated with three independent cohorts including934participants (healthy, chronic hepatitis B, cirrhosis, and HBV-related HCC), recruited between August2008and June2010. First, we used microarray to screen723microRNA in137plasma samples for diagnosing HCC. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) assay was then applied to evaluate the expression of selected microRNA. A logistic regression model was constructed using a training cohort (n=407) and then validated using an independent cohort (n=390). Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy.Results:We identified a microRNA panel (miR-122. miR-192, miR-21. miR-223, miR-26a, miR-27a and miR-801) that provided a high diagnostic accuracy of HCC (AUC=0.864and0.888for training and validation data set, respectively). The satisfactory diagnostic performance of the microRNA panel persisted regardless of disease status (AUCs for Barcelona Clinic Liver Cancer stages0, A. B, and C were0.888,0.888,0.901, and0.881, respectively). The microRNA panel can also differentiate HCC from healthy (AUC=0.941). chronic hepatitis B (AUC=0.842), and cirrhosis (AUC=0.884), respectively.Conclusion:We found a plasma microRNA panel that has considerable clinical value in diagnosing early-stage HCC. Thus, patients who would have otherwise missed the curative treatment window can benefit from optimal therapy. PART TWO Dynamic Changes of Circulating MicroRNA with Pathologic classification of HBV-related Chronic Liver DamagePurpose:We aim to find out the relationship between pathologic classification of HBV related chronic liver damage and expression of plasma microRNA.Methods:First, we used microarray to screen723microRNA with the plasma of22CHB patients and33healthy people. Mann-whitney unpaired test and clustering analysis were used to choose candidate microRNA. qRT-PCR assay was then applied to evaluate the expression of selected microRNA in another118CHB and58control samples. All of the118CHB samples divided into four groups according to their corresponding morphology of liver biopsy tissue. Statistical analysis was carried to find out the connection between microRNA expression level and the morphology change in the process of HBV related chronic liver damage.Results:6microRNA (miR-122, miR-192, miR-148a, miR-194, miR-215. miR-27b) were discovered that the express level changed dramatically with the degree of liver damage. MiR-122, miR-148a. miR-215, miR-27b were identified as a panel to diagnose CHB from the healthy control with a high accuracy (AUC=0.938). miR-148a and miR-194were formed as a logistic model to distinguish CHB patients with hepatocellular necrosis from those without necrosis (AUC=0.857). MiR-122was selected according to the algorithm to serve as a good indicator to distinguish CHB patients with or without fibrosis (AUC=0.800).Conclusion:The dynamic changes of microRNA can reflect the degree of liver damage. Plasma microRNA could be potential biomarker for HBV-related chronic liver damage.