| Background：Cancer is one of the most common diseases in the world and severelythreatens our health. Malignant tumors in digestive system are predominantworldwide. The incidences of gastrointestinal cancers such as gastric cancer,colon cancer and liver cancer have been increasing. Thus, it is urgent to explorethe molecular mechanism underpinning the development of these three cancers.The RNA-editing enzyme ADAR (adenosine deaminaseacting on RNA) is adouble-stranded RNA (dsRNA) binding protein that modifies cellular and viralRNA sequences though adenosine deamination. There are three members in thefamily of RNA-specific adenosine deaminase, named ADAR1, ADAR2andADAR3. Among them, more focus has been drawn on ADAR1, which has been demonstrated to play important roles in embryonic erythropoiesis, viral response,and RNA interference. ADAR1modifies the coding and noncoding sequences ofcellular and viral RNAs though deamination that converts adenosine to inosine.ADAR1is proposed to play a role in host defense mechanisms, tumordevelopment and inflammatory response. But the roles of ADAR1in tumors, suchas gastric cancer, colon cancer and liver cancer still remain unclear; the aim ofthis study is to investigate the expression and significance of ADAR1in thesegastrointestinal cancers.Objective：1. To prepare and purify the polyclonal antibodies to RNA-dependent adenosinedeaminase l (ADAR1), and to ascertain their titer by ELISA.2. To investigate the expression and localization of RNA-dependent adenosinedeaminase l (ADAR1) in gastric cancer, liver cancer and colon cancer, and toanalyze its correlation with the clinicopathological features of these diseases．3. To investigate the expression and localization of RNA-dependent adenosinedeaminase l (ADAR1) in HBV+hepatitis, HBV-hepatitis and liver cirrhosis,and to examine its relationship to HBV..4. To construct and characterize the overexpression lentivirus vector of ADAR1gene, package them with lentivirus particle and purify the lentivirus particle.Then transfect the gastric cell lines with ADAR1overexpression lentivirus toestablish stable ADAR1-overexpressing cell lines, and to provide a powerful toolfor the subsequent research.Methods：1. Prepare the antigen peptides for ADAR1, immunize a healthy rabbit with them, and purify the antibodies by Affinity Chromatography.2. The expressions of ADAR1in hepatitis, liver cirrhosis, gastric cancer, livercancer, colon cancer and paired adjacent non-cancerous tissues were examined byimmunohistochemistry.3. The relationships between ADAR1expression and clinicopathological features were analyzed statistically.4. RNA overexpression lentivirus vector of ADAR1gene was constructed,and identified by transfecting293T cells, and the efficacy was evaluated by Western blotting. Then we packaged it with lentivirus particle and establishd stable ADAR1-overexpressing cell lines.Results:1. Preparation of poloclonal antibodies to ADAR1The antibodies that recognize epitopes of ADAR1(both of N-terminal andC-terminal) were obtained by immunizing rabbits with different peptides. Thenimmunohistochemistry was performed using the antibody toward the N-terminalpeptide ofADAR1.2. Expression of ADAR1in gastric cancer and normal gastric mucosa.246gastric cancer tissues were immunostained by anti-ADAR1, ADAR1was predominantly located to the cytoplasm, and could also be detected in thecell nucleus in some poorly differentiated gastric cancers. The positivity ofADAR1in gastric cancer was96.7％(238/246) andwas significantly lower thanthat of100％(134/134) in paired adjacent non-cancerous tissues. Of the246cancer tissues,48.3％(119/246) were strongly positive forADAR1, versus89.6％(120/134) in normal gastric tissues, indicating that ADAR1was down-regulatedin gastric cancer (P<0.05). ADAR1expression was also found correlated with TNM stages and differentiation status of gastric cancer (P<0.05).3. Expression of ADAR1in liver cancer, adjacent tissues, hepatitis and cirrhosis.98liver cancer and paired adjacent non-cancerous tissues were examined,ADAR1were expressed in the cytoplasm. The positive rate of ADAR1was87.8％(86/98) in live cancer, versus98％(96/98) in adjacent non-canceroustissues, and there existed a significant difference (P<0.001). ADAR1expressionwas associated with TNM stage and differentiation status of liver cancer, but notwith age and gender. ADAR1were located to the cytoplasm in hepatitis and livercirrhosis. The positive rates of ADAR1in HBV+hepatitis and HBV-hepatitiswere93.1％(27/29) and97.1％(34/35), respectively. And the positive rates ofADAR1in HBV+liver cirrhosis and HBV-liver cirrhosis were86.4％(51/59)and75.8％(25/33), respectively. There was no significant difference betweenthese groups.4. Expression of ADAR1in colon cancer and adjacent tissuesADAR1was located to the cytoplasm in colon cancer and adjacentnon-cancerous tissues. The positive rate of ADAR1in colon cancer was88％(88/100) and was significantly lower than that of98％(98/100) in pairedadjacent issues(P<0.05). ADAR1correlated with their tumor differentiation incolon cancers, but not with age, gender and TNM stages.5. Construction of overexpression lentivirus of ADAR1We successfully constructed and identified the ADAR1overexpressionlentivirus vector pGC-LV/ADAR1P110and pGC-LV/ADAR1P150，temporarily transfecting293T cells with virus, and test the virus concentrationand mRNA levels by Q-PCR. Then we packaged it with lentivirus particle. Andwe successfully constructed gastric cancer cell lines that stably expressingADAR1-P110and ADAR1-P150isoforms of ADAR1. Empty lentivirus vectors will be used as control.Conclusion：1. Antibodies to both C and N terminal epitopes of ADAR1have beensuccessfully generated.2. ADAR1expression was down-regulated in gastric cancer, liver cancer andcolon cancer. There is a correlation between the expressions of ADAR1in thesetypes of cancer with their TNM stages. And there is no relationship between theexpressions of ADAR1in the cancers above with the gender and ages. Thesefoundlings indicate that ADAR1would play an important role in the cancerdevelopment.3. ADAR1-lentivirus transfected gastric cancer cells would provide cell andanimal models for further investigation of the biological functions of ADAR1. |
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