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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。
OBJECTIVE: To screen the shRNA sequence that blocks the expression of prostate cancer specific membrane antigen (PSMA) in prostate cancer cell line LNCaP by genetic engineering and construct the lentiviral vector. To observe the effect of transfection of prostate cancer cells on PSMA mRNA and protein expression. Methods: According to PSMA gene information, three siRNA sequences targeting siRNA cds region of PSMA gene were designed, and 4 pairs of complementary single-stranded DNAs corresponding to shRNA were designed, including the sense and antisense strands of siRNA. The order of 3 was: BamHI, interference sequence (19bp), loop loop (TTCAAGAGA), reverse complement of interference sequence (19bp), termination signal (TTTTT), restriction enzyme site (EcoRI) . After the transfection of prostate cancer cells, the expression of PSMA mRNA was detected by real-time PCR and the expression of PSMA protein was detected by Western blotting. Results: The second one of the three sequences targeting PSMA had the best inhibitory effect. The target sequence was located at 1207-1226 of PSMA (NM_004476) and the stem-loop sequence was 5-GATCC GTCT-CAAAGTGCCCTACAA TTCAAGAGA TTGTAGGGCACTTTGAGAC TTTTTG-3. The inhibition rate of PSMA mR-NA expression in prostate cancer cell lines was 60%, and the inhibition rate of PSMA protein expression was 86%. After transfection, cells stably expressed PSMA stably. CONCLUSIONS: The shRNA sequence blocking the expression of PSMA in prostate cancer cell line LNCaP was successfully obtained and the lentiviral vector was constructed. The inhibition rate of PSMA mRNA expression in pSIH-PSMA-siRNA2 transfected prostate cancer cells was 60% Inhibition rate of 86%. This study provides the experimental basis for the study of the mechanism of PSMA in the pathogenesis of prostate cancer and immune-directed therapy.