The Expression Of LRIG3 In Bladder Urothelial Carcinoma And The Effects Of LRIG3 Gene Recombinated Adenovirus Plasmid On T24

PartⅠ. The expression of the LRIG3 gene in Urothelial carcinoma of thebladder and its clinical significanceObjective: To investigate the relationship between the expression of leucine-richrepeats and immunoglobulin-like domain protein 3 (LRIG3) gene and the biologicalbehavior of bladder urothelial carcinoma (BUC).Methods: Expressions of LRIG3 in 64 specimens of bladder urothelial carcinoma and12 specimens of normal bladder tissues were detected by SP of immunohistochemistry andRT-PCR. The results were analyzed in relation to the clinical data.Results :The expression of LRIG3 protein and mRNA in bladder cancer weresignificantly lower than that of normal bladder epithelium. The expression of LRIG3protein and mRNA were significantly decreased in higher staging and grading tumorscomparing to lower ones.Conclusions: The expression of LRIG3 gene was lost or reduced in bladder urothelialcarcinoma, which indicates that the LRIG3 gene may play an important role in suppressingthe tumorigenesis and development in bladder urothelial carcinoma.It may be a new targetfor tumor gene therapy. PartⅡ. The construction and identification of Adenovirusexpression vector targeting LRIG3 geneObjective : To construct and identify adenovirus vector that expresses the LRIG3 gene(rAd-LRIG3) and to establish a monoclone T24 cell line transfected with plasmidrAd-LRIG3, which will enable development of a gene therapy protocol for the treatment ofhuman bladder carcinoma.Methods: Genetic engineering techniques, including plasmid extraction, agarose gelelectrophoresis, restriction enzymolysis, connection, transformation of competent cells,were used to construct the recombinant plasmid of pLRIG3-EGFP from competentescherichia coli DH5α, which is confirmed by restriction enzyme digestion, agarose gelelectrophoresis and sequencing identification. The adenovirus plasmid rAd-LRIG3 wasconstructed by Adeno-x expression system, and the control adenovirus plasmid rAd-HKwas constructed by the same system. After verification by enzymolysis and PCR, thepAd-LRIG3 vector was cotransfected into 293 cells where they were packed as thereplication-deficient adenovirus rAd-LRIG3, rAd-LRIG3 was abundantly amplified andthen virus titer was evaluated. The whole experiment was divided into 3 groups. TherAd-LRIG3 was used to infect the human bladder cancer T24 cells. For controlexperiments, the vector rAd-HK was also transfected into T24 cells and nontransfected T24cells. RT-PCR and Western Blotting were used to detect the LRIG3mRNA and proteinexpression in T24 cell lines.Results:rAd-LRIG3 was successfully constructed and verified by enzymolysis andsequencing. RT-PCR and W-B analysis showed that LRIG3mRNA and protein level inrAd-LRIG3 transfected T24 cells was significantly higher than that in rAd-HK transfectedand nontransfected T24 cells; the difference was significant(P＜0.05).Conclusions: Successful construction of the recombinant adenovirus vector containingthe LRIG3 gene was achieved. A T24 cell line transfected with rAd-LRIG3 was obtained, which could stably express the LRIG3 protein in cells, and it will establish a foundation forthe future research about bladder cancer gene therapy.PartⅢ. The Effects of LRIG3 Gene on the Biological Behaviors ofHuman Bladder Cancer Cell (T24)Objective: To investigate whether the expression of gene LRIG3 can effect thegrowth, proliferation and apoptosis of bladder cancer T24 cells.Methods: The whole experiment was divided into 3 groups. The rAd-LRIG3 was usedto infect the human bladder cancer T24 cells. For control experiments, the vector rAd-HKwas also transfected into T24 cells and nontransfected T24 cells. The effect of LRIG3 onthe cellular proliferation capacity of T24 cells was assayed by the growth curve. The cellapoptosis was detected by Hoechst33258 staining, electron microscope, and flow cytometryanalysis. The cell migration assay was used to detect the difference of invasion andmetastases between transfected and non-transfected cells.Results: MTT showed the cell proliferation was markedly inhibited compared with thecontrol cells. Partial cancer cells presented morphological changes of apoptosis byHoechst33258 staining , electron microscope and flow cytometry analysis. Contrast tocontrol cells and rAd-HK transfected T24, the invasion ability of rAd-LRIG3 transfectedcells decreased obviously.Conclusion: LRIG3 gene can suppress T24 cell growth and proliferation, in additionto acceleration of its apoptosis. LRIG3 may act as a tumours suppressor gene in bladdercancer progression. This implied the possibility of LRIG3 as the potential method for genetherapy of bladder cancer.