| Research Background:Bladder cancer is reported to be one of the ten most common tumors in the whole body,and also the most common malignant tumor in urinary system diseases.It occupies the first place in the incidence of urogenital system tumors in China.In recent years,with the continuous improvement of various treatment methods and the application of various surgical methods and drugs,treatment strategies for bladder cancer are becoming more and more abundant.However,the prognosis of patients with bladder cancer is still not optimistic,and the 5-year survival rate of patients has not been significantly improved.Bladder tumors are classified as urothelial cell carcinoma(transitional epithelial carcinoma),squamous cell carcinoma,adeno-carcinoma,and rare pathological classifications such as small cell carcinoma,mixed carcinoma,carcinosarcoma,and metastatic carcinoma.Among them,the vast majority of bladder tumors are transitional cell carcinoma(TCC),which accounts for over 90%of all bladder tumors.However,the relevant mechanism of its regulation of invasion has not been fully understood so far.Therefore,the research on its occurrence and development mechanism is crucial to find new targets for the treatment of bladder cancer.The transient receptor potential(TRP)superfamily was first discovered in the visual system of fruit flies as a kind of non-selective cationic channel family,which is widely distributed in lower and higher organisms.Currently,more than 30 members have been found.According to their homology,these proteins could be divided into 7 different subfamilies:TRPC(canonical),vanilloid(TRPV),TRPM(melastatin),TRPA(ankyrin transmembrane protein),TRPML(mucolipin),TRPP(polycystin)and TRPN(nompc-like).Among them,TRPV is the largest and most diverse important subfamily,including six members of TRPV1-6,which can participate in the regulation of a variety of cell functions.TRPV4 channels are extensively expressed in feeling,digestive,respiratory and cardiovascular system and participate in a variety of physiological and pathological process,TRPV4 can be activated by physical and chemical stimulus such as temperature,shear stress,osmotic pressure,in order to participate in the regulation of the body’s physiological functions.In recent years,researchers have found that the abnormal expression of TRPV4 plays an important role in promoting or inhibiting tumor development,and TRPV4 has gradually become the focus of tumor research.Activation of TRPV4 channels result in increase of intracellular Ca2+,Ca2+ is the important intracellular signal which may contribute to the pathogenesis of cancer.Therefore,we hypothesized that the abnormal expression of TRPV4 channel might play a role in the development of bladder cancer.Research Objective:The role of TRPV4 in the development of bladder cancer was explored by comparing the function and protein expression of TRPV4 channels in normal bladder epithelial cells and bladder cancer cell lines.Research Method:Human normal bladder epithelial cell line SV-HUC-1,human bladder cancer 5637,T24,J82,SW1710 cell lines were cultured in vitro.Under the fluorescence microscope using calcium imaging method to TRPV4 channels agonist GSK 1016790 a activate TRPV4 channels,record the five cell lines within the intracellular calcium ion flow amplitude and a reaction cell percentage,the dose-response curve,it is concluded that half the effect of concentration(EC50)and maximum response amplitude(Max)and compare the EC50 and Max is the difference between the cell line,in order to observe difference of TRPV4 channels function expression.ATP was used to activate urothelial purine receptors,and the cell activity of the five cell lines was observed and recorded.TRPV4 expression at protein levels in normal bladder epithelial cell line SV-HUC-1 and bladder cancer cell lines 5637,T24,J82 and SW1710 was examined by immunohistochemical method.So does human bladder cancer adjacent tissues and tumor tissues.ANOVA and Dunn’s Method statistics were used to analysis the calcium influx induced amplitude difference caused by GSK in each cell lines;The chi-square test was used to calculate the difference in the percentage of cells responding to GSK.Research Result:TRPV4 channel receptor agonist GSK evoked intracellular increase were recorded in the nnormal bladder epithelial cell line(SV-HUC-1)and bladder cancer cell lines 5637,T24 and J82 with calcium imaging method.The amplitude of intracellular calcium increase evoked by GSK1016790A of normal bladder epithelial cells(NBC)were significantly higher than those of low-grade bladder cancer cell line 5637 and high-grade bladder cancer cell line T24.GSK did not evoked any change of intracellular Ca2+ in J82 and SW1710 bladder cancer cells.The immunohistochemical test showed that the expression of TRPV4 channel in normal bladder epithelial cells(NBC)was higher than that in other bladder cancer cells.The results of cell culture showed that all the cells in the normal bladder epithelial cell line SV-HUC-1 and the four bladder cancer cell lines showed polygonal growth,suggesting that the cells have been differentiated and there is no significant difference in morphology.Calcium imaging results showed that GSK induced a concentration-dependent increase of intracellular calcium in normal bladder epithelial cell line SV-HUC-1,bladder cancer cell line 5637 and T24 cells,with EC50 of 38.25nM,1.29nM,0.97nM,respectively.;the maximum amplitude of calcium influx was 1.39±0.02,0.40 ±0.08,and 0.63 + 0.03,respectively.However,there was no increase in intracellular calcium in any of the bladder cancer cell lines SW1710 and J82 stimulated by GSK,and EC50 and Max could not be calculated.When the intracellular calcium ion increase caused by a single concentration of 30nM GSK stimulation was compared,it was found that the intracellular calcium influx of the normal bladder epithelial cell line SV-HUC-1 was significantly higher than that of the bladder cancer cell lines 5637 and T24.The maximal increase of intracellular calcium influx was 1.07±0.07,0.37±0.04 and 0.60+ 0.12,and there was no change of intracellular calcium concentration in bladder cancer cell lines J82 and SW1710.Compared normal cell line with other four cancer cell lines,the significant difference(ANOVA,P<0.01),the normal cell line and other cell lines showed a difference between the two(each P<0.01).When stimulated by 30nM GSK,the percentage of cells in the normal epithelial cell line SV-HUC-1,bladder cancer cell lines 5637,T24,SW1710 and J82 cells with calcium influx was 52.20%,1%9 25%,0 and 0,the normal cell line was significantly different from the other four cancer cell lines(chi-square test,P<0.01).The ATP response was used as an indicator of cell viability.The results showed that there was no significant difference in the response of 100uM ATP to each cell line(P>0.05),which indicated that the difference in calcium influx caused by GSK in each cell was not caused by the difference in cell viability.The results of immunohistochemistry showed that the TRPV4 channel receptor proteins of these five cell lines were expressed in different degrees,but compared with the normal bladder epithelial cell line SV-HUC-1,the expression of bladder cancer cell lines was weak,indicating Protein expression of the TRPV4 channel receptor is reduced in bladder cancer cell lines compared to normal bladder epithelial cell lines.At the same time,we also compared the expression of TRPV4 channel protein in human bladder cancer tissues and human tumor tissues,and obtained the same results..Research Conclusion:Our results indicate that the TRPV4 channels of the four bladder cancer cell lines are lower in functional expression level or protein expression than normal bladder epithelial cell lines,suggesting that TRPV4 may play a role in the development of urothelial carcinoma,and TRPV4 channels may become Future targets for the treatment of bladder cancer. |
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