The Preliminary Study Of Biological Function And Regulation Mechanisms Of FAM83D In Colorectal Carcinoma

In recent years,new cases of colorectal cancer(CRC)have gradually increased in China,and it is one of the most common malignant tumors of the digestive system.The mortality rate of CRC is high,and this is ascribed to frequent tumor recurrence after surgical resection.In addition,colorectal cancer patients might be asymptomatic for many months before presentation and missed the most ideal window of opportunity for cancer treatment.Therefore,it is of great significance to explore and understand the exact biomolecular mechanisms of colorectal cancer development and search for the biomarkers expressed during the early stages of cancer,and its discovery would optimize the procedures for patient's diagnosis and treatment.Studies have confirmed that the differential gene expression is closely related to the occurrence and development of colorectal cancer,including the gene of a family with sequence similarity 83,member D(FAM83D).FAM83D,locating on chromosome 20q,is part of FAM83.Its main function is encoding a mitotic spindle-associated protein which is associated with mitosis.In addition,FAM83D is differentially expressed in a variety of cancers,including breast cancer,hepatocellular cancer,and lung cancer.FAM83D is not just a potential candidate for the improved diagnosis and prognosis in various cancers,but is also involved in regulating cell proliferation,apoptosis,invasion,and migration in various cancer cells.Therefore,FAM83D may be a new biomolecular target for cancer treatment.Until now,the biological function of FAM83D in colorectal cancer and its molecular mechanism has only been studied with limited results.A study was conducted to explore whether FAM83D is a novel biomolecular target for CRC treatment.In the study,mRNA expression of FAM83D was detected by quantitative reverse transcription polymerase chain reaction(qRT-PCR)in colorectal cancer tissues(n=36)and paired adjacent normal colon tissues(n=36).In addition,the mRNA and protein expression of FAM83D was detected using qRT-PCR and Western blotting methods in 7 colorectal cancer cells(Caco-2,RKO,DLD-1,HT-29,LoVo,SW480 and HCT116)and a normal colonic epithelial cell(NCM460).Our findings showed that FAM83D expression was dramatically up-regulated in colorectal cancer tissues and cells.Furthermore,we observed the effect of FAM83D on cell proliferation,apoptosis,invasion,and epithelial-mesenchymal transition(EMT),and explored the molecular mechanisms of FAM83D in regulating biological function of colorectal cancer cells SW480 and HCT116.Part One:The Expression Level of FAM83D in Colorectal Cancer Tissues and CellsMethods1.Clinical sample collection:colorectal cancer tissues and paired adjacent normal colorectal tissues(n=36).2.Cell culture:human colorectal cancer cell lines(Caco-2,RKO,DLD-1,HT-29,LoVo,SW480,HCT116),and human normal colon epithelial cell lines(NCM460).3.qRT-PCR was used to examine the FAM83D mRNA expression in colorectal cancer tissues and paired adjacent normal colorectal tissues.4.qRT-PCR and Western blotting were utilized to detect the FAM83D mRNA and protein expression in human colorectal cancer cell lines and human normal colon epithelial cell lines.5.Statistical analysis:Data were presented as mean ± SD.Statistical analysis was performed using SPSS 17.0 software.The differences were evaluated using one-way analysis of variance(ANOVA)and the student's t-test,P<0.05 was regarded as statistically significant.Results1.The mRNA level of FAM83D was remarkably up-regulated in colorectal cancerous tissues when compared with that of adjacent normal colon tissues.2.The protein and mRNA levels of FAM83D was notably up-regulated in colorectal cancer cell lines(Caco-2,RKO,DLD-1,HT-29,LoVo,SW480,HCT116)when contrasted with that of human normal colon epithelial cell lines(NCM460),especially in SW480 and HCT116 cells,both at the protein and mRNA expression levels(P<0.01).Part Two:The Effect of FAM83D on Biological Function of Colorectal CarcinomaMethods1.The SW480 and HCT116 cells were transfected with the recombinant plasmid pcDNA3.1-FAM83D,the empty plasmid pcDNA3.1 as negative control,FAM83D siRNA,and negative control siRNA to construct the stable overexpression and knockdown of FAM83D in colorectal cancer cells(SW480 and HCT116).After transfection,qRT-PCR and Western blotting were used to examine the mRNA and protein expression level of FAM83D.2.Experiment group:control:the untreated colorectal cancer cells(SW480 and HCT116)were cultured in RPMI-1640 medium;pcFAM83D:the colorectal cancer cells(SW480 and HCT116)were transfected with pcDNA3.1-FAM83D;siFAM83D:the colorectal cancer cells(SW480 and HCT116)were transfected with FAM83D siRNA;pcNC:the colorectal cancer cells(SW480 and HCT116)were transfected with empty plasmid pcDNA3.1;siNC:the colorectal cancer cells(SW480 and HCTI16)were transfected with siRNA negative control.3.MTT assay was used to examine the cell proliferation in overexpressed-and knockdowned-FAM83D colorectal cancer cells(SW480 and HCT116).4.Colony forming assay was used to detect the ability of colony formation in overexpressed-and knockdowned-FAM83D colorectal cancer cells(SW480 and HCT116).5.Cell Death Detection ELISA assay was used to examine the apoptosis rate in overexpressed-and knockdowned-FAM83D colorectal cancer cells(SW480 and HCT116).6.Transwell assay was used to examine the ability of cell migration and invasion in overexpressed-and knockdowned-FAM83D colorectal cancer cells(SW480 and HCT116).7.Western blotting and qRT-PCR were used to examine the protein and mRNA expression of apoptosis-related and EMT-related in overexpressed-and knockdowned-FAM83D colorectal cancer cells(SW480 and HCT116).8.Statistical analysis:Data were presented as mean±SD.Statistical analysis was performed using SPSS 17.0 software.The differences were evaluated using one-way analysis of variance(ANOVA)and the student's t-test,P<0.05 was considered to be statistically significant.Results1.The results of qRT-PCR showed that the mRNA expression level of FAM83D was notably upregulated in colorectal cancer cells(SW480 and HCT116)transfected with the recombinant plasmid pcDNA3.1-FAM83D when compared with that of the empty plasmid pcDNA3.1 negative control(P<0.05).In addition,the FAM83D mRNA expression level was remarkably decreased in colorectal cancer cells(SW480 and HCT116)transfected with FAM83D siRNA when compared with that of negative control siRNA group(P<0.05).Therefore,the results of Western blotting showed that the protein expression level of FAM83D was notably upregulated in colorectal cancer cells(SW480 and HCT116)transfected with the recombinant plasmid pcDNA3.1-FAM83D(P<0.05).In addition,the FAM83D protein expression level was remarkably decreased in colorectal cancer cells(SW480 and HCT116)transfected with FAM83D siRNA(P<0.05).2.The results of MTT showed that FAM83D knockdown by siRNA suppressed cell proliferation in colorectal cancer cells(SW480 and HCT116).In addition,FAM83D overexpression promoted cell proliferation in colorectal cancer cells(SW480 and HCT116).3.The results of colony forming assay showed that FAM83D knockdown by siRNA,suppressed colony formation in colorectal cancer cells(SW480 and HCT116).In addition,FAM83D overexpression promoted colony formation in colorectal cancer cells(SW480 and HCT116).4.The results of Cell Death Detection ELISA assay showed that the silencing of FAM83D in colorectal cancer cells(SW480 and HCT116)notably increased cell apoptosis.In addition,cell apoptosis was notably decreased by FAM83D overexpression in colorectal cancer cells(SW480 and HCT116).5.The results of transwell assay showed that by knockdowning FAM83D would notably decreased the number of cell invasion in colorectal cancer cells(SW480 and HCT116)(P<0.05).FAM83D overexpression dramatically increased the number of cell invasion in colorectal cancer cells(SW480 and HCT116)(P<0.05).6.FAM83D knockdown remarkably increased the protein and mRNA expression of Bax,caspase-3 and caspase-9,but decreased the protein and mRNA expression level of Bcl-2 and Bcl-xl in colorectal cancer cells(SW480 and HCT116)(P<0.05).FAM83D overexpression notably increased the protein and mRNA expression of Bcl-2 and Bcl-xl but decreased the protein and mRNA expression level of Bax,caspase-3 and caspase-9 in colorectal cancer cells(SW480 and HCT116).In addition,FAM83D knockdown remarkably promoted the protein and mRNA expression of E-cadherin,but decreased the protein and mRNA expression level of P-catenin,N-cadherin,vimentin and Snail in colorectal cancer cells(SW480 and HCT116)(P<0.05).FAM83D overexpression notably diminished the protein and mRNA expression of E-cadherin,but dramatically promoted the protein and mRNA expression level of P-catenin,N-cadherin,vimentin and Snail in colorectal cancer cells(SW480 and HCT116)(P<0.05).ConclusionFAM83D knockdown inhibited cell proliferation and colony formation,promoted cell apoptosis,diminished cell invasion,and suppressed EMT process in colorectal cancer cells(SW480 and HCT116).FAM83D overexpression promoted cell proliferation and colony formation,inhibited cell apoptosis,increased cell invasion,and induced EMT process in colorectal cancer cells(SW480 and HCT116).Part Three:The molecular mechanisms of FAM83D in regulating biological function of colorectal carcinomaMethods1.qRT-PCR was used to examine the FBXW7 mRNA expression in colorectal cancer tissues and paired adjacent normal colon tissues.The qRT-PCR and western blotting were used to detect the FBXW7 mRNA and protein expression in human colorectal cancer cell lines and human normal colon epithelial cell lines.2.Cell transfection and experimental group:pcFAM83D:the colorectal cancer cells(SW480 and HCT116)were transfected with pcDNA3.1-FAM83D;pcNC:the colorectal cancer cells(SW480 and HCT116)were transfected with empty plasmid pcDNA3.1;siFAM83D:the colorectal cancer cells(SW480 and HCT116)were transfected with FAM83D siRNA;siFBXW7:the colorectal cancer cells(SW480 and HCT116)were transfected with FBXW7 siRNA;siNC:the colorectal cancer cells(SW480 and HCT116)were transfected with siRNA negative control;siFAM83D+siFBXW7:the colorectal cancer cells(SW480 and HCT116)were transfected with FAM83D siRNA and FBXW7 siRNA;siFAM83D+pcNotch-1:The colorectal cancer cells(SW480 and HCT116)were transfected with FAM83D siRNA and pcDNA3.1-Notch-1.3.Western blotting and qRT-PCR were used to detect the protein and mRNA expression levels of FBXW7 and Notchl in FAM83D knockdown and overexpression in colorectal cancer cells(SW480 and HCT116).In addition,it was also used to examine the protein and mRNA expression levels of Notchl in FAM83D knockdown and FBXW7 knockdown in colorectal cancer cells(SW480 and HCT116).4.MTT assay was used to examine to cell proliferation in Notch overexpression and FAM83D knockdown in colorectal cancer cells(SW480 and HCT116).5.Colony forming assay was used to detect the ability of colony formation in Notch overexpression and FAM83D knockdown in colorectal cancer cells(SW480 and HCT116).6.Cell Death Detection ELISA assay was used to examine the apoptosis rate in Notch overexpression and FAM83D knockdown in colorectal cancer cells(SW480 and HCT116).7.Transwell assay was used to examine the ability of cell invasion in Notch overexpression and FAM83D knockdown in HCT116 and SW480 cells.8.Western blotting and qRT-PCR were used to examine the protein and mRNA expression of apoptosis-related and EMT-related in Notch overexpression and FAM83D knockdown in colorectal cancer cells(SW480 and HCT116).9.Statistical analysis:Data was presented as mean ± SD.The SPSS 17.0 software was used to perform statistical analysis.One-way analysis of variance(ANOVA)and the student's t-test were used to evaluate the differences,P<0.05 was considered statistically significant.Results1.The mRNA level of FBXW7 was remarkably decreased in colorectal cancerous tissues when compared with that of adjacent normal colon tissues.The protein and mRNA levels of FBXW7 was notably reduced in colorectal cancer cell lines(Caco-2,RKO,DLD-1,HT-29,LoVo,SW480,HCT116)when compared with that of human normal colon epithelial cell lines(NCM460),especially in SW480 and HCT116 cells,both for the protein and mRNA expression levels(P<0.01).When compared with that of negative control siRNA,the protein and mRNA expression level of FBXW7 was notably upregulated and the Notchl protein and mRNA expression level was remarkably decreased in colorectal cancer cells(SW480 and HCT116)transfected with FAM83D siRNA(P<0.05).When compared with that of the empty plasmid pcDNA3.1 control,the protein expression level of FAM83D was notably decreased and the Notchl protein and mRNA expression level was remarkably increased in colorectal cancer cells(SW480 and HCT116)transfected with the recombinant plasmid pcDNA3.1-FAM83D(P<0.05).In addition,when compared with siFAM83D group,the Notch1 protein and mRNA expression level was remarkably upregulated in siFAM83D+siFBXW7 group(P<0.05).2.The results of MTT assay showed that FAM83D knockdown by siRNA in colorectal cancer cells(SW480 and HCT116)suppressed cell proliferation.In addition,when compared with siFAM83D group,cell proliferation was remarkably upregulated in siFAM83D+pcNotchl group(P<0.05).3.The results of colony forming assay showed that FAM83D silence by siRNA suppressed colony formation in colorectal cancer cells(SW480 and HCT116).In addition,when compared with siFAM83D group,the number of cell colonies was remarkably increased in siFAM83D+pcNotchl group(P<0.05).4.The results of Cell Death Detection ELISA assay showed that the silencing of FAM83D in colorectal cancer cells(SW480 and HCT116)notably increased cell apoptosis.In addition,when compared with siFAM83D group,cell apoptosis was notably decreased in siFAM83D+pcNotchl group(P<0.05).5.FAM83D knockdown remarkably increased the protein expression of Bax,caspase-3 and caspase-9,but decreased the protein expression level of Bcl-2 and Bcl-xl in colorectal cancer cells(SW480 and HCT116)(P<0.05).In addition,when compared with siFAM83D group,the protein expression level of Bax,caspase-3 and caspase-9 was notably decreased,but the protein expression level of Bcl-2 and Bcl-xl was increased in siFAM83D+pcNotchl group(P<0.05).6.The results of transwell assay showed that FAM83D knockdown notably decreased the number of cell invasion in colorectal cancer cells(SW480 and HCT116)(P<0.05).In addition,the number of cell invasion were dramatically increased in siFAM83D+pcNotchl group(P<0.05).7.FAM83D knockdown remarkably promoted the protein expression of E-cadherin,but decreased the protein expression level of ?-catenin,N-cadherin,vimentin and Snail in colorectal cancer cells(SW480 and HCT116)(P<0.05).In addition,when compared with siFAM83D group,the protein expression of E-cadherin was diminished,but the protein expression level of ?-catenin,N-cadherin,vimentin and Snail was dramatically upregulated in siFAM83D+pcNotchl group(P<0.05).Conclusion1.FAM83D knockdown promoted the protein and mRNA expression level of FBXW7 in colorectal cancer cells.FAM83D overexpression suppressed the protein and mRNA expression level of FBXW7 in colorectal cancer cells.2.FAM83D knockdown promoted the protein and mRNA expression level of Notchl in colorectal cancer cells.FAM83D overexpression suppressed the protein and mRNA expression level of Notch 1 in colorectal cancer cells.3.FBXW7 siRNA reverses the effect of FAM83D knockdown on Notchl protein and mRNA expression in colorectal cancer cells.4.FAM83D knockdown inhibited cell proliferation and colony formation,promoted cell apoptosis,diminished cell invasion,and suppressed EMT process in colorectal cancer cells through regulating FBXW7/Notch-1 signal pathway.