Study On Relationship Between TRAIL Expression And Multidrug Resistance Gene SIRT1

Esophageal Carcinoma is a malignant tumor that occurr on esophageal epithelial tissue, it accounts for 2% in all malignant tumor. Our country is one of the most esophageal carcinoma, every year, there are 150 thousand people died of Esophageal Carcinoma. Postop chemotherapy is one of the most important treatments for Esophageal Carcinoma.In these years, as the non-standard use of drugs, genetic instability of tumor cells, heterogeneity of expression, high mutation rates, individuals with different chemotherapy regimens have great differences in the sensitivity and other reasons, which leads to poor efficacy of many patients.Silent information regulator type 1 (SIRT1) is a human class III histone deacetylase,which allocates on human's chromosome-10q22.1, its total length is 33kb and molecular weight is 60KD, it has NAD+-dependent deacetylase activity. It plays an important role in the DNA damage repair, cell cycle controlling, inhibit apoptosis, against oxidative stress and the extension of cells, so SIRT1 is known as the "longevity genes".Tumor necrosis factor (TNF) -related apoptosis-inducing ligand(TRAIL) has higher sequence homology with FasL and TNF, also known as Apo2L, which allocates on human's chromosome-3q26, its total length is 1769bp and molecular weight is 32.5KD. It codes 281 amino acid sequences. TRAIL has higher anticancer against many cancer cells, however, it has lowest cytotoxicity against normal cell. Just because of this character, TRAIL causes so many attentions. It has reported that TRAIL could produce a synergism effect with radiotherapy, chemotherapy and other methods, thereby could achieved to reverse tolerance and multidrug resistance of tumor.However, the relationship between TRAIL and drug-resistance gene SIRTl has been few studied and has not been reported. In this study, we constructed a eukaryotic expression vector pcDNA3.1 (+)-TRAIL, used liposome transfect esophageal carcinoma cell and studied more on the expression and biological function of TRAIL in esophageal carcinoma cell,which also studied on TRAIL against drug tolerance gene SIRT1, which is the basis for follow-up experiment. Objects:Constructing a eukaryotic expression vector pcDNA3.1 (+)-TRAIL, detecting its expression and biologic activity and studying on TRAIL against drug tolerance gene SIRT1, which is the basis for follow-up experiment. Methods:We attained TRAIL from the prokaryotic plasmid pCA13-TRAIL by being digested with HindⅢand BamHⅠ, and attained 848bp fragment; At the same time, pcDNA3.1 (+) was digested with HindⅢand BamHⅠ, and linked by using T4 ligase. So we have constructed recombinant eukaryotic expression vector pcDNA3.1 (+)-TRAIL, identifying with incision enzyme. We transfect the recombinant eukaryotic expression vector into the esophageal carcinoma cell, after 48h, we detected its expression of mRNA and protein in cells by using RT-PCR and Immunocytochemistry, we observed cells' biological change in the apoptosis progress by using light microscope. Then we detected recombinant plasmids'apoptosis in esophageal carcinoma cells by using flow cytometry after 24h and 48h. We also detected recombinant plasmids'expression and biologic activity against drug tolerance gene SIRT1 by using RT-PCR and Immunocytochemistry.Results:1 Construction and identification of recombinant eukaryotic expression vector pcDNA3.1(+)-TRAIL: We attained TRAIL from the prokaryotic plasmid pCA13-TRAIL by being digested with HindⅢand BamHⅠand attained 848bp fragment, pcDNA3.1(+) was also digested then linked by using T4 ligase, so we have constructed recombinant plasmid. Digested with HindⅢand BamHⅠ, we attained 5400bp and 800bp fragments, the result confirmed that TRAIL gene has been embedded successfully to the plasmid pcDNA3.1 (+).2 Identify the expression of TRAIL gene in esophageal carcinoma cell:The recombinant plasmid was transfect into the esophageal carcinoma cell, after 48h, agarose gel electrophoresis showed that there is more TRAIL gene expression product in transfect group than un-transfect group. Electrophoresis results were detected by the software showing the Gray Value of un-transfect group and transfect group were: 1.05±0.33 and 1.80±0.35, the results showed that the expression of TRAIL gene in transfect group was significantly higher than in un-transfect group (P<0.05). Immunocytochemistry results showed that the expression of TRAIL gene located in the cell membrane and cytoplasm, results were detected by the software showing the Gray Value of transfect group and un-transfect group were:118.23±0.46 and 78.19±0.41, the expression of TRAIL gene in transfect group was significantly higher than in un-transfect group.3 Apoptosis activity of TRAIL in the esophageal carcinoma cell:Flow cytometry results displayed that there has apoptosis curve after 48h but not after 24h. This reported that apoptosis activity of TRAIL has reached to peak time after 48h(P<0.05).4 The effect of recombinant plasmid against drug tolerance gene SIRT1:The recombinant plasmid was transfect into the esophageal carcinoma cell, after 48h, agarose gel electrophoresis showed that there is lower SIRT1 gene expression product in transfect group than un-transfect group. Electrophoresis results were detected by the software showing the Gray Value of un-transfect group and transfect group were 1.79±0.35 and 1.06±0.32, the results showed that the expression of SIRT1 gene in the transfect group was significantly lower than in the un-transfect group (P<0.05). Immunocytochemistry results showed that the expression of SIRT1 gene located in the cell nucleus and the expression of SIRT1 gene in the transfect group was significantly lower than in the un-transfect group, results were detected by the software showing the Gray Value of the transfect group and the un-transfect group were 78.18±0.42 and 118.24±0.46, which reported that TRAIL gene can inhibit the expression of drug tolerance gene SIRT1 mRNA and protein (P<0.05). Conclusions:1 Recombinant eukaryotic expression vector pcDNA3.1 (+)-TRAIL has been constructed successfully and it can express great quantity of TRAIL mRNA and protein product in human esophageal carcinoma cell.2 TRAILGene can cause apoptosis effect in human esophageal carcinoma cell.3 Recombinant plasmid TRAIL Gene can inhibit the expression of drug tolerance gene SIRT1 mRNA and protein, which can decrease drug resistance...