MiR-495-3p Facilitates Colon Cancer Cell Growth Via Wnt/β-catenin Signaling Pathway By Inhibiting Wnt Inhibitory Factor

BackgroundColorectal cancer is a heavy burden around the world.Its incidence has declined in a number of Western countries,mainly because of early detection,prevention,and treatment measures.Therefore,the underlying etiologies impacting colon cancer tumorigenesis has to be classified to help in monitoring the progress of therapy.MiR-495 plays an important role in the occurrence and development of a variety of tumors,and is considered as a promising target for tumor therapy.MiR-495 was previously considered to be a breast cancer cell promoter that targeted the regulation of E-cadherin and REDD1.This miRNA was also observed to be decreased in MLL-rearranged leukemia.However,little else is known about the reverse chain of miR-495,miR-495-3p,the implications of its function and expression in colon cancer.ObjectTo demonstrate that miR-495-3p appears to be a novel oncogene miRNA,which promotes colon cancer tumorigenesis and facilitates the stemness of colon cancer cells via suppressing Wnt inhibitory factor(WIF1).We investigated the role of miR-495-3p in colon cancer cell growth and its possible mechanism.In order to provide a new perspective for the pathogenesis of colon cancer and find out the potential new direction of colon cancer treatment.MethodsThe level of miRNA and mRNA in cell were tested by real-time polymerase chain reaction.Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay.Cell spheroid formation was measured by colony assay.Expression protein was tested using Western blotting.β-catenin binding ability was detected by chromatin immunoprecipitation assay.MiRNA target gene was definited by luciferase assay.Results(1)MiR-495-3p increased in colon cancer cell lines and tissuesTo investigate miR-495-3p expression in human colon cancer cells,we selected 6 colon cancer cell lines and found that miR-495-3p levels were obviously increased compared with normal colon cell line CCD18Co(p<0.05).Next,10 paired our hospital colon cancer and corresponding para-carcinoma tissues were detected by real-time PCR.It was observed that miR-495-3p expression in colon cancer tissues was markedly higher than that of normal tissues,which confirmed its overexpression in colorectal cancer(p<0.05).(2)MiR-495-3p facilitated HCT-116 cell proliferation and spheroid formationAs miR-495-3p was upregulated in colon cancer,we speculated that miR-495-3p was involved in facilitating colon cancer proliferation.MiR-495-3p expression was regulated by miR-495-3p mimics or inhibitor transfection.Next,MTT assay demonstrated that miR-495-3p mimics elevated,whereas miR-495-3p inhibitor significantly declined HCT-116 cell viability after four days’ incubation.At the same time,spheroid formation assay revealed that miR-495-3p mimics significantly enhanced spheres formation compared with control,while miR-495-3p inhibitor reduced the amount of spheres.It indicates that miR-495-3p may affect cell proliferation of HCT-116 and spheroid formation.(3)MiR-495-3p promoted Wnt signaling pathwayPrevious studies revealed that Wnt signaling pathway may be involved in colon cancer cell proliferation and sternness,thus we speculated that miR-495-3p may achieve its impact on colon cancer cells via Wnt signaling pathway.TOP/FOP ratio was significantly elevated in HCT-116 cells after miR-495-3p overexpression,while it reduced after miR-495-3p-in transfection(p<0.05).We further assessed the influence of miR-495-3p on β-catenin nuclear translocation.After its extraction from the cytoplasm and the nucleus,the protein was detected using Western blot.It was shown that β-catenin content in the nucleus was markedly increased in the miR-495-3p group(p<0.05).In addition,ChIP assay was applied to evaluate Wnt signaling pathway activation.It was found that miR-495-3p overexpression facilitated nuclear β-catenin binding with the promoter of MMP-9 and RUNX2,enhancing gene transcription and help colon cancer cell proliferation(p<0.05).(4)MiR-495-3p targeted WIF1 in spheroid formationAfter checked the miRNA database,it was found that miR-495-3p may complimentarily bind with WIF1 mRNA.Luciferase assay demonstrated that miR-495-3p markedly reduced the luciferase activity of WIF1 3’-UTR with dose dependence,whereas it exhibited no significant impact on WIF1 3’-UTR mutation.On the other hand,knockdown of WIF1 enhanced spheroid formation ability of HCT-116 cells decreased by miR-495-3p inhibitor(p<0.05).Simultaneously,WIFI level was negatively correlated with miR-495-3p,further indicating their relationship.ConclusionMiR-495-3p is elevated in colon cancer.This oncogene is a negative controller of Wnt/β-catenin signaling inhibitor,and its abnormal overexpression in colon cancer enhances Wnt/β-catenin signaling pathway.This aberrant signaling further allows cells to accelerate spheroid formation and sternness,resulting in colon cell proliferation and tumorigenesis.The results in this study provide new insight i nto colon cancer pathogenesis and a potential direction for colon cancer therapy.