PMEPA1在胰腺癌中的表达及对胰腺癌细胞生物学行为的影响
发布时间：2018-09-11 14:22
【摘要】：背景胰腺癌发病隐匿,预后极差,是致死率最高的恶性肿瘤之一。在我国,胰腺癌的发病率居所有恶性肿瘤的第9位,死亡率居第7位,其5年生存率仅为5%左右。除了早期根治性手术治疗,目前尚无其他有效的治疗方法。因此,深入探索胰腺癌的发生和发展机制,发现潜在的治疗靶点和预后相关生物标记物,对胰腺癌的治疗有重要的意义。前列腺跨膜雄激素诱导蛋白 1 (prostate transmembrane protein, androgen i nduced 1,PMEPA1),又称实体瘤相关基因1,是近年来发现的肿瘤相关蛋白编码基因。研究显示:PMEPA1在乳腺癌、肺癌和前列腺癌等恶性肿瘤中存在异常表达,能通过转化生长因子β (transforming growth factor-beta, TGF-β)、表皮生长因子(epidermal growth factor, EGF)、雄激素受体、蛋白激酶 B (protein kinase B,PKB/Akt)、缺氧等信号通路,参与恶性肿瘤细胞的增殖、凋亡、迁移、侵袭等生物学行为,并与肿瘤的预后相关。然而,PMEPA1在胰腺癌中的表达情况及其在胰腺癌中的作用尚未明确。目的明确PMEPA1在胰腺癌组织中的表达情况及其与临床病理学指标和预后的相关性;研究PMEPA1异常表达对胰腺癌细胞生物学行为的影响;探讨胰腺癌细胞中PMEPA1与PTEN/Akt通路的调控机制。方法1. 1)收集15对胰腺癌组织及其配对癌旁组织,检测胰腺癌及癌旁组织中PMEPA1 mRNA水平;2)收集74对胰腺癌组织及其配对癌旁组织,以及18例胰腺癌组织,应用免疫组化染色检测PMEPA1蛋白在胰腺癌组织及癌旁组织中的表达情况;3)统计临床病理资料,应用Pearson χ2检验分析胰腺癌中PMEPA1表达与胰腺癌病人的临床病理学指标之间的关系;4)应用Kaplan-Meier法及Cox回归风险比例模型分析胰腺癌中PMEPA1表达与胰腺癌病人术后生存的相关性。2. 1)通过Western blot方法检测胰腺癌细胞株与胰腺导管上皮细胞中PMEPA1蛋白的表达情况;2)构建慢病毒shRNA载体和PMEPA1表达载体,对胰腺癌细胞中PMEPA1基因进行敲减和过表达,筛选稳定表达shRNA和PMEPA1过表达序列的胰腺癌细胞;3)通过CCK-8、平板克隆形成实验及裸鼠皮下移植瘤实验,检测PMEPA1表达改变对胰腺癌细胞增殖能力的影响;4)通过Transwell细胞迁移实验和侵袭实验,检测PMEPA1表达改变对胰腺癌细胞迁移和侵袭能力的影响。3. 1)通过Western blot方法检测PMEPA1过表达后以及转染PTEN表达载体后,胰腺癌细胞 PTEN/Akt 通路中 PTEN、Akt、pAkt、p27kip1、CyclinD1 蛋白的变化;2)通过CCK-8实验检测PMEPA1过表达后以及转染PTEN表达载体后,胰腺癌细胞增殖活力的变化;3)通过qPCR方法检测PMEPA1过表达后以及转染PTEN表达载体后,胰腺癌细胞EMT相关标记物的变化。结果1. 1)胰腺癌组织中PMEPA1的mRNA水平显著高于配对癌旁组织(P0. 001);2)免疫组化显示:PMEPA1蛋白在胰腺癌组织及癌旁组织主要表达于细胞浆内;胰腺癌组织中高表达PMEPA1蛋白的比例显著高于其配对癌旁组织(63. 51 % vs.10.81%, P0. 001);3) PMEPA1蛋白的表达水平与胰腺癌的组织学分级(χ2=4. 552,P=0.033)、淋巴结转移(χ2=5. 902,P=0.015)相关;4)Cox回归多因素分析显示:PMEPA1高表达(HR=1.956,P=0.015)是胰腺癌病人术后预后的独立危险因素;5) PMEPA1蛋白高表达的病人的生存时间显著短于PMEPA1蛋白低表达的病人(中位生存期:7. 7个月vs. 23个月),生存时间存在统计学差异(χ2==6. 979,P=0.008)。2. 1) PMEPA1蛋白在人胰腺癌细胞株AsPC-1,BxPC-3,CFPAC-1,SW1990中的表达水平高于人胰管上皮细胞株HPDE6-c7;2)与阴性对照组和空白对照组相比,稳定转染靶向PMEPA1shRNA的细胞中PMEPA1蛋白表达减少,稳定转染PMEPA1表达序列的细胞中PMEPA1蛋白表达增加;3)与阴性对照组相比,过表达PMEPA1的BxPC-3细胞的增殖活力提高(P0.001)、克隆形成数目增加(P0. 001)、Transwell迁移实验和侵袭实验中穿透小室膜的细胞数增加(P0. 01);4)与阴性对照组相比,敲减PMEPA1的AsPC-1细胞的增殖活力降低(P0.001)、克隆形成数目减少(P0. 01)、裸鼠皮下成瘤的速度减慢(P0. 001)、Transwell迁移实验和侵袭实验中穿透小室膜的细胞数减少(P0. 01)。3. 1)与阴性对照组相比,过表达PMEPA1的BxPC-3细胞的PTEN蛋白表达减少,磷酸化Akt表达增加,总Akt蛋白表达无明显改变;Akt信号下游的细胞周期蛋白D1表达增加,p27kip1表达减少;转染PTEN表达载体后,过表达PMEPA1的BxPC-3细胞中磷酸化Akt表达减少,细胞周期蛋白D1表达减少,p27kip1表达增加;2)过表达PMEPA1的BxPC-3细胞转染PTEN表达载体后,细胞增殖活力降低(P0. 001)。3)与阴性对照组相比,PMEPA1过表达后,Snail、N-cadherin和Vimentin的mRNA水平升高,E-cadherin的mRNA水平下降(P0. 001); PMEPA1过表达并转染PTEN表达载体后,Snail、N-cadherin和Vimentin的mRNA水平降低,E-cadherin 的 mRNA 水平升高(P0. 001 )。结论1. PMEPA1在胰腺癌组织中表达高于癌旁组织;PMEPA1高表达与胰腺癌组织学分级和淋巴结转移相关,是胰腺癌病人术后生存的独立风险因素;2. PMEPA1能促进胰腺癌细胞的增殖、迁移和侵袭;3. PMEPA1通过抑制PTEN,激活Akt,促进细胞周期相关蛋白的表达,并诱导胰腺癌细胞中EMT相关分子标志物改变。
[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.
【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】：R735.9
本文编号：2236941
[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.
【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】：R735.9
【参考文献】
相关期刊论文 前2条
1 雷振伟;张瑜;张旭;;miR-181a在人类恶性肿瘤中作用机制的研究进展[J];解放军医学院学报;2016年11期
2 郭晓川;石燕;陈丽;施伟伟;韩雅琳;秦锐;戴广海;;缺氧诱导因子对肿瘤细胞上皮-间质转化的诱导机制[J];解放军医学院学报;2015年01期
，本文编号：2236941
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2236941.html
