RNA干扰抑制HMGB1基因表达对子宫内膜癌细胞增殖作用的研究

发布时间：2018-04-03 10:56

本文选题：高迁移率族蛋白1　切入点：子宫内膜癌　出处：《中南大学》2014年硕士论文

【摘要】：目的：通过RNA干扰抑制子宫内膜癌细胞中高迁移率族蛋白1(High mobility group box1, HMGB1)的表达,研究HMGB1对子宫内膜癌细胞的增殖及细胞周期、细胞凋亡的影响,并探讨其分子机制。方法：构建靶向HMGB1基因的慢病毒shRNA载体,转染子宫内膜癌细胞株HEC-1A,同时利用实时荧光定量聚合酶链反应法(Real time-PCR)和免疫印迹法(Western Blot)检测转染HMGB1-siRNA后细胞HMGB1mRNA和蛋白表达水平的变化。MTT法和流式细胞术分别检测转染HMGB1-siRNA后HEC-1A细胞增殖、细胞周期和细胞凋亡的变化。Western Blot法检测转染HMGB1-siRNA后细胞AKT、pAKT和CyclinD1蛋白的表达水平。结果： 1、子宫内膜癌细胞株HEC-1A转染慢病毒重组质粒后,荧光显微镜下可见细胞表达较强的荧光信号,转染效率达80%左右,说明转染成功。 2、子宫内膜癌细胞转染慢病毒72h后,Real time-PCR结果显示：实验组细胞中HMGB1mRNA表达较空白对照组和阴性对照组降低(P0.05); Western Blot检测细胞中HMGB1蛋白表达显示：实验组明显低于空白对照组和阴性对照组(P0.01)。 3、MTT检测结果显示：实验组细胞增殖率约为72.03%,较空白对照组和阴性对照组显著降低(P0.01)；流式细胞术检测细胞周期分布结果显示：实验组G0/G1期细胞比例较空白对照组和阴性对照组明显增加(P0.01),而实验组S期和G2/M期细胞比例均显著低于空白对照组和阴性对照组(P0.01)。 4、Annexin V-FITCPI双染法检测细胞凋亡情况显示：实验组早期凋亡率、中晚期细胞凋亡率以及细胞总凋亡率均高于空白对照组和阴性对照组(P0.01)。 5、三组细胞中AKT蛋白表达无明显差异(P0.05),而实验组细胞中pAKT和CyclinD1蛋白表达水平均较空白对照组和阴性对照组降低(P0.01)。结论：慢病毒介导的HMGB1shRNA重组质粒可有效抑制子宫内膜癌细胞中HMGB1mRNA和蛋白的表达；沉默子宫内膜癌细胞中HMGB1基因表达,可抑制细胞增殖,且使细胞周期阻滞于G0/G1期,并诱导细胞凋亡；在子宫内膜癌中,HMGB1可能通过PI3K/AKT信号通路影响细胞增殖过程。
[Abstract]:Aim: to study the effect of HMGB1 on the proliferation, cell cycle and apoptosis of endometrial carcinoma cells by inhibiting the expression of high mobility group 1(High mobility group box1 (HMGB1) in endometrial carcinoma cells by RNA interference, and to explore its molecular mechanism.Methods: a lentivirus shRNA vector targeting HMGB1 gene was constructed.Transfection of endometrial carcinoma cell line HEC-1A, real-time fluorescent quantitative polymerase chain reaction (Real time-PCR) and Western blotting were used to detect the changes of HMGB1mRNA and protein expression after transfection of HMGB1-siRNA. MTT assay and flow cytometry were used to detect the expression of HMGB1mRNA and protein, respectively.HEC-1A cells proliferated after HMGB1-siRNA staining.The changes of cell cycle and apoptosis. Western Blot assay was used to detect the expression of AKT pAKT and CyclinD1 protein after HMGB1-siRNA transfection.Results:1. After transfection of lentivirus recombinant plasmid into endometrial cancer cell line HEC-1A, strong fluorescent signal could be observed under fluorescence microscope, and the transfection efficiency was about 80%, which indicated that the transfection was successful.2. After 72 hours of lentivirus transfection of endometrial cancer cells, the results of Real time-PCR showed that the expression of HMGB1mRNA in the experimental group was lower than that in the blank control group and the negative control group, and the expression of HMGB1 protein in the experimental group was significantly lower than that in the blank control group and the negative control group.Control group and negative control group (P 0.01).The cell proliferation rate of the experimental group was about 72.03, which was significantly lower than that of the blank control group and the negative control group, while the cell cycle distribution of the experimental group in G0/G1 phase was significantly lower than that of the blank control group and the control group by flow cytometry.The percentage of cells in S phase and G 2 / M phase in the negative control group was significantly lower than that in the blank control group and the negative control group.4Annexin V-FITCPI double staining showed that the early apoptosis rate, the late apoptosis rate and the total apoptosis rate in the experimental group were higher than those in the blank control group and the negative control group (P 0.01).There was no significant difference in the expression of AKT protein among the three groups, but the expression levels of pAKT and CyclinD1 in the experimental group were lower than those in the blank control group and the negative control group.Conclusion: lentivirus-mediated HMGB1shRNA recombinant plasmid can effectively inhibit the expression of HMGB1mRNA and protein in endometrial cancer cells, silence the expression of HMGB1 gene in endometrial cancer cells, inhibit cell proliferation, and block cell cycle at G0/G1 stage.In endometrial carcinoma, HMGB1 may affect cell proliferation through PI3K/AKT signaling pathway.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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