针对IER3IP1基因的shRNA真核表达载体的构建和鉴定

目的:构建特异性针对IER3IP1基因的shRNA(Short Hairpin RNA)的真核表达载体,观察对IER3IP1基因表达的沉默作用,筛选出抑制效果较好的干扰片段。方法:针对IER3IP1基因,设计合成2对特异性DNA片段以及1对无关序列的阴性对照DNA片断,将它们插入真核表达载体pGenesil-1中构建重组质粒,质粒转染L02正常肝脏细胞株后,以半定量RT-PCR法检测转染后24、48、72hIER3IP1基因的抑制效果。结果:成功构建2个特异性针对IER3IP1的shRNA真核表达载体;所构建的载体中有1对能够特异性的降低L02细胞中IER3IP1的mRNA表达水平,且干扰效果显著,抑制率较高,与空载对照组比在转染后24、48、72h抑制率分别为64%、79%、69%。结论:设计构建的真核表达载体可以特异性干扰IER3IP1基因的表达,为进一步研究IER3IP1基因的功能奠定了基础。

Effect on Proliferation and Erythroid Differentiation of K562 Cells by IER3IP1-Knockdown

Objective： To investigate the effect on erythroid differentiation and proliferation of K562 cells by IER3IP1-knockdown with RNA interference targeting at IER3IP1 gene. Methods： The shRNA eukaryotic expression vectors targeting at IER3IP1 gene were designed and constructed. Inhibitory effect was detected by semiquantitative RT-PCR. The impacts on K562 cells by RNAi were studied by MTT assay, benzidine staining, light microscope and electron microscopy observation, cell cycles analysis, colony formation assay and RT-PCR. The expressions of erythroid differentiation correlated genes Gfi-lB, GPA and 7-globin were studied after being exposed to 0.2μmol/L imatinib for two days. Results： The shRNA eukaryotic expression vectors were successfully constructed. The expression of IER3IP1 gene was significantly inhibited with an inhibition efficiency of 76% （P〈0.01）. Compared with the control groups, bcr/abl mRNA level was increased in K562/shRNA-IER3IP1 group （P〈0.01）. The proliferation ability was enhanced （P〈0.01） and the proportion of cells at G0/G1 phase decreased but S phase increased （P〈0.05） in K562/shRNA-IER3IP1 group. Under electron microscopy, the amount of euchromatin increased but heterochromatin decreased. There were structural abnomalities in endocytoplasmic reticulum and clusters of vesicular. The percentage of benzidine staining positive cells and mRNA expression levels of Gfi-1B, GPA and γ-globin were all decreased after being exposed to 0.2 μmol/L STI571 for two days in K562/shRNA-IER3IP1 group （P〈0.01）. Conclusion： IER3IP1-knockdown can hinder the erythroid differentiation and elevate the proliferation level of K562 cells. IER3IP1 may play a role in erythroid differentiation and proliferation of K562 cells.

苦参碱处理的K562细胞中IER3IP1基因表达及其对细胞增殖的影响

目的研究苦参碱诱导 K562细胞分化过程中 IER3IP1基因的表达,以明确 IER3IP1基因表达与苦参碱作用的量效及时效关系,并初步探讨该基因在 K562细胞中的功能。方法锥虫蓝染色分析苦参碱对 K562细胞的生长抑制作用;用 RT-PCR 半定量方法观察 K562细胞在不同时间、不同剂量苦参碱作用下 IER3IP1基因的表达情况;对 IER3IP1基因重组质粒转染的 K562细胞(K562/eYFP-IER3IP1)作细胞形态学及细胞增殖变化的观察,同时进行细胞周期检测以及超微结构观察。结果苦参碱对 K562细胞有增殖抑制作用,在苦参碱作用3 h 后 IER3IP1基因表达可升高3～4倍,并呈剂量依赖性,其后6～48 h 表达下降,低于非苦参碱处理组。K562/eYFP-IER3IP1细胞的增殖速度显著减缓,G_0/G_1期细胞数升高(P<0.05);且苦参碱作用24 h 后在光镜和电镜下均可见红系分化细胞增多。结论苦参碱抑制 K562细胞生长,同时使 IER3IP1基因表达以剂量依赖的方式瞬时升高,转染了 IER3IP1基因的 K562细胞对苦参碱敏感性增高,提示该基因可能参与了苦参碱作用于 K562细胞的早期反应,并在后续的红系分化中发挥作用。

shRNA干扰IER3IP1基因表达对K562细胞增殖和红系分化的影响

目的探讨靶向干扰IER3IP1基因对K562细胞增殖和红系分化的影响。方法针对IER3IP1基因设计并构建小发夹状（shRNA）真核表达载体，转染K562细胞后用RT—PCR方法鉴定沉默效果。采用MTT实验、联苯胺染色、光镜和电镜观察、流式细胞术以及RT—PCR，观察抑制IER3IP1基因表达后对1（562细胞的影响；采用0．2μmol／L伊马替尼诱导K562细胞2d，观察诱导分化过程中IER3IP1基因表达变化情况，抑制IER3IPl基因表达后对红系分化相关基因Gfi—1B、GPA和γ—globin以及细胞表面GPA蛋白表达的影响。结果成功构建针对IER3IP1基因的shRNA真核表达载体，转染至K562细胞中，该基因mRNA表达水平明显降低，抑制率达76％（P〈0．01）。与对照组细胞相比，K562-shRNA—IER3IP1组bcr—ablmRNA表达升高（P〈0．05）；MTT检测结果显示细胞增殖能力增强（P〈0．01）；流式细胞术检测结果显示S期细胞比例增加，G0／G1期细胞比例减少（P〈0．05）；电镜可见细胞常染色质增加、异染色质减少，内质网部分扩张，囊泡样结构滞留。0．2μmol／L伊马替尼作用48h后，K562-shRNA—IER3IP1组联苯胺染色阳性率由作用前的（44．7±2．5）％降低至（22．7±1．5）％（P〈0．01），且红系分化相关基因Gfi-1B、GPA和γ—globin的mRNA表达水平明显降低，细胞表面GPA蛋白表达水平降低。同时伊马替尼作用过程中IER3IP1基因在作用3h时表达明显升高，6h时降低，12～48h表达水平基本不变。结论干扰IER3IP1基因表达后，K562细胞增殖加快，红系分化过程受阻，提示该基因可能在K562细胞增殖和向红系分化过程中发挥作用。