ΔNp63 shRNA表达质粒的构建及在膀胱癌中的作用

Construction of deltaNp63 specific small hairpin RNA expressing plasmid and its role in bladder cancer——a preliminary study

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 deltaNp63 specific small hairpin RNA （shRNA） expressing plasmid,to examine its inhibitory effect to the expression of deltaNp63 protein and mRNA in transitional cell carcinoma of the bladder （TCCB） , its effect on TCCB cells cycle and proliferation. Methods DeltaNp63 specific oligonucleotides were designed and synthesized. These oligonucleotides were annealed to form double strand DNA fragments and this fragment was cloned into Pgenesil-1 plasmid. The recombinant deltaNp63- shRNA expression construct was confirmed by using Pst Ⅰ ＋ Sal Ⅰdouble digestion and by sequencing. Fluorescence staining was used to confirm the success of transfection in TCCB cells under the fluorescence microscope. The inhibitory effect of deltaNp63-shRNA construct was examined with semi-quantitative reverse transcriptase-polymerase chain reaction （RT-PCR） and immunohistochemical staining assay. The cell cycle of TCCB cells was assayed by flow cytometry （FCM）. The cellular proliferation of TCCB cells was assayed by tetrazolium bromide （MTT） colorimetry. Results The deltaNp63-shRNA expression plasmid was successfully constructed and transfected into TCCB cells. It can effectively reduce the expression of deltaNp63 protein and mRNA. The reduction rate of deltaNp63 mRNA was 63.0% , and the G0/G1 ratio was increased and S phase was decreased in transfected TCCB cells. The cellular proliferation was also lower in transfected 5637 cells in comparrison with that of non-transfected TCCB cells. Conclusion A deltaNp63- shRNA expression plasmid, constructed from Pgenesil-1 plasmid, can successfully be transfected into TCCB cells and can effectively inhibit the expression of deltaNp63 protein and mRNA. It also can take part in regulation of the cell cycling and inhibit the cellular proliferation of TCCB cells.