稳定阻断前列腺癌细胞中p65的shRNA基因序列的获得及蔓病毒载体的构建

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

目的 获得稳定阻断前列腺癌细胞株Lncap中核因子[NF-κB（p65）]表达的shRNA序列,并构建蔓病毒载体.方法 根据p65基因信息,设计siRNA1、siRNA2、siRNA3 3条针对p65基因cds区的siRNA序列,组建shRNA对应的4对互补的单链DNA,包括siRNA的正义链和反义链;正义链序列按5＇向3＇顺序依次为：酶切位点（BamH Ⅰ）、干扰序列（19bp）、loop环（TTCAAGAGA）、干扰序列的反向互补序列（19 bp）、中止信号（TTTTT）、酶切位点（EcoR Ⅰ）.将合成的序列插入空载体pSI H1-H1-copGFP shRNA Vector中,转染前列腺癌细胞后,通过real-time聚合酶链反应（PCR）检测不同序列片段对p65的mRNA抑制效果,通过Western blot检测对p65蛋白表达的抑制效果.从而获得可以稳定高效抑制前列腺癌细胞中p65表达的shRNA序列.结果 设计的3条针对p65的序列中第3条的抑制效果最好,目的序列位于p65（NM_021975）的1096～1113,茎环序列为5＇-GATCC GCCCTATCCCTTTACGTCA TTCAAGAGA TGACGTAAAGGGATAGGGC TTTTT G-3＇.其对前列腺癌细胞株中p65的mRNA的干扰效率为59%,对其蛋白表达的抑制率为81%.转染细胞后,细胞可以稳定低表达p65.结论 成功获得稳定阻断前列腺癌细胞株Lncap中p65表达的shRNA序列,并构建蔓病毒载体.

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.