靶向siRNA干扰HOXA5对Jurkat细胞Livin、Smac蛋白表达的影响
发布时间：2019-06-26 22:26
【摘要】：目的:本课题旨在构建针对HOXA5sh RNA真核表达载体p RNAT-GFP-Neo-HOXA5沉默HOXA5基因表达,探讨其对Jurkat细胞Livin蛋白、Smac蛋白表达的影响,为白血病的靶向治疗提供理论依据和线索。方法:1、根据HOXA5基因的m RNA序列,设计合成三条针对HOXA5基因不同位点的si RNA序列,并筛选有效干扰HOXA5基因表达的特异性si RNA序列,构建靶向HOXA5的sh RNA真核表达载体p RNAT-GFP-Neo-si HOXA5。2、实验分组:取对数期细胞(约3x107/ml),将Jurkat细胞分为三组:实验组(脂质体转染靶向HOXA5的si RNA)、阴性对照组(脂质体转染阴性对照si RNA)和空白对照组(仅加等量细胞及培养基)。3、转染后FQ-PCR:通过脂质体LipofectamineTM2000介导si RNA转染Jurkat细胞,48h后通过FQ-PCR和Western blot测定实验组、阴性对照组和空白对照组三组Jurkat细胞HOXA5m RNA和蛋白以及Livin、Smac蛋白相对表达水平。结果:1、设计并构建针对HOXA5基因的短发卡RNA(Short hairpin,sh RNA),与空白对照组和阴性对照组作比较,HOXA5基因的表达量在实验组明显下调,转染后实验组各组Jurkat细胞基因表达受抑制,抑制率分别为:p R N A T-G F P-N e o-H O X A 5 A(2 4.6 2±2.3 4)%,p RNAT-GFP-Neo-HOXA5B(35.07±3.21)%,p RNAT-GFP-Neo-HOXA5C(70.89±6.41)%,其中序列p RNAT-GFP-Neo-HOXA5C的效果最为显著。2、成功构建了有效沉默HOXA5基因的质粒表达载体;与阴性对照组(1.34±0.16)%和空白对照组(1.29±0.21)%相比,实验组(p RNAT-GFP-Neo-HOXA5C)HOXA5m RNA表达水平(0.39±0.01)%明显下降(P0.05)。与阴性对照组(0.84±0.02)及空白对照组(0.85±0.01)相比,实验组(p RNAT-GFP-Neo-HOXA5C)Jurkat细胞HOXA5蛋白表达水平(0.18±0.01)较明显下降(P0.05)。3、实验组livin蛋白表达量(0.20±0.02)明显低于阴性对照组(1.45±0.01)和空白对照组(1.33±0.01)(P0.05),而阴性对照组和空白对照组比较无明显差异(PO.05);4、实验组Smac蛋白表达量(1.26±0.03)明显高于阴性对照组(0.87±0.03)和空白对照组(0.86±0.03)(P0.05),而阴性对照组和空白对照组比较无明显差异(PO.05)。结论:1、成功构建的针对HOXA5基因的si RNA表达载体,能够有效沉默HOXA5基因的表达,下调HOXA5表达能直接诱导Livin蛋白表达下调和Smac蛋白表达上调,并促进细胞凋亡;2、本研究为急性淋巴细胞白血病靶向治疗提供了新的理论依据。
[Abstract]:Objective: to construct HOXA5sh RNA eukaryotic expression vector p RNAT-GFP-Neo-HOXA5 to silence HOXA5 gene expression, to explore its effect on the expression of Livin protein and Smac protein in Jurkat cells, and to provide theoretical basis and clue for targeted therapy of leukemia. Methods: 1. According to the m RNA sequence of HOXA5 gene, three si RNA sequences targeting different sites of HOXA5 gene were designed and synthesized, and the specific si RNA sequences which effectively interfered with the expression of HOXA5 gene were screened. The sh RNA eukaryotic expression vector pRNAT-GFP-Neo-si HOXA5.2, targeting HOXA5 was constructed. Logarithmic cells (about 3x107/ml) were selected and divided into three groups: experimental group (si RNA), targeting HOXA5 by liposomes). Negative control group (negative control group si RNA) and blank control group (only add the same amount of cells and culture medium). 3. The relative expression of HOXA5m RNA, protein and Livin,Smac protein in Jurkat cells was measured by FQ-PCR and Western blot 48 hours later, and the relative expression of HOXA5m RNA, protein and Livin,Smac protein in Jurkat cells was measured by FQ-PCR and Western blot 48 hours later. Results: 1. Compared with the blank control group and the negative control group, the expression of HOXA5 gene was significantly down-regulated in the experimental group. After transfection, the gene expression of Jurkat cells in the experimental group was inhibited, and the inhibition rate was (24.62 卤2.34)%, respectively. P RNAT-GFP-Neo-HOXA5B (35.07 卤3.21)%, p RNAT-GFP-Neo-HOXA5C (70.89 卤6.41)%, among which the effect of sequence p RNAT-GFP-Neo-HOXA5C was the most significant. 2. The plasmid expression vector which effectively silenced HOXA5 gene was successfully constructed. Compared with the negative control group (1.34 卤0.16)% and the blank control group (1.29 卤0.21)%, the expression level of HOXA5m RNA in the experimental group (p RNAT-GFP-Neo-HOXA5C) was significantly lower than that in the negative control group (1.34 卤0.16)% and the blank control group (1.29 卤0.21)% (P 0.05). Compared with the negative control group (0.84 卤0.002) and the blank control group (0.85 卤0.001), the expression of HOXA5 protein in Jurkat cells in the experimental group (p RNAT-GFP-Neo-HOXA5C) was significantly lower than that in the negative control group (0.84 卤0.002) and the blank control group (0.85 卤0.001). 3. The expression of livin protein in the experimental group was significantly lower than that in the negative control group (1.45 卤0.001) and the blank control group (1.33 卤0.001). There was no significant difference between the negative control group and the blank control group (PO.05). 4. The expression of Smac protein in the experimental group (1.26 卤0.03) was significantly higher than that in the negative control group (0.87 卤0.03) and the blank control group (0.86 卤0.03) (P 0.05), but there was no significant difference between the negative control group and the blank control group (PO.05). Conclusion: 1. The successfully constructed si RNA expression vector targeting HOXA5 gene can effectively silence the expression of HOXA5 gene, down-regulate the expression of HOXA5 can directly induce the down-regulation of Livin protein expression and the up-regulation of Smac protein expression, and promote apoptosis. 2, this study provides a new theoretical basis for targeted therapy of acute lymphoblastic leukemia.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
[Abstract]:Objective: to construct HOXA5sh RNA eukaryotic expression vector p RNAT-GFP-Neo-HOXA5 to silence HOXA5 gene expression, to explore its effect on the expression of Livin protein and Smac protein in Jurkat cells, and to provide theoretical basis and clue for targeted therapy of leukemia. Methods: 1. According to the m RNA sequence of HOXA5 gene, three si RNA sequences targeting different sites of HOXA5 gene were designed and synthesized, and the specific si RNA sequences which effectively interfered with the expression of HOXA5 gene were screened. The sh RNA eukaryotic expression vector pRNAT-GFP-Neo-si HOXA5.2, targeting HOXA5 was constructed. Logarithmic cells (about 3x107/ml) were selected and divided into three groups: experimental group (si RNA), targeting HOXA5 by liposomes). Negative control group (negative control group si RNA) and blank control group (only add the same amount of cells and culture medium). 3. The relative expression of HOXA5m RNA, protein and Livin,Smac protein in Jurkat cells was measured by FQ-PCR and Western blot 48 hours later, and the relative expression of HOXA5m RNA, protein and Livin,Smac protein in Jurkat cells was measured by FQ-PCR and Western blot 48 hours later. Results: 1. Compared with the blank control group and the negative control group, the expression of HOXA5 gene was significantly down-regulated in the experimental group. After transfection, the gene expression of Jurkat cells in the experimental group was inhibited, and the inhibition rate was (24.62 卤2.34)%, respectively. P RNAT-GFP-Neo-HOXA5B (35.07 卤3.21)%, p RNAT-GFP-Neo-HOXA5C (70.89 卤6.41)%, among which the effect of sequence p RNAT-GFP-Neo-HOXA5C was the most significant. 2. The plasmid expression vector which effectively silenced HOXA5 gene was successfully constructed. Compared with the negative control group (1.34 卤0.16)% and the blank control group (1.29 卤0.21)%, the expression level of HOXA5m RNA in the experimental group (p RNAT-GFP-Neo-HOXA5C) was significantly lower than that in the negative control group (1.34 卤0.16)% and the blank control group (1.29 卤0.21)% (P 0.05). Compared with the negative control group (0.84 卤0.002) and the blank control group (0.85 卤0.001), the expression of HOXA5 protein in Jurkat cells in the experimental group (p RNAT-GFP-Neo-HOXA5C) was significantly lower than that in the negative control group (0.84 卤0.002) and the blank control group (0.85 卤0.001). 3. The expression of livin protein in the experimental group was significantly lower than that in the negative control group (1.45 卤0.001) and the blank control group (1.33 卤0.001). There was no significant difference between the negative control group and the blank control group (PO.05). 4. The expression of Smac protein in the experimental group (1.26 卤0.03) was significantly higher than that in the negative control group (0.87 卤0.03) and the blank control group (0.86 卤0.03) (P 0.05), but there was no significant difference between the negative control group and the blank control group (PO.05). Conclusion: 1. The successfully constructed si RNA expression vector targeting HOXA5 gene can effectively silence the expression of HOXA5 gene, down-regulate the expression of HOXA5 can directly induce the down-regulation of Livin protein expression and the up-regulation of Smac protein expression, and promote apoptosis. 2, this study provides a new theoretical basis for targeted therapy of acute lymphoblastic leukemia.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
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