Study On Expression Silencing And Inhibition Metastasis Properties Of 15-Hydroxyprostaglandin Dehydrogenase Gene In Human Colorectal Cancer

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.