The Function And Mechanisms Of CircFBXL5 Promoting Breast Cancer Metastasis To Lung

BackgroudThe morbidity of breast cancer is the first malignant tumors of women in the world.But in China,the morbidity of breast cancer ranks first,and the mortality ranks fifth,and the morbidity is increased gradually and become younger.Over 90% deaths of breast cancer were induced by widespread metastasis,particularly the prognosis of lung metastasis is the worst.However,the mechanisms of breast cancer metastasis to lung are still needed to be investigated.The possible mechanisms include: epithelial-mesenchymal transition?EMT?,breast cancer stem cells,tumor microenvironment,circulating tumor cells,tumor associated macrophages,and non-coding RNA.Circular RNAs?circRNAs?are a variety of single covalent circularion without 5' cap and3' tail.It's reported that a number of circRNA were dysregulated and played roles in carcinogenesis,proliferation,apoptosis,reagent resistance and metastasis in breast cancer.Because breast cancer metastasis is organ specificity,scientists screened differential expressed circRNA betweenbrain metastasized cells and wildtype cells in breast cancer using RNA sequencing.However,the mechanisms of the differential expression of circRNAs between primary cancer and metastatic cancer needs to be explained.Objective and significanceIn this study,differential expressed circRNAs were screened between primary lesion and lung metastasis lesion in breast cancer,the function and mechanisms of the most differential expressed circRNA was selected,and was analyzed its effect and mechanisms of circRNA in regulating breast cancer metastasis to lung.This study will identify potential prognosis and treatment biomarkers in lung metastasis of breast cancer and supply novel idea for lung metastasis of breast cancer treatment.MethodsThe differential expressed cirRNAs between primary lesion and lung metastasis lesion in breast cancer were screened by circRNA microassay and selected the most 20 differential expressed circRNAs to verify.The expression of circFBXL5,miR-660 and SRSF6 mRNA was detected by qRT-PCR.The correlation between circFBXL5 expression and the prognosis of breast cancer patients was analyzed by Kaplan-Meier assay.The cell vability was detected by CCK8.The capacity of clony formation was tested by colony formation assay.The breast cancer cells transplanted nude mice model and breast cancer lung metastasis nude mice model were constructed.The ability of tumor formation and lung metastasis was analyzed after inhibiting circFBXL5 using those two constructed mice models.MiRNAs binding to cirFXBL5 and targets of miR-660 were predicted by online software Targetscan.The combination of miR-660 to circFBXL5,SRSF6 to miR-660,SRSF6 to circFBXL5 was analyzed by luciferase reporter assay.The enrichment to Ago2 fo circFBXL5,miR-660,SRSF6 was detected by RIP.Results1.CircRNAs differential expressed between primary lesion and lung metastasis lesion in breast cancerCompared to breast cancer primary lesion,the most upregulated circRNAs in lung metastasis lesion were as follows: hsa_circ₀125597,hsa-circ₃336-9 and hsa-circ₃617-3 et al;the most downregulated circRNAs were as follows: hsa-circ₅044-5,hsa_circ₀002018 and hsa_circ₀010438 et al.2.CircFBXL5 was upregulated in breast cancer cells and negatively related to prognosisCircFBXL5 was expressed mainly in the cytoplasm ofMDA-MB-453 and MDA-MB-231 cells,and the expression of nucleus was lower than that of cytoplasm?P<0.05?.The expression of circFBXL5 was increased in several breast cancer cell lines compared to normal breast epidermal MCF10 A cells by qRT-PCR.The expression of circFBXL5 was the highest in MDA-MB-231 cells and the second in MDA-MB-453 cells,but not increased in MCF7 and BT474 cells?P <0.05?.The overall survival time in circFBXL5 low-expression group was longer than in circFBXL5 high-expression group by Kaplan-Meier analysis?P<0.05?.3.CircFBXL5 acted as an oncogene in breast cancerThe viability of MDA-MB-453 and MDA-MB-231 cells was remarkably decreased compared to the control after transfected with circFBXL5 siRNA detected by CCK8 assay?P<0.05?.The clone number of circFBXL5 siRNA group was less than the control group.The tumor weight of circFBXL5 siRNA group was less than the control group in MDA-MB-453 and MDA-MB-231 cells transplanted nude mice models?P<0.05?.In breast cancer lung metastasis nude mice model,the number of metastasis lesion in circFBXL5 siRNA group was decreased compared to the control group?P<0.05?.4.circFBXL5 combined to SRSF6 by competing with miR-660 in breast cancerCircFBXL5 was predicted to bind with miR-660 by online softwareTargetscan.MiR-660 was found to downregulated in breast cancer cell lines compared to MCF-10 A cells by qRT-PCR.The expression of miR-660 was the lowest in MDA-MB-231 cells?P < 0.05?.CircFBXL5 was identified to bind to miR-660 by luciferase reporter asay.SRSF6 was predicted to be a target gene of miR-660 by online software Targetscan.SRSF6 was found to upregulated in breast cancer cell lines compared to MCF-10 A cells by qRT-PCR.The expression of SRSF6 was the highest in MDA-MB-231 cells?P<0.05?.The combination to Ago2 of circFBXL5,miR-660 and SRSF6 was detected by RIP.The enrichment of circFBXL5,miR-660 and SRSF6 in Ago2 was higher than IgG group either in MDA-MB-453 or in MDA-MB-231 cells.In MDA-MB-453 or in MDA-MB-231 cells,inhibition of circFBXL5 could increase the enrichment of SRSF6 in Ago2 detected by RIP?P < 0.05?.When inhibition circFBXL5 expression in MDA-MB-453 or in MDA-MB-231 cells,the expression of SRSF6 was decreased,but suppressing miR-660 at the same time,the expressin of SRSF6 was recovered?P<0.05?.Conclusions1.The expression of circFBXL5 was upregulated in breast cancer tissues and cells.The expression of circFBXL5 was negatively correlated with worse outcome of breast cancer patients.2.Inhibition of circFBXL5 could suppress proliferation,clony formation,tumor genesis and lung metastasis.3.SRSF6 was a directly target gene of miR-660.4.CircFBXL5 probably act as a ceRNA of miR-660 to combine with SRSF6 competitively and promote progression in breast cancer.