The Study Of Tumor Suppressor PRSS8 In Colorectal Cancer

BackgroundColorectal cancer(CRC)is the third most common malignant disease and the second leading cause of cancer-related death in the United States,which is majorly due to cancer metastasis.Several decades effort have revealed that the development and progression of colorectal cancer is linked to inactivation or downregulation of tumor suppressors,and activation or upregulation of oncogenes,leading to oncogenic signaling activation such as Wnt/?-catenin,inflammatory signaling etc.Human protease serine 8,also known as Prostasin(PRSS8),was preliminarily found highly expressed in normal prostate gland and seminal fluid.Recent studies have demonstrated PRSS8 play an important tumor suppressive roles of development and progression on prostate,breast,bladder and gastric cancers.Using gene expression analysis we have found that PRSS8 was significantly reduced in colorectal cancer,but its role in colorectal cancer is largely unknown.ObjectivesThe study focused on colorectal tissues,colorectal cancer cells and nude mouse and gene knock out mouse,discussed the role of PRSS8 in development and progression of colorectal cancer.Methods1.Immunohistochemistry,staining intensity evaluation and survival analysis: weconducted immunohistochemical staining in a colorectal cancer tissue microarray(TMA)that had 282 cases of colorectal cancer and adjacent non-cancer tissues with follow-up information.In addition,to determine the levels of PRSS8 in other cancers,we used 4 sets of TMAs that contained 72 pairs of esophageal cancers and theiradjacent non-cancer tissues,117 pairs of liver cancers and their adjacent non-cancer tissues,46 pairs of prostate cancers and their adjacent adenoma or non-cancer tissues,and 75 pairs of breastcancers and their adjacent adenoma or non-cancer tissues,respectively.The staining intensities were scored as 0(no staining),1(weak staining),2(moderate staining and 3(strong staining).Cancer patient survival analysis was performed using Kaplan-Meier method and GraphPad Prism 5.0 software.2.Quantitative reverse-transcriptional polymerase chain reaction(qRT-PCR):Taken the total mRNA extracted from 38 colorectal cancer samples and their paired normal samples,the expression level of PRSS8 were detected by qRT-PCR.3.Cell culture,plasmid construction,siRNA synthesis and transfection:PRSS8expression plasmids were constructed and siRNA of PRSS8 were synthesized.Human embryonic kidney cells HEK293 and human colorectal cancer cell lines HCT116,SW480,HCT8 and Caco2 were cultured and transfected with PRSS8 expression plasmids and siRNA.The relationship of PRSS8 with Wnt/?-catenin signal pathway,Sphk1/S1P/Stat3/Akt signal pathway,EMT signal pathway was detected by western blot.The proliferation of colorectal cancer cells were detected by MTT assay.Cell cycle was analyzed by flow cytometry.The ability of migration and motility were detected by wound healing and transwell assay.Cell morphology changes were determined by staining the cells with phalloidin solution.The effects of PRSS8 on Wnt/?-catenin transcriptional activities were determined using TOP/FOP reporter system.The interaction between PRSS8 and Sphk1 was determined by co-immunoprecipitation(co-IP)and co-localization analysis.4.Cancer cell transplantation(xenograft)in nude mice: 1.5x106 HCT116 stably transfected with pFlag-PRSS8 or negative control plasmid were injected subcutaneously into the flank of the normal nude mice at age about 8 weeks(10 mice per group).30 days after inoculation,the animals were sacrificed and the xenografts were isolated and observed,the weight(g)and size(cm3)of the xenografts were determined.The xenografts were detected by immunohistochemistry and western blot.5.sphk1+/+ and-/-mouse:The Sphk1+/+ and-/-mice were sacrificed at about 8 weeks,and colon tissues were collected and fixed in formalinfor immunohistochemical staining using anti-PRSS8 antibody.All immunohistochemical staining slides were imaged using Aperio ImageScope software for immunostaining intensity evaluation.Results1.The results of immunohistochemistry showed that the expression level of PRSS8 were reduced in colorectal cancer and positively associated with differentiation and survival of colorectal cancer.Similar to colorectal cancer,PRSS8 expression levels were reduced in esophageal cancers and liver cancers compared to normal adjacent tissues.Consistent with previous studies,PRSS8 expression was reduced in breast and prostate cancers,compared to breast and prostate adenomas.Moreover,PRSS8 expression was positively associated with differentiation of esophageal,liver,breast and prostate cancers.2.The results of qRT-PCR showed that PRSS8 mRNA levels were reduced in colorectal adenoma and adenocarcinoma tissues.3.The results of western blot showed that overexpression of PRSS8 inhibited EMT signal pathway,Wnt/?-catenin signal pathway,Sphk1/S1P/Stat3/Akt signal pathway;Knockdown of PRSS8 activated EMT signal pathway,Wnt/?-catenin signal pathway,Sphk1/S1P/Stat3/Akt signal pathway.4.1The result of MTT indicated that compared with negative control,proliferation of cells in the PRSS8 overexpression group were suppressed,while they were enhanced in the PRSS8 knockdown group.2 The result of wound healing and transwell showed that compared with negative group,the cell ability of migration and motility in PRSS8 overexpression group were suppressed,while they were enhanced in the PRSS8 knockdown group.Flow cytometry analysis showed that increased expression of PRSS8 led to cell cycle arrest at G1/G2 phase at both SW480 and HCT116 cells,and also caused cytoskeleton and morphologychanges,as observed via Red-Phalloidin staining.The results of co-immunoprecipitation(co-IP)and co-localization analysis showed PRSS8 can interact with sphk1 as a complex.5.Overexpression of PRSS8 inhibited tumor growth in nude mice,resulting in significant retardation of tumor size and tumor weight.6.PRSS8 expression was increased in the colon of Sphk1-/-mice,compared to Sphk1+/+ mice,assayed by immunohistochemical staining.Concludes1.As a tumor suppressor gene,PRSS8 could inhibit the cell ability of proliferation and migration,and EMT signal pathway,Wnt/?-catenin signal pathway,Sphk1/S1P/Stat3/Akt signal pathway in colorectal cancer cells,which were related with the development and progression of colorectal cancer.2.PRSS8 was positively associated with differentiation and survival of colorectal cancer,which could be a useful biomarker for monitoring colorectal carcinogenesis and progression and for predicting outcomes.