Research of the p65 gene function in the prostate cancer cell by the obtaining of shRNA sequences blocking the expression of nuclear factor kappa- B (p65) stably and construction of lentivirus vector / 中华泌尿外科杂志

ABSTRACT

Objective To obtain shRNA sequences that can stably block the expression of Nuclear Factor kappa- B (p65) in the prostate cancer cell line LNCaP and construct the lentivirus vector.And validate the gene function of p65 in the cell line. Methods According to p65 genetic information, we design siRNA1, siRNA2, siRNA3 those three siRNA sequences targeting the ods area of p65 gene and then form the corresponding four pairs of complementary single strand DNA of shRNA, including the sense strand and the antisense strand. The synthetic shRNA sequence was inserted into the empty pSIH1-H1-copGFP shRNA Vector, and after transfecting the prostate cancer cells , the inhibitory effect of p65 mRNA by different sequences was detected through real-time PCR, and the inhibitory effect of p65 protein expression was detected by Western-blotting. Thus we can obtain highly effective shRNA sequences in the inhibition of p65 in prostate cancer cells. MTT, flow cytometry, transwell were chosen to test the cell growth, migration and invasive power in vitro to compare the difference of the experimental group, control group and negative group. Results The third shRNA sequence had the best inhibitory effect and the inhibitory effect of p65 mRNA in prostate cancer cell line was 59 % and the protein was 81%. It's position locates in p65 (NM_021975 ) 1096-1113 and it's stemloop sequence is 5'-GATCCGCCCTATCCCTTTACGTCATTCAAGAGATGACGTAAAGGGATAGGGCTTTTTG-3'. After transfecting, the prostate cancer cell line had the low expression of p65 stably. Through MTT, we got the growth curve, which showed that the growth ability of experimental group was significantly decreased compared with the control group and the Logarithmic growth didn't appear in the first 96 hours. Flow cytometry test displayed that the percentage of G0-G1-phase cells in experimental group was 61.49%, and the control group was 44.89%, idle group was 41.52%, which was increasing oberviously. The S-phase cells in the experimental group was 28.58%, compared with the 47.36% and 46. 10% diminished. The results of transwell showed that the experimental group had 16. 5000±6. 62076 cells and the other two groups had 45. 6333 13. 54159 and 36. 8333±5. 68412 cells, which showed the invasive power of experimental group was significantly declined(P＜0.05).Conclusions It's successful to obtain shRNA sequences that can stably block the expression of p65 in the prostate cancer cell line LNCaP and construct the lentivirus vector. p65 can positively regulates the biological behavior of prostate cancer LNCaP cell line in the cell growth, migration and invasive power.