The Study Of Circulating Nucleic Acids And DNA Methylation In HCC

Objective: Hepatocellular carcinoma (HCC) is one of the most common neoplasms worldwide and has been the 2nd cancer killer in China since the 21st century. One of the important risk factors for HCC is chronic infection by hepatitis B or C virus, although there are many other risk factors have been implicated in its pathogenesis. Chronic hepatitis B is one of the most common infectious liver diaeases which had high morbidity and mortality. The percentage of HBsAg (+) in our contury is 7.18% from data, and the total amount of HBsAg (+) is about 120 million. And there are also about 20-30 million patients which are infected by hepatitis B. Chronic hepatitis B may develop to cirrhosis or hepatocellular carcinoma (HCC) without diagnosis and treatment in time. So it is very importmant to control the pathogenetic condition of the chronic hepatitis B and to screening as well as diagnosis the hepatocellular carcinoma (HCC) in time .Although progress has been made in clinical aspect of liver cancer, and some patients with primary liver cancer who was diagnosed and treated at an early stage yielded a curable outcome, however, the overall dismal prognosis has not completely changed. The poor prognosis of liver cancer is largely due to a high rate of recurrence after operation which is mainly resulted from intra-hepatic metastases or vascular invasion. So identifying a biomarker with prognostic significance, especially for predicting recurrence has become an important aspect in the hepatocellular carcinoma (HCC).Free circulating nucleic acids is an cell-free acids including cell-free DNA and cell-free RNA which existing in the serum or plasma. Since patients with cancers, particularly with high metastatic potential, tend to have higer levels of DNA in plasma, and the molecular biologic findings of circulating DNA in cancer patients is mimicked to that of cancer cells, plasma DNA may be a candidate because of less invasiveness, simple manipulation, and prognostic information available before operation.The research of methylation in DNA is well studied in cancers including hepatocellular carcinoma (HCC). An activated methyl group was combinated covalencyly to the 5th carbon atom in cytosine which conver to 5-methylcytosine (m~5C) by DNA methyltransferases (DNMTs) during the process of methylation in DNA. The aberrant methylation in DNA may affect the development and progress of hepatocellular carcinoma (HCC). So it is very important to explore the relationship between methylation and hepatocellular carcinoma (HCC) for its early diagnosis and therapy. In our study, we want to explore a fast method of extracting cell-free DNA in serum which could be used in the next step. We could use the selected method to extract the cell-free DNA in serum of hepatocellular carcinoma (HCC) patients, HBV carriers and healthy people. Then we analyze the quantity of cell-free DNA in serum of hepatocellular carcinoma (HCC) patients, HBV carriers and healthy people by real-time PCR. We also explore the effect of surgery in hepatocellular carcinoma (HCC) patients to cell-free DNA in serum. And we also try to find that the relationship between DNA methylation and hepatocellular carcinoma (HCC) patients, as well as the character of pathology in hepatocellular carcinoma (HCC) patients.Method: 1. Use ten methods of extracting cell-free DNA in serum prepared to extract the cell-free DNA from different compounded serum. Real-time PCR was used to analyze the quantity of cell-free DNA. Choose a best extracting methods by the quantity of cell-free DNA from each method of extracting cell-free DNA.2. 50 hepatocellular carcinoma (HCC) patients, 50 HBV carriers and 50 healthy people was selected from which cell-free DNA was extracted. Then use Real-time PCR to analyze the quantity of cell-free DNA to find the difference between different groups as well as the 14 hepatocellular carcinoma (HCC) patients which were operated or not.3. 35 cases of hepatocellular carcinoma (HCC) and 35 cases of paired non-tumor tissues were examined for the study of condition of DNA methylation. Then we analyzed the relationship between the condition of DNA methylation and hepatocellular carcinoma (HCC), as well as the character of pathology in hepatocellular carcinoma (HCC) patients.Results: 1. One of methods of extracting cell-free DNA by phenol-chloroform is the best method to extract cell-free DNA in our study.2. The quantity of cell-free DNA in hepatocellular carcinoma (HCC) patients was higher than HBV carriers and healthy people, and the difference was statistically significant (P<0.01). The quantity of cell-free DNA in hepatocellular carcinoma (HCC) patients was lower after surgery, and the difference was also statistically significant (P<0.01). The integrity of cell-free DNA in hepatocellular carcinoma (HCC) patients was related to the APF and cirrhosis, and the difference was also statistically significant (P<0.05).3. The level of DNA methylation in hepatocellular carcinoma (HCC) tumor tissues was higher than paired non-tumor tissues, and the difference was statistically significant (P<0.05). The total level of DNA methylation in hepatocellular carcinoma (HCC) patients was related to the GGT, and the difference was also statistically significant (P<0.05).Conclusion: Cell-free DNA and the level of DNA methylation could both be used for a good marker for the early screening and diagnosis in hepatocellular carcinoma (HCC). And they are also related to some characters of pathology in hepatocellular carcinoma (HCC) patients.