發(fā)布時間：2018-09-11 14:22

【摘要】：背景胰腺癌發(fā)病隱匿,預后極差,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)展機制,發(fā)現(xiàn)潛在的治療靶點和預后相關生物標記物,對胰腺癌的治療有重要的意義。前列腺跨膜雄激素誘導蛋白 1 (prostate transmembrane protein, androgen i nduced 1,PMEPA1),又稱實體瘤相關基因1,是近年來發(fā)現(xiàn)的腫瘤相關蛋白編碼基因。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,能通過轉化生長因子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)、缺氧等信號通路,參與惡性腫瘤細胞的增殖、凋亡、遷移、侵襲等生物學行為,并與腫瘤的預后相關。然而,PMEPA1在胰腺癌中的表達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情況及其與臨床病理學指標和預后的相關性;研究PMEPA1異常表達對胰腺癌細胞生物學行為的影響;探討胰腺癌細胞中PMEPA1與PTEN/Akt通路的調控機制。方法1. 1)收集15對胰腺癌組織及其配對癌旁組織,檢測胰腺癌及癌旁組織中PMEPA1 mRNA水平;2)收集74對胰腺癌組織及其配對癌旁組織,以及18例胰腺癌組織,應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情況;3)統(tǒng)計臨床病理資料,應用Pearson χ2檢驗分析胰腺癌中PMEPA1表達與胰腺癌病人的臨床病理學指標之間的關系;4)應用Kaplan-Meier法及Cox回歸風險比例模型分析胰腺癌中PMEPA1表達與胰腺癌病人術后生存的相關性。2. 1)通過Western blot方法檢測胰腺癌細胞株與胰腺導管上皮細胞中PMEPA1蛋白的表達情況;2)構建慢病毒shRNA載體和PMEPA1表達載體,對胰腺癌細胞中PMEPA1基因進行敲減和過表達,篩選穩(wěn)定表達shRNA和PMEPA1過表達序列的胰腺癌細胞;3)通過CCK-8、平板克隆形成實驗及裸鼠皮下移植瘤實驗,檢測PMEPA1表達改變對胰腺癌細胞增殖能力的影響;4)通過Transwell細胞遷移實驗和侵襲實驗,檢測PMEPA1表達改變對胰腺癌細胞遷移和侵襲能力的影響。3. 1)通過Western blot方法檢測PMEPA1過表達后以及轉染PTEN表達載體后,胰腺癌細胞 PTEN/Akt 通路中 PTEN、Akt、pAkt、p27kip1、CyclinD1 蛋白的變化;2)通過CCK-8實驗檢測PMEPA1過表達后以及轉染PTEN表達載體后,胰腺癌細胞增殖活力的變化;3)通過qPCR方法檢測PMEPA1過表達后以及轉染PTEN表達載體后,胰腺癌細胞EMT相關標記物的變化。結果1. 1)胰腺癌組織中PMEPA1的mRNA水平顯著高于配對癌旁組織(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于細胞漿內;胰腺癌組織中高表達PMEPA1蛋白的比例顯著高于其配對癌旁組織(63. 51 % vs.10.81%, P0. 001);3) PMEPA1蛋白的表達水平與胰腺癌的組織學分級(χ2=4. 552,P=0.033)、淋巴結轉移(χ2=5. 902,P=0.015)相關;4)Cox回歸多因素分析顯示:PMEPA1高表達(HR=1.956,P=0.015)是胰腺癌病人術后預后的獨立危險因素;5) PMEPA1蛋白高表達的病人的生存時間顯著短于PMEPA1蛋白低表達的病人(中位生存期:7. 7個月vs. 23個月),生存時間存在統(tǒng)計學差異(χ2==6. 979,P=0.008)。2. 1) PMEPA1蛋白在人胰腺癌細胞株AsPC-1,BxPC-3,CFPAC-1,SW1990中的表達水平高于人胰管上皮細胞株HPDE6-c7;2)與陰性對照組和空白對照組相比,穩(wěn)定轉染靶向PMEPA1shRNA的細胞中PMEPA1蛋白表達減少,穩(wěn)定轉染PMEPA1表達序列的細胞中PMEPA1蛋白表達增加;3)與陰性對照組相比,過表達PMEPA1的BxPC-3細胞的增殖活力提高(P0.001)、克隆形成數(shù)目增加(P0. 001)、Transwell遷移實驗和侵襲實驗中穿透小室膜的細胞數(shù)增加(P0. 01);4)與陰性對照組相比,敲減PMEPA1的AsPC-1細胞的增殖活力降低(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遷移實驗和侵襲實驗中穿透小室膜的細胞數(shù)減少(P0. 01)。3. 1)與陰性對照組相比,過表達PMEPA1的BxPC-3細胞的PTEN蛋白表達減少,磷酸化Akt表達增加,總Akt蛋白表達無明顯改變;Akt信號下游的細胞周期蛋白D1表達增加,p27kip1表達減少;轉染PTEN表達載體后,過表達PMEPA1的BxPC-3細胞中磷酸化Akt表達減少,細胞周期蛋白D1表達減少,p27kip1表達增加;2)過表達PMEPA1的BxPC-3細胞轉染PTEN表達載體后,細胞增殖活力降低(P0. 001)。3)與陰性對照組相比,PMEPA1過表達后,Snail、N-cadherin和Vimentin的mRNA水平升高,E-cadherin的mRNA水平下降(P0. 001); PMEPA1過表達并轉染PTEN表達載體后,Snail、N-cadherin和Vimentin的mRNA水平降低,E-cadherin 的 mRNA 水平升高(P0. 001 )。結論1. PMEPA1在胰腺癌組織中表達高于癌旁組織;PMEPA1高表達與胰腺癌組織學分級和淋巴結轉移相關,是胰腺癌病人術后生存的獨立風險因素;2. PMEPA1能促進胰腺癌細胞的增殖、遷移和侵襲;3. PMEPA1通過抑制PTEN,激活Akt,促進細胞周期相關蛋白的表達,并誘導胰腺癌細胞中EMT相關分子標志物改變。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.9

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