The Regulatory Mechanism And Function Of STIM1 In Pancreatic Cancer Under Hypoxia

BackgroundPancreatic ductal adenocarcinoma(PDAC,referred to as pancreatic cancer)is a type of malignant tumor with a very poor prognosis,which incidence is parallel to the mortality[1].The median survival time in patients with pancreatic cancer is 3-5months in China,and the 1-year survival is as low as 10%.While,5-year survival remains as low as 6% in the United States [2-3].Most patients with pancreatic cancer have no obvious symptom until the disease develops to an advanced stage.The probability of surgery is less than 20% and the 5-year survival rate after complete resection is only 25% [1-2].The biological characteristics of pancreatic cancer and the resistance to chemoradiotherapy are favorable factors for recurrence and metastasis [4].A large number of studies have confirmed that calcium channels and calcium transient play a very important role in tumorigenesis and development [5-8].It is a key molecule in regulating calcium channel and biological behaviors such as proliferation,apoptosis and migration of tumor cells [9-13].Stromal interaction molecule 1(STIM1),a calcium ion sensor located on the endoplasmic reticulum membrane,has been implicated in cancer progression [14].These processes occur in many tumors including nephroblastoma,rhabdomyosarcoma,adrenal carcinoma,breast cancer,liver cancer,bladder cancer,lung cancer,prostate cancer,colorectal cancer,ovarian cancer,testicular cancer and pancreatic cancer [15-16].Hypoxia is another essential feature of carcinogenesis.Studies have confirmed that the expression of hypoxia-inducible factor-1α(HIF-1α)is elevated in liver cancer which can promote the formation of hepatocellular carcinoma.HIF-1α can also regulates the calcium flow which is mediated by STIM1[17].In the preliminary work of this study,we found that STIM1 was significantly higher in pancreatic cancer cells than that of normal pancreatic cells,which is similary to the expression of HIF-1α.But the mechanism between HIF-1α and STIM1 in pancreatic cancer has not been fully understood.Therefore,we aimed to explore the correlation between HIF-1α and STIM1,and to explain its regulatory mechanisms in pancreatic cancer.Method1.Immunohistochemical staining of HIF-1α and STIMM1 was performed on surgical specimens of pancreatic ductal adenocarcinoma to analyze the relationship between STIM 1 and HIF-1α.ALL patients were followed up and the relationship between STIM1 and survival time was statisticed.2.The basal expression levels of HIF-1α and STIM1 in normal pancreatic ductal epithelial cells and various pancreatic cancer cell lines were detected by Western blot;We construct the plasmid of STIM1 down-expressing with molecular cloning method and transfected it to the pancreatic cancer cells;Next,we detect the expression level of STIM1.3.Transwell and Western-blot analysis of EMT-related protein expression were performed using the cell lines constructed above,to observe the effect of STIM1 on migration and invasion ability of pancreatic cancer cells.4.The effect of HIF-1α on the expression level of STIM1 at the mRNA was verified by RT-PCR.5.CHIP and Double Luciferase experiments to used to verify the regulatory relationship between HIF-1α and STIM1.Results1.The expression of STIM1 in pancreatic cancer tissue and its correlation with HIF-1α.Immunohistochemical staining of STIM1 and HIF-1α was performed on the tissues: STIM1 expression was low in normal tissues and high in pancreatic ductal adenocarcinoma.HIF-1α was positively correlated with STIM1 expression,and the poorer the tumor tissue differentiation,the higher the expression level of HIF-1α and STIM1,and the worse the prognosis,which was statistically significant.2.Western-blot was used to verify the relationship between HIF-1α and STIM1 at protein level.Four pairs of pancreatic cancer and adjacent tissues were selected,it can be seen that both HIF-1α and STIM1 were up-regulated in pancreatic cancer cells.Seven cell lines of L3.7-2,SW1990,Miapaca2,PANC-1,CFPAC-1,BxPC-3 and AsPC-1 were selected,Western-blot confirmed the basic expression of HIF-1a andSTIM1,which were up-regulated simultaneously in L3.7-2,SW1990,Miapaca2,PANC-1 and CFPAC-1 cell lines.Three highly expressed cell lines,PANC-1,CFPAC-1 and SW1990 were selected as STIM1 knockout stable lines and their expression was verified.It can be concluded that the expression of HIF-1a and STIM1 at protein level is also positively correlated.3.In cell function experiments certified that STIM1 overexpression could enhance the invasion and migration of pancreatic cancer cells.The SW1990 and SW1990 KO STIM1 cell lines were selected for Transwell experiments,and it was found that the invasion and migration ability of SW1990 KO STIM1 cell line was significantly reduced.Western-blot revealed that the expression of E-cadherin was down-regulated and vimentin was up-regulated,indicating that STIM1 was involved in the progression of EMT.In conclusion,up-regulation of STIM1 expression could enhance the invasion and migration of pancreatic cancer cell lines.Conversely,STIM1 down-regulation significantly inhibited the invasion and migration of pancreatic cancer cell lines,and which was statistically significant.4.The correlation between HIF-1α and STIM1 was verified at the mRNA level.A positive correlation between HIF-1α and STIM1 expression was confirmed by immunohistochemistry and Western-blot.We further confirmed by qPCR experiments that when the HIF-1α gene was knocked down,the expression level of STIM1 was significantly decreased,which was statistically significant.5.Chromatin immunoprecipitation(ChIP)experiment and Double luciferase experiment were used to verify the regulatory between HIF-1α and STIM1.Bioinformatics analysis indicated that there was a targeting sequence that HIF-1α can binding in the promoter regions of STIM1.We further verified that there were two binding sites in STIM1 promoter regions by ChIP.HIF-1α and a luciferase reporter plasmid containing the STIM1 promoter were co-transfected into PANC-1 cells,it was confirmed that STIM1 was a direct target gene of HIF-1α;after shHIF-1α was transfected into PANC-1 cells,the expression of HIF-1a and STIM1 was significantly inhibited at the level of protein and mRNA.It was confirmed that STIM1 expression was directly regulated by HIF-1α in pancreatic cancer cells.Conclusion1.The expression level of STIM1 is significantly related to the survival of patients with pancreatic cancer.2.The up-regulation of STIM1 can promote invasion and metastasis of pancreatic cancer cells.3.HIF-1α can bind to the promoter regions of STIM1 and regulate the expression of STIM1.