MiR-5188 Regulates Human Breast Cancer Prolife Ration,Metastasis And Stemness Through A Loop With FOXO1/β-catenin/c-Jun Signal Pathway

The incidence and mortality of breast cancer in the world have been increasing and younger.MiRNA plays an important role in self-renewal and invasion and metastasis of breast cancer stem cells.MicroRNA-5188(miR-5188)as the newly discovered have not been studied.This study have demonstrated that miR-5188 was notably upregulated in Cancer Stem Cells(CSCs)derived from breast cancer cells,and can be used as oncogenes in the cancer proliferation,metastasis and stemness in the breast cancer.The mechanism study revealed that miR-5188 directly targeted the classic tumor-suppressor gene-FOXO1.The interaction between FOXO1 andβ-catenin regulated the effect of β-catenin on c-Jun activation,and the activation induced miR-5188 expression.miR-5188 promote cancer proliferation,metastasis and stemness through Wnt/β-catenin/-c-Jun signal pathway.In this study,we explored the function and mechanism of miR-5188 play in breast cancer,and reveals miR-5188 promote cancer proliferation,metastasis and stemness through Wnt/β-catenin/-c-Jun signal pathway,and provides a theoretical basis for the treatment of breast cancer.Contents and methods1.The effect on cell biology of breast cancer cell by miR-5188.(1)We used qPCR to examine the expression of miR-5188 in breast cancer cell lines.(2)We used MTT assay,colony formation,Edu incorporation assays and xenograft tumor to examine the effect of miR-5188 on breast cancer cell proliferation.(3)We used migration and invasion assays,Transwell assays and Caudal vein metastasis model in nude mice to examine the effect of miR-5188 on breast cancer cell metastasis.(4)We used Side Population assays,flow cytometry assay,immunofluorescence assay and the mammosphere formation assay to examine the effect of miR-5188 on breast cancer cell stemness.2.The molecular mechanism of miR-5188 promote cancer proliferation,metastasis and stemness.To determine the mechanisms by which miR-5188 promoted breast cancer cells proliferation,metastasis and cancer stemness,We used western blot to examine whether the expression of N-Ca,E-Ca,Vimentin,Snail,Slug,Claudin-1,Sox2,Oct4,Nanog,β-catenin,c-Jun,CCND1 were effect by miR-5188.3.miR-5188 directly targets FOXO1.(1)Through bioinformatics software,there was predicted that miR-5188 direct target FOXO1 in breast cancer cells.(2)Western blots of FOXO1 in miR-5188-overexpressing or miR-5188-inhibited breast cancer cells.(3)We detected expression of FOXO1 by qPCR assay in miR-5188 overexpressed or miR-5188 inhibited breast cancer cells.(4)We detected expression of FOXO1 by immunohistochemistry in orthotopic xenografts results from breast cancer cells.(5)We detected miR-5188 targeting the FOXO1 3’UTR by luciferase reporter assay.(6)We used Co-IP assay and immunofluorescence assay to determine the relationship of FOXO1 and β-catenin in miR-5188-overexpressing breast cancer cells.(7)western blot to examine whether the expression of N-Ca,Vimentin,Sox2,Oct4,Nanog,c-Jun,CCND1,β-catenin were influenced by si-FOXO1.4.The promoter region of miR-5188 was binds with c-Jun,c-Jun promotes miR-5188 transcription.(1)Through bioinformatics software to explore the transcriptional regulatory mechanisms of miR-5188 expression,we found a 3-kb region upstream of the transcription start site(TSS)of miR-5188 promoter region,and there are three c-Jun-binding motifs where in-1424 to-1412,-631 to-625 and-250 to-237.(2)We detected expression of miR-5188 when knocking down c-Jun expression byqPCR.(3)We determined that c-Jun protein was recruited to the putative miR-5188 promoter including all the three binding sites by chromatin immunoprecipitation(ChIP)assays in breast cancer cells.(4)We determined whether the c-Jun binding sites in the promoter region of miR-5188 including all the three binding sites by the EMSA assay.(5)We detected that c-Jun directly binds to promoter of miR-5188 and promotes its transcription by luciferase assay.Results1.miR-5188 is upregulated in breast cancer.2.miR-5188 promote cancer proliferation,metastasis and stemness.3.miR-5188 promote cancer proliferation,metastasis and stemness through Wnt/β-catenin signal pathway.4.miR-5188 directly targets FOXO1.5.c-Jun directly binds to promoter of miR-5188,and can promote its transcription.ConclusionsmiR-5188 promote cancer proliferation,metastasis and sternness through Wnt/β-catenin/-c-Jun signal pathway.