Effect And Mechanism Of Hsa＿circ＿0000851 On Proliferation Of Breast Cancer

Backgrounds and purposeBreast cancer is one of the most common malignant tumors in women all over the world.The incidence of breast cancer ranked first in the world.Breast cancer is the leading cause of cancer death in women.The social and family burden caused by breast cancer is gradually increasing.It is urgent to improve the level of diagnosis and treatment of breast cancer.Early diagnosis and treatment of breast cancer is the fundamental way to improve the prognosis of breast cancer.Abnormal gene expression caused by gene mutation and epigenetic changes is considered to play a crucial role in the occurrence of breast cancer.Further exploration of the molecular biological mechanism of breast cancer has important clinical significance.Circular RNAs(circRNAs)are members of noncoding regulatory RNAs.Unlike linear RNAs that are terminated with 5’caps and 3’tails,circRNAs are single-stranded covalently closed circular transcripts.Circ RNAs are considered to be closely related to the formation,progression and prognosis of various malignant tumors.To study the role of circRNAs in breast cancer is helpful to improve the molecular mechanism network of breast cancer formation and development,and to provide potential targets for the diagnosis and treatment of breast cancer.Based on the previous results of gene sequencing,bioinformatics analysis and pre-experiment,a new hsa＿circ＿0000851 was selected as the research object.This study first detected the expression of hsa＿circ＿0000851 in triple-negative breast cancer(TNBC),and analyzed the correlation between the expression of hsa＿circ＿0000851 and clinicopathological features.Then the closed-loop structure,expression stability and subcellular localization of hsa＿circ＿0000851 were analyzed.The biological function of hsa＿circ＿0000851 in breast cancer was studied in vivo and in vitro.In the aspect of mechanism research,this study explored the regulatory mechanism of hsa＿circ＿0000851 on breast cancer cells from the perspective of competitive endogenous RNA(ceRNA).Finally,the expression,function and possible mechanism of hsa＿circ＿0000851 in breast cancer were clarified,which provided a reference for exploring the pathogenesis network of breast cancer and a potential target and direction for targeted therapy of breast cancer.Research methods1.RT-qPCR was used to detect the expression of hsa＿circ＿0000851 in TNBC tissues and cell lines.The correlation between hsa＿circ＿0000851 expression and clinicopathological data of TNBC was analyzed by statistical method.RNase R and actinomycin D treatment experiments were used to identify the ring structure and stability of hsa＿circ＿0000851.The subcellular localization of hsa＿circ＿0000851 in breast cancer cells was determined quantitatively and qualitatively by using plasmon nuclear separation and fluorescence in situ hybridization.2.The expression levels of hsa＿circ＿0000851 in breast cancer cells MDA-MB-231 and BT-549 were down regulated by si RNA and up regulated by lentiviral plasmid transfection,respectively.The function of hsa＿circ＿0000851 was analyzed by MTT cell proliferation assay,cell plate clone formation assay and subcutaneous tumor formation assay in nude mice.The expression of PCNA was detected by Western blot.3.The miR-1183 interacting with hsa＿circ＿0000851 was predicted and screened by bioinformatics website.The direct binding relationship between hsa＿circ＿0000851and miR-1183 was verified by cell transfection,RT-qPCR and dual luciferase reporter gene detection.4.RT-qPCR was used to detect the expression of miR-1183 in TNBC tissues and cell lines.The function of miR-1183 was analyzed by MTT cell proliferation assay and cell plate clone formation assay.The functional recovery of miR-1183 inhibitor on si-hsa＿circ＿0000851 was explored by co-transfection.Finally,we searched and verified the downstream target genes of miR-1183,and further explored and improved the mechanism of hsa＿circ＿0000851-miR-1183-mRNA axis.Results1.Hsa＿circ＿0000851 was highly expressed in TNBC tissues and cell lines,and its expression level was positively correlated with tumor diameter,Ki-67 level and lymph node metastasis.Hsa＿circ＿0000851 showed a ring structure and stable expression.The subcellular localization of hsa＿circ＿0000851 in breast cancer cells MDA-MB-231 and BT-549 was mainly cytoplasmic.The experimental results provide a basis for the follow-up study of specific functions and deep mechanism.2.Functional studies showed that hsa＿circ＿0000851 could promote the proliferation and clone formation of breast cancer cells in vitro,and promote the expression of proliferation related protein PCNA.In vivo experiments showed that hsa＿circ＿0000851 could enhance the subcutaneous tumorigenicity of breast cancer cells.3.The bioinformatics website was used to predict and screen the miR-1183 interacting with hsa＿circ＿0000851,and further verified the direct binding relationship between hsa＿circ＿0000851 and miR-1183,which laid the foundation for further improving the mechanism of hsa＿circ＿0000851.4.miR-1183 is low expressed in TNBC,which can inhibit the proliferation of breast cancer cells and play an anti-cancer role.Hsa＿circ＿0000851 can play the role of ceRNA,act as the molecular sponge of miRNA,adsorb miR-1183,and indirectly regulate the target genes PDK1 and pAKT,forming the regulatory axis of hsa＿circ＿0000851-miR-1183-PDK1/pAKT,thus promoting the proliferation of breast cancer.Conclusion Hsa＿circ＿0000851 is highly expressed in TNBC and plays a role in promoting cancer,which is closely related to the clinicopathological characteristics of TNBC patients.The hsa＿circ＿0000851-miR-1183-PDK1/pAKT regulatory axis may be one of the mechanisms of hsa＿circ＿0000851 involved in the regulation of breast cancer.Results of this study provide a reference for exploring the pathogenesis network of breast cancer,and provide potential targets and directions for targeted therapy of breast cancer.Hsa＿circ＿0000851 is expected to be a potential biomarker and therapeutic target for breast cancer.