Studies On The Roles And Underlying Mechanisms Of CircHIF1A In Regulating TNBC Progression And Metastasis

BackgroundBreast cancer is the most common malignant tumor among female around the world,and its incidence is still on the rise,seriously threatening the lives and health of female patients.Triple-negative breast cancer（TNBC）is a class of aggressive breast cancer subtype.TNBC patients are prone to early recurrence and metastasis,and have a poor prognosis.Nowadays,chemotherapy still dominates in the systemic therapy of TNBC.However,most patients face the problem of drug resistance during treatment,which severely limits the efficacy of chemotherapy,leading to recurrence,metastasis and death in the patients.Therefore,it is urgent and significant to deeply explore the molecular mechanism of TNBC progression and look for novel targets of TNBC treatment.In recent years,the roles and potential mechanisms of circular RNA（circular RNA,circRNA）in the development of human cancer have attracted much attention of researchers.Unlike linear RNAs,circRNAs form a covalently closed continuous loop without a 5’ cap and 3’polyadenylated tail.Initially,circRNA was considered as a junk product resulting from intracellular alternative splicing,but with the development of RNA high-throughput sequencing technology in recent years,an increasing number of circRNAs with biological functions have been discovered.Aberrant expression of circRNAs and the roles of circRNAs in modulating the biological behaviors of tumors have been reported in a variety of human cancer.In the present study,we identified that hsa_circ₀004623 was significantly upregulated in breast cancer tissues,especially in the TNBC subtype.Besides,high expression level of hsa_circ₀004623 was associated with poor prognosis of breast cancer patients.Hsa_circ₀004623 is generated by the back-splicing of its host gene HIF1A and composed of five exons（exons 2-6）.We renamed hsa_circ₀004623 as circHIF1A hereafter.Through literature search,we found that the biological functions of circHIF1A had not been reported before.In the present study,we aimed to explore the biological functions and potential mechanisms of circHIF1A in TNBC progression and metastasis.Methods and resultsOur present study comprises three sections,and the research methods and main findings of each section are as follows:Part 1 The expression and clinical values of circHIF1A in breast cancerMethods1.RT-qPCR and ISH assays were conducted to detect the expression of circHIF1A in breast cancer tissues and surrounding normal mammary tissues.Besides,the expression levels of circHIF1A in TNBC and non-triple negative breast cancer（non-TNBC）tissues were compared.2.Student’s t test,log-rank test and Cox proportional hazards regression model were used to analyze the relationship between circHIF1A expression and clinicopathological features,such as lymph node metastasis and hormone receptor status,as well as the impact of circHIF1A levels on the prognosis of breast cancer patients.3.The circular structure of circHIF1A was verified by Sanger sequencing,RNase R treatment and actinomycin D treatment in TNBC cells.Results1.Higher expression level of circHIF1A was detected in breast cancer tissues compared to adjacent normal breast tissues,and circHIF1A expression in TNBC tissues was significantly higher than that in non-TNBC tissues.2.The expression level of circHIF1A was positively correlated with lymph node metastasis,and negatively correlated with the expression of ER,PR and HER-2.Higher level of circHIF1A was an independent predictor of poor prognosis in breast cancer patients.3.CircHIF1A was indeed a circular RNA with high stability that existed in TNBC cells.Part 2 circHIF1A significantly promoted the proliferation and metastasis of TNBCMethods1.RNA high throughput sequencing analysis（RNA-seq）was used to screen differentially expressed genes（DEGs）in circHIF1A overexpressing TNBC cells and control cells.Then Gene Ontology（GO）analysis was performed to preliminarily determine the biological processes that circHIF1A might regulate.2.The effects of circHIF1A overexpression or knockdown on the proliferation of TNBC were detected by MTT,clone formation and EdU assays.In vitro experiments such as Transwell and wound healing assays were used to verify the impacts of circHIF1A on the migration ability of TNBC.The effects of circHIF1A on apoptotic levels of TNBC cells were detected by flow cytometry.3.Subcutaneous breast cancer xenograft model and breast cancer lung metastasis mouse model were constructed respectively to study the effects of circHIF1A on the growth and metastasis of TNBC in vivo.Results1.Go analysis showed that circHIF1A might regulate the cell growth,cell adhesion,immune response and other biological processes in TNBC cells.2.In vitro experiments showed that circHIF1A overexpression could significantly promote the proliferation,DNA synthesis and migration of TNBC cells,and significantly inhibit the level of apoptosis;Silencing circHIF1A by siRNAs inhibited the proliferation,metastasis and DNA synthesis,and enhanced apoptosis in TNBC cells.3.In vivo experiments showed that TNBC cells overexpressing circHIF1A had stronger tumorigenicity and lung metastasis abilities,while knockdown of circHIF1A had the opposite effect.Part 3 Studies on the molecular mechanisms of circHIF1A functions and biogenesisMethods1.The subcellular localization of circHIF1A in TNBC cells was determined by RNA nucleocytoplasmic separation experiment and fluorescence in situ hybridization（FISH）assay.2.Bioinformatics tools were utilized to predict the downstream targeted genes of circHIF1A,and the interaction between circHIF1A and candidate targets were verified by RNA pull-down,RNA immunoprecipitation（RIP）and dual luciferase report assays.3.The effects of circHIF1A on the expression and subcellular localization of targeted genes were detected by RT-qPCR,Western blotting,immunofluorescence and protein nucleocytoplasmic separation assays.4.Rescue experiments based on MTT,EdU and Transwell assays were carried out to explore the mediating role of the targeted genes in the functions of circHIF1A.5.Bioinformatics analysis,RNA pull-down,RIP and chromatin immunoprecipitation（ChIP）assays were performed to explore the molecular mechanisms of circHIF1A cyclization and dysregulation.Results1.CircHIF1A was mainly located in the cytoplasm.According to previous reports on the mechanisms of circRNAs,we speculated that circHIF1A might function by interacting with microRNAs（miRNAs）or cytoplasmic proteins.2.CircHIF1A could interact with AGO2,suggesting that circHIF1A might act as a "sponge"for miRNAs.Further investigations revealed that the tumor suppressor miR-149-5p was a downstream target of circHIF1A.The binding between miR-149-5p and circHIF1A was validated.3.miRwalk,Pictar,TargetScan and RNA22 databases were used to predict the targeted mRNAs of miR-149-5p and the oncogene NFIB was selected.The interaction between miR-149-5p and 3’ UTR region of NFIB was verified by dual luciferase report assay.4.miR-149-5p could downregulate the expression of NFIB at mRNA and protein levels,while circHIF1A increased the levels of NFIB.In addition,circHIF1A could promote the nuclear transport of NFIB protein by binding to NFIB proteins.5.The regulation of circHIF1A on miR-149-5p and NFIB mediated its oncogenic roles in TNBC.6.The RNA binding protein FUS could interact with the intron 1 of circHIF1A precursor RNA（pre-HIF1A）and promote the biogenesis of circHIF1A.7.FUS was highly expressed in TNBC and was significantly correlated with poor prognosis of TNBC patients.FUS overexpression significantly elevated the proliferation and migration abilities of TNBC cells,while silencing FUS significantly inhibited the progression of TNBC.8.NFIB interacted with the promoter region of FUS gene and promote its transcriptional activation,leading to the upregulation of circHIF1A.Therefore,a positive feedback regulatory pathway that drived TNBC progression and metastasis was formed by FUS/circHIF1A/NFIB axis.Conclusions1.CircHIF1A is highly expressed in TNBC,and its dysregulation plays an important role in the progression and metastasis of TNBC.2.CircHIF1A can not only promote the expression of the oncogene NFIB through competitively binding with miR-149-5p,but also bind to NFIB protein to promote its nuclear translocation.3.The biogenesis of circHIF1A is positively regulated by the RNA binding protein FUS,and NFIB can promote the transcriptional activation of FUS,thus forming a positive feedback loop to promote the progression of TNBC.FUS/circHIF1A/NFIB axis is expected to be a novel target for TNBC treatment.4.CircHIF1A is remarkably upregulated in the tumor tissues of breast cancer patients,and is significantly associated with poor prognosis.CircHIF1A is expected to be a novel biomarker for diagnosis and prognosis prediction of breast cancer.