| BackgroundColorectal cancer(CRC)is one of the most common malignancies worldwide and metastasis is a major threat to CRC patients.Therefore,identifying metatastasis-associated molecules and uncovering molecular mechanisms responsible for CRC metastasis are important for preventing and treating CRC metastasis.We have reported that peroxiredoxin-2(PRDX2)is associated with CRC invasion and metastasis.However,the mechanisms regulating PRDX2 expression remain unclear.It is a breakthrough scientific discovery that microRNAs(miRNAs)regulate gene expression at the post-transcriptional level.It has been reported that miRNAs are involved in regulation of cancer progression by directly inhibiting target gene.However,miRNAs regulating PRDX2 expression in CRC metastasis are not identified.In this study,we investigate whether miRNAs regulate PRDX2 expression and the molecules involved in regulation of mi RNA expression in CRC progression,which will be to provide potential targets preventing and treating CRC metastasis.Methods1.Screening and identification of miRNAs targeting PRDX2 in CRC1)Bioinformatic algorithms,including TargetscanHuman.7.0,DIANA LAB and miRanda,were used to screen for the candidate miRNAstargeting PRDX2.2)Western blot and quantitative real-time PCR(qPCR)were used to detect the expression of PRDX2 protein and the candidate miRNAs in 6CRC cell lines with different metastatic potentials,respectively.Pearson's correlation analysis was used to evaluate the correlation between the PRDX2 protein levels and the candidate mi RNAs' expression in 6 CRC cells.3)Dual-luciferase reporter assay was used to assess the effect of the candidate miRNA on the luciferase activities of PRDX2 mRNA 3'UTR.2.MiR-200b-3p inhibits invasion and metastasis of CRC cells by directly repressing PRDX2 expression1)Lentiviral vectors expressing miR-200b-3p or silencing miR-200b-3p or expressing PRDX2 were used to establish LoVo/miR cells stably expressing mi R-200b-3p,LoVo/miR + PRDX2 cells stably co-expressing miR-200b-3p and nontargetable PRDX2 and SW480/Zip-mi R cells stably silencing miR-200b-3p.2)Western blot and in vitro invasion assay were used to evaluate the effect of miR-200b-3p over-expression,miR-200b-3p silencing and co-expression of miR-200b-3p with nontargetable PRDX2 on the epithelial-mesenchymal transition(EMT)and in vitro invasion of CRC cells.3)Nude-mouse subcutaneous tumor model and nude-mouse cecum transplanted tumor model were used to assess the effect of mi R-200b-3p over-expression,miR-200b-3p silencing and co-expression of miR-200b-3p with nontargetable PRDX2 on growth and metastasis of CRC cells.3.The c-Myc-miR-200b-3p feedback loop regulates invasion andmetastasis of CRC cells1)On-line database UCSC-Ensembl was used to search for the promoter of mi R-200b-3p.On-line database Consite was used to predict the potential transcription factors binding to the promoter of miR-200b-3p.2)Western blot was used to evaluate the expression levels of the transcription factor in 6 CRC cells with different metastatic potentials.Pearson's correlation analysis was used to evaluate the correlation between the transcription factor expression and mi R-200b-3p expression in 6 CRC cells.Luciferase reporter assay was used to assess the effect of the transcription factor on the luciferase activities of the mi R-200b-3p promoter.Chromatin immunoprecipitation(ChIP)assay was used to evaluate the binding of the transcription factor to the miR-200b-3p promoter.3)Lentiviral vectors expressing c-Myc or miR-200b-3p were used to establish SW480/c-Myc cells stably expressing c-Myc or SW480/c-Myc+mi R cells co-expressing c-Myc and mi R-200b-3p.4)Western blot,in vitro invasion assay,nude-mouse subcutaneous tumor model and nude-mouse cecum transplanted tumor model were used to evaluate the effect of c-Myc over-expression and co-expression of c-Myc with miR-200b-3p on EMT,in vitro and in vivo invasion and metastasis in SW480 cell.5)Western blot was used to assess the effect of miR-200b-3p over-expression,miR-200b-3p silencing and combination of miR-200b-3p silencing with GSK3?inhibitor(TWS119)treatment on the protein expression levels of GSK3,phosphorylated GSK3?(Ser9),c-Myc,phosphorylated c-Myc(T58)and phosphorylated c-Myc(S62)in CRCcells.6)Dual-luciferase reporter assay was used to assess the effect of miR-200b-3p on the luciferase activity of AKT2 mRNA 3'UTR.7)Western blot was used to evaluate the effect of miR-200b-3p over-expression on the protein expression levels of AKT2,phosphorylated AKT2(Ser474),GSK3?,phosphorylated GSK3?(Ser9),phosphorylated c-Myc(T58),phosphorylated c-Myc(S62)and c-Myc in LoVo cell.4.Analysis for the profiles of c-Myc,miR-200b-3p and PRDX2 in CRC tissues samples and the contribution of the three molecules to patients' clinicopathologic phenotype and outcome.1)qPCR was used to detect miR-200b-3p expression prolife in 97 cases of fresh CRC tissue samples and the paired normal colon mucosa(PNCM)tissues.Kaplan–Meier method was used to analyze the effect of miR-200b-3p on the overall survival of CRC patients.We also performed the contributions of miR-200b-3p expression to patients' clinicopathologic phenotypes.2)Immunohistochemistry(IHC)was used to detect c-Myc and PRDX2 protein expression profiles in 97 cases of paraffin-embedding CRC tissue samples.Kaplan–Meier method was used to analyze the effect of c-Myc and PRDX2 on the overall survival of CRC patients.We also performed the contributions of c-Myc and PRDX2 protein expressions to patients' clinicopathologic phenotypes.3)Pearson's correlation analysis was used to evaluate the correlation between c-Myc protein expression and miR-200b-3p expression,and miR-200b-3p expression and PRDX2 protein expression in 97 CRC tissuesamples.5.The c-Myc/miR-200b-3p/PRDX2 feedback loop regulates chemotherapeutic resistance of CRC cells1)Cell Counting Kit-8(CCK8)assay was used to assess the resistance of CRC cells to chemotherapy2)Flow cytometre was used to detect the drug-induced cytotoxicity.Results1.Screening and identification of miRNAs targeting PRDX2 in CRC1)MiR-1228-3p,mi R-200b-3p,miR-200c-3p and miR-429 were agreed by all three algorithms,including Targetscan Human.7.0,DIANA LAB and mi Randa,to be candidates.2)Western blot showed that PRDX2 protein level was significantly higher in the metastatic cell lines(SW620 and LoVo)than in the pre-invasive cell lines(SW480,Caco2,HT29 and HCT116).Conversely,qPCR showed that miR-200b-3p had lower expression levels in the metastatic cell lines than in the pre-invasive cell lines.Pearson's correlation analysis showed that miR-200b-3p expression levels were significantly inverse correlation with PRDX2 protein levels in 6 CRC cell lines(r =-0.977,p = 0.001).3)Dual-luciferase reporter assays showed that miR-200b-3p over-expression suppressed the luciferase activity of PRDX2 mRNA3'UTR in 293 T and LoVo cells.Also,ectopic miR-200b-3p expression reduced PRDX2 protein levels in SW620 and LoVo cells.2.MiR-200b-3p inhibits EMT,invasion and metastasis of CRC cells by directly repressing PRDX2 expression1)Invasion in vitro assays showed that mi R-200b-3p silencingincreased the invasive behavior of SW480 cell,while miR-200b-3p over-expression inhibited the invasiveness of LoVo cell.Mi R-200b-3p silencing increased frequencies of cells with fibroblastic or spindle-like morphology and concomitantly decreased frequencies of cobblestone-like cells.In contrast,miR-200b-3p over-expression showed the opposite effect.Similarly,miR-200b-3p silencing decreased the expression of the epithelial marker E-cadherin and increased the expression of the mesenchymal markers N-cadherin and vimentin in SW480,while miR-200b-3p over-expression increased the expression of the epithelial marker E-cadherin and decreased the expression of the mesenchymal markers N-cadherin and vimentin in LoVo.Importantly,the suppressive effects of miR-200b-3p on in vitro invasion and EMT were substantially reversed by the nontargetable PRDX2.2)Nude-mouse subcutaneous tumor model showed mice with LoVo/miR cells injection displayed inhibited tumor growth,while mice with SW480/Zip-miR cells injection showed the opposite effect.Intriguingly,the subcutaneous tumors derived from Lo Vo/miR cells were well encapsulated in false fibrous membrane compared to those derived from LoVo/NC cells.Nude-mouse cecum transplanted tumor model showed that mice with miR-200b-3p overexpressing tumors developed fewer metastatic nodules in intestine and liver than control mice with tumors of similar size.In contrast,mice with miR-200b-3p silencing tumors showed the more metastatic nodules than control mice.Importantly,the decreased effects of miR-200b-3p over-expression on in vivo growth,invasion and metastasis of LoVo were rescued by nontargetable PRDX2.3.The c-Myc-miR-200b-3p feedback loop regulates EMT,invasion and metastasis of CRC cells1)c-Myc was screened to be a possible transcription factor of miR-200b-3p using Consite on-line database.Western blot analysis showed that c-Myc protein level was significantly higher in the metastatic cell lines(SW620 and LoVo)than in the pre-invasive cell lines(SW480,Caco2 and HT29).An inverse correlation between c-Myc protein and miR-200b-3p was observed using Pearson's correlation analysis in 6 CRC cells(r =-0.908,p = 0.012).Luciferase report assay showed that c-Myc repressed the luciferase activities of the miR-200b-3p promoter in 293 T and SW480 cells.ChIP assay showed that c-Myc bound to the two region(-371 to-210 bp and-993 to-830 bp)of the miR-200b-3p promoter.Moreover,over-expression of c-Myc inhibited miR-200b-3p expression levels of SW480 and Caco2 cells.2)invasion assays in vitro showed that over-expression of c-Myc enhanced the invasiveness of SW480 cell.c-Myc over-expression increased frequencies of cells with fibroblastic or spindle-like morphology and concomitantly decreased frequencies of cobblestone-like cells.Similarly,c-Myc over-expression decreased the expression of the epithelial marker E-cadherin and increased the expression of the mesenchymal markers N-cadherin and vimentin.Importantly,these increased effects of c-Myc on in vitro invasion and EMT were abolished by re-expression of miR-200b-3p,3)Nude-mouse subcutaneous tumor model showed mice with SW480/c-Myc cells injection displayed increased tumor growth.Intriguingly,the subcutaneous tumors derived from SW480/c-Myc cells were not well encapsulated in false fibrous membrane compared to those derived from SW480/mock cells.Nude-mouse cecum transplanted tumor model showed that mice with c-Myc over-expressing tumors developedmore metastatic nodules in intestine and liver than control mice with tumors of similar size.Importantly,the increased effects of c-Myc on growth,invasion and metastasis in vivo were abolished by re-expression of miR-200b-3p.4)Western blot showed that miR-200b-3p silencing increased Ser9 phosphorylation of GSK3? without altering total GSK3? protein level.MiR-200b-3p silencing also decreased T58 phosphorylation but increased S62 phosphorylation of c-Myc protein and c-Myc protein level.Conversely,miR-200b-3p over-expression showed the opposite changes of phosphorylation in GSK-3? and c-Myc proteins.Intriguingly,following Tws119 treatment,a selective GSK-3? inhibitor,the increased Ser9 phosphorylation of GSK3?,S62 phosphorylation of c-Myc and c-Myc protein level,and the decreased T58 phosphorylation of c-Myc induced by miR-200b-3p silencing were well abolished5)Bioinformatics analysis showed that AKT2 was a potential target of miR-200b-3p.Dual-luciferase reporter assays showed that mi R-200b-3p over-expression suppressed the luciferase activity of AKT2 mRNA 3?UTR in 293 T cell.Moreover,western blot showed that miR-200b-3p over-expression decreased total AKT2 protein level,which contributed to decreased levels of AKT2 Ser474 phosphorylation,Ser9 phosphorylation of GSK3?,S62 phosphorylation of c-Myc and c-Myc protein,and increased level of T58 phosphorylation of c-Myc in LoVo cell.4.Analysis for the profiles of c-Myc?miR-200b-3p and PRDX2 in CRC tissues samples and the contribution of the three molecules to patients' clinicopathologic phenotype and outcome.1)q PCR showed that miR-200b-3p expression was dramaticallydown-regulated in most CRC tissues compared to in the PNCM tissues.Aberrant mi R-200b-3p expression levels were well associated with tumor differentiation,tumor size,infiltration depth of tumor and tumor metastasis.Kaplan–Meier analysis showed that CRC patients with high miR-200b-3p expression had a longer overall survival than CRC patients with low miR-200b-3p expression.2)IHC results showed PRDX2 protein levels were frequently higher in CRC tissues than in adjacent normal mucosa tissues.Aberrant PRDX2 protein levels were well associated with tumor differentiation,tumor size,infiltration depth of tumor and tumor metastasis.Kaplan–Meier analysis results showed that CRC patients with high PRDX2 expression had a shorter overall survival than CRC patients with low PRDX2 expression.Similarly,c-Myc protein levels were frequently higher in CRC tissues than in adjacent normal mucosa tissues.Aberrant c-Myc protein levels were well associated with tumor size,infiltration depth of tumor and tumor metastasis.Kaplan–Meier analysis results showed that CRC patients with high c-Myc expression had a shorter overall survival than CRC patients with low c-Myc expression.3)Pearson's correlation analysis showed there was an inverse correlation between c-Myc protein levels and mi R-200b-3p expression levels(r=-0.464,p<0.001),and miR-200b-3p expression levels and PRDX2 protein levels(r=-0.524,p<0.001)in 97 cases of CRC tissue samples.5.The c-Myc/miR-200b-3p/PRDX2 feedback loop regulates chemotherapeutic resistance of CRC cells1)The half inhibitory concentration(IC50)of SW480/c-Myc cells to oxaliplatin(OXL)was dramatically higher than that of the control SW480cells(10.39 ?M vs.6.365 ?M),while the increased IC50 was abolished by re-expression of miR-200b-3p(4.859 ?M vs.8.521 ?M).On the other hand,The IC50 of LoVo/miR cells to OXL was dramatically lower than that of the control LoVo cells(10.39 ?M vs.6.365 ?M),while the decreased IC50 was restored by nontargetable PRDX2(4.859 ?M vs.7.325 ?M).2)After oxaliplatin treatment,SW480/c-Myc cell had reduced apoptosis ratios compared to the control,while re-expression of miR-200b-3p abrogated the reduced effects.On the other hand,LoVo/miR cell had increased apoptosis ratios,while the increased effect was rescued by nontargetable PRDX2Conclusion1.mi R-200b-3p is a regulator of PRDX2 and represses PRDX2 protein level at post-transcriptional level in CRC cells.2.c-Myc suppresses miR-200b-3p expression at transcriptional level,whereas miR-200b-3p counteracts c-Myc to disrupt the stability of c-Myc protein in CRC cells.3.The expression levels of c? Myc,mi R? 200b? 3p and PRDX2 are disrupted in human CRC tissues and the aberrant expression levels are associated with clinicopathologic phenotypes and outcome of CRC patients.4.Disruption of the c? Myc/miR? 200b? 3p/PRDX2 feedback loop enhances tumor cell EMT,invasion,metastasis and chemotherapeutic resistance in CRC. |
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