靶向YWHAE基因shRNA慢病毒表达质粒的构建及功能验证

目的 构建靶向YWHAE基因的shRNA慢病毒表达质粒,并验证其对AGS胃癌细胞增殖的影响. 方法 设计并合成5对针对人YWHAE基因的shRNA序列,分别克隆入pLentiLox3.7(pLL3.7)慢病毒表达质粒,接着将重组的慢病毒表达质粒和包装质粒PHR、包膜质粒VSVG一起采用磷酸钙法转染293T细胞包装慢病毒,收集制备的慢病毒感染AGS细胞,用抗生素Puromycine进行筛选.Western-blot检测感染细胞YWHAE蛋白的表达.选取干扰效率最高的慢病毒表达质粒,针对性设计siRNA的点突变引物,重叠延伸PCR法扩增获得点突变的YWHAE基因,克隆入pcDNA3.1/myc-His(-)A载体,构建YWHAE点突变的表达质粒,转染YWHAE沉默效果最好的AGS细胞株,Western-blot检测转染细胞YWHAE蛋白的回复表达.采用MTS法检测YWHAE-shRNA慢病毒对AGS细胞增殖的影响. 结果 5对针对人YWHAE基因的shRNA序列构建的慢病毒中,pLL3.7-siYWHAE-5包装成的慢病毒抑制AGS细胞的YWHAE蛋白表达的效果最明显,仅为对照组相对表达量的(0.269±0.083)倍;pLL3.7-siYWHAE-5包装的慢病毒,其干扰效应可经由YWHAE点突变表达质粒回复.与对照组比较,YWHAE-shRNA组AGS细胞增殖能力明显下降. 结论 成功构建了靶向YWHAE基因的shRNA慢病毒表达质粒,获得YWHAE基因表达显著下调的AGS细胞株;探明YWHAE表达的下调可有效抑制AGS细胞的增殖,提示YWHAE在胃癌中的致癌潜能.

Construction and Function Identification of Short Hairpin RNA Lentiviral Expression Plasmid Targeting YWHAE

Objective To construct a short hairpin RNA (shRNA) lentiviral expression plasmid targeting YWHAE (Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon) gene and then detect the effect of shRNA on the proliferation of AGS cells.Methods Five shRNA interference sequences targeting YWHAE gene were designed, synthesized and cloned into the pLentiLox3.7(pLL3.7) expression vector.Then the expression plasmids, PHR and VSVG were co-transfected into 293T cells using calcium phosphate to package virus.AGS cells were infected with the extracted virus, and screened with Puromycine.Silencing effect was verified via Western-blot.The lentiviral expression plasmid with the best silence of YWHAE was selected, then YWHAE gene with point mutation was constructed by overlap-extension PCR, and cloned into pcDNA3.1/myc-His(-)A vector.The pcDNA3.1-myc-YWHAEm was transfected into AGS cells with the best silence of YWHAE, then re-expression of YWHAE was detected by Western-blot.MTS assay was used to detect the proliferation of AGS cells.Results Among the five packaged lentivirus, the pLL3.7-siYWHAE-5 lentivirus were selected with the best effect.The relative level of YWHAE protein expression (0.269±0.0 83)-fold] of AGS cells infected with the selected virus was significantly lower than that of AGS cells infected with the pLL3.7-siNC lentivirus.After pcDNA3.1-myc-YWHAEm was transfected into the AGS cells with the best silence of YWHAE, however YWHAE protein expression were re-established.Compared to negative control group (infected with NC-shRNA lentivirus), cell proliferation in YWHAE-shRNA group was significantly inhibited.Conclusion The recombinant shRNA lentivirus targeting the YWHAE gene was constructed successfully, and the AGS cells with knock-down of YWHAE were obtained.Knocking down YWHAE gene has noticeable effects on the proliferation inhibition of AGS cells, which implies YWHAE may be a putative oncoprotein in AGS cells.