The Effects Of IRF-2on The Proliferation、 Apoptosis And Chemosensitivity Of Pancreatic Cancer

Pancreatic cancer is one of the most aggressive malignant cancers ranking eighth as the cause of cancer-related death in the world. Although the technology of diagnosis and therapy of pancreatic cancer was improved in recent years, survival rate and median survival of the patients are still pessimistic, which is mainly due to the difficulty in detecting at the early stage and the complicated pathological mechanism. Furthermore,tumorigenesis of pancreatic cancer is attributed to dysregulation of multiple genes. Interferon regulator factor2(IRF-2) belongs to the family of interferon regulatory factors, which has been known to be involved in tumorigenesis of some cancers.However, there is no systemic study about the function of IRF-2in pancreatic cancer.In this study,we studied the function of IRF-2on pancreatic cancer from four aspects. Firstly,we construct the siRNA vector to silence the expression of IRF-2in pancreatic cancer cells. Secondly, we studied the function of IRF-2on the proliferation, tumorgenesis, invasion and metastasis of pancreatic carcinoma cells. Thirdly, we evaluated the effects of IRF-2on chemosensitivity to gemcitabine in human pancreatic cancer cells. Finally, we explored the expression and prognostic significance of IRF-2in pancreatic cancer tissues.Part1The construction of siRN A vector to silence the expression of IRF-2Objective:To construct the siRNA vector of IRF-2and silence the expression of IRF-2in pancreatic cancer cells.Methods:Firstly, we construct the vector of pBS/hU6-land FG12containing the siRNA sequence which targeting the endogenous IRF-2gene. The virus was packaged in293T cells.Then, pancreatic cancer cells were infected by the virus and Western blot assay was used to detect the silencing efficiency. After3-4days,cells silencing the expression of IRF-2stably were sorted by FACS.Results:We constructed the vectors succesfully. The results of Western blot revealed that the expression of IRF-2was knocked down in MIAPaCa-2and PANC-1cells.Conclusions:The expression of IRF-2was knocked down successfully. Part2The effects of IRF-2on the biological character in pancreatic cancer cellsObjective:To study the function of IRF-2on the proliferation, tumorgenesis, invasion and metastasis of pancreatic cancer cells.Methods:The function of IRF-2on the proliferation、 tumorgenesis、 invasion and metastasis of pancreatic cancer cells was investigated by MTT assay、 BrdU incorporaing tassay、 colony formation assay、 soft agar assay、 chamber boyden assay、 tumor xenograft assays and In vivo metastasis assay. Finally, in order to explore the underlying mechanism, we checked the function of IRF-2on cell cycle and cell apoptosis.Results:In comparison with the control group, knock down the expression of IRF-2inhibits cell proliferation and tumorgenesis. But, there were no obvious differences on the cell mobility in vitro and in vivo assay. The FACS assay showed that knockdown the expression of IRF-2promoted apoptosis in comparison with the control group. We further found that this effect was associated with cell cycle arrest in G0-G1phase and S phase. Western blot analysis shown that knock down the expression of IRF-2significantly increased the expression of PARP、 BAX and caspase8and decreased some proliferation-related genes such as cyclinD1and PCNA were significantly down-regulated both in MIAPaCa-2and PANC-1.Conclusions:Silencing the expression of IRF-2inhibited cell growth and promoted cell apoptosis. Part3The effects of IRF-2siRNA on chemosensitivity of gemcitabine in human pancreatic cancer cellsObjective:To evaluate the effects of IRF-2siRNA on chemosensitivity to gemcitabine in human pancreatic cancer cells.Methods:The IRF-2expression was detected by Western blot in PANC-1or MIAPaCa-2cell lines. MTT evaluated IC50value to detect chemotherapy sensitivity of the two cell lines. The changes of IC50value and gemcitabine sensitivity after siRNA IRF-2were examined by MTT assay. The influence of siRNA IRF-2altering gemcitabine sensitivity to pancreatic cancer in virto and in vive was tested by clony formation assay and xenograft in nude mice. Clony numbers、 Tumor growth and final weight of tumor tissues were observed. Cell cycle and cell apoptosis were analysed by FACS.Results:The expression of IRF-2in PANC-1cells was higher than those in MIAPaCa-2cells and the IC50of Gemcitabine to PANC-1cells was16.5±2.lμg/ml, whereas that of MIAPaCa-2was5.6±1.2μg/ml (P<0.05). After72h, under the treatment of gemcitabine, the IC50of Gemcitabine to PANC-1si2#cells was9.5±1.4μg/ml which was significant lower than those of control cells16.3±1.5μg/ml (p<0.05). Furthermore, after treatment of gemcitabine, the tumorgenesis of PANC-1si2#cells was inhibited in contrary to PANC-1si con cells according to the results of clony formation assay and xenograft in nude mice. The G1phase percent of PANC-1si2#cells was higher than PANC-1si con cells, meanwhile, the S phase percent of PANC-lsi2#cells was lower than PANC-lsi con cells (P<0.05).The percentage of apoptotic cells in PANC-lsi2#cells was higher than that in PANC-lsi con cells (P<0.05). Finally, after treatment of gemcitabine in PANC-lsi2#cells, the expression of apoptosis related genes such as PARP、 BAX and caspase-8was higher than that in PANC-1si con cells, but the expression of genes related to cell proliferation such as Cyclin D1and PCNA was lower than that in PANC-lsi con cells.Conclusions:The sensitivity of human pancreatic cancer cells to gemcitabine could be significantly enhanced by RNA interference knocking down the exp-ression of IRF-2. Part4The expression and prognostic significance of IRF-2in pancreatic cancer tissuesObjective:To investigate the expression of IRF-2in pancreatic cancer and study the correlation between the expression of IRF-2and clinicpatholigic characteristics in pancreatic cancers.Methods:We studied the expression of IRF-2in30fresh pancreatic cancer using immunohistochemery, western blot and real-time PCR, the paired normal samples were used as control. We performed the tissue microarray containing156pancreatic cancer specimens, as well as the paired normal pancreatic tissues. The expression of IRF-2was examined by immunohisto-chemical staining. Clinical and pathological data were retrospectively analyzed. We analyzed the correlation between IRF-2 expression with clinicopathologic characteristics and patients survival using X2sequare、 Univariate and mult-iva-riate survival analysis.Results:In30pancreatic cancer paired fresh samples,the expression of IRF-2in tumors was higher than those in matched normal tissues. In the tissue microarray, positive staining of IRF-2was detected in121patients(77.6%) and high IRF-2expression was associated with larger tumor size(X2=14.512; P=0.001), TNM stage(X2=21.297; P<0.001)and as well as poor tumor differentiation(X2=10.197; P=0.006). Furthermore, the patients with low IRF-2expression had much longer median survival time than those with high IRF-2expression(P=0.027). But the results of multivariate survival analyses did not indicate that high IRF-2expression was an independent unfavorable prognostic factor in pancreatic cancer tissues.Conclusions:The expression of IRF-2in pancreatic ductal adenocarcinoma tissues is significantly higher compared with in normal pancreatic tissues. High IRF-2expression may play roles in the progression of pancreatic cancer.