The Experimental Study On Expression Of Runx3 Gene In Gastric Carcinoma And Its Clinical Significance

The gastric cancer which occupied second in the malignant tumor lethal reason, is most commonly in the digestive malignant tumor .The stomach cancer is as same as the other malignant tumor which caused by the environmental factor and the heredity factor, in recent years, following by the research of gene polymorphism, it was demonstrated that the susceptibility of gastric cancer heredity was closed to the immunity correlation gene,the polymorphism of enzyme gene,oncogene and antioncogene. The function change of oncogene andantioncogene played the vital role in the stomach cancer occurrence. The oncogene, which taked variation in cells, played biological function in normal cells. these genes which were important to grows and development of body could play very vital regulative function on proliferation and differentiation of cells. The protein which encode by these genes all existed in the cell each constituent, including cell nucleus, cytoplasm and cell surface. Antioncogene, named recessive oncogene,played very important negativited regulated function on control of auxesis, cell proliferation and differentiation , in the meantime it could inhibit tumor grows potentially. If the abnormal phenomenon of antioncogene function deactivated,or antioncogene deletion and mutation happened,the vicious transformation would take place on cells in order to the development of the malignant tumor. The biological function of antioncogene is contrary to oncogene.They were the plus positive and negative regulative signal in the proliferation, differentiation and apapoptosis of cells on organism life process. The tumor occurrence was multisteps and multi-genes participation biology process. The variation of oncogene included point mutation, augmentation and excess expression taked place on development of the malignant tumor. The variation of antioncogene included deletion, point mutation, low expression and methylation taked place on development of the malignant tumor. The excess expression of gene often happened on the preliminary stage of cell cancerization and gene point mutation maybe the most one milestone on initialization phase of cell cancerization. The reversibility in this stage would turn cancerizated cells to normal cells. Gene augmentation and rearrangement often taked place on promotion stage development of the malignant tumor. Cancerizated cells of this stage started to advanc towards to vary biological behavior differentiation , in the meantime they would show heterogeneity and separate from the cntrol of body. The genome was more serious destroyed by gene deletion and it would induce the variety biological behavior of cancerizated cells and induce the nonreversibility of out of control of proliferation and differentiation of cancerizated cells and development of the malignant tumor.In recently, human runt-related transcription factor 3(Runx3)was the one of runt family gene that attracts the most attention. Runx3 was the basic of runt family of mammal evolution. Runx3gene pitched on human chromosome NO.1 galianconism 1p36. All member of Runt family were endowed with RuntDomain which contained 123 amino acids. Runx protein was closed to occurrence of human disease by transmit TGF-β/activin signal through forming complexment with Smad2 and Smad3. Runx3 had two high degree conservative CpG islands. The big one of CpG islands which riched character of GC promoter located around on promoter P2 of Runx3 gene which could control transcription of Runx3 gene. Transforming growth factor beta(TGF-β)was many cells effective inhibiting factors and its signal disorder would cause many tumors occurrences. In recent years, some study showed that Runx protein could instruct the Smad complexment which activated during TGF-βsignal transduction from cytoplasm into the specific target position intranucleolus and reinforce the bonding strength between the Smad complexment and the target position spot. Furthermore, it could activate the target gene in order to play regulation role in the cell differentiation, regulation and control of the cell cycle, cell apoptosis and vicious transformation. At the present day, Study showed that the closed correlativity between abnormal of human chromosome 1p36 and occurrence of the digestive malignant tumor such asgastric carcinoma, cholangiocarcinoma, pancreatic cancer and hepatocellular carcinoma. Runx3 gene was nominated for the candidacy as the antitumor gene that attracts the most attention.The result of study showed that runx3 gene could inhibit the grows of gastric carcinoma cell line and their oncogenicity in the nude mice. The hyperplasia would happen on gastric mucosa of mice by knocking-down the Runx3 gene.The expression of Runx3 gene were detected on epithelial cells of human normal gastric mucosa by using hybridization in situ. On the other hand, the Runx3 gene were deactivated in about 60 % gastric carcinoma tissues and closed to the stage of gastric carcinoma. Therefore, the Runx3 gene was regard as be one kind of recent discovery antioncogene and play the vital role in the gastric carcinoma occurrence developing process. For that reason, gastric carcinoma and adjacent normal tissues by ectome, gastric carcinoma cell line named SGC-7901 and nude mice loaded gastric carcinoma cell were regarded as object of study in this investigation. Cell culture, animal experiment, immunohistochemistry, pathomorphism observation, RT-PCR, Western-blot and MSP technique were used to detected expression of Runx3 gene mRNA, change of Runx3 gene function and state of methylation on the gastric carcinoma and adjacent normal tissues by ectome, gastric carcinoma cell line SGC-7901 and nude mice loaded gastric carcinoma cell. The result of this study could explain clearly that the closed relationship between the expression change of Runx3 gene on the gastric carcinoma tissues and occurance and growth of gastric carcinoma. Furthermore, to investigate the mechanism of Runx3 gene action in gastric carcinoma gene therapy as the candidacy antioncogene in order to provide the objective observation index of gastric carcinoma patient`s early specificity diagnosis or judgment of prognosis and establish the theory and the foundation of clinical application of gastric carcinoma gene therapy and animal experiment.Part One The experimental study on expression of Runx3 protein in gastric carcinoma and corelations with clinical pathology characterObjective: To investigate the expression of Runx3 protein in human gastric carcinoma and surrounding normal tissue and its clinical significance.Methods: S-P immunohistochemical technique was used to examine the expression of Runx3 protein in 50 cases of gastric carcinoma and the surrounding tissues of 5cm distance of normal tissues. The level of the expression was analyzed combined with clinicopathological features of age and sex of the cases, the size and loci of the tumor, infiltration depth, differentiation degree, lymph node metastasis.Results: The color of the immune positive products of Runx3 was buffy. Runx3 located in nucleus. Low expression of Runx3 revealed in 21/50 samples (42.00%) of carcinoma which showed a negative correlation with tumor differentiation (P<0.01). The expression of Runx3was lower in poor differentiation degree than those in high and moderate differentiation degree and the rate of positive expression was 28.13% (9/32) and 66.67% (12/18) respectively (P<0.01). The expression percent of Runx3 was 20.69% (6/29) in lymph node metastatic cancer whereas only 71.43% (15/21) of Runx3 expression was shown in non-metastatic cancer (P<0.01). The expression of Runx3 was 93.33% (28/30) in the surrounding tissues of 5cm distance which showed significantly higher than that in carcinoma tissues (P<0.05). There were no significant differences in the age and sex of the cases, the size and loci of the tumor of the expression of Runx3 protein (P>0.05).Conclusion: Lower expression of Runx3 exsist in gastric carcinoma and show a good relation with tumor differentiation and lymphnode metastasis. As the antioncogene, Runx3 genes involved in the negative regulatory in the molocular mechanism of gstric carcinogenesis. Part Two The experimental study on expression change of Runx3 mRNA and protein in gastric carcinoma and methylation detection of Runx3 gene promoterObjective: To explore the correlations between methylation of Runx3 gene in promoter region with the expression of Runx3 gene mRNA and its protein in human gastric carcinoma and surrounding normal tissue.Methods: RNA and protein were extracted from 60 human gastric carcinoma and surrounding normal tissue specimens. Reverse transcription polymerase chain reaction (RT-PCR), MSP and Western blot were used to detect the Runx3 mRNA expression, methylation of Runx3 gene in promoter region and Runx3 protein expression. In the same time, analysis of correlations between clinic pathological parameters with the expression of Runx3 gene had been taken.Results: (1)Expression of Runx3 mRNA in human gastric carcinoma and surrounding normal tissue:The gene amplification segment of Runx3 and GAPDH primer were electrophoresis and dyeing by EB. The expression of Runx3 mRNA in human gastric carcinoma and surrounding normal tissue was 25.00% (15/60) and 78.33% (47/60) respectively. The size of amplification segment of Runx3 and GAPDH was as same as the one designed (353bp and 208bp). Relative expression value of Runx3 in human gastric carcinoma and surrounding normal tissue was (0.58±0.11) and (0.83±0.30) respectively (t=6.1435,P<0.01). (2) Expression of Runx3 protein in human gastric carcinoma and surrounding normal tissue:Expression of Runx3 protein in human gastric carcinoma tissue was lower than that in surrounding normal tissue. The integrated light intensity of Runx3 protein in human gastric carcinoma and surrounding normal tissue was (6719.01±343.29) and (36752.51±1205.68) respectively (t=23.9579,P<0.01). (3) Incidence rate of methylation of Runx3 gene in promoter region of human gastric carcinoma and surrounding normal tissue:The methylation expression percent of Runx3 gene in promoter region was 53.33% (32/60) in gastric carcinoma tissue whereas none of runx3 expression was shown in surrounding normal tissue. The negative correlation existed in methylation of Runx3 gene in promoter region and lower expression of Runx3 mRNA (r=-0.4915,P<0.01 ). (4) Correlations between clinic pathological parameters with the expression of Runx3 gene: There were high significant differences in the differentiation degree and lymph node metastatic of the tumor of the expression of Runx3 protein (P<0.05), and no significant differences in the age and sex of the cases, the size and loci of the tumor of the expression of Runx3 protein (P>0.05).Conclusion: Lower expression of Runx3 gene mRNA and its protein in the gasric carcinoma. Abnormal expression of Runx3 gene and its protein probably played an important role in the tumorigenesis and development of gastric carcinoma. Abnormal expression of Runx3 gene followed by hypermethylation of Runx3 gene in promoter region. Part Three The experimental study on re-expression of Runx3 gene in human gastric carcinoma cell line named SGC-7901 which treated by 5-Aza-CdR and effection on grows of SGC-7901 cells by demethylated medicineObjective: RT-PCR, MTT, cell proliferation curve and FCM were used to detected the change of re-expression of Runx3 gene in human gastric carcinoma cell line named SGC-7901 which treated by 5-Aza-CdR and effection on grows of SGC-7901 cells by demethylated medicine. To Analysis of correlations between re-expression of Runx3 gene with the methylation of Runx3 gene in promoter region in order to find the mechanism of Runx3 gene deactivated action and basic of molecular biology in the tumorigenesis and development of gastric carcinoma. Methods: Human gastric carcinoma cell SGC-7901 were cultured by conventional method and the cells in logarithmic phase were selected as the object of study. SGC-7901 cells were divided into two groups by random when cells transfer of culture and cells of experiment group would be treated with 5-Aza-CdR to demethylation culture. Growing state and Cell growing curve of two groups would be recorded. RT-PCR and FCM were used to detecte the expression of Runx3 mRNA and apoptosis rate of carcinoma cells in two groups.Results: (1)The effection on human gastric carcinoma cells SGC-7901 growing by demethylation medicine: Using microscope, the morphocytology changing showed that the cells in experiment group by HE deying appeared the character of apoptosis including karyopyknosis, karyorrhexis, chromatin agglutination nearby arrounding karyotheca, chromatin fragmented, apoptosis bodies forming and so on. It is shown in cells prolife curve that demethylation medicine could inhibit the human gastric carcinoma cells SGC-7901 growing. (2) The effection on apoptosis rate of human gastric carcinoma cells SGC-7901 by demethylation medicine: It is shown by Using FCM that the apoptosis rate of human gastric carcinoma cells SGC-7901 by demethylation medicine in 1μmol/L group (10.42±0.30) %and 5μmol/L group (20.87±0.95) % was higher than control group (2.59±0.12)%. (3) The expression changing of Runx3 mRNA in human gastric carcinoma cells SGC-7901 by demethylation medicine: The re-expression of Runx3 mRNA was detected by using RT-PCR in human gastric carcinoma cells SGC-7901 by demethylation medicine. The integrated light intensity of Runx3 mRNA in experiment group and control group was (0.414±0.02) and (0.19±0.01), respectively.Conclusion: The re-expression of Runx3 mRNA on human gastric carcinoma cells SGC-7901 by demethylation medicine showed that the most reason of Runx3 gene deactivated was the hypermethylation of runx3 gene in promoter region. The Runx3 gene could reexpress by demethylation treat and suppress the human gastric carcinoma cells SGC-7901 and promote their apoptosis.Part Four The experimental study on expression change of Runx3 gene in nude mice which loaded human gastric carcinoma cells demethylation treated by 5-Aza-CdRObjective: Human gastric carcinoma cell SGC-7901 were cultured by conventional method and the cells in logarithmic phase were selected as the object of study. SGC-7901 cells were divided into two groups by random when cells transfer of culture and cells of experiment group would be treated with 5-Aza-CdR to demethylation culture and inoculated subskin of BALB/c nude mice respectively. Difference oncogenicity of human gastric carcinoma cell SGC-7901 in two groups were observed. RT-PCR used to detect the re-expression of Runx3 mRNA in human gastric carcinoma cells SGC-7901 by demethylation medicine. Analysis of result in order to investigate the mechanism of methylation of Runx3 gene in promoter region action in gastric carcinoma and provide the data of gastric carcinoma therapy by demethylation medicine.Methods: 24 female BALB/c nude mice were randomly divided into experiment group and control group (12 per group). Human gastric carcinoma cell SGC-7901 were cultured and the cells in logarithmic phase were selected as the object of study. In experiment group, mice were by subcutaneous (s.c.) inoculation of human gastric carcinoma cell SGC-7901 which treated by 5-Aza-CdR. In control group, mice were by s.c. inoculation of human gastric carcinoma cell SGC-7901 which non-treated by 5-Aza-CdR. The growing situation of two groups were observed until the subskin tumor formed. To compare the incidence rate and bulk of tumor in nude mice loaded gastric carcinoma cells between experiment group and control group.RT-PCR was used to detect the re-expression of Runx3 mRNA in transplantable tumor.Results: (1) Bulk of transplantable tumor in loaded nude mice:The incidence rate of forming transplantable tumor in experiment group and control group was 66.7% and 100%, respectively. In five weeks, the bulk of transplantable tumor in experiment group and control group was (1178.98±228.00) mm3 and (560.65±173.92) mm3, respectively (t=4.8215, P<0.01). (2) The expression of Runx3 mRNA in transplantable tumors of two groups. The size of amplification segment of Runx3 and GAPDH was as same as the one designed (353bp and 208bp). Relative expression value of Runx3 in experiment group and control group was (0.39±0.04) and (0.22±0.02), respectively (t=4.4275, P<0.01).Conclusion: Hypermethylation leads to lose the Runx3 gene expression in human gastric carcinoma. Methylation of Runx3 promoter maybe correlated to on cogenesis, metastasis of gastric carcinoma. Runx3 gene could re-express and inhibit the grows of gastric tumor through treating by demethylated medicine...