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目的构建ΔNp63特异性短发夹RNA(shRNA)的表达载体,探讨其在膀胱移行细胞癌(TCCB)细胞中的作用。方法设计、合成ΔNp63特异性的短链寡核苷酸,克隆到Pgenesil-1质粒载体中,构建ΔNp63特异shRNA表达载体,转染TCCB细胞；免疫组化S-P法检测ΔNp63蛋白表达水平,半定量逆转录-聚合酶链反应(RT-PCR)检测ΔNp63 mRNA的表达水平,流式细胞术(FCM)检测细胞周期,四甲基偶氮唑蓝(MTT)法检测细胞增殖活性。结果经PstⅠ和SalⅠ双酶切和测序鉴定,成功构建ΔNp63-shRNA质粒表达载体,并能够成功转染TCCB细胞。转染后可显著抑制细胞中ΔNp63蛋白和mRNA的表达,其中ΔNp63 mRNA的表达抑制率为63．0%；G_0/G_1期细胞显著增加,为(66．38±3．08)%,S期细胞显著减少,为(33．68±2．06)%；细胞增殖活性明显降低。结论运用Pgenesil-1质粒载体构建的ΔNp63-shRNA表达载体可成功转染TCCB细胞,有效抑制ΔNp63蛋白和mRNA表达,并可调控TCCB细胞的细胞周期,抑制其增殖。
Objective To construct the expression vector of ΔNp63 specific short hairpin RNA (shRNA) and investigate the role of ΔNp63 in bladder transitional cell carcinoma (TCCB). Methods The ΔNp63-specific short-chain oligonucleotide was synthesized and cloned into Pgenesil-1 plasmid vector. The vector of ΔNp63-specific shRNA was constructed and transfected into TCCB cells. The expression of ΔNp63 protein was detected by immunohistochemical SP method and semi-quantitatively reversed The expression of ΔNp63 mRNA was detected by polymerase chain reaction (RT-PCR). The cell cycle was detected by flow cytometry (FCM) and the cell proliferation activity was detected by MTT assay. Results Double enzyme digestion with PstⅠ and SalⅠ and sequencing proved that ΔNp63-shRNA plasmid was successfully constructed and transfected into TCCB cells successfully. After transfection, the expression of ΔNp63 protein and mRNA was significantly inhibited, among which, the expression of ΔNp63 mRNA was inhibited by 63.0% and the percentage of G_0 / G_1 phase was significantly increased (66.38 ± 3.08)%, S phase The number of cells was significantly decreased (33.68 ± 2.06)%. The cell proliferation activity was significantly decreased. CONCLUSION: The ΔNp63-shRNA expression vector constructed by Pgenesil-1 plasmid vector can successfully transfect TCCB cells, effectively inhibiting the expression of ΔNp63 protein and mRNA, and regulating the cell cycle of TCCB cells and inhibiting their proliferation.