Function And Molecular Mechanism Of The Interaction Between CircACTN4 And FUBP1 In Regulating The Occurrence And Development Of Breast Cancer

Objective Recent studies have shown that the abnormal expression of circular RNA(circ RNA)is nearly related to the progresses of cancers,but its mechanism has not been elucidated.The purpose of this study is to explore the expression and function of circ RNA circACTN4 in human breast cancer(BC)tissues and cells,and to clarify the principle of controlling the progresses of breast cancer,so as to provide scientific basis for finding fresh diagnostic and therapeutic targets of BC.Methods 4 pairs of breast cancer and paracancerous clinical specimens were selected for RNA microarray analysis,obtain the differential expression profile of circ RNA.Circ ACTN4 was identified by bioinformatics website,Sanger sequencing,RNA enzyme digestion and actinomycin D digestion.The expression of circACTN4 in 80 pairs of BC and normal tissues and 240 cases of BC were tested by q RT-PCR and ISH,respectively.And analyse the relationship between circACTN4 expression and clinicopathological characteristics and prognosis.Subsequently,the overexpression and knockdown plasmids of circACTN4 were constructed,and a series of gain-and loss-of-function experiments were carried out to verify the effects of circACTN4 abnormal expression on proliferation,metastasis,apoptosis and cell cycle of BC cells.The overexpression of circACTN4 and knockdown of lentivirus supernatant were designed and synthesized,and the cells were infected and stable transformants were screened.A series of in vivo experiments such as tumor formation,overall survival and visible light imaging of nude mice were carried out by subcutaneous or tail vein injection in nude mice to clarify the role of circACTN4 in the occurrence and development of BC.In terms of mechanism,the regulatory effect of Upstream Stimulate Factor 2(USF2)on circACTN4 biogenesis was verified by Ch IP and luciferase reporter gene assay.Subsequently,RNA pull down,RIP,FISH,Ch IP,western blot,immunohistochemistry and Co-IP were used to verify whether circACTN4 can specifically bind to RNA binding protein FUBP1.Thus activate the related signal pathways that regulate the occurrence and development of BC.Results The results of microarray analysis showed that the difference of circACTN4 was the highest.q RT-PCR and ISH results also demonstrated that,circACTN4 was significantly expressed in breast cancer tissues and cells compared with human normal paracancerous tissues and human normal breast epithelial cells MCF-10 A,and its expression level was positively correlated with clinical stage and poor prognosis.A series of function experiments proved that the abnormal high expression of circACTN4 is positively related to the proliferation,migration and invasion of breast cancer cells,and the abnormally low expression of circACTN4 promotes the apoptosis and cell cycle block of BC cells.Animal experiments show that circACTN4 can promote the occurrence and development of BC and metastasis in vivo,and has a positive correlation with the overall survival(OS)of nude mice.The results of mechanism show that USF2 can promote the biogenesis of circACTN4,circACTN4 can specifically bind FUBP1.As a transcription factor,FUBP1 can recruit to the promoter of oncogene MYC,and circACTN4 can promote the binding of FUBP1 and MYC promoter,thus activating the transcription and expression of MYC and promoting the occurrence and development of BC.Conclusion USF2-mediated circACTN4 can specifically bind to RNA binding protein FUBP1 to promote the transcription of oncogene MYC,thus promoting the progress of BC.Hence,circACTN4 can be used as a fresh biomarker and medication target of BC.