| Background and objectiveOncoproteomics,as one of the major focuses of the updated research on cancer,plays important roles in the identification of the signaling network proteins who effect positively or negatively on the tumor progression,to help the screening for potential molecular targets for cancer.Colorectal cancer has been a malignant tumor with a highest mortality as well as an increasing morbidity in China,to screening for tumor metastasis related genes using oncology comparative proteomics and to figuring out the downstream signaling networks the genes participate in with molecular biology methods works positively on the spot of potential molecular targets,and helps keeping the malignant tumor from metastasis and improving clinical survival rate.WTX,Wilms Tumor gene on the X chromosome,also named as FAM123B,is the first tumor suppressor gene spotted on the X chromosome.WTX gene plays an fundamental role in normal physiological function,for instance the differentiation of the mesenchumal tissue.However,WTX is also related to the development of diverse types of tumors.To trigger the colorectal cancer researchers’ interest,WTX gene contains three APC(adenomatous polyposis coli)binding regions and is therefore capable of interacting with APC directly to participate in the regulation of APC expression and cell location.Besides,we previously widely detected the WTX expression in somatic multiple-organ tumors and matched normal tissues,which turned out that WTX showed much less;or even none expression in the colorectal cancer tissues when compared to the normal mucosal tissues,to reveal that WTX plays an important role in colorectal cancer development,which needs to be much more clearly understood.EMT(Epithelial Mesenchymal Transition)has been a most regular way epithelium originated tumors metastasis participate in,we previously extracted the protein of dozens pairs of colorectal cancer tissues and matched normal mucosa tissues to analyze the relationship between WTX and EMT related proteins expression,E-cadherin,N-cadherin and Vimentin,however,it turned out that WTX expression hardly related with these proteins expression,revealing that WTX inhibits the colorectal cancer metastasis through a EMT independent pathway.In that way,what is the exact role WTX plays in colorectal cancer development?Does WTX work on colorectal cancer metastasis or proliferation?If it’s not through the EMT pathway,then what the mechanism of WTX influence on colorectal metastasis would be?Any downstream effector proteins involved?And in which signaling pathway exactly?They are all to be resolved.In this study,we would firstly detect the WTX function on colorectal cancer proliferation,metastasis and invasion,followed by screening for the WTX downstream interaction protein partners,which is hopefully be published known to be capable of influencing tumor metastasis,to preliminary reveal the mechanism that WTX influences colorectal cancer development in an EMT independent pathway eventually.This topic aims to provide new clues and a potential molecular target for clinical colorectal cancer therapy.Methods1、WTX’s influence on colorectal cancer cell biology behavior.(1)To construct WTX over-expression lentivirus vector,and infected the virus to colorectal cancer cell lines SW620 and HT29,which showed lowest level of internal WTX expression to establish WTX stable forced over-expression colorectal cancer cell lines.Using Real-time PCR and western blotting to detect the WTX expression changes in cell lines groups.(2)Three WTX siRNA fragments as well as one negative control fragment were designed and synthesized to transfected into colorectal cancer cell lines SW480 and HCT116,which showed the highest level of internal WTX expression,the siRNA fragment with the best WTX interference efficiency was selected to be constructed into lentivirus vector and infect SW480 and HCT116 cell lines.Using Real-time PCR and western blotting to detect the WTX expression changes in cell line groups.(3)CCK8(cell counting kit 8),cell clone formation assays,transwell cell migration as well as invasion detection assays and wound healing assays were performed to detect the effect WTX forced over-expression or knock down has on the proliferation,invasion and migration ability of colorectal cancer cells.2.Identification of WTX interaction protein partners and analyze the regulating relationship between them(1)To extract appropriate amount of SW620-W+ and HT29-W+ cell protein,using the mono antibody who specially recognize WTX as a "bait" to pull down RhoA,RAC1 and CDC41,the three main members of the Small GTPases family,which would turn out that CDC42 instead of RhoA or RAC1 could-be pulled down by WTX.Re-testify the direct interaction between WTX and CDC42 with con-focal(immunofluorescence).Using immonoprecipitation and western blotting to detect the changes of CDC42 expression and activity after WTX were forced over-expressed or knocked down by siRNA.(2)Preliminary analysis of WTX domain controlling binding with CDC421)Besides of the full length sequence,there are two other WTX splice forms existing in most cells,which are firstly the one with N terminus 50-326AA missing and the other one with C terminus 786-804AA different from the full length sequence and 804-1135AA missing.To detect the splice speciality of WTX interaction with CDC42,three DNA fragments were amplified and sub-cloned into an expression vector GV141,which was a product from JiKai Company.Sequence detection result showed that the tree vectors were successfully established.Transfer the vectors into SW620 cells and extract the cell protein,using mono-antibody to Flag tag as a "bait" to pull down CDC42 and detecting the cell migration ability changes between groups.2)Truncate the mid WTX fragment into 5 more truncates and amplify the fragments with primers specially designed containing specialized enzymes sites,followed by sub-clone of the fragments into GV141 vectors and sequence detection.Transfer the recombinant truncates into SW620 cells and extract the cell protein,using mono-antibody to Flag tag as a "bait" to pull down CDC42 and overlapping analyzing the WTX domain that pulls down CDC42 to get the conclusion of WTX region controlling binding with CDC42.(3)Preliminary analysis of the mechanism WTX inhibits CDC42 activity1)Accordingly,Small GTPases family members work as "molecular switches"cycling between an inactivated GDP-bound form and an activated GTP-bound form,RhoGDI inhibits the RHO protein from transferring from GDP-bound form to GTP-bound form and keeps them inactivated.Coimmunoprecipitation was firstly used to detect the association between WTX and RhoGDI,which interaction would then be retested by con-focal.2)Analysis of WTX domain controlling binding with RhoGDIa.Transfer the recombinant WTX truncates into SW620 cells,use mono antibody to Flag tag as "baits" to pull down RhoGDIα,to overlapping analyze the WTX domain that pulls down RhoGDIa and get the WTX region controlling binding with RhoGDIa.3)Analyze the tripartite association relationship between WTX,CDC42 and RhoGDIa by CO-IP.Three CDC42 and RhoGDIa siRNA fragments as well as one negative control fragment were designed and synthesized to transfected into recombinant colorectal cancer cell lines SW620-WTX+,selecting the siRNA fragment with the best CDC42 or RhoGDIa interference efficiency with Real-time PCR and western blotting(interference efficiency>50%).Grouped seed SW620-WTX+ cells into three 10cm cell culture plates,and transfer the negative control vector,siRNA to CDC42 or siRNA to RhoGDIa into the cells followed by extracting cell protein and using WTX mono-antibody as a "bait"to pull down CDC42 in the RhoGDIα knocked down cells and using WTX mono-antibody to pull down RhoGDIα in the CDC42 knocked down cells so to solve the question of if WTX associates with CDC42/RhoGDIa with RhoGDIa/CDC42 as an "intermediary protein "?3.The role of WTX-CDC42 in colorectal cancer cell migration(1)"Rescue" experiments to verify CDC42 as a downstream effector of WTX1)Based on the suppose that WTX negatively controls CDC42 activity,double force over-express both WTX and CDC42 in SW620 cells by into transferred the WTX and CDC42 expression vectors,using cell counting kit8 and transwell cell migration ability detection assay to detect cell proliferation and migration ability differences among SW620-NC,SW620-WTX+ and SW620-W+C+ cell groups.2)Double knock down both WTX and CDC42 in SW480 cells by into transferred the siRNAs to WTX and CDC42 respectively,using cell counting kit8 and transwell cell migration ability detection assay to detect cell proliferation and migration ability differences among SW480-NC,SW480-WTX-and SW480-W-C-cell groups.(2)Sta:in F-actin in colorectal cancer cells(SW620-W+/N1 and SW480-W-/N1)with rhodamine labeled phalloidine and use immunofluoscence to observe cell actin cytoskeleton morphology changes after WTX was forced over-expressed or knocked down.Double stain filopodia with mDia2/Fmn12 and F-actin,lamellipodia with Arp2/3 and F-actin,stress fibers with a-actinin and myosinⅡA,and focal adhesion with paxillin and myosinⅡA to observe the actin cytoskeleton morphology changes after WTX was forced over-expressed or knocked down in colorectal cancer cells and analyze the exact way WTX influence actin cytoskeleton organization by immunofluoscence,(3)The signaling pathway WTX participate in to regulate the dynamics of actin cytoskeleton in colorectal cancerUsing western blotting to detect the expression changes of MRCK、LIMK1/2、p-Cofilin、p-moesin and p-MLC downstream of CDC42 in colorectal cancer cells after WTX was forced over-expressed or knocked down to screen for the potential signaling pathway member proteins and summarize the signaling network WTX participate in to regulate the dynamics of actin cytoskeleton.4.Statistical analysisSPSS 13.0 was used for data statistical analysis.Using independent-Samples T test for the analysis of quantitative PCR results(displayed in the format of 2-△△Ct value)as well as transwell cell migration detection assay results and cell colonies formation assay results.Factorial design analysis of variance was used to statistically analyze the data of CCK8 which was used to detect the cell proliferation ability.P<0.05 was considered as a threshold for statistically significant.Results1、WTX’s influence on colorectal cancer cell biology behavior.(1)Establish the WTX stable over-expression colorectal cancer cell lines and matched negative control cell lines HT29-W+/HT29-N1 and SW620-W+/SW620-N1 by lentivirus infection.Using Real-time PCR to detect the mRNA expression differences of WTX between the cell groups,which showed that WTX expresses statistically higher in SW620-W+ and HT29-W+ cell lines when compared with that in SW620-N1 and HT29-N1 cell lines.Using western blotting to detect the WTX protein expression differences between cell groups and it showed that WTX expresses statistically higher in SW620-W+ and HT29-W+ cell lines when compared to SW620-N1 and HT29-N1 cell lines indicating that WTX stable over-expression colorectal cancer cell lines successfully established,named as HT29-W+/HT29-N1 and SW620-W+/SW620-N1.(2)Three WTX siRNA fragments named as WTX SI-2,SI-2,SI-3 were designed,synthesized and transiently transferred into colorectal cancer SW480 and HCT116 cell lines.Using quantitative PCR to detect WTX mRNA expression level in grouped cells and it showed that WTX was better knocked down by SI-1 than SI-2 or SI-3 with a interference efficiency>50%.In consist with the QPCR result,western blotting showed that WTX was best knocked down by SI-1,which was selected therefore as the candidate siRNA to be packaged into lentivirus supernatant and used as a lentivirus vector.Establish the WTX stable knocked down colorectal cancer cell lines and matched negative control cell lines SW480-W-/SW480-N1 and HCT116-W-/HCT116-N1 by lentivirus infection.Using Real-time PCR to detect the WTX mRNA expression differences between cell groups,which showed that WTX expresses statistically lower in SW480-W-and HCT116-W-cell lines when compared with that in SW480-N1 and HCT116-N1 cell lines.Using western blotting to detect the WTX protein expression differences between cell groups and it showed that WTX expresses statistically lower in SW480-W-and HCT116-W-cell lines when compared to SW480-N1 and HCT116-N1 cell lines indicating that WTX stable knocked down colorectal cancer cell lines SW480-W-/SW480-N1 and HCT116-W-/HCT116-N1 successfully established.(3)CCK8 and Cell colonies formation assays to detect cell proliferation ability after WTX was forced over-expressed or knocked downUsing CCK8 to detect cell proliferation ability changes between groups after WTX was knocked down,Factorial analysis results showed that there was statistically significant difference on the level of growth time in SW480 or HCT116 cell lines,and the ability of cell proliferation comparison had significantly differences between groups,Time and group interactional effect also had significant difference(all P<0.001)indicating that WTX knocked down promotes cell proliferation ability in colorectal cancer cells.CCK8 carried out in colorectal cancer cells after WTX was forced over-expressed showed,there was no significant difference on the level of growth time in SW620 or HT29 cell lines,the ability of cell proliferation comparison had statistically significant differences between groups,Time and group interactional effect also had significant difference(all P<0.001)indicating that WTX forced over-expression reduces cell proliferation ability in colorectal cancer cells.Cell colonies formation assays carried out in WTX recombinant stable expression colorectal cancer cells showed that,the cell colonies formation rate in HT29-N1/HT29-W+ and SW620-N1/SW620-W+ cell lines was 61%/14.67%and 45%/22%respectively,which was significantly different between groups.And the the cell colonies formation rate was also significantly different between SW480-N1/SW480-W-and HCT116-N1/HCT116-W-groups,which was 25.2%/61.13%and 29.47%/73.53%respectively.To sum up,the proliferation ability of colorectal cancer cells was significantly reduced after WTX was forced over-expressed,and WTX knocked down by siRNA promotes the proliferation ability of colorectal cancer cells.(4)Transwell cell migration detection assays and wound healing assays to detect cell migration ability changes after WTX was forced over-expressed or knocked downTranswell assays result showed that,after WTX was forced over-expressed,HT29-W+ and SW620-W+ cells showed much slower migration ability when compared to the negative control HT29-N1 and SW620-Nlcell lines,and the reduce was statistically significant.After WTX was knocked down by siRNA,the migration ability of SW480-W-and HCT116-W-cell lines were much higher than SW480-N1 and HCT116-N1 cell lines,the difference was statistically significant(p<0.001).Wound healing assay result showed that,72 hours after the cells were straightly wounded by a thin pipette tip,the gap between HT29-W+ and SW620-W+ cells were much wider than HT29-N1 and SW620-N1 cells.The opposite trend was observed in SW480-W-/HCT116-W-and SW480-N1/HCT116-N1 cell groups.To sum up,the forced over-expression of WTX in SW620 and HT29 cell lines reduced the cell migration ability,and in consist,the knock down of WTX promotes the migration ability of colorectal cancer cells SW480 and HCT116.2、Identification of WTX interaction protein partners and analyze the regulating relationship between them(1)Using the mono antibody to WTX as a "bait" to pull down CDC42,RHOA and RAC 1,who are the three main members of Small GTPases family,and it turned out that CDC42,but not RhoA or RAC1 could be pulled down by WTX indicating that WTX interacts directly with CDC42 instead of RhoA or RAC1.To verify the association between WTX and CDC42,which turned out that WTX co-localized with CDC42 in the plasma of SW620 and SW480 cells.And the co-localization turned much stronger when WTX was over-expressed in SW620 cells,meanwhile it seems weaker when the WTX was knocked down in SW480 cells.There was not obvious difference in CDC42 total protein expression in colorectal cancer cells after WTX was over-expressed or knocked down measured by western blotting,but the CDC42 activity quantified by the amount of GTP-bound CDC42 was reduced after WTX over-expression in SW620 cells and promoted after WTX was knocked down in SW480 cells indicating that WTX inhibits the activation of CDC42.(2)Preliminary analysis of WTX domain controlling binding with CDC421)Three DNA fragments representing WTX splices existing in colorectal cancer cells were amplified and sub-cloned into GV141 vectors,and the agarose gel electrophoresis result after double inter-restricted enzymes digestion showed that the length of all the amplification products accord with predication.Transforming the recombinant truncates of WTX into DHSabacteria followed by propagating and extracting the plasmids DNA,whose sequence results showed 100%matching with NCBI announcement indicating that the WTX truncates numbered 1,2 and 3 were successfully constructed.2)Transiently transfer the three WTX splicing vectors into SW620 cells and detect the cell migration ability changes among cell groups by transwell cell migration detection assay,which showed that the migration ability of all three groups of SW620 cells that were transferred with WTX splicing truncates numbered 1,2 and 3 were reduced when compared with the negative control group,and the reduce was statistically significant.Extract the cell protein for CO-IP with mono antibody to WTX as "baits" to pull down CDC42 and it showed that all three splices pulled down CDC42,but the splice numbered 3 pulled much less than splice 1 and 2.3)Truncate WTX mid region into 5 more truncates,and followed by being amplified and sub-cloned into GV141 vectors,the agarose gel electrophoresis result of which after double inter-restricted enzymes digestion showed that the length of all the truncates accord with predication.Transforming the mid WTX truncates into DH5abacteria followed by propagating and extracting the plasmids DNA,whose sequence results showed 100%matching with NCBI announcement indicating that the WTX truncates numbered 5,6,7,8,9 and 10 were successfully constructed.4)Transfer the truncates into SW620 cells and extract cell protein for CO-IP using mono antibody to WTX as "baits" to pull down CDC42,the mapping analysis of which indicates that WTX interacts with CDC42 through N terminus 327-716AA domain.(3)Preliminary analysis of the mechanism WTX inhibits CDC42 activity1)CO-IP result showed that,WTX pulls down RhoGDIa verifying RhoGDIa another direct interaction partner of WTX.2)Three siRNAs to CDC42 and three to RhoGDIa named as CDC42 SI-1,SI-2,SI-3 and RhoGDIa SI-1,SI-2,SI-3 respectively were designed as well as synthesized and then being transiently transferred to SW480 and SW620 cells,quantitative PCR result showed that SI-1 to CDC42 and both SI-1 and SI-2 to RhoGDIa have a interference efficiency of over 50%,the western blotting result was in consist with PCR data.Choose SI-1 to CDC42 and SI-1 to RhoGDIa for followed up researches.The CO-IP result between WTX,CDC42 and RhoGDIa turned out that there was not obvious change in the WTX interaction with RhoGDIa after CDC42 was knocked down in SW620-W+ cells.However,after the RhoGDIa was knocked down,WTX pulled down less CDC42 if there was any.Combine with the "truncates" result,these experiments indicate that,in colorectal cancer cells,part of the WTX interaction with CDC42 is through RhoGDIa,namely RhoGDIa works as a intermediary protein in the WTX association with CDC42.3)The mapping analysis result of WTX truncates turned out that WTX binds with RhoGDIa through the same region it binds with CDC42,indicating the existence of a WTX-CDC42/RhoGDIa tripartite complex in colorectal cancer cells.After all,all the researches above indicartes that WTX interacts with CDC42 with RhoGDIa as a intermediary protein.(We suppose that WTX inhibits the activation of CDC42 partly through stabilizing the interaction between CDC42 and RhoGDIa,which reduces the transformation of CDC42 from GDP-bound form to GTP-bound form).4.The role of WTX-CDC42 in colorectal cancer cell migration(1)"Rescue" experiments to verify CDC42 as a downstream effector of WTX1)After both WTX and CDC42 were double over-expressed in SW620 cells,using CCK8 to compare the proliferation ability among groups,Factorial analysis results of which showed that there was significant difference on the level of growth time in SW620-WW+C+,SW620-N1 and SW620-W+ cells(F=1557.996,P<0.001),and the ability of cell proliferation comparence had significantly differences between groups(F=148.127,P<0.001),Time and group interactional effect also had statistically significant difference(F=1 8.913 P<0.001)indicating that WTX over-expression reduces the proliferation ability of SW620 cells,yet the reduction could restored by CDC42 over-expression.The result of transwell cell migration detection assay turned out that,SW620W+ cell showed the weakest migration ability,the negative control SW620-N1 cells showed the strongest,and the SW620W+C+ cells came between them indicating that WTX over-expression reduces the migration ability of SW620 cells,yet the reduction could restored by CDC42 over-expression.2)After both WTX and CDC42 were double knocked down in SW480 cells,using CCK8 to compare the proliferation ability among groups,Factorial analysis results of which showed that there was significant difference on the level of growth time in SW480-W-C-,SW480-N1 and SW480-W-cells(F=672.235,P<0.001),and the ability of cell proliferation comparence had significantly differences between groups(F=362.501,P<0.001),Time and group interactional effect also had significant differences(F=57.727,P<0.001)indicating that WTX knock down promotes the proliferation ability of SW480 cells,yet the promotion could rescued by knock down CDC42.The result of transwell cell migration detection assay turned out that,SW480W-cell showed the strongest migration ability,the negative control SW480-N1 cells showed the weakest,and the SW480W-C-cells came between them indicating that WTX knock down promotes the migration ability of SW480 cells,yet the promotion could rescued by the knock down of CDC42.In the summary,CDC42 works as a downstream effector of WTX and its expression changes could rescue the colorectal cancer cell proliferation or migration ability changes caused by the dysfunction of upstream WTX protein.(2)It was observed with immunofluoscence that there were more thin and bundled actin filaments showed up after WTX was knocked down by siRNA in SW480 cells.And when compared to the negative control cells,the red actin filaments at the periphery of SW620 cells were less after WTX over-expressed.Verify that WTX negatively regulates the organization of actin cytoskeleton in colorectal cancer cells.(3)After the double staining of filopodia,lamellipodia,stress fibers and focal adhesion in SW620N1/SW620W+ and SW480N1/SW480W-cells,it was observed by immunofluoscence that there was not obvious changes in the formation of filopodia or lamellipodia,but the formation of cell stress fibers and focal adhesion was much stronger after WTX was knocked down in SW480-W-cells when compared with the negative control SW480-N1 cells,the opposite tread was observed in SW620NI1 and SW620W+ cell groups.(4)Western blotting result showed that,after WTX was knocked down in SW480 and HCT116 cells,the expression of MRCK、p-LIMKl/2 and p-cofilin was higher when compared to the negative control cells and after WTX over-expression in SW620 and HT29 cells,the expression of MRCK、p-LIMK1/2 and p-cofilin became much lower,while there was not obvious changes in the expression of p-MLC and P-moesin.Moreover,QPCR results carried out in pairs of matched colorectal cancer tissues and normal mucosa showed that WTX mRNA expression negatively relates with MRCKα expression with a 0.72 relative rate indicating that WTX regulates the dynamics of actin cytoskeleton in colorectal cancer cells through a WTX-GTP-CDC42-MRCK-LIMK1/2-p-Cofilin-F-actin polymerization pathway.Conclusions1、WTX inhibits the proliferation and migration ability of colorectal cancer.2、WTX interacts directly with CDC42 and inhibits its activity partly through stablizing the association between RhoGDIa and CDC42.3、WTX inhibits the formation and dynamics of cell stress fibers and focal adhesion partly through a WTX—GTP-CDC42—MRCK—LIMKl/2—p-Cofilin—F-actin polymerization pathway,so to negatively regulates the migration and invasion of colorectal cancer. |
