Human Protease Serine 8 Methylation And Its Significance In Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer is one of the most common cancers worldwide,the incidence is increasing rapidly in recent years in China,and is the fourth most frequent cause of cancer-related deaths in China,five-year survival is very poor,however,the underlying mechanisms are largely unknown.Human protease serine 8(PRSS8),also known as Prostasin,a trypsin-like serine peptidase,was reduced in the cancers of prostate,breast,bladder and stomach,showing tumor suppressive roles.In our present study,we found that the expression of PRSS8 is significantly decreased in esophageal squamous cell carcinomas(ESCC),but its role in ESCC is largely unknown.ObjectivesTo explore the biological function of prostasin in esophageal squamous cell carcinoma.Methods1.To determine the expression levels of PRSS8,a tissue microarray(TMA)containing 362 cases of ESCC tissues was examined using immunohistochemical staining.2.We used Immunoblotting to determined PRSS8 expression levels in ESCC cell lines.3.We treated DAC and small interfering RNA targeting human PRSS8 in ESCC cell lines to explore the function of prostasin.4.We used Immunoblotting to determined the mechanisms of tumor inhibition by PRSS8.Results1.PRSS8 was reduced in ESCC tissues and the reduction of PRSS8 was associatedwith poor differentiation and shorter survival time.2.PRSS8 was differentially expressed in ESCC cell lines,and the reduction of PRSS8 was associated with PRSS8 promoter hypermethylation.3.PRSS8 expression could be restored by demethylation agent decitabine(DAC),and the restoration of PRSS8 could be reversed by small interfering RNA in ESCC cells;DAC led to the inhibition of cell proliferation,motility,migration and cell cycle arrest at G1 phase,which was reversed by siR-PRSS8.4.Overexpression of PRSS8 led to the alterations of cell proliferation-related proteins(increase of P21WAF1 and decrease of Cyclin D1),and led to the alterations of epithelial-mesenchymal transition(EMT)-related proteins(upregulation of E-cadherin and downregulation of Twist and Snail).ConclusionWe found that the expression of PRSS8 was significantly decreased in esophageal squamous cell carcinomas at mRNA and protein levels,the reduction of PRSS8 was well correlated with poor differentiation and shorter survival time.More interestingly,ESCC stromal expression levels of PRSS8 was significantly correlated with stromal lymphocyte infiltration and cancer progression.PRSS8 was hypermethylated associated with downregulation of PRSS8 expression and decreased activities of PRSS8 promoter.De-methylation agent decitabine was able to restore PRSS8 expression,leading to the inhibition of cancer cell proliferation,motility,migration and cell cycle arrest.However,the restored PRSS8 and its tumor inhibition could be reversed by small interfering RNA targeting PRSS8.Mechanistic study showed that tumor inhibition of PRSS8 may be associated with proliferation-and epithelial mesenchymal transition-related proteins.