The Correlated Mechanism Research On The Regulation Of Proliferation And Apoptosis By HIF-1α In Pancreatic Ductal Adenocarcinoma

The tumor has the characteristics of infinite proliferation and avoidance of apoptosis.The occurrence and development of tumors are the result of multiple factors and links,involving tumor metabolism,and tumor immunity.Currently,it is confirmed that abnormal glucose metabolism in tumors can meet the energy for tumor growth and provide the raw materials needed for tumor cell proliferation,as well as acidic microenvironments which can inhibit the response of effector T cells,monocyte migration,and alter the secretion of cytokines.In addition,because PDAC is a typical form of hypovascular tumor,the tumor is often in a hypoxic microenvironment.HIF-1α,as an important hypoxia regulating factor of tumors,can regulate multiple pathways involved in tumor growth including glycolysis.Hypoxia can induce non-specific CD4+T cells to differentiate into regulatory T cells or T helper cells,and HIF-la can also regulate the transcription factor FOXP3,regulate T cell immune checkpoints and so on.Therefore,abnormal tumor glucose metabolism,hypoxic microenvironment and tumor immunity are inseparable promotional factors for cancer.Hypoxia inducible factor(HIF-1α)promotes the apoptosis of tumor cells by regulating factors such as Bcl-2 and Bax.Mitochondrial apoptosis,as the central link during cell apoptosis,is closely related to mitochondrial morphology and especially,mitochondrial division is the initiation step of mitochondrial apoptosis.Therefore,the present study was designed to investigate the regulation mechanisms of HIF-1α on the proliferation of pancreatic ductal adenocarcinoma(PDAC)from the perspetives of glycolysis and immunosuppression and on the apoptosis of PDAC from the persptives of mitochondrial apoptosis pathway.Accordingly,this study was mainly divided into four parts:Part1Objectives:1.To analysis the expression of HIF-la,p-Akt and p-mTOR in PDAC.2.To analysis the correlation between HIF-la and p-mTOR.3.To study the regulation of mTOR by HIF-1a in PANC-1.Methods:1.50 patients with PDAC were divided into two groups,namely the high-medium differentiation group and the low differentiation group,according to the degree of histological differentiation,and PDAC tissue specimens were immunohistochemically stained.2.PANC-1 cells were treated with sirolimus,LY294002 inhibitor and CoCl2,respectively,and expression of HIF-la and mTOR was analyzed by PCR.Results:1.HIF-la,p-Akt and p-mTOR were highly expressed in pancreatic cancer tissues,and their expression levels increased as the degree of differentiation decreased;2.The expression of HIF-la was positively correlated with that of mTOR in pancreatic cancer tissues(R=0.26,P=0.01);3.COCl2 upregulated the expression of HIF-la in PANC-1 cells,which was significantly increased compared with that of the untreated cells(p<0.05);4.Sirolimus and LY294002 down-regulated the expression of mTOR in PANC-1 cells;5.After the downregulation of mTOR in PANC-1 cells,the expression of HIF-1α was reduced compared with that of the control group(p<0.05).Conclusion:The expression of HIF-1α is related to the degree of differentiation of PDAC and mTOR regulates the expression of HIF-la.Part2Objectives:l.To analysis the expression of GLUT-1 and LDHA in PDAC.2.To research the regulation of GLUT-1 and LDHA by HIF-la in PANC-1.Methods:1.Immunohistochemical staining of PDAC tissues was performed and the correlation with HIF-la was analyzed;2.Effect of HIF-la on the expression of GLUT-1 and LDHA was analyzed by PCR method.Results:(1)GLUT-1 and LDHA were highly expressed in PDAC tissue and positively correlated with the degree of differentiation;(2)The expression of HIF-la was positively correlated with that of GLUT-1 and LDHA,with correlation coefficients of 0.31(P=0.003)and 0.37(P=0.001),respectively.(3)The results of PCR analysis showed that the expression of GLUT-1 and LDHA was increased when HIF-la was highly induced by CoCl2,and significantly reduced when HIF-1α was inhibited.Conclusion:GLUT-1 and LDHA,the key enzymes in glycolysis pathway,are highly expressed in PDAC,and HIF-la can regulate the expression of GLUT-1 and LDHA,which is contributive to the proliferation of pancreatic cancer.Part3Objectives:1.To analysis the expression of FoxP3 and PD-L1 in PDAC.2.To research the regulation of FoxP3 by HIF-1a in PANC-1.3.To analysis the differences of FoxP3+ T Cells and neutrophils in peripheral blood in PDAC.4.To study the regulation of FoxP3+T cell by HIF-la in PDACMethods:1.PDAC tissue specimens were immunohistochemically stained;2.Effect of HIF-la on the expression of FoxP3 was analyzed by PCR method;3.Difference between FoxP3+ T cells and neutrophils in the peripheral blood of PDAC patients were analyzed by using flow cytometry;and 4.CD4+T cells and CD8+T cells were isolated with magnetic beads,and then co-cultured with PANC-1 cells.Cell proliferation was detected by the MTS assayResults:(1)FoxP3 and PD-L1 were highly expressed in PDAC and positively correlated with the degree of differentiation;(2)the PCR results showed that the expression profiles of HIF-la and FoxP3 were not consistent with the results of immunohistochemistry;(3)Flow cytometry analysis showed that compared with the high differentiation group,the proportion of FoxP3+T cells in the low differentiation group was increased(P<0.05);and the proportion of CD62LCD274+ neutrophils was increased,with a higher NLR(neutrophil-to-lymphocyte ratio)(P<0.05);(4)The MTS results showed that the inhibition effect of FoxP3+T cells on tumor proliferation was blocked when the expression of HIF-la was suppressed.Conclusion:Immunosuppressive molecules are expressed in PDAC.FoxP3+T cells inhibit the proliferation of PDAC,and HIF-la suppresses the function of FoxP3+T cells to promote the proliferation of pancreatic cancer.Part4Objectives:1.To analysis the regulation of apoptosis by HIF-1a in PANC-1.2.To research the regulation of apoptosis induced by mitochondria by HIF-1a in PANC-1.3.To study the relationship between mitochondria fission and apoptosis.Methods:1.Expression of HIF-1α was silenced by siRNA interference technique,and the morphology of mitochondria in PANC-1 cells was observed by electron microscopy.2.MiR-125a was overexpressed or inhibited by chemical transient transfection,and the viability,apoptosis,and expression of apoptotic proteins in PANC-1 cells were analyzed by CCK-8 assay,TUNEL assay,and Western blot,respectively.3.Mitochondrial morphology was observed by Immunofluorescence method after the expression of miR-125a was interfered,and the expression of mitochondrial fission-related proteins was analyzed by Western blot.Results:(1)Abnormal morphology of mitochondria,i.e.,fragmented mitochondria,was observed after HIF-1α was silenced;(2)HIF-1α could negatively regulate the expression of miR-125a;(3)The CCK-8 assay showed that miR-125a inhibited the viability of PANC-1 cells,the TUNEL assay showed that miR-125a promoted the mitochondrial apoptosis,and Western blot showed that the expression of proapoptotic proteins,including caspase-9,Bax,and Bad,was upregulated and that of the anti-apoptotic proteins,including Bcl-2 and x-IAP,was down-regulated after the overexpression of miR-125.(4)The immunofluorescence staining revealed morphologically abnormal,or "fragmented" mitochondria and abnormal expression of the mitochondrial division-related proteins after the overexpression of miR-125a.Conclusion:MiR-125a promotes mitochondrial apoptosis by regulating mitochondrial fission,and HIF-1α negatively regulates miR-125a to enhance the apoptosis of PANC-1 cells.