ARRDC3及ARRDC3-ITGβ4通路在人类前列腺癌中的表达及其作用的研究

发布时间：2018-06-21 18:53

本文选题：前列腺癌 + ARRDC3　；参考：《南方医科大学》2017年博士论文

【摘要】：研究背景及目的:前列腺癌(prostate cancer,PCa)是常见的泌尿系恶性肿瘤之一,目前我国的发病率和病死率呈现逐年上升的趋势。前列腺癌根治术是早期阶段的优选治疗方法,内分泌治疗可作为肿瘤晚期的主要治疗手段。近年来以阿比特龙(Abiraterone)为代表的内分泌治疗及分子靶向治疗显现出一定优势,但疗效尚未确定。然而几乎所有患者最终发展成激素非依赖性前列腺癌,目前尚无有效的治疗方法。既往的研究和我们先前的实验已经证实了 miR-30d在前列腺癌中表达上调,并发挥关键的促癌作用。因此,我们利用基因表达谱芯片技术对过表达miR-30d的LNCaP和DU145细胞株进行基因表达谱分析,获得一批miR-30d调控的差异表达下调的基因,其中Arrestin domain-containing protein 3(ARRDC3)基因表达水平明显下调。含有Arrestin结构域的蛋白3(ARRDC3)是哺乳动物a-抑制蛋白家族的成员,已被确定为肿瘤抑制基因,但其在人类前列腺癌中的功能仍未见报道。DraheimKM等一项研究显示ARRDC3直接与磷酸化形式的ITGβ4结合,导致其内化,泛素化并最终降解,抑制乳腺癌的进展。本研究旨在确定ARRDC3在前列腺癌中的表达情况及ARRDC3与前列腺癌的进展和预后的相关性,探索ARRDC3-ITGβ4通路在前列腺癌中的作用。方法:1.构建miR-30d抑制表达和过表达的LNCaP细胞株,通过qRT-PCR和蛋白质印迹法分别验证miR-30d和ARRDC3之间的表达相关性。2.免疫组织化学分析ARRDC3在临床前列腺癌组织芯片中的表达情况。利用统计学方法,分析Taylor数据库中ARRDC3表达水平与前列腺癌患者的临床特征和预后的相关性,通过生存曲线分析及Cox回归分析研究ARRDC3的表达与总体生存率、无生化复发生存率的关系及是否可作为预后的预测因子。3.构建ARRDC3抑制表达和过表达的DU145和LNCaP细胞株,细胞功能实验分析ARRDC3对前列腺癌细胞功能的影响。4.过表达DU145细胞株接种至裸鼠皮下组织构建皮下移植瘤模型,用于分析ARRDC3在体内对前列腺癌成瘤的影响。5.免疫组织化学分析裸鼠皮下肿瘤组织中Ki67,MMP-9和ITGβ4的表达情况。蛋白质印迹法检测裸鼠皮下肿瘤细胞中ITGB4的表达情况。结果:1.在过表达miR-30d的基因表达谱分析中,我们获得一批差异性表达下调的基因,其中ARRDC3明显下调。qRT-PCR和蛋白质印迹法分析结果显示ARRDC3和miR-30d存在负相关性。2.组织芯片免疫染色分析显示前列腺癌组织标本的ARRDC3低表达与高Gleason评分显著相关(P = 0.045)。Taylor数据库分析ARRDC3结果显示ARRDC3的下调表达与Gleason评分、术后发生转移和生化复发呈相关性(P分别为0.0001,0.01 和 0.02)。3.Kaplan-Meier法和Log rank法分析结果显示低表达ARRDC3与前列腺癌患者不良预后相关(P = 0.010),在前列腺癌中作为抑癌基因。COX回归模型单因素和多因素分析显示,ARRDC3是前列腺癌患者无生化复发生存的独立预测因子。4.细胞功能实验显示过表达的ARRDC3能降低DU145和LNCaP细胞株的增殖、迁移和侵袭能力(P0.05),ARRDC3的抑制表达能增强细胞的增殖、迁移和侵袭能力(P0.05)。5.动物实验结果显示过表达ARRDC3能够显著抑制裸鼠皮下移植瘤的生长速度(P0.05)。与对照组相比,Ki67和MMP-9的免疫组化评分明显降低(P0.05),证实过表达ARRDC3抑制肿瘤细胞的增殖和侵袭。6.蛋白质印迹和免疫组织化学结果显示ARRDC3和ITGβ4呈负相关性(P0.05)。结论:1.ARRDC3在前列腺癌中起抑癌作用,抑制前列腺癌进展,并作为独立预测指标预测术后生化复发和转移的风险。2.ARRDC3可通过ARRDC3-ITGβ4通路抑制前列腺癌的进展,ARRDC3-ITGβ4通路可作为前列腺癌新的治疗目标。
[Abstract]:Background and purpose: prostate cancer (PCa) is one of the common malignant tumors of the urinary system. The incidence and fatality rate of our country are increasing year by year. Radical prostatectomy is the preferred treatment method at the early stage, and endocrine therapy can be used as the main treatment for advanced cancer. (Abiraterone) there are some advantages for the representation of endocrine therapy and molecular targeting therapy, but the effect is not yet determined. However, almost all patients eventually develop into hormone non dependent prostate cancer. There is no effective treatment yet. Previous studies and our previous tests have confirmed the expression of miR-30d in prostate cancer. Therefore, we use gene expression chip technology to analyze the gene expression profiles of LNCaP and DU145 cells overexpressing miR-30d, and obtain a number of differentially expressed genes regulated by miR-30d, in which the expression level of Arrestin domain-containing protein 3 (ARRDC3) gene is obviously downregulated. The protein 3 (ARRDC3) of the estin domain is a member of the mammalian a- suppressor family and has been identified as a tumor suppressor, but its function in human prostate cancer has not been reported in a study of.DraheimKM, such as.DraheimKM, which shows that ARRDC3 is directly associated with the form of phosphorylation of ITG beta 4, leading to its internalization, ubiquitination, and final degradation and inhibition of breast cancer. The purpose of this study is to determine the expression of ARRDC3 in prostate cancer and the correlation between ARRDC3 and the progression and prognosis of prostate cancer. The purpose of this study is to explore the role of ARRDC3-ITG beta 4 pathway in prostate cancer. Method: 1. to construct the LNCaP cell lines with miR-30d inhibition and overexpression, and to verify miR-30d and A by qRT-PCR and Western blotting, respectively. Expression correlation between RRDC3 and.2. immunohistochemical analysis of the expression of ARRDC3 in the clinical prostate cancer tissue microarray. The correlation between the expression level of ARRDC3 in the Taylor database and the clinical characteristics and prognosis of the prostate cancer patients was analyzed by statistical method. The expression of ARRDC3 was analyzed by the survival curve analysis and the Cox regression analysis. The relationship between the overall survival rate, the relationship of no biochemical recurrence survival and the predictor of the prognosis.3. construction of ARRDC3 inhibition expression and overexpression of DU145 and LNCaP cell lines, the effect of ARRDC3 on the function of prostate cancer cells by cell function test,.4. overexpressed DU145 cell lines to subcutaneous tissue of nude mice to construct subcutaneous transplantation tumor model Type, used to analyze the effect of ARRDC3 on the tumor formation of prostate cancer in the body.5. immunohistochemical analysis of the expression of Ki67, MMP-9 and ITG beta 4 in subcutaneous tumor tissues of nude mice. The expression of ITGB4 in subcutaneous tumor cells of nude mice was detected by Western blot. Results: 1. in the analysis of gene expression profiles of over expressed miR-30d, we obtained a group of differences. Down-regulation of heterosexual genes, in which ARRDC3 was obviously down regulated by.QRT-PCR and the results of Western blot analysis showed that there was a negative correlation between ARRDC3 and miR-30d,.2. tissue chip immunostaining analysis showed that the low ARRDC3 expression of prostate cancer tissue specimens was significantly correlated with the high Gleason score (P = 0.045).Taylor database analysis ARRDC3 results showed ARR. Down regulated expression of DC3, Gleason score, postoperative metastasis and biochemical recurrence (P respectively 0.0001,0.01 and 0.02).3.Kaplan-Meier and Log rank analysis results showed that low expression ARRDC3 was associated with poor prognosis of prostate cancer patients (P = 0.010). In prostate cancer as a single factor and multiple cause of the tumor suppressor gene.COX regression model ARRDC3 is an independent predictor of the survival of the prostate cancer patients without biochemical recurrence..4. cell function experiments show that the overexpressed ARRDC3 can reduce the proliferation, migration and invasion ability (P0.05) of DU145 and LNCaP cell lines, and the inhibition of ARRDC3 can enhance cell proliferation, migration and invasion (P0.05).5. animal experiment results The expression of ARRDC3 could significantly inhibit the growth rate of subcutaneous xenografts in nude mice (P0.05). Compared with the control group, the immunohistochemical score of Ki67 and MMP-9 was significantly lower (P0.05). It was confirmed that the overexpression of ARRDC3 inhibited the proliferation of tumor cells and the invasion of.6. protein blots and immunohistochemical results showed that ARRDC3 and ITG beta 4 were negatively correlated (P0.05). Conclusion: 1.ARRDC3 plays an inhibitory role in prostate cancer and inhibits the progress of prostate cancer and is an independent predictor for predicting the risk of postoperative biochemical recurrence and metastasis..2.ARRDC3 can inhibit the progression of prostate cancer through the ARRDC3-ITG beta 4 pathway. The ARRDC3-ITG beta 4 pathway can be used as a new therapeutic target for prostate cancer.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25

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