PLCε shRNA质粒构建及功能鉴定

Construction and functional identification of phospholipase C epsilon shRNA expression vector in bladder cancer cell line T24

目的构建PLCε基因的shRNA表达载体及其功能鉴定。方法设计并合成针对PLCε基因mRNA的寡核苷酸序列,插入绿色荧光蛋白质粒真核表达载体pGenesil中构建重组载体pGenesil-PLCε。重组载体经酶切、测序鉴定后转染膀胱癌细胞,RT-PCR及Western blot检测对膀胱癌细胞株PLCε的抑制作用,流式细胞术(FCM)及MTT检测其对膀胱癌细胞株增殖活力影响。结果成功构建了针对2个PLCε基因的shRNA表达载体。RT-PCR、Western blot检测结果显示:2个重组质粒载体转染T24细胞株后对PLCεmRNA及对PLCε蛋白表达均有明显抑制,流式细胞术结果表明细胞阻滞于G0/G1期,MTT检测表明重组的载体质粒明显抑制T24细胞增殖活力。结论针对PLCε基因的shRNA能有效抑制膀胱癌T24细胞中PLCε基因的表达及膀胱癌细胞增殖。

Objective To construct a shRNA expression plasmid targeting phospholipase C epsilon （PLCε） and identify its function in bladder cancer cell line T24. Methods Potential RNAi oligonueleotides of PLCε were selected with the aid of online program. Then these oligonucleotides were synthesized and pGenesil- PLCε plasmids were constructed using pGenesil vector containing green fluorescent protein （GFP）. The plasraids were digested by Sal I and sequenced to confirm the inserted sequence. RT-PCR and Western blot analysis were taken to detect the expression of PLCε at RNA and protein levels. The function of this PLCε RNAi in T24 cells was analyzed by MTT assay and flow cytometry. Results It was confirmed by digesting and sequencing that the 2 recombinant plasmids had been constructed successfully. The result of RT-PCR and Western blotting showed that pGenesil-PLCε plasmids inhibited PLCε expression in T24 cells obviously, and flow cytometry showed that the cell cycle was arrested at G0/G1 phase. MTT assay showed that proliferation of T24 cells was obviously inhibited. Conclusion pGenesil-PLCε effectively inhibits PLCε expression and cell proliferation in the bladder cancer cells.