The Research On The Apoptosis-related Molecular Mechanism In The Development Of Rectal Adenocarcinomas

Rectal cancer is one of the most common tumors in digestive system.Rectal cancer is a disease of high mortality and poor prognosis.Most of these patients are in an advanced stage when they come to hospital and the exprerience a relapse and metastasis soon after operation. The cancer incidence is rising and younger trend in China. The occurrence of rectal cancer, development and prognosis are closely related with cell apoptosis. Improper regulation of apoptosis can lead to abnormal cell proliferation, which is often regarded as one of the mechanisms of tumor formation. The identification of the relationship between cell apoptosis related genes and the risk of rectal cancer deeply will not only contribute to the understanding of the function of the molecules involved in rectal carcinogenesis but also offer novel molecular tools to diagnosis and reveal potential targets for therapeutic intervention. Apoptosis in the cancer research has become a hot basic and clinical research, which has shown good prospects in clinic.In this study, we compared the gene expression differences between the rectal adenocarcinoma and normal rectal mucosal with Gene chip technology to find significantly different expression of apoptosis-related genes between tumor tissue and normal tissue.First part:A: Study on gene expression profiles related to apoptosis of rectal adenocarcinoma using cDNA Microarray in normal rectal mucosa and rectal adenocarcinoma tissuesAims: To investigate the gene expression profiles of apoptosis related to rectal cancer.Methods:Between January 1995 and May 2006 , 18 samples of rectal adenocarcinoma and corresponding normal rectum tissue were harvested during the resection for rectal caner in our department. TRIzol and Rneasy Kit were used to extract and purify the total RNA. Polymerase chain reaction (PCR) was employed to amplify 458 apoptosis-related genes in cDNA microarray. Three chips of apoptosis-related gene cDNA microarray (SBC-R-HC-101-10) were prepared by PixSys 7500 DNA Microarray System. We extracted mRNA from both the rectal adenocarcinoma and corresponding normal rectum tissue and produced probes for hybridization wih the cDNA microarray. The 18 samples were divided into three groups in randomization. After the hybridization between cDNA microarray and mixed-probe, Axon scanner (Scan resolution as a resolution of 10μm, PMT 100%) was used to observe the result, and data was analyzed by ImaGene 3.0. Results: Of the 458 apoptosis-related genes, 38 genes were detected having≥2-fold difference of expression between the cancerous and paracancerous tissue, with 27 genes up-regulated and 11 genes down-regulated. Conclusions: The 27genes involved in the whole apotosis pathway might be relevant to the pathogenesis of gastric adenocarcinoma, and further studies are warranted.B:The mehtod of Real-time fluorescence quantitative PCR was performed to detect the expression of TNFRSF7,TNFRSF7 mRNA in the cancerous and paracancerous tissue.Aims: To verify the apoptosis-related gene chip test results in former study, we analyzed the expression of TNFRSF7,TNFSF7 mRNA in the cancerous and paracancerous tissue by Real-time fluorescence quantitative PCR.Methods: The cancerous and paracancerous tissue were collectted from 9 patients with rectal cancer. The mehtod of Real-time fluorescence quantitative PCR was performed to detect the expression of TNFRSF7,TNFRSF7 mRNA in the cancerous and paracancerous tissue.Results: The relative gene copy number of TNFRSF7,TNFSF7 and Actin mRNA in cancerous tissue was significant higher than that of paracancerous tissue(p≤0.01). There was no significant difference in the initial relative gene copy number of TNFRSF7 mRNA between cancerous and paracancerous tissue, when we calculate the ratio of the initial relative gene copy number of TNFRSF7,TNFSF7 mRNA according to that of Actin mRNA.Conclusions: The TNFRSF7 and TNFRSF7 play a key role in the oncogenesis development and lymph node metastases of rectal cancer and their expressions could be as useful indexes to assess the malignant level of rectal carcinoma and its clinical prognosis.C:The method of in situ hybridization was performed to detect the expression of TNFRSF7,TNFSF7 mRNA in the cancerous and paracancerous tissue.Aims: To verify the apoptosis-related gene chip test results in former study, we analyzed the expression of TNFRSF7,TNFSF7 mRNA in the cancerous and paracancerous tissue by Fluorescence in situ hybridization FISH. Methods:The cancerous and paracancerous tissue were collectted from 9 patients with rectal cancer. The method of in situ hybridization was performed to detect the expression of TNFRSF7,TNFSF7 mRNA in the cancerous and paracancerous tissue. Results: the hybridization signal of biotinylated probe of TNFRSF7 and TNFSF7 could be detected in all kinds of rectal adenocarcinoma and epithelium mucosae of 10cm length adjacent to the tumor, in which the hybridization signal of TNFRSF7 is inferior to that of TNFSF7. The analysis of hybridization results by means of paired t-test demostrated that the signal of mRNA of TNFSF7 and TNFRSF7 was much higher than that of the normal epithelium mucosae 10cm adjacent to the tumor(p<0.01).Conlusions: The TNFRSF7 and TNFRSF7 play a key role in the oncogenesis,development and lymph node metastases of rectal cancer and their expression could be as useful indexes to assess the malignant level of rectal carcinoma and its clinical prognosis.D:Expression of CD27(TNFRSF7),CD70(TNFSF7)in rectal cancer tissue and their clinical significanceAims:To explore the expression of TNFRSF7 and TNFSF in rectal cancer tissue and its clinical significance. Methods: The TNFRSF7 and TNFSF were detected with immunohistochemical stain method in 60 rectal cancer tissues and adjacent non-tumor normal tissues, the relationship between TNFRSF7 and TNFSF and some clinicopathological factors were analyzed withχ2. Results: The positive TNFRSF7 was mostly located in plasmo-cells with rates of 1.67%. While TNFSF7 was mostly located in plasmo-cells with rates of 33.33%. TNFRSF7 expression had no correlation with rectal cancer tissues and adjacent non-tumor normal tissues. The expressions of TNFSF7in rectal carcinoma was significantly higher than that in adjacent non-tumor normal rectal tissues (p<0.05). Conclusions: The gene products TNFSF7 play a key role in the generation and development of rectal cancer and their expression could be as a useful index to assess the malignant level of rectal carcinoma and its clinical prognosis. Second part:A:Expression of CD70 in rectal carcinoma and its suppressive impact on the proliferation of lymphocytesAims:To investigate the expression of the CD70 gene in rectal carcinoma and its role in the immune escape of rectal carcinoma. Methods:The expression of CD70 was determined by RT-PCR. The eukaryotic expression vector was constructed and transiently transfected into the LOVO cell line. After being co-cultured with Jurkat cells, the effects of CD70 on inhibiting lymphocyte proliferation were assessed by the Cell counting kit8(CCK8) . Results:CD70 gene presented in SW117 and SW620 but not in LOVO. CD70 was positive in three cases of rectal carcinoma while it was negative in two cases of rectal tissues adjacent to cancer tissues. The eukaryotic expression vector was successfully constructed and transiently transfected into the LOVO cell line. The LOVO cell line transfected with the CD70 gene can significantly inhibit the proliferation of PHA-activated Jurkat cells.The prolife ration inhibition rate of Jurkat cells cocultured with LOVO cells transfected by the CD70 gene was 64.99%, which was significantly higher than that of Jurkat cells and Jurka cells coculturedwith LOVO cells (37.98% and 29.96%). Conclusions:The CD70 gene is expressed in rectal carcinoma andcan inhibit the proliferation of lymphocytes. The CD70 gene may play an important role in the immune escape of rectal cells and their expression could be useful indexes to assess the malignant level of rectal carcinoma and its clinical prognosis.B:Silencing of CD70 in rectal carcinoma and its suppressive impact on the proliferation of lymphocytesAims: To investigate the feasibility of Silencing of CD70 gene with interfering RNA , and observe the role in the immuneescape of rectal carcinoma.Methods: CD70 specific siRNA oligonucleotides were designed and synthesized artificially. siRNA was transfected into rectal carcinoma SW117 and SW620 cells to inhibit the expression of CD70 in vitro. The mRNA level of CD70 was detected by RT-PCR. After being co-cultured with Jurkat cells, the effects of CD70 on lymphocyte proliferation were assessed by the CCK-8. Results: The expression of CD70 mRNA in siRNA transfected samples was downregulated greatly. There was a significant difference between the RNAi cells and negative controls (P<0.05). The proliferation inhibition rate of Jurkat cells co-cultured with CD70 Silencing cells was significantly lower than the negative controls ( P< 0.05) . Conclusions: The specific siRNA targeting CD70 can effectively the expression of CD70 mRNA, which induce the proliferation inhibition rate of Jurkat cells co-cultured with silencing groups decreased obviously, in human rectal carcinoma SW117 and SW620 cells. The CD70 gene may play an important role in the immune escape of human rectal carcinoma cells and their expression could be useful indexes to assess the malignant level of rectal carcinoma and its clinical prognosis.