| Background and Objective:Bladder cancer is the most common malignant tumor in urinary system. The incidence and mortality of bladder cancer ranking the first among all urinary tumors.Ubiquitin-proteasome system( UPS) is an important pathway to degrade intracellular proteins.F-associated transcript 10( FAT10) which has a similar structure and function with ubiquitin,moreover,in contrast to ubiquitin,a single FAT10 suffices to bind to the 26 S proteasome and to efficiently mediate proteasomal degradation in a ubiquitin-independent manner, FAT10 plays a significant role in the regulation of cytobiology functions.Survivin is a member of inhibitor of apoptosis proteins gene family, it can inhibit cell apoptosis and promote cell mitosis and regulate cell cycle and increase cell proliferation.In our study, we will first explore the relation between clinical significance and the expression of FAT10 and survivin in bladder cancer tissues; Secondly,we will observe the effect of FAT10 regulating survivin on bladder cancer proliferation by gene transfection and RNA interference technology in vivo and in vitro;Finally, we will clarify the mechanism through which FAT10 regulates survivin by immunoprecipitation, laser scanning confocal microscope and in vivo ubiquitination assay. This study will provide the new theoretical basis for clarifying the mechanism of bladder cancer proliferation and target for bladder cancer gene therapy.Methods:1、133 cases of bladder cancer which were followed up for more than 5 years,the expression of FAT10 and survivin in paraffin blocks of bladder cancer were detected by immunohistochemistry,the correlation between FAT10 and survivin expression were analyzed,and the relationship between clinical significance and prognosis with the expression of FAT10 and survivin in bladder cancer tissues were analyzed.2、Bladder cancer tissue and adjacent tissue in 50 patients with bladder cancer were collected,the expression of FAT10 and survivin in the bladder cancer tissues and the corresponding adjacent tissues were detected by q RT-PCR and Western blot,and whether there is a correlation between each other were analyzed.3、The expression of FAT10 and survivin in the bladder cancer cells T24、J82、5637、UM-UC-3 and normal cell line human ureteral epithelial cells SV-HUC-1 were detected by q RT-PCR and Western blot.4 、 Construction of four sh FAT10 interference plasmid were transfected into bladder cancer cell, and analyze the expression of FAT10 by q RT- PCR and Western blot,we screened the best interference plasmid of sh FAT10.The best sh FAT10 interference plasmid was transfected into 5637, and analysis FAT10 and survivin protein expression by Western blot, at the same time Ed U experiment was used to observe the change of bladder cancer cell proliferation.Over expressed FAT10 plasmid was transfected into UM-UC-3, and analyze the expression of FAT10 and survivin protein by Western blot, at the same time Ed U experiment was used to observe the change of bladder cancer cell proliferation.Nude mice subcutaneous tumor model which was inject with establish FAT10 stable low expression cell lines were used to observe the growth of tumor in nude mice.5、Construction of four shsurvivin interference plasmid were transfected into bladder cancer cell, and analyze the expression of survivin by q RT- PCR and Western blot,we screened the best interference plasmid of shsurvivin.We will increase the expression of survivin in FAT10 knockdown bladder cancer cell and decrease the expression of survivin in FAT10-overexpressing bladder cancer cell, and then we will observe the FAT10 and survivin protein expression levels and cell proliferation by Western blot and Ed U.6 、 Finally, we will clarify the mechanism through which FAT10 regulates survivin by immunoprecipitation, laser scanning confocal microscope and in vivo ubiquitination assay.Result:1、The positive rate of FAT10 was 52.6% and the positive rate of survivin was56.4% in 133 cases of bladder cancer,there was a positive correlation between each other(P<0.001);The expression of FAT10 and survivin in the bladder cancer tissues and the corresponding adjacent tissues were detected by q RT-PCR and Western blot,the results showed that the expression of FAT10 and survivin m RNA and protein in cancer tissue was significantly higher than that in adjacent tissue(P <0.05),FAT10 and survivin were positively correlate with each other by the analysis of the scatter plot(P <0.05);The expression of FAT10 and survivin m RNA and protein in bladder cancer cells T24、J82、5637、UM-UC-3 were significantly higher than that in normal cell line human ureteral epithelial cells SV-HUC-1 which were detected by q RT-PCR and Western blot(P <0.05);The expression of FAT10 and survivin were related to the tumor stage(P <0.05),the 5 years overall survival rate and the 5 years disease-free survival rate of FAT10 positive expression group and survivin positive expression group were lower than those of negative expression group(P<0.05);The expression of FAT10 and survivin of patients was divided into four categories: FAT10(-) survivin(-), FAT10(+) survivin(-) FAT10(-)(+), survivin, FAT10(+) survivin(+),the results showed that the average survival time of FAT10(+)survivin(+) was the shortest(P<0.05); Multivariate Cox regression analysis showed that tumor grade was the influencing factor of independent prognostic factors.2、Transfect sh FAT10 into 5637 cell line found that FAT10 protein expression level was significantly decreased(p<0.05), also found that the expression of survivin also reduced(p<0.05),at the same time, Ed U experiment found that cell proliferation was significantly decreased(p <0.05); Over expressed FAT10 plasmid was transfected into UM-UC-3,found that FAT10 protein expression level was significantly increased(p<0.05), also found that the expression of survivin also raised(p<0.05),at the same time, Ed U experiment found that cell proliferation was significantly increased(p<0.05);in vivo,successfully established tumor model of human bladder cancer in nude mice and found the sh FAT10 group were significantly smaller than the sh NC group(p<0.05),the tumor weight was significantly less than the control group(p<0.05).3、Western blot showed that the downregulation of FAT10 decreased survivin expression, whereas the upregulation of survivin attenuated the loss of survivin expression in FAT10 knockdown 5637 cell. We also found that the knockdown of FAT10 dramatically decreased the proliferation abilities of 5637 cell, whereas the upregulation of survivin rescued the decreased proliferation abilitiesinduced by FAT10 knockdown;Also,Western blot results showed that overexpression of FAT10 significantly increased the expression of survivin, whereas the knockdown of survivin expression dramatically inhibited the increase of survivin expression induced by FAT10 in UM-UC-3 cell. meanwhile, the downregulation of survivin significantly reduced FAT10-enhanced cell proliferation. Thus, these results confirm that survivin is essential for FAT10-mediated bladder cancer proliferation.4、Immune coprecipitation and confocal laser scanning showed that survivin and Ub were directly combined,survivin was degradated through the ubiquitinproteasome pathway in bladder cancer cell; Immune coprecipitation and confocal laser scanning also showed that survivin and FAT10 were directly combined;FAT10stable survivin was by antagonizing the ubiquitination and degradation of survivin expression.Conclusion:FAT10 could affect the proliferation of bladder cancer by antagonizing the ubiquitination and degradation of survivin expression. |
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