shRNA靶向抑制HDAC1对前列腺癌PC-3M细胞RB1与E2F1基因表达的调控研究
发布时间：2018-11-17 11:04
【摘要】：前列腺癌（prostate cancer）是人类特有的肿瘤疾病，是欧美男性最常见的恶性肿瘤之一。中国等亚洲国家的前列腺癌发病率虽远低于欧美，但随着生活以及饮食习惯的改变，近年来我国男性前列腺癌的发病率呈逐年增长趋势。 组蛋白去乙酰化酶抑制剂（HDACIs）作为一类新兴的抗肿瘤药物，具有低毒、高效的特点，通过抑制肿瘤细胞的增殖、阻抑细胞周期、促进凋亡等方式抑制肿瘤的发生与发展。有研究显示，在前列腺癌组织和细胞中检测到HDAC1蛋白过表达。HDAC1与RB1和E2F1联系密切，RB家族蛋白通过募集HDAC1来抑制E2F1的活性，进而调控下游基因转录。但HDAC1在此复合物中的具体调控关系仍不确定。 本研究通过构建针对于HDAC1基因表达的shRNA（short hairpinRNA，短发夹RNA）重组干扰质粒，脂质体转染shRNA HDAC1重组干扰质粒，成功导入PC-3M细胞，抑制HDAC1基因转录水平，运用Real-time PCR检测细胞转染后RB1和E2F1基因mRNA表达水平，运用流式细胞仪检测沉默HDAC1基因表达对PC-3M细胞周期的影响。 研究结果显示，shRNAHDAC1重组干扰质粒转染进入PC-3M细胞后，HDAC1基因mRNA表达水平降低，，RB1和E2F1基因mRNA表达水平虽均有不同程度的下降，但均未达到显著水平。提示HDAC1在复合物中不处于中心调控位置，HDAC1、RB1和E2F1基因分别受其各自的上游基因调控。RB1、E2F1与HDAC1在细胞内以形成复合物的形式来发挥调控细胞周期的作用，且RB1蛋白通过磷酸化与去磷酸化两种形式的改变来参与细胞周期调控。更进一步的蛋白水平研究还将继续。PC-3M细胞中HDAC1基因表达降低后，细胞周期阻抑于G1期，从而促进肿瘤细胞发生凋亡，提示HDAC1可作为有效的肿瘤抑制剂分子靶点。 本研究得到以下结论： 1.实验采用RNA（iRNA interference，RNA干扰）技术，以HDAC1为靶基因，成功设计、构建了两条能够在真核细胞内表达针对HDAC1基因的shRNA表达载体，为前列腺癌的基因治疗提供了物质基础。 2. shRNA HDAC1重组干扰质粒干扰降低PC-3M细胞中HDAC1mRNA的表达水平，对RB1和E2F1基因mRNA的表达无显著影响。 3. shRNA HDAC1重组干扰质粒抑制PC-3M细胞中HDAC1mRNA的表达，能够阻抑PC-3M细胞周期，进而诱导细胞凋亡。
[Abstract]:Prostate cancer (prostate cancer) is one of the most common malignant tumors in Europe and America. Although the incidence of prostate cancer in China and other Asian countries is far lower than that in Europe and America, with the change of life and diet habits, the incidence of male prostate cancer in China has been increasing year by year. Histone deacetylase inhibitor (HDACIs), as a new antitumor drug, has the characteristics of low toxicity and high efficiency. It inhibits the occurrence and development of tumor by inhibiting the proliferation of tumor cells, inhibiting cell cycle and promoting apoptosis. Studies have shown that overexpression of HDAC1 protein has been detected in prostate cancer tissues and cells. HDAC1 is closely related to RB1 and E2F1. RB family proteins inhibit E2F1 activity by recruiting HDAC1 and then regulate downstream gene transcription. However, the specific regulatory relationship of HDAC1 in this complex remains uncertain. In this study, we constructed the shRNA (short hairpinRNA, short hairpin RNA (shRNA (short hairpinRNA, short hairpin RNA) interference plasmid for the expression of HDAC1 gene, transfected the shRNA HDAC1 recombinant interference plasmid with liposome, successfully introduced into PC-3M cells, and inhibited the transcription level of HDAC1 gene. The expression of RB1 and E2F1 gene mRNA was detected by Real-time PCR and the effect of silent HDAC1 gene expression on PC-3M cell cycle was detected by flow cytometry. The results showed that after transfection of shRNAHDAC1 recombinant interference plasmid into PC-3M cells, the mRNA expression level of HDAC1 gene decreased, but the mRNA expression level of RB1 and E2F1 genes decreased in varying degrees, but none of them reached significant level. HDAC1,RB1 and E2F1 genes are regulated by their respective upstream genes respectively. RB1,E2F1 and HDAC1 play a role in regulating cell cycle in the form of complex. RB1 protein participates in cell cycle regulation through phosphorylation and dephosphorylation. The further study of protein level will continue. After the expression of HDAC1 gene in PC-3M cells decreases, the cell cycle is inhibited in G1 phase, thus promoting the apoptosis of tumor cells, suggesting that HDAC1 can be used as an effective molecular target of tumor inhibitor. The conclusions of this study are as follows: 1. Using RNA (iRNA interference,RNA interference technique and HDAC1 as target gene, two shRNA expression vectors which can express HDAC1 gene in eukaryotic cells were successfully designed and constructed, which provided a material basis for gene therapy of prostate cancer. 2. ShRNA HDAC1 recombinant interference plasmid interference reduced the expression of HDAC1mRNA in PC-3M cells, but had no significant effect on the expression of RB1 and E2F1 gene mRNA. 3. The recombinant shRNA HDAC1 interference plasmid inhibited the expression of HDAC1mRNA in PC-3M cells, inhibited the cell cycle and induced apoptosis of PC-3M cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
本文编号：2337554
[Abstract]:Prostate cancer (prostate cancer) is one of the most common malignant tumors in Europe and America. Although the incidence of prostate cancer in China and other Asian countries is far lower than that in Europe and America, with the change of life and diet habits, the incidence of male prostate cancer in China has been increasing year by year. Histone deacetylase inhibitor (HDACIs), as a new antitumor drug, has the characteristics of low toxicity and high efficiency. It inhibits the occurrence and development of tumor by inhibiting the proliferation of tumor cells, inhibiting cell cycle and promoting apoptosis. Studies have shown that overexpression of HDAC1 protein has been detected in prostate cancer tissues and cells. HDAC1 is closely related to RB1 and E2F1. RB family proteins inhibit E2F1 activity by recruiting HDAC1 and then regulate downstream gene transcription. However, the specific regulatory relationship of HDAC1 in this complex remains uncertain. In this study, we constructed the shRNA (short hairpinRNA, short hairpin RNA (shRNA (short hairpinRNA, short hairpin RNA) interference plasmid for the expression of HDAC1 gene, transfected the shRNA HDAC1 recombinant interference plasmid with liposome, successfully introduced into PC-3M cells, and inhibited the transcription level of HDAC1 gene. The expression of RB1 and E2F1 gene mRNA was detected by Real-time PCR and the effect of silent HDAC1 gene expression on PC-3M cell cycle was detected by flow cytometry. The results showed that after transfection of shRNAHDAC1 recombinant interference plasmid into PC-3M cells, the mRNA expression level of HDAC1 gene decreased, but the mRNA expression level of RB1 and E2F1 genes decreased in varying degrees, but none of them reached significant level. HDAC1,RB1 and E2F1 genes are regulated by their respective upstream genes respectively. RB1,E2F1 and HDAC1 play a role in regulating cell cycle in the form of complex. RB1 protein participates in cell cycle regulation through phosphorylation and dephosphorylation. The further study of protein level will continue. After the expression of HDAC1 gene in PC-3M cells decreases, the cell cycle is inhibited in G1 phase, thus promoting the apoptosis of tumor cells, suggesting that HDAC1 can be used as an effective molecular target of tumor inhibitor. The conclusions of this study are as follows: 1. Using RNA (iRNA interference,RNA interference technique and HDAC1 as target gene, two shRNA expression vectors which can express HDAC1 gene in eukaryotic cells were successfully designed and constructed, which provided a material basis for gene therapy of prostate cancer. 2. ShRNA HDAC1 recombinant interference plasmid interference reduced the expression of HDAC1mRNA in PC-3M cells, but had no significant effect on the expression of RB1 and E2F1 gene mRNA. 3. The recombinant shRNA HDAC1 interference plasmid inhibited the expression of HDAC1mRNA in PC-3M cells, inhibited the cell cycle and induced apoptosis of PC-3M cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
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