Roles And Mechanisms Of MicroRNA-149 In Regulating The 5-FU Chemosensitivity Of Colorectal Cancer Cells Through FOXM1

Colorectal carcinoma(CRC)is the third leading cause of cancer-related deaths around the world.Chemotherapeutic agents serve as important adjuvant therapies for CRC treatment.5-Fluorouracil(5-FU)is one of the chemotherapeutic drugs widely used alone or combined with other drugs for patients with advanced CRC,which has yielded clinical benefits to CRC patients.However,90% of CRC patients suffered from 5-FU resistance and disease progression inevitably.Unfortunately,the mechanisms underlining 5-FU resistance in CRC remain poorly understood,thus,elucidation of molecular mechanisms involved in 5-FU resistance will be helpful to develop novel strategies for the treatment of patients with 5-FU-resistant CRC.Micro RNAs(mi RNAs)are a class small non-coding RNAs with 17~25 nucleotides,which suppress gene expression post-translationally by binding to 3’-untranslated regions(3’-UTR)of specific target m RNAs.Increasing evidence has shown that dysregulation of mi RNAs play critical roles in various physiological and pathological processes including growth,differentiation and apoptosis,as well as therapy resistance.Here,we showed that ectopic expression of mi R-149 could reverse the 5-FU resistant phenotype of CRC cells at partially by targeting Fox M1.Our data indicated a novel regulatory pathway for 5-FU resistance involving mi R-149 and FOXM1,and suggested that targeting mi R-149/Fox M1 signaling will be a potential strategy for the treatment of 5-FU-chemoresistant CRC patients.Based on bioinformatics analysis and dual luciferase reporter assay,study results showed that the level of Fox M1 was negatively correlated with mi R-149 expression,in which mi R-149 directly binds to 3’-UTR of FOXM1 m RNAs.Silencing of Fox M1 mimics the effect of mi R-149 upregulation on 5-FU response in 5-FU-resistant CRC cells.Through expression profile analysis and bio-informatics,we identified PHF5 A involving in the downstream of mi R-149/Fox M1 signal pathway.Collectively,this study provided the evidence that reduced mi R-149 is a critical factor in the mechanisms by which CRC cells resist the cytotoxicity of 5-FU.Re-expression of mi R-149 could increase the 5-FU sensitivity of CRC cells via enhancing 5-FU-inducing apoptosis by directly targeting FOXM1.Therefore,targeted therapies to mi R-149/FOXM1 signaling may increase sensitivity to 5-FU treatment and may promise a therapeutic strategy for 5-FU-resistant.Also,PHF5 A was found to play a role in cell proliferation and apoptosis regulating.And PHF5 A may be involved in drug resistance phenotype mediated by Fox M1.Part I The correlation between mi R-149 expression and 5-FU ensitivity in CRCObjective: To investigate the expression of mi R-149 in human CRC and its function in 5-FU resistant CRC cells.Methods: 1.MTT was conducted to measure the IC 50 of the 5-FU-resistant CRC cells(HCT-8/5-FU、Lo Vo/5-FU)and the parental cells(HCT-8、Lo Vo).2.HCT-8/5-FU and Lo Vo/5-FU cells were transiently transfected with mi R-149/mimics or mi R-NC/mimics,respectively.Parental HCT-8 and Lo Vo cells were transiently transfected with anti-mi R-149 or anti-mi R-NC,respectively.3.q RT-PCR was utilized to detected the level of mi R-149 in each CRC cell lines.4.q RT-PCR was utilized to detected the level of mi R-149 in CRC tissues collected from advanced CRC patients(n=24)with different 5-FU sensitivity.Results: 1.The IC50 of HCT-8/5-FU(IC50: 34.8μg/ml)and Lo Vo/5-FU(IC50: 73.7μg/ml)was significantly higher than the IC50 of parental cells HCT-8(IC50: 2.8μg/ml)and Lo Vo(IC50: 11.8μg/ml).2.The resistance index of HCT-8/5-FU and Lo Vo/5-FU was 13.6 and 6.3.respectively.3.The level of mi R-149 in 5-FU-resistant CRC cells was significantly lower than the level in parental cells(p < 0.01).4.Overexpression of mi R-149 could increase the 5-FU sensitive of resistant CRC cells.5.Knockdown of mi R-149 could decrease the sensitivity of parental CRC cells to 5-FU.6.The level of mi R-149 in 5-FU responsive group was significantly higher than in the non-responsive group.Conclusion: Overexpression of mi R-149 in CRC cells could significantly decreased the chemoresistance of CRC to 5-FU and vice versa.Moreover,mi R-149 was significantly downregulated in the 5-FU non-responsive CRC patients.These results revealed that targeting mi R-149 will provide a novel strategy for improving the 5-FU response of CRC.Part II mi R-149 modulated 5-FU sensitivity by targeting Fox M1 in CRC cellsObjective: To investigate the mechanism of mi R-149 on 5-FU sensitivity in CRC cells.Methods: 1.The target gene of mi R-149 was selected by bioinformatics software(Pictarget 、 Target Sca 、 mi Randa).2.Dual-luciferase reporter assays confirmed the functional relationship between mi R-149 and Fox M1.3.q RT-PCR and Western blot were performed to determine the regulation of Fox M1 by mi R-149.4.The previously constructed sh RNA vector targeting Fox M1(p Sil/sh FOXM1)and control vector(p Sil/shcontrol)were stably transfected into HCT-8/5-FU and Lo Vo/5-FU cells,respectively.Then,q RT-PCR and Western blot confirmed the knockdown of Fox M1.5.q RT-PCR was employed to measure the level of Fox M1 m RNA in CRC tissues collected from advanced CRC patients with different 5-FU sensitivity.Results: 1.There is a binding site for mi R-149 in the 3’UTR of Fox M1 based on the bioinformatics software.2.Results from luciferase assay showed that the luciferase activity of p LUC/Fox M1-3’-UTR-wt but not p LUC/Fox M1-3’-UTR-mut was significantly decreased in CRC cells co-transfected with mi R-149 mimics and was significantly increased in CRC cells co-transfected with mi R-149 inhibitors.3.Both the Fox M1 m RNA and protein levels in 5-FU-resistant CRC cells were significantly higher than those in parental CRC cells.After transfected with mi R-149 mimics,the m RNA and protein levels of Fox M1 were decreased in the 5-FU-resistant CRC cells,and vice versa.4.Results from MTT showed that 5-FU-resistant CRC cells(HCT-8/5-FU and Lo Vo/5-FU)transfected with sh Fox M1(shcontrol as control)exhibited reduced 5-FU resistance with IC50 decreasing to 10.5ug/ml(p<0.05)and 29.4ug/ml(p<0.05),respectively.5.Overexpression of Fox M1 could decrease the sensitive of parental CRC cells to 5-FU.6.Linear regression analysis confirmed that the level of mi R-149 was negatively correlated with Fox M1(n=24,r=-1.52,p<0.01).Conclusion: Fox M1 is a target gene of mi R-149.And knockdown Fox M1 could partially reverse the resistamt phenotype of CRC cells.These data together indicated that the effect of mi R-149 on 5-FU resistance in CRC might be through targeting Fox M1.Part III Microarray screening and validation of Fox M1 downstream target genesObjective: To screen the downstream targets of Fox M1 by RNA interference and gene chip technique,and investigate the effect of gene interference on the proliferation of 5-FU resistant colon cancer cells.The genes with proliferative inhibition phenotype werescreened out.Methods: 1.The Fox M1 RNA interference sequence was designed by software and transfected into HCT-8/5-FU cells.Real time PCR was used to validate the interference effect.2.Total RNA was extracted from HCT-8/5-FU cells before and after Fox M1 interference,and the differential gene was screened by Affymetri’s Gene Chip primeview human gene chip.3.Differentially expressed genes were analyzed by Pathway enrichment analysis and Gene Ontology analysis software.Since Fox M1 mainly positively regulate downstream genes,we focused on the genes that were significantly down-regulated after Fox M1 interference.Through literature and Gen Bank information,we selected part of the genes that may be involved in proliferation regulation for next step study.4.The high-content screening(HCS)method was used to compare the effect of gene knockdown on cell proliferation rate.RNA interference sequence were designed for each gene,and after 4 days of sh RNA lentivirus infection,the cells were plated in 96-well plates and celigo was continuously tested for 5 days.5.For the selected genes,flow cytometry,cell colony formation assay and MTT assay were used to verify their effect on cell cycle,apoptosis and proliferation.Results:1.Fox M1 level was successfully knocked down2.Gene chip results showed that there was significant difference between Fox M1 knockdown and control HCT-8/5-FU cells(FC > 1.2,p <0.05),with 554 genes upregulated,and 650 genes downregulated.3.Twenty-five downregulated genes were selected out for further RNAi and HCS screen,and PHF5 A was the one with most significant proliferative alteration.4.Knockdown of PHF5 A suppressed HCT-8/5-FU cell proliferation and colony formation ability,enhanced cell apoptosis rate and 5-FU chemosensitivity.Conclusion: Fox M1 may regulate the PHF5 A expression and influence the chemosensitivity of colorectal cancer cells to 5-FU by effecting cell cycle and apoptosis.