Analysis Of Differential Expression Of Subtractive CDNA Fragments P₀2 And P₅5 In Hepatocellular Carcinoma Tissues

[Background] Hepatocellular carcinoma (HCC) is one of the most familiar malignant tumors which is seriously harmful to health. The incidence of HCC in worldwide keeps the eighth place in all of the malignant tumors, among which keeps the sixth in male and eleventh in femal. The occurrence of HCC is different in different area. China is one of the highest-occurrence areas of HCC and the incidence in China shares 40%-45% of that all over the world. It has been showed in the spot check of the death analysis of malignant tumors in the resident in 27 provinces of China during 1990-1992 that the death rate of HCC shares the 18.8% of all the malignant tumors and keeps the second place, and the mortality hold the first place among the population within the age of 30-40. The patients of HCC with transparent symptoms and signs are often in advanced stage. Because the most of HCC patients are based on the liver cirhosis, it is hard to gain satisfactory curative effect. So far the marker of HCC used most commonly is alpha fetal protein (AFP), but its value in clininc practice is still limited. There is about 30%-40% of HCC patients whose level of AFP in serum is within the normal range and even if the patientshave the positive result, the diagnosis must not be approached before differentiating from the hepatitis, cirrhosis and so on. This bring about the difficulty for clinical diagnosis. It has been showed by modern molecular biology research that there are close correlation between the tumorigenesis and the gene mutation. A lot of work has been done on the genetic mechanisms of tumorigenesis, but it is still not clear on the molecular mechanisms of hepatocarcinogenesis. In order to furtherly find the genes associated with hepatocarcinogenesis and establish the genetic basis of the further studies on the mechanisms of hepatocarcinogenesis and the development of the diagnostic and theraputic methods on HCC, Dr. Zhu Wuling has constructed a compliment DNA (cDNA) library by using suppression subtractive hybridization (SSH) technology and got 93 clones. All of these clones need to be further screened and analysed.[Aim] 1 To analyse the factual abundance of P-02 and P-55 mRNAs in the tissues used in the construction of the cDNA library to exclude the false positive results in the process of SSH. 2 Detecting the abundance of P-02 and P-55 mRNAs in the tissues surgically removed from 10 HCC patients to analyse the difference of the expression in HCC and liver cirrhosis tissues. 3 To speculate the function of P-02 and P-55 in the process of hepatocarcinogenesis.[Methods] By abstracting the total RNA from tissue using acid-phenol methods, and by hybidization with total RNA using HRP-labelled cDNA probes, screen the positive clones. Then analyse the differential expression of mRNA in hepatocellular carcinoma tissues by reverse transcriptase-polymerase chain reaction (RT-PCR). The result are statitically analysed by t-test. Finally homologicallyanalyse in the Genbank database to speculate the function of P-02 and P-55 in hepatocarcinogenesis.[ Result 3 1) The expression values of the hybridization signal intensity of cDNA probe P-02 hybridized with cirrhosis and HCC tissues are 37299.5771 and 148455.8521, respectively, and that of cDNA probe P-55 are 1792.8770 and 2014.8179, respectively. 2) By analysing the results of RT-PCR in cirrhosis and HCC tissues total RNA, the expression values of the RT-PCR products of P-02 mRNA are 1.4416 + 0.2150 and 0.523020 + 0.1647 in cirrhosis and HCC group, and there is significant difference between two groups; while the same values of P-55 mRNA in two groups are 0.5176 + 0.2219 and 0.8329 + 0.2718, respectively and there is significant difference between two groups. 3) By homologically analysing in GenBank nucleic acid database, we found that the P-02 mRNA is homologous to human translationally controlled tumor protein, but there is no known genes homologous to P-55 mRNA. 4) By homologically analysing in GenBank human genome database, we found that P-02 gene localized...