MiR-375-3p Suppresses Tumorigenesis And Partially Reverses 5FU Resistance By Targeting YAP1 And SP1 In Colorectal Cancer Cells

【Background】Colorectal cancer(Colorectal cancer,CRC)is the fourth most fatal cancer in the world.Although chemotherapy is one of the preferred strategies for treatment of patients with the advanced CRC,drug resistance has become the main obstacle to CRC treatment caused by chemotherapy,severely resulting in poor prognosis.Thus,it is urgent to improve anti-cancer drug effects by optimizing multidrug treatment approaches to achieve better therapeutic effect and prognosis for CRC treatment.The aberrant expression of micro RNAs(miRNAs)can modulate various cancer phenotypes.It has been proved that miRNAs also participate in chemoresistance and different miRNAs can lead to different outcomes in chemoresistance,making miRNAs a potential target for drug resistant treatment of tumor.micro RNA-375-3p(miR-375-3p)was reported as an important tumor suppressor that related to chemosensitivity of various cancers.Yes associated protein 1(YAP1)and Specificity protein 1(SP1)are two target genes of miR-375-3p,which were reported highly express in CRC and associated with the poor prognosis of CRC.Meanwhile,YAP1 is an important downstream effector of Hippo pathway,and activation of Hippo-YAP1 pathway ultimately suppresses tumorigenesis.Fluorouracil(5-fluorouracil,5FU)is one of the broad-spectrum anticancer drugs.The latest research demonstrated miR-375-3p served crucial function in stratifying CRC patients to preoperative chemoradiation,and researches have aslo showed that miRNAs mediated-YAP1and/or-SP1(YAP1/SP1)is closely related to cancer drug resistance,whereas it seems unclear about how miR-375-3p modulates therapeutic resistance of CRC cells by targeting YAP1 and/or SP1.Therefore,the purpose of our study was designed to explore the mechanism of miR-375-3p mediated-YAP/SP1 regulating 5FU resistance in CRC,and the combination of miR-375-3p with chemotherapeutic drugs was expected to provide a novel insight into the management of CRC chemoresistance.【Purpose】Our study aims to reveal the molecular mechanism of miR-375-3p mediated YAP1/SP1 in CRC 5FU resistance,through which provides a new therapeutic target for the clinical treatment of CRC chemoresistance.【Methods】1.5FU resistant CRC cell lines HCT116/FU and HCT8/FU were induced by stepwise selection on exposure to increasing does of 5FU in vitro,based on CRC cell lines HCT116 and HCT8,respectively.CCK-8 assay accessed the inhibition growth of parental and resistant cell lines in response to 5FU,and the growth inhibition curves were plotted,the half maximal inhibitory concentration(IC50)value of 5FU was calculated by Graphpad and the resistance index(RI)was abtained.The IC50 value of irinotecan,capecitabine and oxaliplatin were obtained by the same approach.2.CRC and adjacent normal tissues(ANTs),5FU resistant and 5FU sensitive tissues were collected followed by CRC cell lines(HCT116,HCT29,HCT8,SW480,SW620,DLD1 and Ca CO2)and normal intestinal mucosal epithelial cell(FHC)cultured,real-time quantitative polymerase chain reaction(q RT-PCR)was used to verify miR-375-3p expression levels in CRC tissues and cells.Starbase 3.0 database,TCGA database were used to analyze miR-375-3p expression levels in CRC tissues(CRC patients who received5FU-based chemotherapy were dividied into two groups.5FU resistant tissues,patients who responded well to 5FU-based chemotherapeutic treatment;5FU sensitive tissues,patients who relapsed after the first 5FU-based chemotherapeutic treatment).3.After miR-375-3p mimics,NC mimics;miR-375-3p inhibitors,NC inhibitors;small interference RNA(si RNA): si NC,si YAP1,si SP1;YAP1 and SP1 overexpressing plasmids:empty vector,pc DNA3.1-YAP1,pc DNA3.1-SP1 transfected into CRC resistant and nonresistant cells by using lipfectamine 2000,q RT-PCR was used to detecte the expression of miR-375-3p,YAP1 and SP1 in CRC cell lines.4.The proliferation ability and anti-apoptosis ability were detected by MTT assay,colony formation assay and flow cytometry.The subcutaneous tumor formation models of nude mice were constructed to observe the influence of miR-375-3p on tumor growth and drug resistance in animals.5.The downstream target genes of miR-375-3p were predited by online tools(Target Scan 7,Pic Tar and micro T-CDS),detected by q RT-PCR and verified by a dual luciferase reporter assay.Western blot and immunohistochemistry assay(IHC)were applied to detect the expression levels of YAP1 and SP1.The influence of YAP1/SP1 regulated by miR-375-3p exreted on CRC proliferation and chemoresistance was analyzed by MTT assay and flow cytometric.Western blot and IHC assay were applied to analyze the proteins expression levels of Hippo-YAP1 pathway and apoptosis-related proteins.【Results】1.After 6.8 months,the 5FU resistant cell lines(HCT116/FU,HCT8/FU)was successfully established based on the CRC cell lines(HCT116,HCT8)and could keep growing in the culture medium with the concentration of 5FU up to 200μg/ml.2.Compared to ANTs and FHC cells,q RT-PCR showed that miR-375-3p was significantly downregulated in CRC tissues and cell lines,especially much lower in 5FU resistant cell lines and tissues.The Starbase 3.0 and TCGA databases confirmed that miR-375-3p was remarkably decreased in CRC tissues.The results revealed that low expression level of miR-375-3p was negatively correlated with chemoresistance,malignancy and poor prognosis.3.High expression of miR-375-3p markably suppressed proliferation and chemoresistance and promoted 5FU induced apoptosis of CRC cells in vitro.Subcutaneous tumor formation model exhibited that high miR-375-3p expression significantly inhibited tumor growth and5 FU resistance,whereas inhibition of miR-375-3p produced opposite effect in vivo.4.The bioinformatics analysis displayed that YAP1 and SP1 were two target genes of miR-375-3p which was verified by double luciferase reporter assay.Both m RNA and proteins expression levels of YAP1 and SP1 were notably upregulated in CRC tissues and cell lines,especially much higher in 5FU resistant cell lines and tissues compared that in the nonresistance.Furthermore,in vitro and in vivo assays demonstrated that high miR-375-3p expression suppressed YAP1 and SP1 expression,whereas inhibition of miR-375-3p produced opposite effect.5.The MTT assay demonstrated that upregulation of YAP1 and SP1 significantly induced proliferation and enhanced 5FU resistance in CRC parental cells,whereas downregulation of YAP1 and SP1 relatively reduced cell proliferation and drug resistance in CRC 5FU resistant cells.After co-transfection,it was found that silencing YAP1 and SP1 could reverse anti-apoptosis and 5FU resistance induced by miR-375-3p inhibitors with or without drug administration,whereas upregulation of YAP1 and SP1 abolished the apoptosis promotion and 5FU sensitivity induced by miR-375-3p with or without drug administration.Moreover,western blot and IHC assay revealed that miR-375-3p regulated CRC tumorigenesis and chemoresistance through Hippo-YAP1 signaling pathway and targeting SP1.【 Conclusions 】 Our study demonstrates that miR-375-3p suppresses tumorigenesis and reversed 5FU resistance by targeting YAP1 and SP1 in colorectal cancer cells,which provides a promising therapeutic strategy for CRC chemoresistance.