HIF-1α Induces MT2-MMP Expression During Hypoxica And Promotes Invasion And Metastases Of Pancreatic Cancer

Objective:To investigate the relationship of membrane-type matrix metalloproteinase-2 (MT2-MMP) and hypoxia inducible factor-1α(HIF-1α) expression in pancreatic cancer.Methods:We used immunohistochemical staining to examine the expression of MT2-MMP and HIF-1αin 78 cases of pancreatic cancer. Quantitative Real-time PCR and Western blot assays were preformed to investigate the effects of hypoxia on the expression of MT2-MMP and HIF-1αin pancreatic cancer cells. And then we detect the protein levels of MT2-MMP expression in PANC-1 cells when HIF-1αexpression was up-or down-regulated during hypoxia.Results:Immunohistochemical staining showed that the positive rates of MT2-MMP and HIF-1αexpression were 57.7% and 66.7% in 78 cases of pancreatic cancer. Regional distribution of MT2-MMP protein mostly locatedly overlapped with HIF-1αin consecutive sections of same pancreatic cancer tissue. Spearman analysis indicated that MT2-MMP expression strongly correlated with HIF-1αin pancreatic cancer (r=0.619, P<0.001). When cells were subjected to hypoxia (1％O2), the mRNA and protein levels of MT2-MMP had a significant increase in a time-dependent manner. We then used CoCl2 or HIF-1αexpression plasmids to up-regulate HIF-1αexpression in PANC-1 cells, the results showed that the protein levels of MT2-MMP were increased in a dose-dependent manner. When HIF-1αinhibitor YC-1 or HIF-1αsiRNA was used to downregulate HIF-1αexpression, the protein levels of MT2-MMP were significant decreased (P<0.05).Conclusion:Both of MT2-MMP and HIF-1αwere expressed with a different degree in pancreatic cancer tissue and cells, and their expression had a strong relationship. Objective:Tumor progression requires an increased adaptation to hypoxic micro-environment, which is largely mediated by hypoxia inducible factor-1α(HIF-1α) through coordinated regulation of hypoxia-responsive genes. Here, we intend to explore whether hypoxia can induce MT2-MMP expression in pancreatic cancer cells.Methods:In this study, we constructed the full-length MT2-MMP promoter reporter to clear whether HIF-1αdirectly bound to MT2-MMP promoter and started gene transcription. To investigate the HIF-1 binding site in the MT2-MMP promoter, we constructed a series of truncated MT2-MMP promoters. Then the HIF-1αbinding and competition ELISA assays and chromatin immunoprecipitation (ChIP) experiments were performed to further determine whether HIF-1αdirectly bound to the MT2-MMP promoter in vitro and in vivo.Results:The MT2-MMP promoter reporters were constructed successfully and co-transfected with pRL-TK into PANC-1 cells, and the results shown that HIF-1αdirectly bound to the MT2-MMP promoter and started gene transcription. The deletion and mutation assays indicated that nt-264 HRE1 site in the MT2-MMP promoter was essential for MT2-MMP transcriptional activation during hypoxia. Further analysis confirmed that HIF-1αspecifically and directly bound to the functional HRE1 site derived from the MT2-MMP promoter and initiated its transcription under hypoxia in vitro and in vivo.Conclusion:Our results suggested that HIF-1αdirectly bound to HRE1 site in the MT2-MMP promoter and initiated transcription of a variety of genes. These findings disclose a new regulatory mechanism of MT2-MMP expression in caner cells. Objective:To investigate the effects of overexpression of MT2-MMP with MT2-MMP expressing plasmid and knockdown of MT2-MMP with MT2-MMP siRNA plasmid on cell proliferation, apoptosis and invasion in pancreatic cancer.Methods:In this study, we intended to construct MT2-MMP expressing plasmid and MT2-MMP siRNA plasmid, and then stably transfected into PANC-1 cells. The mRNA and protein levels of MT2-MMP in the stable-transfected cells were detected by qRT-PCR and Western blot assays. CCK-8, Flow cytometry (FCM) and Transwell assays were used to examine cell proliferation, apoptosis and invasion of pancreatic cancer cells.Results:We successfully constructed pDNA3.1-His-MT2-MMP plasmid and pGenesil-MT2-MMP-siRNA plasmid, and the mRNA and protein levels of MT2-MMP in the MT2-MMP overexpression cells were significantly increased while the levels were decreased in MT2-MMP knockdown cells. The proliferation of MT2-MMP overexpression cells was increased significantly as compared with MT2-MMP siRNA cells. And the apoptotic rates of MT2-MMP overexpression cells were obviously decreased as compared with that MT2-MMP siRNA cells, while the number of invasion cells was increased (216.4±18.6 and 75.8±8.2 respectively, P<0.05).Conclusion:Down-regulation of MT2-MMP by siRNA could inhibit proliferation and invasion and induce apoptosis, and while up-regulation of MT2-MMP improve proliferation and invasion and inhibit apoptosis in pancreatic cancer PANC-1 cells during hypoxia. The findings suggest that MT2-MMP may be a cancer-promoting gene, and provides a new method to study gene-target treatment of pancreatic cancer.