PIK3CA基因沉默对阿司匹林抗骨髓瘤作用的影响及机制研究

发布时间：2018-03-09 10:19

本文选题：PIK3CA基因　切入点：基因沉默　出处：《南昌大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:1.通过检测阿司匹林(ASA)对人骨髓瘤细胞株RPMI-8226细胞的PIK3CA基因及蛋白表达的影响,探讨PIK3CA基因作为ASA抗骨髓瘤作用靶点的可行性。2.通过沉默PIK3CA基因后检测ASA对RPMI-8226细胞增殖活性的影响,及其下游效应蛋白pAKT的变化,探讨PIK3CA基因在ASA抗骨髓瘤效应中的作用及分子机制。方法:1.采用CCK-8方法,检测不同浓度ASA(0、2.5、5、10mmol/L)处理RPMI-8226细胞24h、48h、72h后细胞增殖变化。2.采用实时荧光定量PCR和Western blot法检测不同浓度ASA对RPMI-8226细胞PIK3CA基因及蛋白表达水平。3.利用siRNA干扰技术,将PIK3CAsiRNA转染RPMI-8226细胞,荧光显微镜下观测细胞转染效果,实时荧光定量PCR检测PIK3CA m RNA表达以检测基因沉默效果。4.采用CCK-8方法,检测沉默PIK3CA基因后,ASA处理后RPMI-8226细胞增殖抑制变化。5.采用流式细胞仪,检测沉默PIK3CA基因后,ASA处理后RPMI-8226细胞凋亡与周期变化。6.采用Western blot法,检测沉默PIK3CA基因后,ASA处理后RPMI-8226细胞AKT与pAKT蛋白表达的变化。结果:1.在2.5~10mmol/L范围内,ASA以剂量依赖性抑制骨髓瘤细胞株RPMI-8226细胞增殖。在24~72h内,ASA对RPMI-8226细胞的增殖抑制率呈时间依赖性。2.在2.5~10mmol/L范围内,ASA以剂量依赖性抑制RPMI-8226细胞的PIK3CA基因与蛋白表达。3.PIK3CAsiRNA转染RPMI-8226细胞后,PIK3CA基因表达下降83.2%,表明基因沉默效果较好。4.与对照组相比,PIK3CAsiRNA转染后,ASA对RPMI-8226细胞的抗增殖活性明显下降,且并不随ASA浓度增大而变化。5.与对照组相比,PIK3CAsiRNA转染后,ASA对RPMI-8226细胞的凋亡诱导活性明显下降。6.与对照组相比,PIK3CAsiRNA转染后,ASA对RPMI-8226细胞的G1期阻滞作用明显下降。7.ASA以剂量依赖性抑制RPMI-8226细胞pAKT蛋白表达,但不影响总AKT蛋白表达。沉默PIK3CA基因后,ASA对RPMI-8226细胞pAKT蛋白抑制作用明显减弱。结论:1.ASA以浓度依赖性抑制骨髓瘤细胞株RPMI-8226细胞的PIK3CA基因及蛋白表达。2.PIK3CA基因沉默可明显减弱ASA对RPMI-8226细胞的增殖抑制、凋亡诱导效应及细胞周期G1期阻滞作用。3.沉默PIK3CA基因显著降低ASA对RPMI-8226细胞AKT磷酸化的抑制作用。4.ASA通过抑制PIK3CA基因及下游效应分子AKT磷酸化而起抗骨髓瘤作用。
[Abstract]:Objective to investigate the effect of aspirin on the expression of PIK3CA gene and protein in human myeloma cell line RPMI-8226. To explore the feasibility of PIK3CA gene as a target of ASA anti-myeloma. (2) to detect the effect of ASA on the proliferation of RPMI-8226 cells and the change of downstream effector protein pAKT by silencing PIK3CA gene. To investigate the role and molecular mechanism of PIK3CA gene in the anti-myeloma effect of ASA. The proliferation of RPMI-8226 cells was detected after treatment with different concentrations of ASA for 24 h or 48 h or 72 h. Real-time quantitative PCR and Western blot were used to detect the PIK3CA gene and protein expression level of RPMI-8226 cells with different concentrations of ASA. PIK3CAsiRNA was transfected into RPMI-8226 cells by siRNA interference technique. The effect of cell transfection was observed under fluorescence microscope, and the expression of PIK3CA m RNA was detected by real-time fluorescence quantitative PCR to detect the effect of gene silencing. 4. CCK-8 method was used to detect the inhibition of RPMI-8226 cell proliferation after PIK3CA gene silencing. 5. Flow cytometry was used. Apoptosis and cell cycle changes of RPMI-8226 cells treated with silencing PIK3CA gene were detected. 6. Western blot assay was used to detect apoptosis and cell cycle change of RPMI-8226 cells. The changes of AKT and pAKT protein expression in RPMI-8226 cells treated with silencing PIK3CA gene were detected. Results 1. The proliferation of RPMI-8226 cells was inhibited in a dose-dependent manner in the range of 2.510 mmol / L. The inhibitory rate of AKT and pAKT protein on RPMI-8226 cells was observed within 2472 h. In a dose-dependent manner, the expression of PIK3CA gene and protein in RPMI-8226 cells was inhibited in a dose-dependent manner. 3. PIK3CAsiRNA transfection into RPMI-8226 cells decreased the expression of PIK3CA gene by 83.2%, which indicated that gene silencing effect was better than that of control group. The antiproliferative activity of RPMI-8226 cells decreased significantly. Compared with the control group, the apoptosis-inducing activity of PIK3CAsiRNA on RPMI-8226 cells decreased significantly. 6. Compared with the control group, the G 1 phase arrest of RPMI-8226 cells was significantly decreased by PIK3CAsiRNA transfection. The expression of pAKT protein in RPMI-8226 cells was inhibited in a dependent manner. After silencing the PIK3CA gene, the inhibition of pAKT protein in RPMI-8226 cells was obviously weakened. Conclusion: 1. The inhibition of PIK3CA gene and protein expression in RPMI-8226 cell line by ASA in a concentration dependent manner. 2. PIK3CA gene silencing can make it clear. The inhibitory effect of ASA on the proliferation of RPMI-8226 cells was significantly decreased. Apoptosis-induced effect and G1 phase arrest of cell cycle .3. silencing PIK3CA gene significantly reduced the inhibitory effect of ASA on AKT phosphorylation in RPMI-8226 cells. 4. ASA played an anti-myeloma effect by inhibiting the phosphorylation of PIK3CA gene and downstream effector molecule AKT.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.3

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