FBXW7抑制上皮性卵巢癌生长的作用及机制研究

发布时间：2018-05-31 02:32

本文选题：GEO + 上皮性卵巢癌　；参考：《暨南大学》2017年博士论文

【摘要】：第一章FBXW7在上皮性卵巢癌和正常卵巢上皮细胞中的表达分析目的:明确FBXW7在上皮性卵巢癌组织和正常卵巢上皮组织中的表达情况方法:(1)依托隶属于NCBI(美国国立卫生研究院)的GEO(Gene Expression Omnibus,基因表达数据库),以“上皮性卵巢癌”为关键字搜索出编号为GDS3592上皮性卵巢癌表达谱,然后用其检测数据中FBXW7的表达情况。在数据库中再利用Affymetrix 2.0芯片对12例人正常卵巢上皮细胞和12例人卵巢癌上皮细胞中的表达谱情况进行了分析。抽取FBXW7在正常人卵巢上皮细胞和卵巢癌上皮细胞中的表达数值,进而进行统计学分析。(2)培养包括A2780cp、A2780s、SKOV3和CAOV3上皮性卵巢癌细胞以及正常卵巢上皮细胞HOEC。(3)收集细胞总蛋白和总m RNA,利用WB和real-time PCR检测FBXW7基因在蛋白和m RNA水平上的表达情况。结果:(1)FBXW7在12例正常人卵巢上皮细胞中的表达值为907.0±76.36;FBXW7在12例人卵巢癌上皮细胞中的表达值为599.9±54.31;FBXW7在正常人卵巢上皮细胞中的表达情况显著高于上皮性卵巢癌上皮细胞中的表达情况。(2)于蛋白水平上FBXW7在正常卵巢上皮细胞HOEC中高表达;在A2780cp、SKOV3和CAOV3细胞中无表达;在A2780s细胞中低表达。(3)于m RNA水平上,相比于卵巢上皮细胞HOEC,FBXW7在A2780cp、A2780s、SKOV3和CAOV3细胞中均处于低表达的情况。结论:FBXW7在正常人卵巢上皮细胞中的表达显著高于上皮性卵巢癌上皮细胞中的表达第二章体外细胞实验研究FBXW7对上皮性卵巢癌细胞生长的作用及机理目的:研究FBXW7对上皮性卵巢癌细胞生长作用的影响方法:(1)构建pc DNA3.1载体介导的人FBXW7过表达质粒,然后用于转染A2780cp和A2780s细胞,用WB方法检测蛋白水平的FBXW7的表达。(2)利用CCK8实验检测细胞增殖情况、利用克隆形成实验检测细胞克隆形成能力。(3)PI染色结合流式分析对p-FBXW7质粒转染组和对照质粒转染组上皮性卵巢癌细胞的周期分布情况进行检测。(4)利用WB对p-FBXW7质粒转染后经嘌呤霉素筛选得到的p-FBXW7质粒稳定过表达组和对照组细胞中凋亡相关蛋白的表达情况进行检测。(5)利用WB对FBXW7过表达上皮性卵巢癌细胞和对照组细胞中凋亡相关蛋白Bcl-2、Bax、Bcl-x L、Caspase-3和周期相关蛋白Cyclin B1、Cyclin D1和Cyclin E1的表达情况进行检测,进而明确FBXW7调控上皮性卵巢癌细胞生物学特性的分子机制。结果:(1)过表达FBXW7能显著抑制A2780cp及A2780s细胞细胞的增殖,其抑制率分别为56.2%及58.7%。(2)过表达FBXW7能显著抑制A2780cp及A2780s细胞的克隆形成,对克隆形成的抑制率分别为75.6%及77.3%。(3)过表达FBXW7显著促进A2780cp细胞凋亡的发生(Ctrl组细胞的凋亡率为:3.9±0.6%;FBXW7过表达组细胞的凋亡率为13.4±0.9%);过表达FBXW7显著促进A2780s细胞凋亡的发生(Ctrl组细胞的凋亡率为:4.1±0.49%;FBXW7过表达组细胞的凋亡率为14.8±2.2%)。(4)过表达FBXW7有效将A2780cp细胞阻滞在G1期(Ctrl组细胞的G1期细胞率为:46.7±6.5%;FBXW7过表达组细胞的G1期细胞率为68.3±7.3%);2)过表达FBXW7有效将A2780s细胞阻滞在G1期(Ctrl组细胞的G1期细胞率为:52.1±3.8%;FBXW7过表达组细胞的G1期细胞率为70.1±8.2%);(5)在A2780cp和A2780s细胞中过表达FBXW7能显著诱导凋亡促进蛋白Bax和Caspase 3的表达并且能显著下调凋亡抑制蛋白Bcl-2和Bcl-x L的表达。抑制周期检查点蛋白Cyclin E1和Cyclin D1的表达,但对Cyclin B1的表达无显著的调控作用。结论:过表达FBXW7能显著抑制上皮性卵巢癌细胞的增殖、克隆形成,促进凋亡的发生。同时,过表达FBXW7能有效将上皮性卵巢癌细胞阻滞在G1期,抑制细胞周期进行。对以上凋亡促进蛋白及抑制蛋白的研究结果提示在上皮性卵巢癌细胞中过表达FBXW7调控了线粒体依赖的凋亡发生途径。第三章动物实验研究FBXW7对上皮性卵巢癌皮下移植瘤生长的作用及机理研究目的:从动物实验层面上研究FBXW7对上皮性卵巢癌皮下移植瘤生长的作用及机理方法:将所筛选得到的FBXW7稳定过表达A2780cp细胞和对照组细胞接种到裸鼠右腹侧皮下,建立皮下移植瘤。(1)绘制肿瘤生长曲线,进而明确FBXW7对上皮性卵巢癌皮下移植瘤生长的调控作用。(2)石蜡包埋和切片后利用免疫组化染色检测FBXW7的表达情况,明确FBXW7在上皮性卵巢癌皮下移植瘤组织中高表达。(3)利用免疫组化染色检测增殖相关指标PCNA和肿瘤血管生成相关指标CD31的表达情况;(4)利用TUNEL检测肿瘤组织中细胞凋亡情况。结果:(1)过表达FBXW7能显著抑制A2780cp皮下移植瘤生长,对肿瘤体积的抑制率为70.6%(Ctrl组肿瘤体积为:1463.2±175.3mm3;FBXW7组肿瘤体积为:432.5±65.3mm3);对肿瘤重量的抑制率为69.5%(Ctrl组肿瘤体积为:1.38±0.21g;FBXW7组肿瘤体积为:0.42±0.08g)。(2)在p-FBXW7质粒稳定转染组的皮下移植瘤组织中FBXW7显著高表达;p-FBXW7质粒稳定转染组的皮下移植瘤组织中FBXW7的阳性率为62.3±4.8%。(3)在A2780cp的Ctrl组与FBXW7组相比,其皮下移植瘤组织中处于增殖期的细胞数分别为为70.2±8.2%及18.3±4.2%。(4)比较A2780cp的Ctrl组及p-FBXW7组皮下移植瘤组织中处于凋亡的细胞分别为5.3±1.5%及24.3±4.4%。(5)在Ctrl组A2780cp皮下移植瘤组织中血管密度为43.8±8.7;在FBXW7组A2780cp皮下移植瘤组织中血管密度为8.4±3.2。结论:过表达FBXW7抑制A2780cp皮下移植瘤生长,显著抑制肿瘤细胞的增殖活性促进细胞凋亡的发生并且能显著抑制肿瘤血管生成。
[Abstract]:The expression of FBXW7 in epithelial ovarian cancer and normal ovarian epithelial cells was analyzed in Chapter 1: to identify the expression of FBXW7 in epithelial ovarian cancer and normal ovarian epithelial tissue: (1) relying on the GEO (Gene Expression Omnibus, gene expression database) belonging to NCBI (National Institutes of health of the United States), "epithelial" Ovarian cancer "searched the expression profile of GDS3592 epithelial ovarian cancer for the keyword, and then used it to detect the expression of FBXW7 in the data. In the database, the expression profiles of 12 normal ovarian epithelial cells and 12 human ovarian cancer epithelial cells were analyzed by Affymetrix 2 chip. The FBXW7 was extracted in normal human eggs. A2780cp, A2780s, SKOV3 and CAOV3 epithelial ovarian cancer cells, and normal ovarian epithelial cells HOEC. (3) collected the total cell protein and total m RNA, and the WB and real-time PCR were used to detect the table of FBXW7 gene at the level of protein and M. (2) Results: (1) the expression of FBXW7 in 12 normal human ovarian epithelial cells was 907 + 76.36, and the expression of FBXW7 in the epithelial cells of 12 human ovarian cancer cells was 599.9 + 54.31. The expression of FBXW7 in normal human ovarian epithelial cells was significantly higher than that in epithelial ovarian carcinoma. (2) FBXW7 at protein level. High expression in normal ovarian epithelial cells HOEC; no expression in A2780cp, SKOV3 and CAOV3 cells; low expression in A2780s cells. (3) at m RNA level, compared to HOEC in ovarian epithelial cells, FBXW7 is low in A2780cp, A2780s, SKOV3, and cells. Conclusion: expression in normal human ovarian epithelial cells Expression in epithelial ovarian cancer epithelial cells second chapters in vitro study on the effect and mechanism of FBXW7 on the growth of epithelial ovarian cancer cells: the study of the effect of FBXW7 on the growth of epithelial ovarian cancer cells: (1) construction of human FBXW7 overexpressed plasmid mediated by PC DNA3.1 vector, and then used to transfect A2780cp and A 2780s cells were used to detect the expression of FBXW7 at the protein level by WB. (2) the cell proliferation was detected by CCK8 test and the clone formation test was used to detect the cell clone formation ability. (3) PI staining combined flow analysis was used to detect the periodic distribution of epithelial ovarian cancer cells in the p-FBXW7 plasmid transfected group and the control plasmid transfected group (4). WB was used to detect the expression of apoptosis related proteins in the p-FBXW7 plasmid stable overexpression group and the control group after transfection of the p-FBXW7 plasmid. (5) the apoptosis related egg white Bcl-2, Bax, Bcl-x L, Caspase-3 and cycle related eggs of FBXW7 overexpressed epithelial ovarian cancer cells and control group cells were used by WB. The expression of white Cyclin B1, Cyclin D1 and Cyclin E1 was detected, and the molecular mechanism of FBXW7 regulating the biological characteristics of epithelial ovarian cancer cells was identified. Results: (1) over expression of FBXW7 could significantly inhibit the proliferation of A2780cp and A2780s cell cells, and the inhibition rates were 56.2% and 58.7%. (2), respectively. The inhibition rates of s cells were 75.6% and 77.3%. (3), respectively. The expression of FBXW7 significantly promoted the apoptosis of A2780cp cells (the apoptosis rate of Ctrl group was 3.9 + 0.6%; the apoptosis rate of FBXW7 overexpressed group was 13.4 + 0.9%); overexpression of FBXW7 significantly promoted the apoptosis of A2780s cells (the apoptotic rate of Ctrl group cells) It was 4.1 + 0.49%; the apoptosis rate of FBXW7 overexpression group was 14.8 + 2.2%. (4) overexpression of FBXW7 effectively blocked A2780cp cells at G1 stage (the G1 stage rate of Ctrl group cells was 46.7 + 6.5%, the G1 stage cell rate of FBXW7 overexpression group was 68.3 + 7.3%); 2) FBXW7 effectively blocked A2780s cells in G1 phase (Ctrl group cells) The rate of FBXW7 was 52.1 + 3.8%; the cell rate of G1 cells in the overexpression group was 70.1 + 8.2%. (5) the overexpression of FBXW7 in A2780cp and A2780s cells could significantly induce the expression of apoptosis promoting protein Bax and Caspase 3, and significantly downregulated the expression of apoptosis inhibitor Bcl-2 and Bcl-x L. But the expression of Cyclin B1 has no significant regulatory effect. Conclusion: overexpression of FBXW7 can significantly inhibit the proliferation of epithelial ovarian cancer cells, clone and promote the occurrence of apoptosis. At the same time, overexpression of FBXW7 can effectively block epithelial ovarian cancer cells in G1 stage and inhibit cell cycle. The results suggest that overexpression of FBXW7 in epithelial ovarian cancer cells regulates the apoptotic pathway of mitochondrial dependence. The third chapter studies the effect and mechanism of FBXW7 on the growth of epithelial ovarian cancer subcutaneous xenografts: the effect of FBXW7 on the growth of subcutaneous xenografts of the epithelial ovarian cancer from the animal experimental level. The mechanism method: the selected FBXW7 stable overexpression A2780cp cells and the control group cells were inoculated into the right ventral subcutaneous of the nude mice to establish subcutaneous transplantation tumor. (1) the tumor growth curve was plotted, and then the regulation of FBXW7 on the growth of the epithelial ovarian cancer subcutaneous transplanted tumor was determined. (2) the paraffin embedding and sectioning were used to detect F by immunohistochemical staining. The expression of BXW7 showed high expression of FBXW7 in the subcutaneous tissue of epithelial ovarian cancer. (3) the expression of proliferation related index PCNA and tumor angiogenesis related index CD31 were detected by immunohistochemical staining; (4) the cell apoptosis in tumor tissues was detected by TUNEL. Results: (1) overexpression of FBXW7 could significantly inhibit the A2780cp skin. The growth inhibition rate of tumor growth was 70.6% (group Ctrl tumor volume was 1463.2 + 175.3mm3; FBXW7 group tumor volume was 432.5 + 65.3mm3); the tumor weight inhibition rate was 69.5% (Ctrl group tumor volume was 1.38 + 0.21g; FBXW7 group tumor volume was 0.42 + 0.08g). (2) in the subcutaneous transplantation group of the stable transfection group of p-FBXW7 plasmid The positive rate of FBXW7 in the tissue of the p-FBXW7 plasmids stable transfection group was 62.3 + 4.8%. (3) in the Ctrl group of A2780cp and the FBXW7 group. The number of cells in the proliferation stage of the subcutaneous tumor tissue was 70.2 + 8.2% and 18.3 + 4.2%. (4) in Ctrl and p-FBXW7 subcutaneous xenografts. The apoptotic cells in the tissue were 5.3 + 1.5% and 24.3 + 4.4%. (5) in the Ctrl group A2780cp subcutaneous tumor tissue, the vascular density was 43.8 + 8.7, and the vascular density in the A2780cp subcutaneous tissue of group FBXW7 was 8.4 + 3.2. conclusion: overexpression of FBXW7 inhibits the growth of the subcutaneous transplantation tumor of A2780cp and significantly inhibits the proliferation activity of the tumor cells. Apoptosis can significantly inhibit tumor angiogenesis.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.31

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