靶向shRNA沉默HOXA5的表达对白血病细胞系U937细胞增殖、凋亡影响的研究

发布时间：2018-04-23 13:35

本文选题：白血病 + HOXA5　；参考：《滨州医学院》2015年硕士论文

【摘要】：目的：同源盒基因(homeobox gene,HOX)是一组在进化过程中高度保守的基因,通过编码重要的转录因子特异性结合、调控靶基因,在胚胎早期发育和造血细胞增殖、分化、凋亡等过程中发挥重要的调控作用。HOXA5是HOX家族的一员,定位于7号染色体,其编码的DNA结合转录因子,在调节基因的表达、细胞的分化和机体形态的发生中发挥重要作用。本研究采用RNAi技术,构建并筛选有效干扰HOXA5基因表达的重组质粒载体pGPU6/GFP/Neo-HOXA5 shRNA,观察HOXA5基因表达抑制后对人白血病细胞株U937增殖、凋亡的影响。方法：1.靶向设计合成3对针对HOXA5基因不同位点的siRNA序列,构建构建3种pGPU6/GFP/Neo-HOXA5 shRNA-1,-2,-3重组质粒载体并进行测序及鉴定;载体转染人白血病细胞株U93748 h后,荧光定量PCR、Western blotting法检测HOXA5 mRNA及蛋白表达,筛选出沉默HOXA5效果最佳的重组质粒载体。2.将已筛选出的HOXA5 shRNA重组质粒载体转染U937细胞,CCK-8法和流式细胞术检测pGPU6/GFP/Neo-HOXA5 shRNA对U937细胞增殖能力和细胞周期、细胞凋亡的影响,Western blotting检测细胞凋亡相关蛋白Survivin, Caspase-3的表达变化。结果：1.成功构建3种pGPU6/GFP/Neo-HOXA5重组质粒载体,转染48后荧光定量RT-PCR及Western blotting分析检测发现3种重组质粒载体均能抑制HOXA5的表达,其中shRNA3的沉默效率最高。2.将筛选出的重组质粒载体转染U937细胞,Western blotting和荧光定量RT-PCR法检测结果显示,相较于空白对照组和阴性对照组相比,转染pGPU6/GFP/Neo-HOXA5 shRNA-3重组质粒载体的U937细胞能明显抑制HOXA5基因的表达,且沉默效率可达70%(P0.05)。CCK-8法检测发现分别转染U937细胞0,24,48,72 h, HOXA5 shRNA能明显抑制U937细胞增殖,且生长抑制率随转染时间的延长逐渐增加(P0.05)。流式细胞术检测显示实验组G0/G1期细胞数明显多于空白对照组和阴性对照组,S期细胞数则减少。Annexin V-FITC双染法检测结果发现,空白对照组、阴性对照组、HOXA5shRNA组的细胞凋亡率分别为(7.36±1.63)%、(8.10±1.39)%、(26.52±2.35)%, HOXA5 shRNA组的细胞凋亡率明显高于其余两组(P0.05)。Western blotting检测显示：HOXA5 shRNA干扰U937细胞48 h后,HOXA5 shRNA组Survivin蛋白的表达量明显低于阴性对照组和空白对照组,而Caspase-3蛋白的表达量明显高于其余两组(P0.05)。结论：1.成功构建了靶向沉默HOXA5的重组质粒表达载体pGPU6/GFP/Neo-HOXA5 shRNA-1,-2,-3,且筛选出沉默HOXA5效果最佳的重组质粒载体。2.重组表达载体pGPU6/GFP/Neo-HOXA5-3,转染U937细胞后能有效沉默HOXA5基因,抑制HOXA5 mRNA和蛋白的表达：RNA干扰HOXA5基因的表达能显著抑制U937细胞增殖,诱导周期阻滞,促进凋亡,后者可能与RNA干扰引起抑制凋亡基因Survivin蛋白表达量减少、Caspase-3蛋白表达量增多相关。
[Abstract]:Objective: homeobox gene (HOX) is a group of highly conserved genes in the process of evolution. By coding important transcription factor specific binding and regulating target genes,.HOXA5 plays an important role in the process of early embryonic development and hematopoietic cell proliferation, differentiation and apoptosis..HOXA5 is a member of the HOX family and is located in No. 7 staining. In vivo, its encoded DNA combined with transcription factors play an important role in regulating gene expression, cell differentiation and the occurrence of body morphology. This study uses RNAi technology to construct and screen the recombinant plasmid vector pGPU6/GFP/Neo-HOXA5 shRNA that effectively interferes with the expression of HOXA5 gene, and observes the inhibition of HOXA5 gene expression to human leukemia cell line U9 37 the effect of proliferation and apoptosis. Method: 1. target design and synthesis of 3 pairs of siRNA sequences against HOXA5 gene, construction and construction of 3 pGPU6/GFP/Neo-HOXA5 shRNA-1, -2, -3 recombinant plasmid carrier and sequencing and identification; after transfection of human leukemia cell line U93748 h, fluorescein quantitative PCR, Western blotting method to detect HOXA5 and protein The recombinant plasmid vector,.2., was screened out for the best effect of silent HOXA5, and the screened HOXA5 shRNA recombinant plasmid was transfected into U937 cells. CCK-8 and flow cytometry were used to detect the proliferation ability and cell cycle of U937 cells, cell cycle, apoptosis and Western blotting detection of apoptosis related protein Surviv. The expression changes of in and Caspase-3. Results 1. successfully constructed 3 pGPU6/GFP/Neo-HOXA5 recombinant plasmid carriers. After transfection of 48 fluorescent quantitative RT-PCR and Western blotting analysis, it was found that all 3 recombinant plasmid vectors could inhibit the expression of HOXA5, and shRNA3's silencing efficiency was the highest, and the recombinant plasmid vector was screened to transfect U937 cells, West The results of ern blotting and fluorescence quantitative RT-PCR assay showed that compared to the blank control group and the negative control group, the U937 cells transfected with the recombinant plasmid vector of pGPU6/GFP/Neo-HOXA5 shRNA-3 could obviously inhibit the expression of HOXA5 gene, and the silence efficiency could reach 70% (P0.05).CCK-8 method and found that U937 cells were transfected into 0,24,48,72 h respectively. HRNA could obviously inhibit the proliferation of U937 cells, and the growth inhibition rate increased gradually with the prolongation of transfection time (P0.05). Flow cytometry showed that the number of G0/G1 cells in the experimental group was more than that of the blank control group and the negative control group. The number of S phase cells decreased by.Annexin V-FITC double staining, and the blank control group, negative control group, HOXA5. The apoptosis rate of shRNA group was (7.36 + 1.63)%, (8.10 + 1.39)% and (26.52 + 2.35)%. The apoptosis rate of HOXA5 shRNA group was significantly higher than that of the other two groups (P0.05).Western blotting detection: HOXA5 shRNA interfered U937 cell 48 h, and the expression of HOXA5 shRNA group was significantly lower than that of negative control group and blank control group, and the expression of HOXA5 shRNA group was significantly lower than that of the negative control group and the blank control group. The expression of aspase-3 protein was significantly higher than that in the other two groups (P0.05). Conclusion: 1. the recombinant plasmid expressing vector pGPU6/GFP/Neo-HOXA5 shRNA-1, -2, -3 is successfully constructed, and the recombinant plasmid carrier,.2. recombinant expression vector pGPU6/ GFP/Neo-HOXA5-3, can be effectively silenced after transfection of U937 cells. The 5 gene inhibits the expression of HOXA5 mRNA and protein: RNA interference with the expression of HOXA5 gene can significantly inhibit the proliferation of U937 cells, induce cycle arrest and promote apoptosis. The latter may be related to the decrease of the expression of Survivin protein and the increase of the expression of Caspase-3 protein in the inhibition of the apoptosis gene Survivin.

【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R3416

【参考文献】