發(fā)布時(shí)間：2019-01-23 19:54

【摘要】：[背景]胃癌是我國(guó)第三大類(lèi)最常見(jiàn)的惡性腫瘤,居于我國(guó)癌癥相關(guān)致死原因的第三位。轉(zhuǎn)移性患者的5年生存率只有20%～30%。APPL1在多種組織和細(xì)胞中都有表達(dá),是細(xì)胞內(nèi)具有多種重要生物學(xué)功能的銜接蛋白,由多個(gè)功能結(jié)構(gòu)域組成,包括BAR結(jié)構(gòu)域、PH結(jié)構(gòu)域、PTB結(jié)構(gòu)域。通過(guò)這些結(jié)構(gòu)域,APPL1能夠與多種細(xì)胞膜受體、信號(hào)轉(zhuǎn)導(dǎo)蛋白、細(xì)胞核因子等相結(jié)合,從而調(diào)控多種細(xì)胞生物學(xué)活動(dòng)與過(guò)程,與多種疾病的關(guān)系極為密切。[目的]闡明APPL1表達(dá)與胃癌臨床病理特征及預(yù)后的相關(guān)性,揭示APPL1對(duì)胃癌細(xì)胞增殖的影響及機(jī)制,明確APPL1對(duì)胃癌miRNA表達(dá)譜的影響。[方法]采用免疫組織化學(xué)染色和逆轉(zhuǎn)錄PCR的方法檢測(cè)APPL1在胃癌組織與非癌性胃組織中的蛋白及mRNA表達(dá)水平,統(tǒng)計(jì)分析APPL1在胃癌組織與非癌性胃組織中的表達(dá)差異,APPL1表達(dá)與胃癌臨床病理特征和預(yù)后的相關(guān)性。構(gòu)建過(guò)表達(dá)及干涉APPL1的慢病毒載體,感染體外培養(yǎng)的胃癌細(xì)胞并建立穩(wěn)定細(xì)胞系,采用MTT實(shí)驗(yàn)、BrdU摻入實(shí)驗(yàn)、流式細(xì)胞術(shù)、蛋白質(zhì)免疫印跡等分析過(guò)表達(dá)或干涉APPL1對(duì)胃癌細(xì)胞增殖、周期、凋亡的影響及分子機(jī)制。采用miRNA芯片技術(shù)分析干涉APPL1對(duì)胃癌細(xì)胞miRNA表達(dá)譜的影響,采用實(shí)時(shí)定量PCR對(duì)差異表達(dá)miRNA分子進(jìn)行鑒定,并分析差異miRNA分子對(duì)APPL1影響胃癌細(xì)胞增殖的影響。[結(jié)果]與非癌性胃組織相比,APPL1蛋白和mRNA的表達(dá)水平在胃癌組織中顯著升高,APPL1表達(dá)增高與胃癌的浸潤(rùn)深度、TNM分期、淋巴結(jié)轉(zhuǎn)移密切相關(guān),而與胃癌的組織病理類(lèi)型無(wú)關(guān),同時(shí)發(fā)現(xiàn)APPL1表達(dá)增高是導(dǎo)致胃癌患者預(yù)后不良的風(fēng)險(xiǎn)因素。過(guò)表達(dá)APPL1可導(dǎo)致SGC-7901胃癌細(xì)胞G1期比例降低、S期細(xì)胞比例增高,而干涉APPL1則導(dǎo)致SGC-7901胃癌細(xì)胞G1期比例增高、S期細(xì)胞比例降低,其分子機(jī)制主要是過(guò)表達(dá)APPL1促進(jìn)了 Cyclin D1的表達(dá),抑制了 p16、p27的表達(dá),而干涉APPL1的作用則相反。APPL1能夠影響胃癌細(xì)胞的miRNA表達(dá)譜,共篩選鑒定得到8個(gè)差異miRNA分子,其中miR-139的變化最為顯著,且APPL1對(duì)胃癌細(xì)胞增殖的影響及機(jī)制與miR-139的表達(dá)變化密切相關(guān)。[結(jié)論]APPL1表達(dá)與胃癌的浸潤(rùn)深度、TNM分期、淋巴結(jié)轉(zhuǎn)移相關(guān),是胃癌預(yù)后不良的風(fēng)險(xiǎn)因素。APPL1能夠通過(guò)調(diào)控細(xì)胞周期蛋白表達(dá)而影響胃癌細(xì)胞的增殖。APPL1能夠影響胃癌細(xì)胞的miRNA表達(dá)譜,其對(duì)胃癌細(xì)胞增殖的影響與調(diào)控miR-139的表達(dá)相關(guān)。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.2

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