【作者】 李美宁；

【导师】 程牛亮； 牛勃； 解军；

【作者基本信息】 山西医科大学 ， 生理学， 2008， 博士

【摘要】 研究背景:大肠癌是一种常见的消化系统恶性肿瘤。在西方经济发达国家,大肠癌的死亡率位于恶性肿瘤的第二位;在我国,其死亡率位于第四位左右。近二十年来,大肠癌在我国的发病率总体呈上升趋势,并且随着人们饮食结构和生活方式的改变,大肠癌的发病率还会继续上升。虽然大肠癌诊疗技术有着长足发展,但大肠癌的总体5年存活率并无显著提高,其主要原因是术后复发与肝转移,这也是制约大肠癌预后的主要因素。因此如何早期预测、早期发现、早期诊断与治疗大肠癌,预防复发与肝转移,是提高5年生存率的关键。因而积极开展大肠癌发生发展机制的研究,对于大肠癌的防治具有很重要的现实意义。15-羟基前列腺素脱氢酶(15-Hydroxyprostaglandin dehydrogenase,PGDH)是催化前列腺素降解的关键酶,亦是环氧化酶-2(cyclooxygenase-2)的抑制剂。最近研究发现PGDH是一个新的候选抑癌基因,在人体多种组织器官如胃、肠道、肺、肾脏及前列腺中广泛表达,但在多种人类肿瘤细胞系或肿瘤组织,如乳腺癌、肺癌、胃癌、前列腺癌中表达下调或缺失,而PGDH基因启动子区高甲基化是导致其在乳腺癌和前列腺癌中表达失活的主要原因之一,但PGDH基因在大肠癌中表达情况、启动子区甲基化状态及其生物学功能等研究工作国内尚无开展。因此深入探讨大肠癌中PGDH基因的表达情况及对其生物学功能的研究,将有助于进一步明确PGDH在大肠癌发生、发展中的作用,为大肠癌的预防和治疗提供切入点。研究目的:1.探讨PGDH基因在大肠癌中的表达情况,分析其与临床病理参数间的关系,评判PGDH基因是否为大肠癌的候选抑癌基因;2.检测PGDH基因启动子区甲基化状况,分析其与临床病理参数间的关系,探讨PGDH基因在大肠癌中表达失活的可能机制;3.构建PGDH基因真核表达载体——pcDNA3.1-PGDH,并转染细胞鉴定其表达及活性;4.研究PGDH基因对大肠癌SW480细胞株生长增殖的影响及相关基因的表达改变情况,初步分析其发挥作用的可能机制;5.观察PGDH基因对高转移大肠癌细胞Colo205侵袭转移能力的影响,以期进一步探讨PGDH基因可能的作用机理并寻找肿瘤治疗的新靶点。研究方法:1.应用免疫组织化学染色法分析30例大肠癌组织及癌旁正常组织中PGDH蛋白的表达差异,并分析其与临床病理参数之间的关系;2.采用MSP(甲基化特异PCR)技术检测30例大肠癌及癌旁正常组织中PGDH基因启动子区甲基化状况,并分析其与临床病理参数之间的关系;3. Trizol法提取大肠黏膜组织总RNA,利用RT-PCR扩增PGDH基因cDNA片段,采用基因工程的技术,以pcDNA3.1(+)为表达载体,定向克隆构建真核表达质粒pcDNA3.1(+)-PGDH,经限制性内切酶酶切与PCR方法初步鉴定阳性克隆后送生物公司进行序列测定。通过脂质体介导转染SW480肿瘤细胞,Western Blot分析PGDH蛋白的表达情况;进一步采用ELISA检测细胞培养液中PGE2的改变情况,观察转染表达的PGDH蛋白是否具有活性;4.在脂质体介导下转染到大肠癌SW480细胞,经平板集落形成、软琼脂克隆形成及MTT等试验体外观察PGDH基因对大肠癌细胞SW480恶性增殖的影响;流式细胞仪分析细胞周期及凋亡的改变情况,并应用Western Blot检测相关基因的表达改变情况,探讨可能的作用机理;5.在脂质体介导下转染到高转移大肠癌Colo205细胞,应用划痕擦伤试验、Transwell迁移试验和Transwell侵袭试验体外观察PGDH基因对Colo205细胞侵袭转移能力的影响;利用黏附实验研究PGDH对细胞与细胞外基质间黏附能力的改变;通过Western Blot和明胶酶谱试验初步探讨上述变化的可能机制。研究结果:1.免疫组织化学染色结果显示PGDH蛋白在大肠癌组织中存在广泛的表达下调:30例大肠癌组织中有19例(63.3%)PGDH呈弱阳性染色,11例(36.7%)PGDH表达缺失;而癌旁5cm正常对照组织中有26例(86.7%)PGDH呈强阳性表达,3例(10%)呈中等阳性表达,1例(3.3%)呈弱阳性表达。PGDH在癌组织和癌旁正常组织中的蛋白表达存在显著的统计学差异(P<0.01);PGDH表达缺失与淋巴结转移呈正相关,淋巴结转移组的PGDH阳性表达率(33.3%)明显低于无淋巴结转移组(83.3%),两者差别具有统计学意义(P<0.01);PGDH表达与肿瘤发生部位、年龄及性别无关;2. MSP分析结果表明的大肠癌组织中PGDH基因启动子区存在高甲基化(16/30,53.3%),而相应癌旁正常组织高甲基化不明显(6/30,20%),启动子甲基化率在癌及癌旁正常组织比较中有显著统计学差异(P<0.05);启动子高甲基化与淋巴结转移密切相关(P<0.01),无淋巴结转移组PGDH启动子甲基化率为33.3%,而淋巴结转移组为83.3%,二者相比有显著统计学差异性;PGDH启动子高甲基化与肿瘤发生部位、年龄及性别无关;3.提取总RNA经电泳检测完整性良好,RT-PCR扩增出845bp的PGDH基因cDNA片段。,经酶切与PCR鉴定目的片段已定向克隆入pcDNA3.1载体,送上海生工测序,序列测定结果表明其插入方向及碱基排列均正确;转染SW480细胞后,PGDH蛋白表达升高,并且培养液中的PGE2下降,提示真核表达载体构建成功并具有生物学活性;4.体外抑制增殖的研究发现,转染表达PGDH蛋白后SW480肿瘤细胞生长缓慢、细胞叠堆状或多层明显减少,集落形成能力明显降低,实验组平板集落形成率为16% ,而对照组为58%,两者间差异显著( P <0.05) ;软琼脂克隆形成能力降低,实验组软琼脂克隆体积小且形成个数(7±1.68)明显少于对照组(16.3±3.63) (P<0.05);流式细胞仪分析发现实验组静止期细胞所占比例增加,DNA合成期所占细胞比例减少,凋亡率升高; Western Blot结果表明,PGDH基因转染后P53、P21表达升高,Bcl-2表达降低;5.抑制肿瘤侵袭转移的研究发现,Colo205细胞转染表达PGDH蛋白后体外侵袭转移能力下降:划痕擦伤后实验组细胞平面迁移能力明显降低;Transwell游走实验结果发现对照组约有107.6±7.5个细胞穿过了聚碳酸酯膜,远远多于实验组的62.0±4.6,差异显著(P<0.05);Transwell侵袭实验显示对照组638.5±78.2个细胞穿过了聚碳酸酯膜,远远多于实验组的76.7±7.3,差异具有统计学意义;黏附实验结果发现空载体对照组OD490为0.57±0.06,而转染PGDH的实验组细胞OD490为0.21±0.02,细胞的黏附率降低了40-60%。明胶酶谱和Western Blot结果显示,黏附因子CD44表达降低,基质金属蛋白酶2(MMP-2)分泌降低,并且其活性下降。研究结论:1. PGDH蛋白在大肠癌组织中存在表达缺失或下调,基因启动子区高甲基化是其表达失活的主要原因之一,并且在大肠癌的发生发展中起着重要作用,因而PGDH是大肠癌的新型抑癌基因;2. PGDH基因可能通过依赖P53的P21、Bcl-2途径使肿瘤细胞增殖减慢并且凋亡增加,从而抑制肿瘤细胞的恶性增殖能力;PGDH基因能够抑制肿瘤细胞与细胞外基质的黏附能力和侵袭转移能力,可能是PGDH蛋白抑制了CD44表达与MMP-2的分泌及活化,从而抑制肿瘤细胞的侵袭转移。更多还原

【Abstract】 BackgroudColorectal cancer (CRC) is one of the most common gastrointestinal malignancies that severely threaten the health of human. In western countries, colorectal cancer is the second cause of cancer-related death, only next to lung cancer. In China, the mortality of colorectal carcinoma ranks from the 4th to 6th among all the cancer-related death. Furthermore, the mortality is still increasing, especially in the urban and developed rural area of China, due to the changes of life style and diet elements. Because of recurrence and liver metastasis, the 5 years survival rates of CRC have not been improved yet, although the techniques of diagnosis and therapy have highly developed. Therefore, it is the important measure to improve CRC patient survival through early detection and early therapy. Thus, it is of great significance to study the underlying mechanisms of CRC for its prevention and treatment.Colorectal carcinogenesis is believed to be a long-term and multi-step process involving the activation of oncogenes and inactivation of tumor suppressor genes. Although many genes are reported to be closely related to the colorectal carcinogenesis,there are still a lot of other known or unknown genes which remained to be discovered.Several studies have shown that cyclooxygenases2 (COX-2), which catalyzes dioxygenation and cyclization of arachidonic acid to prostaglandin E2 (PGE2), increase in expression in several tumors. Similarly, accumulating evidence show an important role for PGE2 in the development of CRC. High PGE2 levels within colon tumors are associated with increased proliferation, and angiogenesis. However, the level of PGE2 is controlled not only by synthesis but also by degradation, a fact that has been overlooked in studying prostaglandin and cancer.The first and key enzyme involved in prostaglandin catabolism is NAD+-linked 15-Hydroxyprostaglandin dehydrogenase (PGDH), which is reported as a new tumor suppressor gene. PGDH is widely distributed in various mammalian tissues such as lung, liver, kidney, prostate, etc. Among which colon and rectum are the most active tissues. Previous researches have shown that PGDH is highly expressed in these normal epithelia, while is loss or down-expression in the corresponding malignant tissues and tumor cells. Recent data suggest PGDH could inhibit lung cells proliferation and induced apoptosis; In breast cancer and prostate cancer, the promoter hypermethylation is one of the reasons of PGDH expression silencing. Howerve, in our country, there has not reported PGDH expression state and bio-function in CRC. Thus,elucidating clearly the mechanisms underlying aberrant PGDH expression in CRC could contribute to clarify the role of PGDH in colorectal carcinogenesis,which maybe beneficial to the diagnosis and treatment of CRC.Objiective1. To evaluate expression of the PGDH gene and potential clinical implications in 30 CRC tissues and matched 30 adjacent tissues.2. To detect promoter hypermethylation of the PGDH gene and potential clinical implications in 30 CRC tissues and corresponding normal tissues.3. To further explore the PGDH gene bio-function in tumor cells, we constructed the eukaryotic expression vector pcDNA3.1-PGDH.4. To investigate the inhibitory proliferation behaviors in SW480 cell line and detect the states of the related proliferation genes after transfected expression PGDH.5. To study the role of the PGDH gene in the metastasis and invasion process of CRC cell line Colo205 and potential mechanisms.Methods1. To detect expression of PGDH protein by immunohistochemistry in 30 primary CRC tissues and corresponding normal tissues and the results were compared with the clinicopathollogical data.2. Methylation-specific PCR (MSP) was operated to detect promoter region methylation status of the PGDH gene in CRC. The results were compared with the clinicopathological data.3. We designed and synthesized specific primers for PGDH by primer premier 5.0. Total RNA was isolated from human normal colonic epithelia. Then the PGDH cDNA gene was amplified by reverse transcript polymerase chain reaction (RT-PCR). The amplified fragment was orientationly linked into the eukaryotic expression vector pcDNA3.1(+) by T4 DNA ligase. The recombinant plasmid pcDNA3.1(+)-PGDH was constructed and identified by the sequence analysis. To study the PGDH vector activity, we applied ELISA assay to detect the level of PGE2 in the culture medium after transfected 72h. To further investigate the activity of PGDH vector, PGE2 level in cell medium was measured using ELISA assay.4. To further explore the inhibitory functions of PGDH in cancer, pcDNA3.1- PGDH vector was transiently transfected into SW480 cells cells by Liperfectamine 2000, which were negative for the genes. The effects on proliferation and malignant of PGDH over-expression on SW480 cells were evaluated by growth characteristics such as growth in monolayer culture, growth curve with MTT assay and anchorage-independent growth in soft agar. Flow cytometry was used to analyze the cell cycle and the rates of apoptosis. We detected the expression alteration of the related proliferation genes suah as P53, P21, BCL-2 and BAX through Western Blot.5. To further evaluate the role of PGDH in cancer metastasis, pcDNA3.1- PGDH vector was transiently transfected into Colo205 cells by Liperfectamine 2000, which were negative for the genes. Then, we took use of methods to exam PGDH inhibitor faculty such as scrape-wound-migration assay, Transwell migration assay, Transwell invasion assay and cell adhesion ability to extracellular matrix (ECM) by MTT test. To test the hypothesis that PGDH affects proteases and inactivates ECM, western blot and gelatin zymography were performed by using serum-free conditioned medium.Results1. Loss expression or down regulation expression of PGDH protein was detected in 11(36.7%) or 19 (63.3%) of 30 CRC tissues by IHC, whereas PGDH protein was detectable to highly expresse in 29 (96.7%) of the matched 30 adjacent normal tissues (P<0.01). Loss and down regulation of PGDH was significantly correlated with lymph node metastasis (P<0.05). No relationship was observed between expression of PGDH and sex, age and tumor location (P>0.05).2. MSP analysis demonstrated that 53.3% (16/30) of CRC but 20% (6/30) of adjacent normal tissues was hypermethylated in the promoter of the PGDH gene. There was siginifecant difference of promoter hypermethylation of the PGDH gene between CRC tissues and adjacent normal tissues (P<0.05). The clinicopatnological analysis showed that the PGDH gene promoter hypermethylation was closely associated with lymph node metastasis (P<0.01). Compared with 33.5% of hypermethylation in group without lymph node metastasis, it was significantly higher in those with lymph node metastasis (83.3%).3. The total RNA was isolated and had integrity. The PGDH cDNA fragment was correctly amplified by RT-PCR. The sequence analysis result indicated the recombinant plasmid pcDNA3.1(+)-PGDH was constructed successfully. With the expression of PGDH protein, PGE2 level was significantly decreased in cell medium.4. The PGDH overexpressing cells showed a significantly slower growth rate and a more degree of apoptosis compared with those of the vector control cells. Colony formation activity of SW480/pcDNA3.1-PGDH was 16% while that of the control cell was 58% (P < 0.05). The activity of anchorage-independent proliferation of SW480 /pcDNA3.1PGDH (7±1.68) was lower than that of SW480/pcDNA3.1 (16.3±3.63) in soft agar. Endogenous PGDH expression was negatively correlated with neoplastic potential as evidenced by attenuated anchorage-independent growth.Otherwise, the results of flow cytometry showed that the cell cycle was arrested in G1 and the rate of apoptosis increased. In mechanistic studies, we found that p53 protein and p21 protein, the negative regulate factors in cell cycle, could be induced higher expression than those of the vector control cells. The apoptosis inhibitor BCL-2 could be reduced.5. After transfected express PGDH protein, the metastasis ability was highly weakened. Over-expression of PGDH decreased cell migration and cell invasion approximately 1.9 and 8.4-fold, respectively. The ability of cells adhesion to ECM decreased. Zymography and Western Blot results demonstrated that PGDH protein cound inhibit matrix metalloproteinase-2 (MMP2) synthesis and secretion. In addition, the analysis of the MMP2 activity indicated that expression of PGDH could inhibit activation of MMP2. Furthermore, we also found that PGDH inhibited cells adhesion to ECM and reduced CD44 expression in Colo205 cell. Taken together, PGDH protein might decrease the abilities of metastasis of Colo205 cells by restrainingMMP-2 activated.Conclusion1. Loss or down regulation expression of PGDH gene is frequent in CRC, which is closely associated with lymph node metastasis, PGDH is a putative tumor suppressor gene in CRC. Our findings suggest that epigenetic silencing of PGDH gene expression by promoter hypermethylation could play an important role in CRC. The detection of PGDH hypermethylation by MSP could be a new useful molecular marker of CRC.2. These data indicate that PGDH might partly reverse malignant behavior of CRC cells by suppressing cell proliferation and inducing apoptosis. The suppressing cell proliferation function may be resulted from through p53 pathway;These results demonstrate that induced PGDH gene expression may contribute to inhibiting the invasiveness and metastatic ability of colon cancer cells. The probable mechanism is though decreasing MMP-2 and CD44 expression.This finding may be helpful in delineating molecular mechanism of tumorigenicity and providing a potential route for CRC gene therapy in the future.更多还原