Research On The Role And Mechanism Of TSPAN7 In The Occurrence And Development Of Bladder Cancer

Part 1 The expression and clinical significance of TSPAN7 in bladder cancer tissues and cell linesObjective: To explore the expression of TSPAN7 in bladder cancer tissues and adjacent tissues as well as in the cell lines,further to investigate the relationship between TSPAN7 expression and the prognosis of patients with bladder cancer.Methods: In this study,we screened out the target gene TSPAN7 using bioinformatics methods from TCGA database at first,and then detected the expression levels of TSPAN7 in 34 pairs of bladder cancer tissues and adjacent tissues through q RT-PCR,WB,and IHC.At the same time,the chi-square test was used to analyze the clinicopathological characteristics of each patient and their relationship with the expression level of TSPAN7,and to evaluate the prognosis of each patient.In addition,we also detected the expression of TSPAN7 in 3 kinds of human bladder cancer cell lines and 1 kind of human normal bladder epithelial cell line.Results: 16 key marker genes were selected through bioinformatics analysis,and TSPAN7 was selected as the research gene through comparison of p value and q RT-PCR verification.The results of q RT-PCR,WB,and IHC suggest that TSPAN7 is in BCa tissue specimens and cell lines The expression of TSPAN7 was down-regulated,and the expression level of TSPAN7 was related to the tumor grade(p=0.03)and tumor stage(p=0.01)of patients with BCa.The decreased TSPAN7 expression was significantly related to the overall survival of the patient.Conclusion: TSPAN7 is lowly expressed in bladder cancer and maybe play a tumor suppressor role in the progression of bladder cancer.The expression level may be a molecular marker for evaluating the clinical prognosis of patients with bladder cancer.Part 2 TSPAN7 inhibits the proliferation,migration and invasion of bladder cancer cellsObjective: To evaluate the relationship between TSPAN7 and bladder cancer cells,and to further explore the ways in which changes in TSPAN7 expression levels affect the progression of bladder cancer.Methods: Firstly,a TSPAN7 overexpression bladder cancer cell line was constructed by transfection with lentivirus.CCK-8 detection,wound healing,clone formation and transwell analysis were performed in vitro to detect the effect of TSPAN7 on the proliferation and migration of bladder cancer cell lines.Flow cytometry analysis of the effects of TSPAN7 on the apoptosis and cell cycle of T24,5637 and EJ cell lines,as well as WB detection of the changes of related apoptotic proteins.Finally,in nude mice,the effect of TSPAN7 overexpression on the growth of subcutaneous tumors in nude mice was tested.Results: In vitro,TSPAN7 overexpression inhibited the proliferation,viability,migration and invasion ability of BCa cells.Overexpression of TSPAN7 induced the higher percentage of apoptotic cells than that in the control group.Further WB was performed to detect some apoptosis-related proteins.The results suggested that in the TSPAN7 overexpression group,the expression of cleaved Caspase-3 and Bax were up-regulated,while the expression of Bcl-2 was downregulated.At the same time,TSPAN7 overexpression can induce cell cycle arrest in G1/S phase,the expression of CDK2 and cyclin E in the TSPAN7 overexpression group is down-regulated.The results show that compared with the control group.Finally,TSPAN7 can inhibit bladder tumor growth in vivo.Conclusion: TSPAN7 can inhibit the proliferation,migration and invasion of T24,5637 and EJ cell lines,and at the same time promote tumor cell apoptosis and induce cell cycle arrest in G1/S phase.Part 3 The molecular mechanism of TSPAN7 inhibiting the growth of bladder cancerObjective: To explore the molecular mechanism of TSPAN7 regulating the growth of bladder tumors.Methods: Firstly,the changes of PTEN/PI3K/AKT pathway related proteins were detected by WB,and whether the addition of the AKT agonist SC79 could reverse the effect of TSPAN7 overexpression on T24 cells,and the PTEN inhibitor VO-Ohpic trihydrate was used to verify whether it passed PTEN inhibits the PI3K/AKT pathway and leads to a decrease in the proliferation of the BCa cell line,and then performs cck-8,clone formation and transwell invasion to complete the capillary experiment.Results: After SC79 treatment,the p-AKT level in T24 cells increased significantly,while after VO-Ohpic trihydrate treatment,PTEN expression was significantly inhibited.Compared with the untreated group,the cells treated with SC79 or VO-Ohpic trihydrate had a corresponding effect on the expression of related proteins,and the proliferation,migration and invasion ability of T24 cells were also affected.SC79 and VO-Ohpic trihydrate could partly Reverse the inhibitory effect of TSPAN7 overexpression on T24 cells.Conclusion: TSPAN7 regulates the PTEN/PI3K/AKT pathway by activating PTEN while inhibiting the activity of PI3 K upstream of AKT.