Obtaining of shRNA sequences blocking the expression of nuclear factor-κB ( p65 ) stably and construction of lentivirus vector

Abstract

Objective To obtain shRNA sequences that can stably block the expression of nuclear factor κB[NF-κB ( p65 )]in the prostate cancer cell line Lncap and construct the lentivirus vector. Methods According to p65 genetic information,we design siRNA1, siRNA2, siRNA3 those three siRNA sequences targeting the cds 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 sequence of sense strand from 5' to 3' was: enzyme digestion site ( BamH Ⅰ ) ,interference sequence ( 19 bp) ,the loop-stem structure (TTCAAGAGA), the reverse complementary sequence of interference sequence ( 19 bp) , the ending signal (TTTTT) ,enzyme digestion site (EcoR Ⅰ ). 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 polymerase chain reaction (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. Results The third shRNA sequence had the best inhibitory effect. It' s position locates in p65 ( NM_021975 ) 1096-1113 and it's stem-loop sequence is 5'-GATCC GCCCTATCCCTTTACGTCA TTCAAGAGA TGACGTAAAGGGATAGGGC TTTTT G-3'. The inhibitory effect of p65 mRNA in prostate cancer cell line was 59%and the protein was 81%. After Transfecting, the prostate cancer cell line had the low expression of p65 stably. Conclusion 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. Key words: Nuclear factor; Prostate carcinoma; RNA interference