| Pancreatic cancer is one of the most common gastrointestinal malignant tumors, which has become the fourth cause of cancer death in western countries. Recently, the incidence of pancreatic cancer is increasing in China, which are ranked the7th among all of the cancers. For the low early diagnosis rate, half of the patients have developed systemic metastasis. Overall, pancreatic cancer carries an unfavorable prognosis with short survival rate and poor quality life due to lacking of effective treatment. The5-year overall survival rate is less than6%and the median survival time is4-6months.It is known that tumor angiogenesis is critical to tumor growth and metastasis. When the tumor is growing with a size larger than1-2mm3, its growth is angiogenesis-dependent. Many studies showed that a number of signaling molecules were involved in tumor angiogenesis, including CXCL8and VEGF as pro-angiogenesis factors. Furthermore, some chemokines and their receptors could regulate tumor angiogenesis.ARHI, a maternally imprinted gene belongs to the Ras superfamily is a tumor suppressor gene, which appeared high expression in ovarian and breast normal tissues, but down-regulated expression in ovarian and breast carcinomas. A series of studies in our group showed that ARHI was a tumor suppressor gene in pancreatic cancer. Re-expression of ARHI in pancreatic cancer cells can inhibit cell proliferation and invasion, induce apoptosis, autophagy and cell cycle arrest, and inhibit cell mobility. ARHI is an importment tumor suprressor gene in the occurrence and development of pancreatic cancer. Actually, the growth and metastasis of tumor cells is regulated by the tumor microenvironment, in which angiogenesis play a key role. Angiogenesis has become the important target for the current and future treatment strategy. Therefore, the study for ARHI, angiogenesis and related mechanisms has the important significance. Until now, there is no report to study ARHI-induced angiogenesis in pancreatic cancer.The aim of this study was to disclose the effect of ARHI on angiogenesis and related mechanism in pancreatic cancer and to analyze the impact of ARHI on chemokines and receptors in tumor microenvironment by human pancreatic cancer tissues, cell lines, and pancreatic cancer xenografted in Nude Mice. According to the above researches, we hope to find more theoretical basis of pathogenesis and feasibility of antiangiogenic therapy in pancreatic cancer for the further study.PART I:ARHI and angiogenesis-related protein in pancreatic cancer and its clinicopathological significance.Aim:To explore the relationship between pancreatic cancer and angiogenesis, and illustrate the role of ARHI in the pancreatic angiogenesis.Methods:1.23cases of pancreatic cancer and27cases of normal pancreas with clinical data were collected and recorded.2. Immuohistochemical technique was performed to detect the expression of ARHI, CD34, VEGF and CXCL8in the specimens.3. The relationship between the expression of above proteins and clinicopathological factors of pancreatic cancer was analyzed.4. The relationship among ARHI, MVD, VEGF and CXCL8analyzed.Results:1. The positive protein expression rate of ARHI in normal pancreas and pancreatic cancer tissue was88.9%and21.7%, there was a significant difference between the two groups. MVD in normal pancreas and pancreatic cancer are27.84±2.01and43.47±6.44, there was a significant difference. The positive expression rate of VEGF in normal pancreas and pancreatic cancer was44.4%and100%, there was a significant difference. The positive expression rate of CXCL8in normal pancreas and pancreatic cancer was44.4%and100%, there was a significant difference.2. ARHI expression showed significant negative correlation with tumor differentiation. CXCL8expression showed significant positive correlation with CA199and tumor differentiation. The expression of VEGF protein showed a significant correlation with age, gender, smoking history, drinking history, history of diabetes, serum CA199level, serum CA242level, clinical stage, tumor differentiation, tumor size, tumor location, lymphatic metastasis and vessel infiltration.3. In pancreatic cancer cases, MVD showed significant positive correlation with CXCL8, and there were no significant correlation on MVD with VEGF, CXCL8with VEGF, ARHI with MVD, ARHI with VEGF, ARHI with CXCL8.4. In all cases, CXCL8showed significant positive correlation with VEGF, ARHI showed significant negative correlation with MVD, VEGF and CXCL8.Summary:1) ARHI expression in pancreatic cancer was loss or significantly down-regulated, however, MVD, VEGF and CXCL8showed a significant high expression in pancreatic cancer.2) ARHI expression had significant correlation with tumor differentiation. MVD showed a significant correlation with serum CA199level, clinical stage, tumor differentiation, tumor size and lymphatic metastasis.3) In all cases, CXCL8showed significant positive correlation with VEGF, and ARHI showed significant negative correlation with MVD, VEGF and CXCL8. PART II:The effect of ARHI on angiogenesis of pancreatic cancer cell lines and xenografted in nude mice and the related mechanisms Aim:To investigate the effect of ARHI on angiogenesis of stably transfected PANC-1cell line and pancreatic cancer xenografted in Nude Mice, and to explore the related mechanisms.Methods:1. Both plasmids of pIRES2-ARHI-EGFP and pLNCX2-ARHI-EGFP were transfected into human pancreatic cancer cell line PANC-1by lipofectamine2000, the polyclonal stable PANC-1cell line with G418was selected.2. The expressions of ARHI mRNA and protein in pLNCX2-ARHI-EGFP group were analyzed by RT-PCR and Western blot methods respectively. MTT assay were used to determine the effect of ARHI on the cell proliferation.3. The expressions of mTOR, SOSC3, Stat3, NF-κB, VEGF and CXCL8were detected at the mRNA level by Real-time PCR.4. The cells with pLNCX2-ARHI-EGFP, pLNCX2-EGFP and control were injected subcutaneously into nude mice to establish the pancreatic cancer xenografted model. The tumor growth, size, and body weight change of nude mice were recorded and compared.5. The protein expression of ARHI, Ki-67, CD34, VEGF and CXCL8protein in xenografted tumor tissues were studied by immunohistochemistry. The expressions of mTOR, SOSC3, Stat3, NF-κB, VEGF and CXCL8were detected at the mRNA level in xenografted tumor tissues by Real-time PCR.Results:1. After4weeks of G418selection, the polyclonal stable transfected PANC-1cell line was identified with ARHI mRNA and protein expression by RT-PCR and western blot.2. The stable cell lines transfected with lentivirus plasmid pLNCX2-ARHI-EGFP were chose for further studies due to the stronger fluorescence density.3. The MTT assay showed that ARHI could inhibit cell proliferation in pLNCX2-ARHI-EGFP group with a significant difference compared with that in vector group at the third day.4. The expression of mTOR, Stat3, NF-κB, VEGF and CXCL8at the mRNA level were significantly down-regulated in pLNCX2-ARHI-EGFP group compared with the polyclonal vector stable cell line, while the SOSC3gene was significantly up-regulated. Western blot analysis showed that the expression of p-mTOR, p-Stat3, NF-κB and CXCL8proteins were significantly down-regulated in pLNCX2-ARHI-EGFP group.5. The growth of tumor in nude mice was much slower in pLNCX2-ARHI-EGFP group than the vector group and control group, meanwhile, the size of tumors of ARHI group were also smaller than the other two groups, and the body weight change of vector group and control group were more obvious than pLNCX2-ARHI-EGFP group.6. Pancreatic cancer xenografted models was successfully established with ARHI positive expression.7. In pLNCX2-ARHI-EGFP group xenografted model, the ARHI protein expressed in the higher level than that in vector group and control group, while the Ki-67index, MVD (characterized with CD34), VEGF and CXCL8protein expressed in lower level inversely.8. The expression of mTOR, Stat3, NF-κB, VEGF and CXCL8at the mRNA level were significantly down-regulated in pLNCX2-ARHI-EGFP group xenografted model than the vector group, while while the SOSC3gene was significantly up-regulated, and the result was in accordance with that in the cell level.Summary:1) The polyclonal stable transfected PANC-1cell line which could express ARHI was successfully established.2) Re-expression of ARHI in stable transfected PANC-1cell line could inhibit proliferation of pancreatic cancer cells.3) The pancreatic cancer xenografted nude mice model was successfully established, and the data in the animal levels showed that ARHI gene had an inhibition effect on tumor growth and angiogenesis in pancreatic cancer.4) ARHI gene may inhibit the expression of CXCL8and VEGF by means of mTOR/NF-KB and STAT3signaling pathway, eventually inhibit angiogenesis of pancreatic cancer.Part Ⅲ:The effect of ARHI transfection on chemokines and receptors gene expression profileAim:To explore the effect of ARHI transfection on the chemokines and receptors gene expression profile of PANC-1cells.Methods:1. Expression profile, including84chemokines and receptors related genes was detected by using quantitative real-time PCR based RT2Profiler(?) PCR Array.2. Real-time PCR was performed to detect mRNA expression of CXCL8, CXCL1, CXCR4, CXCR3and CXCR2in stable transfected cell lines and xenografted cancer tissues.Results:1. PCR Array:1) In PANC-1cells transfected with pLNCX2-ARHI-EGFP, the expression levels of mRNA of36genes were down-regulated, and9genes were up-regulated.2) Among these genes related with tumor angiogenesis, CCL2,CXCL1,CXCL2,CXCL8,CXCL12,CXCR4were significantly down-regulated, CXCL3,CCR1,CCR2were slightly down-regulated. CXCL9,CXCL10,CXCL11,CXCR3were significantly up-regulated, and the increase of CXCL9was the most significant (-27.8fold).3) Among these genes related with tumor metastasis and invasion, CXCL12and CXCR4were significantly down-regulated, MMP-2was slightly down-regulated. Among these genes related with location of organ infiltration and latency, CXCL12,CXCR4,CCR7were significantly down-regulated, CXCR5was slightly down-regulated. Among these gene with tumor immunity, CXCL8,CXCR1,CCR7were significantly down-regulated, and decrease of CXCR1was the most significant (-41.3fold).2. The expression of CXCL8, CXCL1and CXCR4at the mRNA level were significantly down-regulated in pLNCX2-ARHI-EGFP group compared with the vector group, while the CXCR3gene was significantly up-regulated, the result was the same as PCR Array. We also found the expression of CXCR2was down-regulated in pLNCX2-ARHI-EGFP group compared with the vector group.3. In xenografted cancer tissues, the expression of CXCL8, CXCL1, CXCR4and CXCR2at the mRNA level were significantly down-regulated in pLNCX2-ARHI-EGFP group compared with the vector group, while the CXCR3gene was significantly up-regulated, and the result was in accordance with that in the cell level.Summary:ARHI gene could influence the expression of many chemokines and receptors, which suggests that the mechanisms of ARHI gene inhibiting tumor growth partly correlate with chemokine and receptors to regulating tumor angiogenesis and immunity in microenvironment in pancreatic cancer.Conclusions:1) The expression of ARHI gene in pancreatic cancer was loss or significantly down-regulated, which verified the inhibition effect of ARHI gene. ARHI showed significant negative correlation with MVD, VEGF and CXCL8.2) Re-expression of ARHI gene could inhibit proliferation of pancreatic cancer cells. ARHI gene could inhibit the growth and angiogenesis of xenografted cancer tissues, as well as the expression of VEGF and CXCL8.3) ARHI gene may inhibit the expression of CXCL8and VEGF by means of mTOR/NF-KB and STAT3signaling pathway, eventually inhibit angiogenesis of pancreatic cancer.4) ARHI gene could influence expression of many chemokines and receptors, and then inhibit tumor growth and metastasis. |
