【摘要】：背景胰腺癌發(fā)病隱匿,預(yù)后極差,是致死率最高的惡性腫瘤之一。在我國,胰腺癌的發(fā)病率居所有惡性腫瘤的第9位,死亡率居第7位,其5年生存率僅為5%左右。除了早期根治性手術(shù)治療,目前尚無其他有效的治療方法。因此,深入探索胰腺癌的發(fā)生和發(fā)展機(jī)制,發(fā)現(xiàn)潛在的治療靶點(diǎn)和預(yù)后相關(guān)生物標(biāo)記物,對(duì)胰腺癌的治療有重要的意義。前列腺跨膜雄激素誘導(dǎo)蛋白 1 (prostate transmembrane protein, androgen i nduced 1,PMEPA1),又稱實(shí)體瘤相關(guān)基因1,是近年來發(fā)現(xiàn)的腫瘤相關(guān)蛋白編碼基因。研究顯示:PMEPA1在乳腺癌、肺癌和前列腺癌等惡性腫瘤中存在異常表達(dá),能通過轉(zhuǎn)化生長因子β (transforming growth factor-beta, TGF-β)、表皮生長因子(epidermal growth factor, EGF)、雄激素受體、蛋白激酶 B (protein kinase B,PKB/Akt)、缺氧等信號(hào)通路,參與惡性腫瘤細(xì)胞的增殖、凋亡、遷移、侵襲等生物學(xué)行為,并與腫瘤的預(yù)后相關(guān)。然而,PMEPA1在胰腺癌中的表達(dá)情況及其在胰腺癌中的作用尚未明確。目的明確PMEPA1在胰腺癌組織中的表達(dá)情況及其與臨床病理學(xué)指標(biāo)和預(yù)后的相關(guān)性;研究PMEPA1異常表達(dá)對(duì)胰腺癌細(xì)胞生物學(xué)行為的影響;探討胰腺癌細(xì)胞中PMEPA1與PTEN/Akt通路的調(diào)控機(jī)制。方法1. 1)收集15對(duì)胰腺癌組織及其配對(duì)癌旁組織,檢測胰腺癌及癌旁組織中PMEPA1 mRNA水平;2)收集74對(duì)胰腺癌組織及其配對(duì)癌旁組織,以及18例胰腺癌組織,應(yīng)用免疫組化染色檢測PMEPA1蛋白在胰腺癌組織及癌旁組織中的表達(dá)情況;3)統(tǒng)計(jì)臨床病理資料,應(yīng)用Pearson χ2檢驗(yàn)分析胰腺癌中PMEPA1表達(dá)與胰腺癌病人的臨床病理學(xué)指標(biāo)之間的關(guān)系;4)應(yīng)用Kaplan-Meier法及Cox回歸風(fēng)險(xiǎn)比例模型分析胰腺癌中PMEPA1表達(dá)與胰腺癌病人術(shù)后生存的相關(guān)性。2. 1)通過Western blot方法檢測胰腺癌細(xì)胞株與胰腺導(dǎo)管上皮細(xì)胞中PMEPA1蛋白的表達(dá)情況;2)構(gòu)建慢病毒shRNA載體和PMEPA1表達(dá)載體,對(duì)胰腺癌細(xì)胞中PMEPA1基因進(jìn)行敲減和過表達(dá),篩選穩(wěn)定表達(dá)shRNA和PMEPA1過表達(dá)序列的胰腺癌細(xì)胞;3)通過CCK-8、平板克隆形成實(shí)驗(yàn)及裸鼠皮下移植瘤實(shí)驗(yàn),檢測PMEPA1表達(dá)改變對(duì)胰腺癌細(xì)胞增殖能力的影響;4)通過Transwell細(xì)胞遷移實(shí)驗(yàn)和侵襲實(shí)驗(yàn),檢測PMEPA1表達(dá)改變對(duì)胰腺癌細(xì)胞遷移和侵襲能力的影響。3. 1)通過Western blot方法檢測PMEPA1過表達(dá)后以及轉(zhuǎn)染PTEN表達(dá)載體后,胰腺癌細(xì)胞 PTEN/Akt 通路中 PTEN、Akt、pAkt、p27kip1、CyclinD1 蛋白的變化;2)通過CCK-8實(shí)驗(yàn)檢測PMEPA1過表達(dá)后以及轉(zhuǎn)染PTEN表達(dá)載體后,胰腺癌細(xì)胞增殖活力的變化;3)通過qPCR方法檢測PMEPA1過表達(dá)后以及轉(zhuǎn)染PTEN表達(dá)載體后,胰腺癌細(xì)胞EMT相關(guān)標(biāo)記物的變化。結(jié)果1. 1)胰腺癌組織中PMEPA1的mRNA水平顯著高于配對(duì)癌旁組織(P0. 001);2)免疫組化顯示:PMEPA1蛋白在胰腺癌組織及癌旁組織主要表達(dá)于細(xì)胞漿內(nèi);胰腺癌組織中高表達(dá)PMEPA1蛋白的比例顯著高于其配對(duì)癌旁組織(63. 51 % vs.10.81%, P0. 001);3) PMEPA1蛋白的表達(dá)水平與胰腺癌的組織學(xué)分級(jí)(χ2=4. 552,P=0.033)、淋巴結(jié)轉(zhuǎn)移(χ2=5. 902,P=0.015)相關(guān);4)Cox回歸多因素分析顯示:PMEPA1高表達(dá)(HR=1.956,P=0.015)是胰腺癌病人術(shù)后預(yù)后的獨(dú)立危險(xiǎn)因素;5) PMEPA1蛋白高表達(dá)的病人的生存時(shí)間顯著短于PMEPA1蛋白低表達(dá)的病人(中位生存期:7. 7個(gè)月vs. 23個(gè)月),生存時(shí)間存在統(tǒng)計(jì)學(xué)差異(χ2==6. 979,P=0.008)。2. 1) PMEPA1蛋白在人胰腺癌細(xì)胞株AsPC-1,BxPC-3,CFPAC-1,SW1990中的表達(dá)水平高于人胰管上皮細(xì)胞株HPDE6-c7;2)與陰性對(duì)照組和空白對(duì)照組相比,穩(wěn)定轉(zhuǎn)染靶向PMEPA1shRNA的細(xì)胞中PMEPA1蛋白表達(dá)減少,穩(wěn)定轉(zhuǎn)染PMEPA1表達(dá)序列的細(xì)胞中PMEPA1蛋白表達(dá)增加;3)與陰性對(duì)照組相比,過表達(dá)PMEPA1的BxPC-3細(xì)胞的增殖活力提高(P0.001)、克隆形成數(shù)目增加(P0. 001)、Transwell遷移實(shí)驗(yàn)和侵襲實(shí)驗(yàn)中穿透小室膜的細(xì)胞數(shù)增加(P0. 01);4)與陰性對(duì)照組相比,敲減PMEPA1的AsPC-1細(xì)胞的增殖活力降低(P0.001)、克隆形成數(shù)目減少(P0. 01)、裸鼠皮下成瘤的速度減慢(P0. 001)、Transwell遷移實(shí)驗(yàn)和侵襲實(shí)驗(yàn)中穿透小室膜的細(xì)胞數(shù)減少(P0. 01)。3. 1)與陰性對(duì)照組相比,過表達(dá)PMEPA1的BxPC-3細(xì)胞的PTEN蛋白表達(dá)減少,磷酸化Akt表達(dá)增加,總Akt蛋白表達(dá)無明顯改變;Akt信號(hào)下游的細(xì)胞周期蛋白D1表達(dá)增加,p27kip1表達(dá)減少;轉(zhuǎn)染PTEN表達(dá)載體后,過表達(dá)PMEPA1的BxPC-3細(xì)胞中磷酸化Akt表達(dá)減少,細(xì)胞周期蛋白D1表達(dá)減少,p27kip1表達(dá)增加;2)過表達(dá)PMEPA1的BxPC-3細(xì)胞轉(zhuǎn)染PTEN表達(dá)載體后,細(xì)胞增殖活力降低(P0. 001)。3)與陰性對(duì)照組相比,PMEPA1過表達(dá)后,Snail、N-cadherin和Vimentin的mRNA水平升高,E-cadherin的mRNA水平下降(P0. 001); PMEPA1過表達(dá)并轉(zhuǎn)染PTEN表達(dá)載體后,Snail、N-cadherin和Vimentin的mRNA水平降低,E-cadherin 的 mRNA 水平升高(P0. 001 )。結(jié)論1. PMEPA1在胰腺癌組織中表達(dá)高于癌旁組織;PMEPA1高表達(dá)與胰腺癌組織學(xué)分級(jí)和淋巴結(jié)轉(zhuǎn)移相關(guān),是胰腺癌病人術(shù)后生存的獨(dú)立風(fēng)險(xiǎn)因素;2. PMEPA1能促進(jìn)胰腺癌細(xì)胞的增殖、遷移和侵襲;3. PMEPA1通過抑制PTEN,激活A(yù)kt,促進(jìn)細(xì)胞周期相關(guān)蛋白的表達(dá),并誘導(dǎo)胰腺癌細(xì)胞中EMT相關(guān)分子標(biāo)志物改變。
[Abstract]:Background Pancreatic cancer is one of the most lethal malignancies in China. The incidence of pancreatic cancer ranks ninth in all malignancies, the mortality rate ranks seventh, and the 5-year survival rate is only about 5%. There is no effective treatment for pancreatic cancer except early radical surgery. The discovery of potential therapeutic targets and prognostic biomarkers is of great significance in the treatment of pancreatic cancer. Prostate transmembrane androgen-inducible protein 1 (PMEPA1), also known as solid tumor-related gene 1, is a tumor-related protein code discovered in recent years. Gene. Studies have shown that PMEPA1 is abnormally expressed in breast cancer, lung cancer, prostate cancer and other malignant tumors. It can be signaled by transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), androgen receptor, protein kinase B (PKB/Akt), and hypoxia. However, the expression of PMEPA1 in pancreatic cancer and its role in pancreatic cancer have not been clearly defined. Methods 1.1) 15 pairs of pancreatic cancer tissues and their matched adjacent tissues were collected to detect the level of PMEPA1 mRNA in pancreatic cancer and adjacent tissues; 2) 74 pairs of pancreatic cancer tissues and their matched adjacent tissues, and 18 pairs of adjacent tissues were collected. Immunohistochemical staining was used to detect the expression of PMEPA1 protein in pancreatic cancer tissues and adjacent tissues; 3) Statistical analysis of clinicopathological data; Pearson_2 test was used to analyze the relationship between the expression of PMEPA1 in pancreatic cancer and clinicopathological parameters of pancreatic cancer patients; 4) Kaplan-Meier method and Cox regression risk ratio. The correlation between the expression of PMEPA1 in pancreatic cancer and the survival of pancreatic cancer patients was analyzed. 2.1) The expression of PMEPA1 in pancreatic cancer cell lines and pancreatic ductal epithelial cells was detected by Western blot; 2) Lentivirus shRNA vector and PMEPA1 expression vector were constructed to knock down and overexpress PMEPA1 gene in pancreatic cancer cells. Screening of pancreatic cancer cells stably expressing shRNA and PMEPA1 overexpression sequence; 3) Detecting the effect of PMEPA1 expression on the proliferation of pancreatic cancer cells by CCK-8, plate cloning and subcutaneous tumor transplantation in nude mice; 4) Detecting the effect of PMEPA1 expression on the migration of pancreatic cancer cells by Transwell cell migration and invasion experiments. 3.1) Western blot was used to detect the changes of PTEN, Akt, pAkt, p27kip1, CyclinD1 proteins in the PTEN/Akt pathway of pancreatic cancer cells after overexpression of PMEPA1 and transfection of PTEN expression vector; 2) CCK-8 assay was used to detect the proliferation activity of pancreatic cancer cells after overexpression of PMEPA1 and transfection of PTEN expression vector. Results 1.1) The mRNA level of PMEPA1 in pancreatic cancer tissue was significantly higher than that in matched adjacent tissues (P 0.001); 2) Immunohistochemistry showed that PMEPA1 protein was mainly expressed in pancreatic cancer tissue and adjacent tissues. In plasma, the expression of PMEPA1 protein in pancreatic cancer was significantly higher than that in adjacent tissues (63.51% vs. 10.81%, P 0.001); 3) The expression level of PMEPA1 protein was correlated with histological grade (_2 = 4.552, P = 0.033) and lymph node metastasis (_2 = 5.902, P = 0.015); 4) Cox regression analysis showed that the expression of PMEPA1 was high (HR = 1.956, P = 0.033). P = 0.015) was an independent risk factor for the prognosis of pancreatic cancer patients; 5) The survival time of patients with high expression of PMEPA1 protein was significantly shorter than that of patients with low expression of PMEPA1 protein (median survival time: 7.7 months vs. 23 months), and the survival time was statistically different (2 = = 6.979, P = 0.008).2.1) PMEPA1 protein in human pancreatic cancer cell lines AsPC-1, BxPC-1. - 3, CFPAC-1, SW1990 expression levels were higher than those of human pancreatic duct epithelial cell line HPDE6-c7; 2) Compared with the negative control group and blank control group, the expression of PMEPA1 protein in stably transfected PMEPA1 shRNA cells decreased, and the expression of PMEPA1 protein in stably transfected PMEPA1 cells increased. 3) Compared with the negative control group, the expression of Bx EPA1 protein in stably transfected PMEPA1 shRNA cells increased. The proliferation activity of PC-3 cells increased (P 0.001), the number of clone formation increased (P 0.001), the number of cells penetrating the ventricular membrane increased (P 0.01) in the experiment of Transwell migration and invasion; 4) Compared with the negative control group, the proliferation activity of ASPC-1 cells knocking down PMEPA1 decreased (P 0.001), the number of clone formation decreased (P 0.01), and the rate of subcutaneous tumorigenesis in nude mice decreased (P 0.001). Compared with the negative control group, the expression of PTEN protein and phosphorylated Akt protein in BxPC-3 cells overexpressing PMEPA1 were decreased, while the expression of total Akt protein was not significantly changed. After transfection with PTEN expression vector, the expression of phosphorylated Akt, cyclin D1 and p27kip1 in BxPC-3 cells overexpressing PMEPA1 decreased, and the expression of p27kip1 increased. 2) After transfection with PTEN expression vector, the proliferation activity of BxPC-3 cells overexpressing PMEPA1 decreased (P 0.001). The mRNA levels of dherin and Vimentin increased, while the mRNA levels of E-cadherin decreased (P 0.001). After overexpression of PMEPA1 and transfection of PTEN expression vector, the mRNA levels of Snail, N-cadherin and Vimentin decreased, while the mRNA levels of E-cadherin increased (P 0.001). Conclusion 1. The expression of PMEPA1 in pancreatic cancer tissue was higher than that in adjacent tissues. Histological grading is associated with lymph node metastasis and is an independent risk factor for survival in pancreatic cancer patients after surgery. 2. PMEPA1 can promote the proliferation, migration and invasion of pancreatic cancer cells. 3. PMEPA1 promotes the expression of cell cycle-related proteins by inhibiting PTEN and activating Akt, and induces the changes of EMT-related molecular markers in pancreatic cancer cells.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.9

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本文編號(hào)：2236941