高效抑制核因子κ—B的茎环RNA基因序列的获得

目的获得高效抑制前列腺癌细胞株Lncap中核因子kappa—B表达的shRNA序列。方法根据核因子kappa—B基因信息，设计siRNA1、siRNA2、siRNA3三条针对核因子kappa-B基因cds区的siRNA序列及无意义的对照序列，组建与之对应的4对互补的单链DNA序列，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（TFCAAGAGA）、干扰序列的反向互补序列（19bp）、中止信号（TTTTT）、酶切位点（EcoR Ⅰ）。将合成的序列插入空载体pSIH1-H1-copGFP shRNA Vector中，转染前列腺癌细胞后，通过real—timePCR检测不同序列片断对核因子kappa—B的mRNA抑制效果。结果设计的3条针对核因子kappa—B的序列中第3条的抑制效果最好，目的序列位于核因子KAPPA—B（NM_021975）的1096到1113，茎环序列为5＇-GATCC GCCCTATCCCTITACGTCA TTCAAGAGA TGACGTAAAGGGATAGGGC TTITT G-3’。其对前列腺癌细胞株中核因子kappa—B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核因子kappa—B。结论成功获得高效抑制前列腺癌细胞株Lncap中核因子kappa—B表达的shRNA序列，为后期研究核因子kappa-B在前列腺癌发病中的作用机理等研究提供了基础。

The obtaining of shRNA sequences blocking the expression of nuclear factor kappa-B with hish quality

Objectives To obtain shRNA sequences that can block the expression of nuclear factor kappa-B in the prostate cancer cell line Lncap with high quality. Methods According to nuclear factor kap- pa-B genetic information, we design siRNA1, siRNA2, siRNA3 and negative sequences, and those three siRNA sequences targeting the cds area of nuclear factor kappa-B 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 nuclear factor kappa-B mRNA by different sequences was detected through real-time PCR ,and the inhibitory effect of nuclear factor kappa-B protein expression was detected by Western-blotting. Thus we can obtain highly effective shRNA sequences in the inhibition of nuclear factor kappa-B in prostate cancer cells. Results The third shRNA sequence had the best inhibitory effect. It＇s position locates in nuclear factor kappa-B （NM_021975） 1096-1113 and it＇s stem-loop sequence is 5＇- GATCC GCCCTATCCCTITACGTCA TFCAAGAGA TGACGTAAAGGGATAGGGC TTTTT G-3＇. The inhibitory effect of nuclear factor kappa-B mRNA in prostate cancer cell line was 60.0% and the protein was 86%. After Transfecting, the prostate cancer cell line had the low expression of nuclear factor kappa-B stably. Concluaions It＇s successful to obtain shRNA sequences that can stably block the expression of nuclear factor kappa-B in the prostate cancer cell line Lncap. It provides solid foundation for further experimental studies on the function of nuclear factor kappa-B.