Function And Mechanism Of Hsa＿circ＿0008591 And Hsa＿circ＿0067842 In The Progression Of Breast Cancer

Objective:Breast cancer(BC),as the most common malignant tumor threatening the health of women in the world,ranks the first in both morbidity and mortality of female tumors,with a trend toward younger incidence.Despite complex pathogenesis and multi-factor interaction BC has,exploring the malignant mechanism and finding effective molecular targets remains critical focus and difficulty of current research.Circular RNAs(circRNAs),a special class of non-coding RNAs with wide organismal distribution,play important roles in the occurrence and development of a variety of cancers.Studies have shown that circRNAs with abnormal expression can be widely involved in the development,metastasis and chemotherapy resistance of cancer,but the potential molecular mechanism of circRNAs in BC has not been fully elucidated and needs further investigation.This study aims to explore the molecular biological function and molecular mechanism of novel circRNAs in BC,which is expected to provide a potential molecular target for the diagnosis and treatment of BC.Methods:1.High-throughput microarray analysis was performed on 6 paired BC and paracancerous tissues,and circRNAs differentially expressed in BC tissues were screened.Candidate circRNAs were identified through functional prediction and verification.Real-time quantitative PCR(RT-qPCR)was used to detect the expression abundance of candidate molecules in BC tissues and BC cell lines.2.The expression levels of hsa＿circ＿0008591 and hsa＿circ＿0067842 were up-regulated or down-regulated by transfecting overexpressing plasmid or small interfering RNA(siRNA)into BC cells.Then gain-and loss-of-function assays were conducted to investigate the biological functions of hsa＿circ＿0008591 and hsa＿circ＿0067842 in BC.In addition,peripheral blood mononuclear cells(PBMCs)were isolated and co-cultured with BC cells in different treatment groups to explore the regulatory role of hsa＿circ＿0067842 in the immune escape process of BC.3.The downstream targets of hsa＿circ＿0008591 was predicted using miRanda,etc.Meanwhile,Cytoscape software was used to construct and visualize the circRNA-miRNA-mRNA ceRNA regulatory network.Besides,the mechanism of hsa＿circ＿0067842 was further explored through RNA pull down,silver staining,mass spectrometry,western blot and rescue experiments.Results:1.Two novel circRNAs(hsa＿circ＿0008591 and hsa＿circ＿0067842)were identified via high-throughput circRNA microarray and validated in BC tissues and cells.hsa＿circ＿0008591 exhibited low expression,while hsa＿circ＿0067842 displayed high expression in BC tissues and cells,with the latter being associated with poor prognosis in BC patients.2.In vitro functional experiments showed that,on the one hand,overexpression of hsa＿circ＿0008591 could inhibit the proliferation of BC cells,while knockdown hsa＿circ＿0008591 could promote the proliferation of BC cells.On the other hand,overexpression of hsa＿circ＿0067842 significantly enhanced the migration and invasion ability of BC cells.Otherwise,the opposite result was obtained.Additionally,the proliferation experiments showed that hsa＿circ＿0067842 had no significant regulatory effects on the proliferation of BC cells.After co-culture of PBMCs with BC cells in different treatment groups,the results showed overexpression of hsa＿circ＿0067842 could inhibit the proliferation of PBMCs,reduce the proportion of CD8+T cells and inhibit the cytotoxicity of PBMCs,while knockdown of hsa＿circ＿0067842 had the opposite effects,finally regulating the immune escape process of BC cells.3.In terms of mechanism,hsa＿circ＿0008591 may play roles in anticancer by affecting a variety of cancer-related pathways,such as Wnt,TNF,TGF-β signaling pathways.Hsa＿circ＿0067842 can bind to HuR protein and promote its transport from nucleus to cytoplasm.The cytoplasmic HuR protein can increase the stability of target gene CMTM6,which acts as a regulator of PD-L1 to inhibit PD-L1 ubiquitination and thus regulate the metastasis and immune escape of BC.Conclusion:1.hsa＿circ＿0008591 is low expressed in BC,while hsa＿circ＿0067842 is high expressed,and can be used as an independent prognostic factor in BC patients.2.hsa＿circ＿0008591 inhibits the proliferation of BC cells and may be involved in regulating tumor-related signaling pathways;And hsa＿circ＿0067842 binds to HuR protein and promotes its nuclear translocation,thus promoting BC metastasis and immune escape via the HuR/CMTM6/PD-L1 axis.