CircMYO9B Facilitates Breast Cancer Cell Proliferation And Invasiveness Via Upregulating FOXP4 Expression By Sponging MiR-4316

BACKGROUND:Among all female tumor diseases,the incidence of breast cancer is at a high level,and it is also the main risk factor leading to patient death,which seriously threatens the safety of patients.Circ RNAs as a member of nc RNAs has been confirmed to be involved in the occurrence and development of breast cancer.It plays an important role in breast cancer,but the specific mechanism of action of most circ RNAs in breast cancer is still unclear.More and more research evidences show that the transcription of many circ RNAs can compete with micro RNAs to play the function of competitive endogenous RNAs(ce RNAs).A large number of studies have shown that circMYO9 B correlates with the occurrence and development of many types of tumor diseases,such as lung cancer and esophageal cancer.However,the role and mechanism of circMYO9 B in breast cancer have not yet been elucidated.OBJECTIVE:The aim of this study is to explore the role of circMYO9B/ miR-4316 / FOXP4 axis in regulating breast cancer progression and its related mechanisms.METHODS:1.Firstly,we screened online database to find the circ RNAs that differentially expressed in breast cancer tissues and adjacent normal tissues.To explore the concern between the identitied circ RNAs and clinical prognosis of breast cancer patients,we analyzed the pattern of circMYO9 B distribution in breast cancer samples andadjacent normal tissue samples via q RT-PCR.2.Small interfering RNA(si RNA)or expression plasmids were utilized to knock down or overexpress circMYO9 B in brease cancer cells respectively.Then we investigated the role of circMYO9 B in breast tumor progression by transwell migration and invasion assays.3.The molecules downstream of circMYO9 B were predicted using miRDB.Then we constructed mutants of circMYO9 B and performed RNA pull-down assays to validate interaction of miR-4316 with circMYO9 B.Besides,q RT-PCR were performed to explore expression patterns of miR-4316 in breast tumors.The correlation between miR-4316 and circMYO9 B expression levels were examined using Spearman correlation analysis.4.Mi R-4316 inhibitors were applied to the breast cancer cells with circMYO9 B knockdown,and cell proliferation assay and transwell assay were conducted to confirm the role of miR-4316 in breast cancer progression the regulated by circMYO9 B.5.The downstream target genes of miR-4316 are tested by prediction software,and the dual luciferase test method is used to detect and verify the relationship between miR-4316 and the downstream target FOXP4;Western blot analysis is used to detect the expression level of FOXP4 protein in breast cancer cells,and analyze the expression differences of FOXP4 and circMYO9 B in tumor samples of breast cancer patients,and explore the mechanism of circMYO9B/miR-4316/FOXP4 axis in regulating breast cancer progression.6.MDA-MB-231 cells with circMYO9 B knockdown were inoculated in the fat pad of nude mice to establish a xenograft tumor model.Then the tumor growth was monitored,and the expression levels of circMYO9B/ Mi R-4316/ FOXP4 axis were analyzed by q PCR and immunohistochemistry to explore the role and mechanism of circMYO9 B in regulating breast tumor progression in vivo.RESULTS:1.The level of circMYO9 B in breast cancer tissues was significantly higher than adjacent normal tissues.And the level of circMYO9 B in breast cancer cell lines(BT474,MCF-7,MDA-MB-231,T47 D and MDA-MB-453)was also significantly higher than that in normal breast epithelial cell lines(MCF-10A).In addition,higher circMYO9 B expression was associated with shorter overall survival in breast cancer patients.2.In MCF-7 and MDA-MB-231 cells,knockdown circMYO9 B significantly reduced the ability of breast cancer cell migration and invasion,while overexpression of circMYO9 B promoted the proliferation,migration and invasion of breast cancer cells.3.The overexpression of miR-4316 clearly reduced the expression level of circMYO9 B in breast cancer cells,and miR-4316 specifically enriched circMYO9 B.Moreover,miR-4316 was significantly down-regulated in breast cancer tissues.The expression levels of miR-4316 were negatively correlated with circMYO9 B levels in breast cancer tissues.4.CircMYO9 B knockdown lead to impaired proliferation,migration and invasion of breast cancer cells,while inhibition of miR-4316 eliminated the inhibition effect.5.FOXP4 expression was inhibited in cells of miR-4316 overexpressing or circMYO9 B knockdown,while FOXP4 level recovery in cells with circMYO9 B knockdown and miR-4316 knockdown.Furthermore,FOXP4 expression pattern in breast tumors of patients was similar with circMYO9 B.FOXP4 overexpression restored proliferation,migration and invasion of tumor cells with circMYO9 B knockdown.6.Knockdown circMYO9 B significantly inhibited tumor growth,tumor cell proliferation and FOXP4 expression in vivo.CONCLUSION:1.High expression of circMYO9 B is associated with bad prognosis of breast cancer patients,and may be a potential prognostic marker.2.CircMYO9 B promotes FOXP4 protein expression by inhibiting miR-4316 levels,and then facilitates proliferation,migration and invasion of breast cancer.