Study On The Mechanism Of RNA Hsa＿circ＿0068631 In The Progress Of Breast Cancer

Objective : The purpose of this study was to detect the expression of hsa＿circ＿0068631 which is generated from TFRC in BC and to evaluate the relationship between hsa＿circ＿0068631 expression and the clinical pathological variables of sixty patients.Also the aim of this study was to unveil the potential molecular mechanism of hsa＿circ＿006863 in the progress of BC,which might provide a scientific basis for hsa＿circ＿0068631 as a novel molecular target for clinical diagnosis and treatment of BC.Methods:(1)Hsa＿circ＿0068631 was identified by Sanger sequencing,RNase enzyme digestion and Actinomycin D assay.(2)Reverse Transcription polymerase chain reaction(RT-q PCR)was used to detect the relative expression of hsa＿circ＿0068631 in sixty BC tissues.The relationship between hsa＿circ＿0068631 expression and the clinical pathological variables of sixty patients was analyzed.(3)RT-q PCR was used to detect the relative expression of hsa＿circ＿0068631 in six cell lines(MDA-MB-231、HCC-1937、 BT 549、SKBR3、MCF-7、MCF-10A).FISH analysis and nuclear-cytoplasmic fractionation assay were performed to explore the cellular distribution of hsa＿circ＿0068631.(4)MDA-MB-231 and MCF-7 were chosen for subsequent experiments.The proliferation of BC cells influenced by depletion or overexpression of hsa＿circ＿0068631 was detected by the MTT assay and colony formation assay.The migration of BC cells influenced by depletion or overexpression of hsa＿circ＿0068631 was detected by transwell migration assay and wound healing assays.Cell cycle influenced by depletion of hsa＿circ＿0068631 was further tested by flow cytometry analysis.(5)Xenograft tumor assay was carried out in mice to observe the tumorigenesis of MDA-MB-231 cells stably infected by LV-circ＿0068631 or LV-vector.(6)Bioinformatic methods were employed to analyze the possible binding RBP of hsa＿circ＿0068631 and downstream target of RBP.The binding of hsa＿circ＿0068631 and RBP,RBP and downstream target was detected by RIP and RNA pull down.(7)Western Blot was used to detect the expression of RBP in BC cell lines.RT-q PCR was used to detect the relative expression of downstream m RNA in sixty BC tissues.(8)Western Blot and RT-q PCR were used to detect the effect of depletion or overexpression of hsa＿circ＿0068631 on protein and m RNA levels of RBP and downstream target.(9)RT-q PCR was used to detect the influence on the biding of RBP and target gene by depletion of hsa＿circ＿0068631.(10)Actinomycin D assay was used to detect the influence on the stability of downstream m RNA by depletion of hsa＿circ＿0068631 or RBP.Results:(1)The back-splicing junction site of exon 3 toward exon 2 of hsa＿circ＿0068631 was verified by Sanger sequencing.RNase enzyme digestion and Actinomycin D assay showed that hsa＿circ＿0068631 was indeed circular.(2)The expression of hsa＿circ＿0068631 in BC tissues(n=60)was significantly higher than that in adjacent normal tissues.High expression of hsa＿circ＿0068631 was positively associated with TNM stage,tumor size,lymph node metastasis and distant metastasis,but had no correlation with age.(3)5 BC cell lines(MDA-MB-231,MCF-7,HCC-1937,BT549 and SKBR3)presented higher hsa＿circ＿0068631 expression compared with MCF-10 A.FISH analysis and nuclear-cytoplasmic fractionation assay showed that hsa＿circ＿0068631 was enriched in the cytoplasm.(4)MTT assays and colony formation assay results showed that hsa＿circ＿0068631-overexpression significantly promoted BC cells proliferation.BC cell migration was enhanced by upregulation of hsa＿circ＿0068631.The opposite results were obtained when the expression of hsa＿circ＿0068631 was downregulated.Flow cytometry showed that the downregulation of hsa＿circ＿0068631 caused cell cycle arrest.(5)The results of animal experiment showed that the volume and weight of breast cancer in the LV-circ＿0068631 group were increased.(6)According to the prediction of Circ Interactome,hsa＿circ＿0068631 had a binding site with EIF4A3.The binding of hsa＿circ＿0068631 and EIF4A3 was confirmed by RIP and RNA pull down.According to the systematic analysis of co-expression network of EIF4A3 in cancers,biological pathway showed c-Myc pathway was enriched among EIF4A3 co-expressed partners.The binding of c-Myc and EIF4A3 and was confirmed by RIP and RNA pull down.(7)EIF4A3 was highly expressed in BC cell lines.The expression of c-Myc m RNA in BC tissues was significantly higher than that in adjacent normal tissues.(8)No significant changes were found in both m RNA and protein levels of EIF4A3 with hsa＿circ＿0068631 depletion or overexpression.The protein level of c-Myc was positively regulated by hsa＿circ＿0068631 but the m RNA level of c-Myc had no changes.Given that cyclin E and CDK4 were confirmed target genes of c-Myc,we found that both cyclin E and CDK4 protein levels were positively regulated by hsa＿circ＿0068631.(9)The biding of EIF4A3 and c-Myc decresed with hsa＿circ＿0068631 depletion.(10)In actinomycin D assay c-Myc m RNA stability was reduced due to hsa＿circ＿0068631 depletion or EIF4A3 depletion.EIF4A3 overexpression rescued c-Myc m RNA stability decrease induced by hsa＿circ＿0068631 depletion in BC cells.Conclusion : In conclusion,our results demonstrate that the expression of hsa＿circ＿0068631 in BC tissues and cell lines is up-regulated.The high expression of hsa＿circ＿0068631 is associated with the clinical pathological variables.We prove that hsa＿circ＿0068631 might be a new oncogene.The upregulation of hsa＿circ＿0068631could facilitate the progression of BC by binding to EIF4A3 to maintain c-Myc m RNA stability.Suppressing hsa＿circ＿0068631/EIF4A3/c-Myc axis represents a potential strategy for BC.