CDNA Cloning Of PARs And Study On The Regulation Of HAI-1 Expression By PARs

Malignant tumor is one of the most serious diseases due to its high incidence and mortality. Loss of function of important organs resulted from malignant invasion and metastasis is the main cause of death.It is great importance to investigate the factors associated with metastasis. Serine proteases and their inhibitors are extensively studied recently to reveal their relevance to metastasis,such as matriptase,hepatocyte growth factor activator(HGFA)and hepatocyte growth factor activator inhibitor type 1(HAl-1).These proteases are involved in both the extracellular matrix degradation and modulation of growth factor activities,with close relation to tumor metastasis.Recently more and more studies are focused on the specific inhibitor of HGFA,HAI-1.Human HAI-1 gene locates at choromosome 15q15,and consists of 11 exons which span 12 kbps.The 2.4kb mRNA encodes a protein with 513 amino acid residues.Various studies show that HAI-1 is widely expressed in epithelia of gastrointestinal tracts,lung,bronchial, breast,etc,with significantly decreased expression in cancer tissues of gastric,colorectal and breast.However,there are contradict reports on HAI-1 expression in tumor cells,e.g,35% primary hepatocyte carcinomas(HCC)show positive for HAI-1 expression,with higher expression in tissues of poorly-differentiated HCC than that of well-differentiated HCC,and no HAI-1 expression in adjacent normal tissues;HAI-1 immnoreactivity is strong at the invasive front of colorectal cancer with elevated mRNA level.Recent study showed that the serum level of HAl-1 is higher in prostate cancer patients that that in patients with benign prostate diseases or normal individuals.The serum level of HAI-1 is also higher in patients with distant metastasis or hormone-resistance.The mechanism of HA1-1 expression regulation has not been elucidated up to date.In our previous work,we studied the expression of HAl-1 in various normal and malignant tumor tissues as well as the role HAI-1 plays in tumor cells.We hypothesize that HAI-1 expression is partly regulated by serine proteases according to the HAI-1 expression patterns in tissues with high environmental serine protease levels such as gastrointestinal tracts,breast and invasive front of malignant tumors.Proteases regulate gene expression via membrane protease activated receptors(PARs).PARs are G protein-coupled receptors with 7 transmembrane domains.Four PARs have been identified,PAR1,PAR2,PAR3 and PAR4.PARs are activated by hydrolysis of the N-termini by specific proteases,and trigger a series of signal transductions via their coupled G proteins.Studies have shown that some proteases are involved in tumor development and progression by activating corresponding PARs.We postulate that up-regulation of HAI-1 in invasive front of tumor and some tumors is modulated by high concentration of proteases in microenvironment via PARs,and the elevated HAI-1 can recruit pro-cancerous factors such as HGFA and matriptase via reversible binding.In this work,the cDNAs of four different PARs were cloned and used to construct mammalian expression vectors,and the effects of PARs on HAI-1 expression and cell growth and motility were invigasted by transfection and treatment of four serine proteases.1,Cloning of cDNAs covering full length coding regions of PARs and construction of mammalian expression vectorsTotal RNAs were isolated from human colorectal cancer cell SW620,HT-29 and small round cell tumor tissues and used for templates to clone the cDNAs of PARs by RT-PCR.The resulting PCR products were cloned into pGEM-T Easy or pMD-19 T vectors,and verified by DNA sequencing.The cDNAs were cut with restriction endonucleases and subcloned into pcDNA3.1 plasmid to make mammalian expression vectors.2,Effect of serine proteases on cellular HAI-1 expressionExpression vectors for PAR-2 and PAR-4 were transiently or stably transfected into HT-29 cells by Lipofectamine^TM2000.Western Blot showed that HAI-1 was up-regulated in transfected cells compared to the non-transfected controls.Treatment of the transfectants with elastase, trypsin,chymotrypsin and thrombin showed an earlier up-regulation of HAI-1 than the controls. These suggest that both the over-expressions of PAR-2/PAR-4 and treatment of serine proteases can up-regulate the HAI-1 expression.3,Effect of over-expressions of PAR-2/PAR-4 on cell growth and motilityMTT assay was used to evaluate the effect of over-expressions of PAR-2/PAR-4 on cell growth of HT-29 cells.The results showed that the over-expression of either PAR-2 or PAR-4 has significant influence on the growth curve of HT-29 colorectal cancer cells(P＜0.05).Wound-healing assay was used to evaluate the effect of over-expressions of PAR-2/PAR-4 on cell motility.The results showed that the PAR-2-or PAR-4-transfected cells exhibited higher motility that the controls,which suggested that over-expression of PAR-2 or PAR-4 can significantly promote tumor cell migration in vitro.Conclusions:1,Over-expressions of PAR-2 and PAR-4 can up-regulate HAI-1 expression.2,Four serine proteases used in this study can up-regulate HAI-1 expression.3,Over-expressions of PAR-2 and PAR-4 in HT-29 cells have significant influence on cell growth and motility.