靶向于前列腺癌特异性膜抗原shRNA的筛选与鉴定

Selection and identification of shRNA sequences targeting the prostate cancer-specific membrane antigen

目的获得高效抑制前列腺癌细胞株Lncap中前列腺癌特异性膜抗原（PSMA）表达的shRNA序列。方法根据PSMA基因信息，设计siRNA1、siRNA2、siRNA3三条针对PSMA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（BamHⅠ）、干扰序列（19bp）、loop环（TTCAAGAGA）、干扰序列的反向互补序列（19bp）、中止信号（TTTTT）、酶切位点（EcoRⅠ）。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-time PCR检测不同序列片段对PSMA的mRNA抑制效果并通过Western blot检测对目的蛋白PSMA的抑制效果。结果设计的3条针对PSMA的序列中第2条的抑制效果最好，目的序列位于PSMA（NM_0004476）的1207到1226，茎环序列为5’-GATCC GTCTCAAAGTGCCCTACAA TTCAAGAGA TFGTAGGGCACTTTGAGAC TTTTT G-3’。其对前列腺癌细胞株中PSMA的mRNA的抑制率为60．0％，对其蛋白表达的抑制率为86％。转染细胞后，细胞可以稳定低表达PSMA。结论成功获得高效抑制前列腺癌细胞株Lncap中PSMA表达的shRNA序列。

Objective To obtain shRNA sequences that can block the expression of prostate cancer-specific membrane antigen （PSMA） in the prostate cancer cell line Lncap with high quality. Methods According to PSMA genetic information, siRNA1, siRNA2, siRNA3 and negative sequences, and those three siRNA sequences targeting the cds area of PSMA gene were designed and then the corresponding four pairs of complementary single strand DNA of shRNA ,including the sense strand and the antisense strand ,were formed. 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 PSMA mRNA by different sequences was detected by using real-time PCR, and the inhibitory effect of PSMA protein expression was detected by Western-blotting. Highly effective shRNA sequences inhibiting PSMA expression in prostate cancer cells could be obtained. Results The second shRNA sequence had the best inhibitory effect. Its position located in PSMA （NM_004476） 1207-1226 and its stem-loop sequence was 5 ＇-GATCC GTCTCAAACTGCCCTACAA TTCAAGAGA TTGTAGGGCACTTTGAGAC TTTTT G-3 ＇. The inhibitory rate of PSMA mRNA and protein in prostate cancer cell line was 60.0% and 86% respectively. After transfection ,the prostate cancer cell line had the low expression of PSMA stably. Conclusion shRNA sequences that can stably block the expression of PSMA is successfully obtained in the prostate cancer cell line Lncap.