ARHI基因对卵巢癌细胞的作用及作用机制研究
发布时间：2018-07-28 09:03
【摘要】：研究目的：肿瘤的发生伴随着癌基因的激活或者抑癌基因的失活，研究这些基因的功能，有助于肿瘤的靶向治疗。ARHI是母源性肿瘤抑制印迹基因，在卵巢癌中失表达。本课题研究卵巢癌细胞过表达ARHI对细胞增殖的抑制作用及机制。 方法：本研究以质粒pcDNA3.0-ARHI为模板PCR扩增ARHI基因编码序列，获得重组质粒所需目的基因，选用pIRES2-EGFP质粒为载体，通过EcoRI和BamHI双酶切及T4连接酶连接方法把扩增的目的基因插入到真核生物载体pIRES2-EGFP的MCS区，两者的粘性末端连接，并后续完成构建后酶切、电泳及测序鉴定。QPCR法检测不同卵巢癌细胞株中ARHI基因的表达情况，将pIRES2-EGFP-ARHI重组质粒转染至ARHI基因低表达的人卵巢癌细胞内实现ARHI基因表达，并设立pIRES2-EGFP空载体转染组和未转染组细胞作为对照， CCK-8法检测pIRES2-EGFP-ARHI重组质粒表达对转染细胞的生长抑制率，流式细胞仪分析细胞周期分布并检测细胞凋亡率，并用Western blotting检测微管相关蛋白轻链Ⅱ（LC3-Ⅱ）、细胞内MAPK/ERK1/2及JAK-STAT3信号转导通路功能蛋白P-ERK和P-STAT3的表达水平变化。 结果：QPCR结果显示所检测卵巢癌细胞均出现不同程度的ARHI失表达，SKOV3细胞在所检测卵巢癌细胞株中ARHI表达最低。SKOV3细胞经不同处理后，，实验组细胞培养24h、48h、72h、96h及120h的生长抑制率分别为64.69%、70.17%、67.01%、66.87%、67.70%，均明显高于质粒对照组（P㩳0.01）。培养48h时实验组、质粒对照组、空白对照组S期细胞的比例分别为64.18%、38.43%及15.15%；凋亡率分别为47.97%、26.53%及9.33%；培养72h时实验组、质粒对照组、空白对照组S期细胞的比例分别为43.95%、12.37%及10.89%；凋亡率分别为51.34%、20.55%及4.39%；实验组细胞培养48h时LC3-Ⅱ表达水平增加，而P-ERK和P-STAT3的表达水平下降，实验组与对照组间有明显差异。 结论：ARHI基因在卵巢癌中失表达，过表达ARHI基因可以抑制SKOV3细胞生长，使SKOV3细胞阻滞于S期，诱导细胞凋亡及自体吞噬。可能机制是ARHI抑制了ERK1/2和STAT3的磷酸化激活，阻断了细胞增殖信号通路，从而诱导的SKOV3细胞凋亡。
[Abstract]:Objective: tumorigenesis is accompanied by activation of oncogenes or inactivation of tumor suppressor genes. Studying the function of these genes may be helpful to target therapy of tumor .ARHI is a maternal tumor inhibitory imprinting gene, which is inexpressed in ovarian cancer. The aim of this study was to investigate the inhibitory effect and mechanism of overexpression of ARHI on ovarian cancer cells. Methods: in this study, the coding sequence of ARHI gene was amplified by PCR using plasmid pcDNA3.0-ARHI as template, and the target gene of recombinant plasmid was obtained. PIRES2-EGFP plasmid was selected as vector. The amplified target gene was inserted into the MCS region of eukaryotic vector pIRES2-EGFP by EcoRI and BamHI double digestion and T4 ligase ligation. Electrophoretic and sequencing methods were used to detect the expression of ARHI gene in different ovarian cancer cell lines. PIRES2-EGFP-ARHI recombinant plasmid was transfected into human ovarian cancer cells with low ARHI gene expression to achieve ARHI gene expression. PIRES2-EGFP empty vector transfected cells and untransfected cells were used as control. CCK-8 assay was used to detect the growth inhibition rate of transfected cells by pIRES2-EGFP-ARHI recombinant plasmid expression. Flow cytometry was used to analyze cell cycle distribution and apoptosis rate. The expression levels of microtubule-associated protein light chain 鈪
本文编号：2149594
[Abstract]:Objective: tumorigenesis is accompanied by activation of oncogenes or inactivation of tumor suppressor genes. Studying the function of these genes may be helpful to target therapy of tumor .ARHI is a maternal tumor inhibitory imprinting gene, which is inexpressed in ovarian cancer. The aim of this study was to investigate the inhibitory effect and mechanism of overexpression of ARHI on ovarian cancer cells. Methods: in this study, the coding sequence of ARHI gene was amplified by PCR using plasmid pcDNA3.0-ARHI as template, and the target gene of recombinant plasmid was obtained. PIRES2-EGFP plasmid was selected as vector. The amplified target gene was inserted into the MCS region of eukaryotic vector pIRES2-EGFP by EcoRI and BamHI double digestion and T4 ligase ligation. Electrophoretic and sequencing methods were used to detect the expression of ARHI gene in different ovarian cancer cell lines. PIRES2-EGFP-ARHI recombinant plasmid was transfected into human ovarian cancer cells with low ARHI gene expression to achieve ARHI gene expression. PIRES2-EGFP empty vector transfected cells and untransfected cells were used as control. CCK-8 assay was used to detect the growth inhibition rate of transfected cells by pIRES2-EGFP-ARHI recombinant plasmid expression. Flow cytometry was used to analyze cell cycle distribution and apoptosis rate. The expression levels of microtubule-associated protein light chain 鈪
本文编号：2149594
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