Circular RNA CircWAC Promotes Epithelial-mesenchymal Transition In Triple-negative Breast Cancer By Regulating MiR-1183/SMAD2 Axis

Background: Breast cancer is the malignant tumor with the highest incidence among all female malignant tumors.As a kind of obvious heterogeneity cancer,breast cancer contains four subtypes: luminal A,luminal B,HER2 overexpression and triple-negative.The gene expression profile,metastatic potential,progression rate,treatment method and clinical outcome of breast cancer are different in each subtype.There are several reasons for poor prognosis of triple-negative breast cancer(TNBC),such as lack of effective treatment,rapid tumor progression,easy recurrence,distant organ metastasis,and chemotherapy resistance.A comprehensive understanding of the molecular mechanism of the occurrence and development of this type of breast cancer can provide a basis for finding new therapeutic targets and help to improve the prognosis of patients with triple-negative breast cancer.Compared with other subtypes,epithelial-mesenchymal transition(EMT)of TNBC cells is active abnormally.EMT refers to the transformation of cells from an epithelial cell phenotype to a mesenchymal cell-like phenotype.Morphologically,it turns into a fusiform shape from a round shape and a polygonal shape.At the molecular level,EMT is characterized by the decreased expression of epithelial cell markers(E-cadherin,Claudin,and CK)and the overexpression of mesenchymal cell markers(N-cadherin and Vimentin).EMT can promote the stem cellization,chemotherapy resistance,distant metastasis and recurrence.Active EMT is an important reason for the poor prognosis of TNBC.With the development of epigenetics,the important role of post-transcriptional regulation in the process of TNBC has gradually been recognized.Micro RNAs,long non-coding RNAs and circular RNAs(circ RNAs)and other types of non-coding RNAs are widely involved in post-transcriptional regulation of cells.Among them,circ RNAs are a kind of endogenous non-coding RNAs with a circular structure,which are conservative,tissuespecific and disease-specific.circ RNAs usually consists of exons and or introns of their host gene.Due to the lack of poly A tail structure,circ RNAs are more stable than linear ones,and can accumulate sufficient abundance in cells to perform biological functions with a unique mechanism.circ RNAs can regulate the occurrence and progression of tumors at the transcription,post-transcriptional and even post-translational level.An increasing numbers of researchs have proved that circ RNAs can regulate the malignant phenotypes of tumor cells such as EMT,stem cellization,and drug resistance.Among the various regulatory mechanisms of circ RNAs,the micro RNA sponge is the most common.In addition,some circ RNAs detected in the peripheral blood of patients can be used as biomarkers for disease diagnosis and prognosis.The purpose of this study is to find the key circ RNA regulating the malignant phenotype of triple-negative breast cancer,and to elucidate explore its regulatory mechanisms,so as to provide a scientific basis for finding new diagnostic indicators and therapeutic targets.Methods: Part 1: The high expressed circ RNAs in TNBC were screened out from the Gene Expression Omnibus(GEO)database.circ WAC(hsa?circ?0007503)was chosen as the candidate molecule,which was detected by q RT-PCR in breast cancer tissues and cell lines.Actinomycin D is used to inhibit cellular RNA synthesis to detect the half-life of circ WAC and WAC m RNA.RNase R is used to detect the resistance of circ WAC to RNase.Agarose gel electrophoresis is used to separate common PCR products.In situ fluorescence hybridization(FISH)experiment and nuclear-plasma separation PCR experiment were used to detect the subcellular localization of circ WAC.Following knockdown of circ WAC by si RNA or overexpression via plasmid,the effects of circ WAC expression level on the biological function of TNBC cells were detected by CCK8 assay,Annexin ?/PI assay,cell migration assay and Transwell.Western blot were used to detect the changes of EMT protein markers.Part 2: Target micro RNAs of circ WAC and target genes of mi R-1183 were predicted by bioinformatics analysis.q RT-PCR is used to detect the expression of mi R-1183 and SMAD2 m RNA.Western blot is used to detect the expression of SMAD2 protein and marker proteins of epithelial and mesenchymal cells.FISH was used to detect the subcellular localization of mi R-1183 and circ WAC.The dual-luciferase reporter assay was used to validate the binding relationship of mi R-1183 with circ WAC or SMAD2.RNA binding protein immunoprecipitation(RIP)and RNA pull down experiments are used to verify whether endogenous mi R-1183 can bind to circ WAC or SMAD2 m RNA.For rescue experiments,si-NC,si-circ WAC or si-circ WAC+mi R-1183 inhibitor were transfected into MDA-MB-231 cells.On the other hand,vector+si-NC,circ WAC overexpression plasmids,or irc WAC overexpression plasmids+si-SMAD2 were transfected into HCC1937 cells.Next,phenotypes of transfected cells were detected by CCK8 assay,Annexin ?/PI assay,cell migration assay and Transwell.Western blot were used to detect the changes of EMT protein markers.A circ WAC stable knockdown MDA-MB-231 cell line was generated by lentivirus transfection and a subcutaneous xenograft tumor model was created in nude mice.Immunohistochemistry is used to detect the expression intensity of related proteins in transplanted tumor tissues.Results: Part 1: This study first screened out 82 up-regulated circ RNAs molecules and 62down-regulated circ RNAs molecules in TNBC through GEO.Among the up-regulated circ RNAs,circ WAC is the most up-regulated in TNBC,which expression level was higher than that other types of breast cancer.The expression of circ WAC had positive correlation with lymph node metastasis and vascular tumor thrombus.circ WAC is composed of the4 th,5th,6th and 7th exons of its host gene.The results of the actinomycin D experiment showed that the half-life of circ WAC was 16 hours,and the half-life of WAC m RNA was4 hours.The results of agarose gel electrophoresis experiments show that circ WAC is only derived from the transcriptome and contains the unique reverse splicing point sequence of circular RNA.In addition,circ WAC was resistant to RNase digestion.The above three experiments prove that circ WAC had the characteristics of circ RNAs.The results of FISH experiment and nuclear and cytoplasmic separation PCR experiment showed that circ WAC mainly localized and cytoplasm.The CCK8 experiment showed that the proliferation rate of tumor cells decreased after circ WAC was knocked down,and the cell proliferation rate increased after overexpression of circ WAC.The A/P apoptosis test results showed that the apoptosis rate of tumor cells increased after circ WAC was knocked down,and the apoptosis rate was decreased after overexpression of circ WAC.Scratch repair experiments showed that tumor cell migration ability was weakened after circ WAC knockdown,and tumor cell migration ability was enhanced after circ WAC overexpression.Transwell experiment results showed that tumor cell invasion ability was weakened after circ WAC was knocked down,and tumor cell invasion ability was enhanced after overexpression of circ WAC.Western blot results showed that the expression of mesenchymal cell markers(N-cadherin protein and Vimentin protein)was down-regulated,and the expression of epithelial cell markers(E-cadherin protein)was up-regulated after circ WAC was knocked down.After overexpression of circ WAC,the expression of N-cadherin and Vimentin protein was up-regulated,and the expression of E-cadherin protein was down-regulated.The above cell function experiments show that circ WAC can promote the proliferation,migration,invasion and EMT of TNBC cells,while inhibiting cell apoptosis.Part 2: There were four possible target micro RNAs of circ WAC according to circ Interactome and circ Bank databases.Among them,the expression of mi R-1183 is most obviously regulated by circ WAC.mi R-1183 is low expressed in cancer tissues,and its expression is negatively correlated with circ WAC.The results of the dual luciferase reporter gene experiment showed that the system fluorescence intensity of mi R-1183 mimics+WT circ WAC group decreased significantly.The results of RIP experiments showed that the expression of circ WAC and mi R-1183 could be detected in the immunoprecipitation complex of the AGO2 antibody group.The results of the RNA pull down experiment showed that the probe containing the circ WAC sequence can enrich mi R-1183 molecules.The above three experimental results show that circ WAC can adsorb mi R-1183.The results of rescue experiments showed that knocking down circ WAC and mi R-1183 at the same time can reverse the cell phenotypic changes caused by only knocking down circ WAC,including proliferation,apoptosis,migration,invasion and MET.It proves that mi R-1183 regulates the phenotype of cells downstream of circ WAC.Next,after bioinformatics prediction,SMAD2 was identified as the target gene of mi R-1183.SMAD2 was highly expressed in cancer tissues,and its m RNA and protein levels were regulated by the circ WAC/mi R-1183 axis.The results of dual luciferase reporter gene experiment and RIP experiment showed that mi R-1183 can bind to the 3'UTR of SMAD2 m RNA.The results of cell function recovery experiments showed that knocking down SMAD2 while overexpressing circ WAC can reverse the changes in cell phenotype caused by only overexpressing circ WAC.It proveed that SMAD2 regulates the phenotype of cells downstream of the circ WAC/mi R-1183 axis.The results of subcutaneous xenograft tumor experiments in nude mice showed that the tumor volume and weight of the circ WAC stable knockdown group were smaller than those of the control group.The expression of Ki-67,SMAD2,Ncadherin and Vimentin was weaker than that of the control group,and the expression of Ecadherin was stronger than that of the control group.It proved that stable knockdown of circ WAC can inhibit the growth and EMT of TNBC cells in vivo.Conclusion: Part 1: circ WAC is a circular RNA that is located in the cytoplasm and is significantly upregulated in TNBC.Cases with high circ WAC expression are more prone to metastasis.circ WAC can promote the proliferation,migration,invasion and EMT of TNBC cells,while inhibiting cell apoptosis.Part 2: circ WAC up-regulates the expression of SMAD2 by adsorbing mi R-1183,and promotes the proliferation,migration,invasion and EMT of tumor cells through the mi R-1183/SMAD2 axis,while inhibiting apoptosis,thereby promoting the progression of TNBC.