The Role And Mechanism Of Circular RNA Circ-FOXO3a In Triple-Negative Breast Cancer

Background and Objective:Breast cancer(BC)is one of the most common malignancy in cancer.Breast cancer is classified as Luminal,Her2+,and triple-negative breast cancer(TNBC).TNBC is the most aggressive breast cancer subtype that is negative for both ER/PR and Her2.The incidence rate accounts for 10%-20%of breast cancer.Because of highly invasive and lacking of therapeutic targets and treatments,compared with other breast cancer molecular subtypes,the prognosis of TNBC is poorer.It is essiential to explore the molecular mechanism of invasion and metastasis in TNBC,and find new biological markers and therapeutic targets for TNBC treatment.In recent years,the key position of non-coding RNA has received increasing attention in the occurrence and development of tumors.Circular RNA is a new type of endogenous non-coding RNA which are circularized by joining the 3’end of the RNA and the 5’ end.and then formming a single-stranded closed RNA molecule without 5’caps and 3’tails.Circular RNA is abundant in eukaryotes,through:1)as miRNA Sponges/pre-RNA;2)protein Sponges;3)translated proteins and peptides;4)regulation of parental genes;5)regulation of transcription and other ways to participate in physiological and pathological processes of tumors and diseases.Studies have shown that the abnormal expression of CircRNA is closely related to the occurrence and development of TNBC and can be used as potential biomarkers or therapeutic targets for TNBC.However,CircRNA research is still in the exploratory stage.Its mechanism and pathological significance in the carcinogenesis and development are far from clear which need further in-depth research urgently.In this study,we analyzed the differential expression of circRNAs in 3 samples of TNBC tissues and paracancerous tissues by high-throughput sequencing,screened circRNAs(circ-FOXO3a,hsa-circ-0006404)with significant differences and explored its mechanism and pathological significance in the development of TNBC,providing new targets and ideas for the diagnosis and treatment of TNBC.Methods:1.We investigated the significant differences expression of circRNAs in 3 samples triple-negative breast cancer(TNBC)and their paracancerous nomal tissues by RNA-sequencing.RT-qPCR was used to verify the expression of circ-FOXO3a in different breast cancer tissues and cells.2.The subcellular localization of circFoxo3a in triple negative breast cancer tissues and cells was detected by Fluorescent in situ hybridization(FISH).3.To construct circ-FOXO3a overexpressing vector,the full-length of circ-FOXO3a was inserted into the pLCDH-ciR lentiviral vector,and infect TNBC cells such as BT549,MDA-MB-231,HCC1937,the stable overexpression of circ-FOXO3a cells were constructed by puromycin.4.In vitro,using flow cytometry,MTS,BrdU,plat colone assay,transwell and scratch experiment to detect the effects of circ-FOXO3a on the cell cycle,proliferation,invasion and metastasis of triple negative breast cancer cells.5.In vivo,establishing nude mice subcutaneous transplantation tumor model and nude mouse metastasis models to investigate the effect of overexpression of circ-FOXO3a on TNBC tumor growth and invasion and metastasis.6.RNA pull down-mass spectrometry and ChIP experiment were used to analyze circ-FOXO3 a bindig proteins.7.Western blotting and real-time fluorescence quantitative PCR(qRT-PCR)were conducted to detect the regulatory relationship between circ-FOXO3a and downstream genes.8.The effects of WHSC1(Wolf-Hirschhorn syndrome candidate 1)on the invasion and metastasis of triple negative breast cancer were detected by western blot,transwell,plat colone assay and scratch experiments after overexpression or interference with WHSC1.9.Through functional recovery experiments to analyze the effects of WHSC1 on the regulation of circ-FOXO3a in breast cancer invasion and metastasis.10.We collected 50 samples breast cancer tissues and related clinicopathological data.Using qRT-PCR and fluorescence in situ hybridization(FISH)to detect the expression of circ-FOXO3a in breast cancer tissues.To detect the expression of WHSC1 in breast cancer tissue by Immunohistochemistry(IHC).Combined with the patient’s clinical data to analyze the pathological significance of circ-FOXO3a and WHSC1 expression.The expression and prognosis of WHSC1 in breast cancer were investigated by TCGA website and survival analysis.Results:1.We identified the differential expression profile of CircRNA in 3 samples of TNBC tissues and paired adjacent tissues by CircRNA-seq analysis,a total of 105 circRNAs(46 up-regulated circRNAs and 59 down-regulated circRNAs)with differential expression at least two folds and statistical significance.After detecting the expression of circ-FOXO3a in 18 TNBC tissues and paired adjacent tissues by qRT-PCR,we found circ-FOXO3a was significantly lower expressed in TNBC tissues than in adjacent tissues.At the same time,we found the expression of circ-FOXO3a in TNBC tissues was significantly lower than in non-TNBC tissues.Using qRT-PCR to detect the expression of circ-FOXO3a in breast cancer cell lines,we found the expression of circ-FOXO3a in breast cancer cells was significantly lower than that of normal breast epithelial cells MCF-10A,and the expression in TNBC cell lines was significantly lower than that of non-TNBC cell lines.2.Circ-FOXO3a is mainly distributed in the cytoplasm of triple negative breast cancer tissues and cells.3.The stable overexpression of circ-FOXO3a was constructed.Up-regulation of circ-FOXO3a significantly suppressed TNBC cell proliferation and induced the G2 arrest in vitro,and inhibited epithelial-mesenchymal transition and the migration and invasion.4.The results of nude mice subcutaneous transplantation tumor model showed that overexpression of circ-FOXO3a inhibited the growth of TNBC.The results of nude mouse metastasis models showed overexpression of circ-FOXO3a significantly inhibited the migration of TNBC.5.RNA pull down-mass spectrometry analysis found that circ-FOXO3a can bind to WHSC1 protein.Further analysis revealed that overexpression of circ-FOXO3a suppressed WHSC1 expression,inhibited WHSC1 entry into the nucleus and down-regulate H3K36me2 expression.6.WHSC1 is highly expressed in TNBC cells,and interference with WHSC1 can inhibit the invasion and metastasis of TNBC cells and inhibit epithelial-mesenchymal transition.Overexpression of WHSC1 can reverse the inhibitory effect of circ-FOXO3a on the proliferation and invasion and metastasis of TNBC cells.7.Fluorescence in situ hybridization(FISH)was used to detect the expression of circ-FOXO3a in 50 samples of breast cancer tissues.The expression of circ-FOXO3a was significantly down-regulated and negatively correlated with lymph node metastasis(p=0.0116).8.In 50 samples of breast cancer tissues,the immunohistochemistry was used to detect the breast cancer tiusses.The results showed that WHSC1 was highly expressed in TNBC and positively correlated with lymph node metastasis(p=0.0055).TCGA database analysis showed WHSC1 is highly expressed in breast cancer tissues and negatively correlated with patient prognosis.Conclusion:1.Circ-FOXO3a is down-expressed in TNBC tissues and cells.Overexpression of Circ-FOXO3a inhibits TNBC cell proliferation,invasion and metastasis.2.Circ-FOXO3a inhibits the invasion and metastasis of triple negative breast cancer by inhibiting the expression and function of WHSC1.3.Circ-FOXO3a and WHSC1 can be used as potential molecular markers and therapeutic targets for triple negative breast cancer.