构建PSMA shRNA慢病毒载体转染293T细胞后PSMA mRNA和蛋白的表达变化

Expression of prostate-specific membrane antigen (PSMA) after the transfection of shRNA lentivirus with PSMA construction in 293 cells

目的:利用基因工程技术筛选获得阻断前列腺癌细胞株LNCaP中前列腺癌特异性膜抗原(PSMA)表达的shRNA序列,并构建慢病毒载体。观察转染前列腺癌细胞后对PSMAmRNA和蛋白表达的影响。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,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(BamHⅠ)、干扰序列(19bp)、loop环(TTCAAGAGA)、干扰序列的反向互补序列(19bp)、终止信号(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-timePCR检测对PSMA mRNA表达,通过Western blotting检测PSMA蛋白的表达。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_004476)的1207到1226,茎环序列为5-GATCC GTCT-CAAAGTGCCCTACAA TTCAAGAGA TTGTAGGGCACTTTGAGAC TTTTTG-3。其对前列腺癌细胞株中PSMA mR-NA表达的抑制率为60%,对PSMA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:成功获得阻断前列腺癌细胞株LNCaP PSMA表达的shRNA序列,并构建慢病毒载体,pSIH-PSMA-siRNA2转染前列腺癌细胞后对PSMA mRNA表达的抑制率为60%,对PSMA蛋白表达的抑制率达86%。为后期研究PSMA在前列腺癌发病中的作用机制以及免疫导向治疗等提供了实验基础。

AIM： To obtain shRNA sequences that can stably block the expression of prostate- specific membrane antigen （PSMA） in the prostate cancer cell line LNCaP and construct the lentivirus vector. METHODS： According to genetic information, we design siRNA1, siRNA2 and siRNA3 of PSMA, .the three siRNA sequences targeting the cds area of PSMA gene and then forming 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） and enzyme digestion site （EcoR Ⅰ ）. The synthetic shRNA sequence was inserted into the empty pSIH1 -H1 -copGFP shRNA vector, and transfected into the prostate cancer cells. The inhibitory effect of PSMA mRNA expression by transfeeting different sequences was detected through real -time PCR, and the inhibitory effect of P65 protein expression was also detected by Western blotting. RESULTS ： The second shRNA sequence, located in PSMA （NM_004476） 1207 - 1226 and its stem - loop sequence was 5＇ - GATCC GTCTCAAAGTGC- CCTACAA TTCAAGAGA TTGTAGGGCACTTTGAGAC TTTTTG -3＇, showing the best inhibitory effect on PSMA mRNA expression in prostate cancer cell line was 60% and the protein expression was 86%. After the transfection, the prostate cancer cell line expreesed the low level of PSMA stably. CONCLUSION： It is successful to obtain shRNA sequences that can stably block the expression of PSMA in the prostate cancer cell line LNCaP and obtain the high inhibitory rates for expression of mRNA and protein of PSMA. The construction of the lentivirus vector pSIH - PSMA - siRNA2 provides solid foundation for further experimental studies on the function of PSMA.