| Background and objective:Breast cancer（BC）is one of the most common malignancies and the leading causes of death in women worldwide.According to statistics,about1.2 million people are diagnosed with BC every year,and 500000 people die of BC.The pathogenesis of BC is complex and there are many pathological types,among which triple-negative breast cancer（TNBC）has the highest degree of malignancy（estrogen receptor,progesterone receptor and proto-oncogene HER2 are all negative）,accounting for15%-20% of all BCs.Since the pathogenesis of BC is not clear,it is very difficult to make a breakthrough in BC diagnosis and treatment.Therefore,it is necessary to explore the dysregulation of key genes as well as their potential mechanism in BC occurrence and development,which will provide new clinical insights and therapeutic targets.Circular RNA（circRNA）is a novel type of endogenous single-stranded RNA,which has a closed ring structure,high resistance to exonuclease,stable expression,and is not easy to degrade.Studies have shown that there are a large number of dysregulated circRNA in tumors,which are closely related to tumor proliferation,migration,invasion,drug resistance as well as other malignant biological behaviors.The mechanism of circRNA functioning in tumorigenesis and development has become a novel research focus in the field of tumor biology.However,its expression characteristics and biological implications in BC are still largely unknown.To determine the deregulated circRNAs in BC,we conducted circRNA high-throughput sequencing in 3 paired TNBC and adjacent normal tissues,and found a total of 69815 circRNA,among which 5033 differentially expressed circRNA（fold change ≥ 2,p<0.05）.Through experimental verification,we selected hsacirc0007294（circANKS1B）with the greatest variation in differential expression for subsequent studies,and explored its role on BC biological behavior and specific underlying mechanism,so as to provide a reliable theoretical basis for it to become a new BC biomarker and therapeutic target.This study is mainly divided into the following six parts:Part Ⅰ: Identification and clinical significance of circANKS1BMethods:1.CircRNA high-throughput sequencing was performed in 3 pairs of TNBC and normal adjacent frozen tissues,and circRNA expression was analyzed by Top Hat2,SRPBM,Audics and R language software.2.qRT-PCR analysis in 20 pairs of TNBC and normal adjacent frozen tissues was performed to verify the results of circRNA high-throughput sequencing.3.RT-PCR experiments were carried out with divergent and convergent primers,the amplified product was collected for electrophoresis,followed by Sanger sequencing to confirm the existence of circ ANKS1 B.4.BC cell lines were treated with Actinomycin D and RNase R,respectively,to detect the half-life and stability of circ ANKS1 B.FISH experiments were performed to determine the subcellular localization of circ ANKS1 B.5.The expression levels of circ ANKS1 B were detected in 40 normal and 165 BC paraffin-embedded tissues by qRT-PCR analysis,and the clinicopathological information of BC patients was collected and patients were followed up.The relationships between circ ANKS1 B and BC pathological characteristics as well as outcome were analyzed.Results:1.A large number of circRNAs（69815）were found in breast tissue,87%of which are originated from the back splicing of exons,including 5033 differentially expressed circRNAs（3726 down-regulated and 1307up-regulated）.2.Compared with normal tissues,circ ANKS1 B had the largest expression difference in TNBC tissues,up by 5.8 times.3.Circ ANKS1 B was produced by the back-splicing of exons 5 to 8 of ANKS1 B,with a mature length of 459 bp.4.Circ ANKS1 B had a half-life of more than 24 hours and was highly resistant to RNase R enzyme,which was mainly located in the cytoplasm.5.Compared with other subtypes of BC,circ ANKS1 B was only overexpressed in TNBC,and was closely related to lymph node metastasis and advanced TNM stage of BC patients,which was an independent risk prognostic factor for overall survival of BC patients.Conclusion: CircANKS1 B with cytoplasmic localization was significantly up-regulated in TNBC and predicted poor prognosis.Part Ⅱ: The effect of circ ANKS1 B on biological behavior of BC cellsMethods:1.Circ ANKS1 B expression levels in normal mammary gland epithelium MCF-10 A cells and BC cell lines were tested by qRT-PCR analysis.The stable circANKS1B-overexpressed or-depleted BC cell lines were generated by using lentivirus vector.MCF-7 and T47 D cells with low circ ANKS1 B expression were selected to infect circ ANKS1 B overexpression lentivirus vector,MDA-MB-231 and BT549 cells with high circ ANKS1 B expression were selected to infect circ ANKS1 B knockdown vector.2.CCK-8/EdU and flow cytometry were carried out to determine the effects of circ ANKS1 B on BC cell proliferation and apoptosis;The effects of circ ANKS1 B BC cell migration and invasion were assessed by wound healing and transwell assays.3.BC cells with stable overexpression/knockdown of circ ANKS1 B were injected into the subcutaneous and tail veins of nude mice,respectively,to establish xenograft tumor model and lung metastasis model for assessing the effects of circ ANKS1 B on the growth and metastasis of BC cells in vivo.4.Western blot and immunofluorescence were used to detect the expression levels of E-cadherin,Vimentin and Fibronectin in the BC cell lines with stable circ ANKS1 B overexpression/knockdown.Results:1.Compared with non-TNBC cell lines,circ ANKS1 B was significantly up-regulated in TNBC cell lines.qRT-PCR was used to confirm the successful construction of stable circ ANKS1 B overexpression/knockdown cell lines.2.Circ ANKS1 B promoted in vitro BC cell migration and invasion,but did not affect cell proliferation and apoptosis.3.CircANKS1 B promoted lung metastasis of BC cells in vivo,but had no effect on tumor growth.4.CircANKS1 B elevated Vimentin and Fibronectin,while reduced E-cadherin expression.Conclusion: Circ ANKS1 B promoted BC cell invasion and metastasis and induced EMT.Part Ⅲ: Circ ANKS1 B acts as a molecular sponge for miR-148a/152-3p to regulate USF1Methods:1.Prediction of miRNAs interacting with circ ANKS1 B by using circ Net database.RIP,RNA pull-down and luciferase reporter gene experiments were performed to validate the database prediction results.2.FISH assay was performed to examine whether there was co-localization between circ ANKS1 B and miR-148a-3p/miR-152-3p.3.Transwell assay was conducted to assess the invasiveness of BC cells after overexpression of miR-148a/152-3p or after simultaneous overexpression of circ ANKS1 B.4.The miRWalk database was used to predict the downstream target genes of miR-148a-3p and miR-152-3p,and qRT-PCR and luciferase reporter gene experiments were applied to confirm the results of miRWalk database.5.Western blot analysis was conducted to detect USF1 protein expression level in BC cells after overexpression of miR-148a/152-3p or after simultaneous overexpression of circ ANKS1 B.6.The expression level of USF1 in 40 normal cases and 165 BC paraffin cases was tested by immunohistochemistry,and the relationships between USF1 and circ ANKS1 B expression,BC clinicopathological data or outcome were assessed.Results:1.Circ ANKS1 B directly bound to miR-148a-3p and miR-152-3p in BC cells.2.There was colocalization between Circ ANKS1 B and miR-148a/152-3p.3.Exogenous miR-148a-3p or miR-152-3p expression attenuated BC cell invasion and down-regulated USF1 expression,but this effect was blocked by circ ANKS1 B overexpression.4.USF1 was notably increased in BC tissues and was strongly linked to lymph node metastasis and poor prognosis.5.USF1 was strongly positively correlated with circ ANKS1 B in BC tissues.Conclusion: Circ ANKS1 B up-regulated the expression of USF1 and promoted BC invasion and metastasis by sponging miR-148a-3p or miR-152-3p.Part Ⅳ: USF1 transcriptionally upregulates TGF-β1Methods:1.TGF-β1 m RNA in BC cells with USF1 overexpression/knockdown was tested by qRT-PCR,and TGF-β1 protein was assessed by Western blot.2.The correlation between USF1 and TGF-β 1 expression in TCGA-BC database was analyzed.3.Whether USF1 bound on the TGF-β1 promoter was examined by Ch IP and luciferase reporter assays.4.Transwell assay was applied to analysis the change of invasion ability of BC cells after overexpression/knockdown of USF1 or after simultaneously knockdown/overexpression of TGF-β1.Results:1.Overexpression of USF1 up-regulated TGF-β 1 expression,while knockdown of USF1 down-regulated TGF-β1 expression.2.USF1 was strongly positively correlated with TGF-β 1 in TCGA-BC database.3.USF1 directly bound to the E-box sequence（CACGTG）located on-962 to-956 of TGF-β1 promoter region to enhance the activity of TGF-β1promoter.4.Overexpression/knockdown of USF1 significantly enhanced/weakened the invasion ability of BC cells,but this effect was blocked by TGF-β1knockdown/overexpression.Conclusion: In BC,TGF-β1 is a direct downstream target gene of USF1.Part Ⅴ: ESRP1 promotes the production of circ ANKS1 B and is transcriptionally regulated by USF1Methods:1.siRNA system was used to screen RNA binding proteins that affect circ ANK1 B production.ESRP1-overexpressing vector was constructed and transfected into BC cell lines,and the expression level of circ ANKS1 B was tested by qRT-PCR analysis.2.The minigene vector with mutated ESRP1 binding site in intron regions on both sides of circ ANKS1 B was constructed and transfected into BC cell lines.The binding of ESRP1 was assessed by RIP assay,and the expression level of circ ANKS1 B after simultaneous transfection of ESRP1 siRNA was determined by qRT-PCR experiment.3.ESRP1 protein expression in 165 BC paraffin tissues was determined by immunohistochemistry,and its correlation with circ ANKS1 B was counted.4.The SYT8 and Snail minigene vector with ESRP1 binding site were constructed and transfected into BC cell lines,followed by qRT-PCR analysis,the amplified product was collected for electrophoresis,followed by Sanger sequencing to test whether ESRP1 could promote the formation of other pre-m RNAs.5.ChIP assay was applied to test whether USF1 could directly interactq with ESRP1 promoter region,and ESRP1 protein levels after USF1overexpression/knockdown were assessed by Western blot assay.6.The correlation between USF1 and ESRP1 expression in TCGA-BC database was analyzed.Results:1.Knockdown/overexpression of ESRP1 significantly reduced/increased circ ANKS1 B expression.2.ESRP1 directly bound to intron regions on both sides of circANKS1 B.When the ESRP1 binding site was mutated,knockdown of ESRP1 had no effect on the expression of circ ANKS1 B.3.The expression of ESRP1 and circ ANKS1 B was strongly positively correlated in BC tissues.4.ESRP1 promoted the back splicing of SYT8 exon 4 and Snail exon 2,thereby forming a circRNA.5.USF1 directly bound to the “CACGTG ”located on-555 to-550 of ESRP1 promoter,and USF1 overexpression/knockdown significantly increased/decreased the expression level of ESRP1.6.There was a significant positive correlation between USF1 and ESRP1 expression in TCGA-BC database.Conclusion: In BC,ESRP1 is also a direct downstream target gene of USF1,which could promote the production of circ ANKS1 B.Part Ⅵ: The ESRP1/circANKS1B/ miR-148 a /152-3p/USF1 positive feedback loop promotes EMT,invasion and metastasis of BC cells by activating TGF-β1/Smad signaling pathwayMethods:1.The migration and invasion abilities of stable circANKS1B-overexpressed BC cells were detected by scratching and transwell assays after transfection with si-ESRP1,miR-148a/152-3p mimics,si-USF1 or treatment with LY2109761,respectively.And The migration and invasion abilities of stable circANKS1B-silenced BC cells were detected by scratching and Transwell assays after transfection with ESRP1/USF1/TGF-β 1 expression vector or miR-148a/152-3p inhibitors,respectively.2.Western blot assay was carried out to examine p-Smad2,p-Smad3,Smad2/3,E-cadherin and Vimentin protein expression levels after transfection of stable circANKS1B-overexpressed BC cells with si-ESRP1,miR-148a/152-3p mimics,si-USF1 or treatment with LY2109761.Results:1.Knockdown of ESRP1/USF1,overexpression of miR-148a/152-3p or treatment with LY2109761 could block the enhanced migration and invasion abilities caused by circ ANKS1 B overexpression.Similarly,overexpression of ESRP1/USF1/TGF-β1 or silencing of miR-148a/152-3p could rescue the weakened invasion and migration abilities caused by circ ANKS1 B knockdown.2.Overexpression of circ ANKS1 B increased the expression of p-Smad2,p-Smad3 and Vimentin,while reduced E-cadherin expression.Knockdown of ESRP1/USF1,overexpression of miR-148a/152-3p or treatment with LY2109761 blocked above effects。Conclusion: The ESRP1/circANKS1B/miR-148a/152-3p/USF1 positive feedback loop could activate the TGF-β1/Smad signaling pathway,thereby promoting EMT,invasion and metastasis of BC cells. |
