APPL1在胃癌中的表达与功能研究

发布时间：2019-01-23 19:54

【摘要】：[背景]胃癌是我国第三大类最常见的恶性肿瘤,居于我国癌症相关致死原因的第三位。转移性患者的5年生存率只有20%～30%。APPL1在多种组织和细胞中都有表达,是细胞内具有多种重要生物学功能的衔接蛋白,由多个功能结构域组成,包括BAR结构域、PH结构域、PTB结构域。通过这些结构域,APPL1能够与多种细胞膜受体、信号转导蛋白、细胞核因子等相结合,从而调控多种细胞生物学活动与过程,与多种疾病的关系极为密切。[目的]阐明APPL1表达与胃癌临床病理特征及预后的相关性,揭示APPL1对胃癌细胞增殖的影响及机制,明确APPL1对胃癌miRNA表达谱的影响。[方法]采用免疫组织化学染色和逆转录PCR的方法检测APPL1在胃癌组织与非癌性胃组织中的蛋白及mRNA表达水平,统计分析APPL1在胃癌组织与非癌性胃组织中的表达差异,APPL1表达与胃癌临床病理特征和预后的相关性。构建过表达及干涉APPL1的慢病毒载体,感染体外培养的胃癌细胞并建立稳定细胞系,采用MTT实验、BrdU掺入实验、流式细胞术、蛋白质免疫印迹等分析过表达或干涉APPL1对胃癌细胞增殖、周期、凋亡的影响及分子机制。采用miRNA芯片技术分析干涉APPL1对胃癌细胞miRNA表达谱的影响,采用实时定量PCR对差异表达miRNA分子进行鉴定,并分析差异miRNA分子对APPL1影响胃癌细胞增殖的影响。[结果]与非癌性胃组织相比,APPL1蛋白和mRNA的表达水平在胃癌组织中显著升高,APPL1表达增高与胃癌的浸润深度、TNM分期、淋巴结转移密切相关,而与胃癌的组织病理类型无关,同时发现APPL1表达增高是导致胃癌患者预后不良的风险因素。过表达APPL1可导致SGC-7901胃癌细胞G1期比例降低、S期细胞比例增高,而干涉APPL1则导致SGC-7901胃癌细胞G1期比例增高、S期细胞比例降低,其分子机制主要是过表达APPL1促进了 Cyclin D1的表达,抑制了 p16、p27的表达,而干涉APPL1的作用则相反。APPL1能够影响胃癌细胞的miRNA表达谱,共筛选鉴定得到8个差异miRNA分子,其中miR-139的变化最为显著,且APPL1对胃癌细胞增殖的影响及机制与miR-139的表达变化密切相关。[结论]APPL1表达与胃癌的浸润深度、TNM分期、淋巴结转移相关,是胃癌预后不良的风险因素。APPL1能够通过调控细胞周期蛋白表达而影响胃癌细胞的增殖。APPL1能够影响胃癌细胞的miRNA表达谱,其对胃癌细胞增殖的影响与调控miR-139的表达相关。
[Abstract]:Background gastric cancer is the third most common malignant tumor in China and the third leading cause of cancer-related death in China. The 5-year survival rate of metastatic patients is expressed only in a variety of tissues and cells. It is a bridging protein with many important biological functions in cells and consists of multiple functional domains, including BAR domains. PH domain, PTB domain. Through these domains, APPL1 can bind to a variety of cell membrane receptors, signal transduction proteins, nuclear factors and so on, thus regulating a variety of cellular biological activities and processes, and closely related to many diseases. [objective] to elucidate the relationship between APPL1 expression and clinicopathological features and prognosis of gastric cancer, reveal the effect and mechanism of APPL1 on the proliferation of gastric cancer cells, and clarify the effect of APPL1 on miRNA expression profile of gastric cancer. [methods] Immunohistochemical staining and reverse transcription PCR were used to detect the expression of APPL1 protein and mRNA in gastric cancer and non-cancerous gastric tissues, and the difference of APPL1 expression between gastric cancer and non-cancerous gastric tissues was statistically analyzed. Correlation of APPL1 expression with clinicopathological features and prognosis of gastric cancer. A lentivirus vector containing overexpression and interference with APPL1 was constructed to infect gastric cancer cells cultured in vitro and to establish stable cell lines. MTT assay, BrdU incorporation assay and flow cytometry were used. The effects of overexpression or interference of APPL1 on the proliferation, cycle and apoptosis of gastric cancer cells were analyzed by Western blot. The effect of interference APPL1 on the miRNA expression profile of gastric cancer cells was analyzed by miRNA chip technique, and the differential expression miRNA molecule was identified by real-time quantitative PCR, and the effect of differential miRNA molecule on the proliferation of gastric cancer cell line APPL1 was analyzed. [results] compared with non-cancerous gastric tissues, the expression of APPL1 protein and mRNA was significantly increased in gastric carcinoma. The expression of APPL1 was closely related to the depth of invasion, TNM stage, lymph node metastasis, but not to the histopathological type of gastric cancer. At the same time, high expression of APPL1 was found to be a risk factor for poor prognosis in patients with gastric cancer. Overexpression of APPL1 could decrease the proportion of SGC-7901 gastric cancer cells in G1 phase and increase the proportion of S phase cells, while interfering with APPL1 could increase the proportion of SGC-7901 gastric cancer cells in G1 phase and decrease the proportion of S phase cells. Its molecular mechanism is that overexpression of APPL1 promotes the expression of Cyclin D1 and inhibits the expression of p16 p27, whereas interfering with APPL1 can affect the miRNA expression profile of gastric cancer cells. The effect of APPL1 on the proliferation of gastric cancer cells and its mechanism were closely related to the changes of miR-139 expression. [conclusion] the expression of APPL1 is related to the depth of invasion, TNM stage and lymph node metastasis of gastric cancer. APPL1 can affect the proliferation of gastric cancer cells by regulating the expression of cyclin. APPL1 can influence the expression of miRNA in gastric cancer cells. The effect of APPL1 on the proliferation of gastric cancer cells is related to the regulation of miR-139 expression.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2

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