The Experimental Study Of The RNA Interference Targeting AR Gene On Inducing Apoptosis Of Bladder Cancer Cell Line T24

There is a question about the bladder cancer which has been existing for many years. That is the obvious sexual difference. Morbidity rate in males is two times more than females. For a long time it has been attributed to the higher smoking prevalence and more exposure to dangerous carcinogenic substance. But with increasing females of various trades and professions and smokers nowadays, this sexual difference still exists.Sexual hormones have become more and more attractive on carcinogenic field. Initially researchers put focus on the target organs of sexual hormones, such as lactiferous gland, ovary and prostate. In 1984, Ahmed confirmed that there were Androgen Receptor(AR), Estrogen Receptor(ER) and Progesterone Receptor(PR). It then came into sight that there may be relationship between urinary tumors and sexual hormones. As AR, ER and PR had been found in bladder cancer tissue, it was assumed that sexual hormones had influence the occurrence and progress of bladder cancer through the receptors.AR has extensive distribution in human body. Besides the genital organs, it exits widely in central nervous system, skeletal muscle, kidney and liver and in tumors such as prostatic carcinoma, hepatocellular carcinoma, intestinal cancer and bladder cancer. Li had tested AR in 64 cases of BTCC and the positive rate is much higher than it was in normal bladder tissue. And it suggest that AR positive rate have negative correlation with tumor grade. Chen suggest AR as a new biological marker and it may predicting prognosis in bladder cancers. The intravesical therapy inducing apoptosis has become the main means on preventing postoperative recurrence of bladder cancer. But the recurrence rate is still high due to the resistance to chemotherapeutic drugs. It prevents tumor cells from apoptosis. There are many evidences to prove the caspase activation in tumor cell apoptosis. caspase-3, key point in caspase pathway, has played an important role in the bladder cancer.RNA interference(RNAi), as a efficient technique in gene silencing, has become a hopeful and effective means for gene therapy. Through RNAi, we can silence AR gene and investigate its effect in bladder cancer. Does androgens enhance growth of bladder cancer through AR? What's the relationship between AR and apoptosis of BTCC? And the mechanism?It will possibly draw a new way to prevent and treat the bladder cancer if we solve these problems satisfactorily.In the background above, the study include the following three parts: Purpose:To investigate the effect of DHT to bladder cancer cell line T24 and AR expression.Methods:Through cell culture and RT-PCR, we tested expression of ARmRNA of T24 cells in different DHT concentration(0,10-10M,10-9M,10-8M); we also made cell growth curves using MTT method to observe the effect to cellular proliferation of T24 cells.Results:The amplification of Real-time PCR was acceptable which could be proved by satisfactory dissolution curve without impure peaks or abnormal broadening of main peaks. Through data analysis, we found that there was no significant influence to AR expression in different DHT concentration(P>0.05). And there was no significant difference(P>0.1) in the OD value between the 4th-day and the 5th-day MTT result under different DHT concentration.Conclusion:DHT has no direct effect in promoting cellular proliferation of T24 cells; DHT concentration has no significant influence to AR expression; T24 cells express AR positively. Purpose:To make appropriate lentivirus vectors targeting to AR gene in T24 cells.Methods:According to target AR gene sequences and design principles for RNAi, we designed two target sequences. After coupling of RNAi vectors by dsDNA oligo, the products were transferred to competent cells. We tested positive clones by sequence analysis and prepared AR RNAi lentivirus vectors. After packing, production and T24 cell infection of these two lentivirus vectors, RT-PCR was used to test AR expression to find out if they can suppress ARmRNA.Results:Through sequence analysis, the two vectors proved to be successful. The titer were 1E+9,2E+9 TU/mL respectively. Observed by fluorescence, the infection efficiency was up to 80%. In T24 cells, group KD1 and group KD2 had significant knock-down effect to AR gene. Compared to group NC, the knock-down efficiency of group KD1 and group KD2 was up to 85% respectively.Conclusion:We has successfully prepared two kind of lentivirus vector targeting to AR gene of T24 cells Purpose:To evaluate the improvement of sensitivity to chemotherapeutical drugs in T24 cell line of bladder cancer when the expression of AR has been inhibited by RNA interference.Methods:We constructed RNA interference vectors with pGCSIL-GFP against AR and then transferred them into the T24 cell line. the expression of AR was determined by RT-PCR, and FCM was employed to examine the apoptosis index of T24 cell line induced by MMC.Results:By data analysis, the expression level of ARmRNA declined while caspase-3mRNA up-regulated after transfection significantly (p<0.05). The apoptosis index was (35.38±1.54)% in the transferred group, (15.67±1.23)%in the control group and (17.43±1.45)% in the liposome group. The sensitivity of T24 cells to MMC increased after AR had been inhibited and the apoptosis index examined by FCM was significantly increased (P＜0.05). The expression level of AR has negative correlation with the apoptosis index (P<0.05).Conclusion:ARsiRNA can impel the apoptosis induced by MMC which may be through caspase pathway.