Downregulation Of Circ＿0003045 And Its Tumor-suppressor Function In Breast Cancer

Background and purpose of the studyBreast cancer is the most common malignant tumor in women.Currently,neoadjuvant chemotherapy,surgery for operable tumors,radiotherapy,adjuvant chemotherapy,endocrine therapy,and targeted therapy are all used in the treatment of breast cancer.Due to the high prevalence of metastasis,recurrence,and treatment resistance,the prognosis of breast cancer patients has not considerably improved despite significant improvements in surgical procedures and medicinal management for the disease.More seriously,the 5-year survival rate for patients diagnosed with breast cancer at an early stage is close to 100%,but the patients with metastatic breast cancer drops dramatically to 26%.Therefore,there is an urgent need to obtain new clinical tools for accurate diagnosis and prognosis prediction,as well as new targets for developing therapeutic agents for breast cancer.Circular RNA(circRNA)is a kind of endogenous non-coding RNA(ncRNA),which is produced by the reverse splicing of precursor mRNA(pre mRNA).Unlike classical linear RNA,circRNA have a continuous,stable,covalently closed cyclic structure that lacks a 5’-cap and a 3’-poly(A)tail;circRNA are therefore resistant to nucleic acid exonuclease or RNase R digestion.With the rapid development of deep sequencing technology and novel bioinformatic approaches,circRNA have been identified to possess multiple biological functions,such as regulating transcriptional and post-transcriptional levels,binding miRNA and protein,modulating parental gene expression,and coding protein in the physiological and pathological development of many organisms,and indirectly contribute to the emergence and growth of cancerous tumors.In this study,we first assessed the level of circ＿0003045 expression in breast cancer tissue and adjacent tissue samples,respectively,and then validated the relationship between the expression of circ 0003045 and the clinicopathological features of breast cancer patients.Secondly,we conducted a series of cellular functional experiments by overexpressing circ＿0003045 in breast cancer cells and observing the effect on proliferation,colony formation,invasion,cell cycle and apoptosis of breast cancer cells.Further bioinformatics methods were adopted to predict the upstream and downstream molecular mechanisms of circ＿0003045’s role in breast cancer.Methods1.Using qRT-PCR,the differential expression of circ＿0003045 in 163 pairs of breast cancer and their corresponding adjacent tissues was examined.The diagnostic value of circ＿0003045 was then established using ROC curve analysis.Using the Student’s t test or the Chi-square test,the association between circ＿0003045 expression and clinicopathological features of the patients was examined.2.MDA-MB-231 and BT474 breast cancer cells were transfected with circ＿0003045 overexpression vector,as confirmed by qRT-PCR.The effects of overexpression of circ＿0003045 on proliferation,migration,invasion,and cell cycle progression in breast cancer cell lines were investigated with the Cell Counting Kit-8,clone formation assay,transwell assay,wound healing assay,and flow cytometry,respectively.3.To explore the underlying molecular mechanism of circ＿0003045 in breast cancer,we determined the subcellular location of circ＿0003045 in MDA-MB-231 cells with fluorescence in situ hybridization(FISH).The circbank and the circinteractome were used to predict the potential downstream target miRNA of circ＿0003045.Next,we used mirDIP database to predict the downstream target genes of selected miRNA and constructed a coexpression network of circRNA-miRNA-mRNA using Cytoscape.GO function and KEGG pathway enrichment analyses of target genes were done with R software,and the protein interaction network of target genes in the regulatory network was built with the STRING database.4.Then the potential binding proteins of circ＿0003045 was predicted by catRAPID and RBPDB database.5.N6-methyladenosine(m6A)modification sites were predicted by SRAMP and confirmed by Methylated RNA immunoprecipitation(MeRIP)assay.We searched the TNM plot database to screen for regulatory genes of m6A RNA methylation associated with breast cancer.RNA interference(RNAi)experiments were then conducted on regulatory genes of m6A to evaluate the expression of circ＿0003045.6.RNA binding proteins(RBP)are a group of proteins associated with RNA regulation and metabolism,and play an essential role in mediating the maturation,transport,localization and translation of RNA.We speculate that the down-regulation of circ＿0003045 expression in breast cancer may be associated with RBP.Next,we sought to explore if these circ＿0003045 is influenced by RNA binding proteins(RBP)like ADAR1 and DHX9.Results1.QRT-PCR showed that the expression of circ 0003045 was significantly lower in breast cancer tissues than in their adjacent tissues.ROC analysis indicated that circ＿0003045 could distinguish breast cancer from adjacent tissue,T-test and chi-square test showed that the low circ 0003045 expression levels are correlated with larger tumor size,lower progesterone receptor(PR)expression levels,higher ki-67 expression levels and higher histological grade.Moreover,the expression level of circ＿0003045 was significantly downregulated in the nonluminal phenotype compared to luminal phenotype.2.Function assays demonstrated that overexpression of circ＿0003045 inhibited proliferation,invasion and migration ability,but had no significant effects on cell cycle and facilitated apoptosis.3.FISH result showed that circ＿0003045 was located both in the cytoplasm and nucleus.It has been reported that circRNA,which mainly located in the cytoplasm,always function as a sponge of miRNA.Circbank and circinteractome analysis predicted 3 miRNA(miR-654-3p,miR-643 and miR-577)that are potential circ＿0003045 targets.The target genes of 3 miRNA were predicted using the online database mirDIP,and the top 30 predicted targets were selected to construct ceRNA network using Cytoscape.GO function and KEGG pathway enrichment analyses of target genes were done with R software,and the protein interaction network of target genes in the regulatory network was built with the STRING database.These results suggest that circ＿0003045 may regulate breast cancer progression through circ＿0003045-miR-654-3p/miR-643/miR-577-SRSF1-Wnt pathway.4.We predicted the RBP that may bind to circ＿0003045 using catRAPID and RBPBD website,and a total of 10 candidate proteins were identified.5.The online bioinformatic tool SRAMP revealed that there were multiple m6A modification sites in the circ＿0003045 sequence.Subsequent MeRIP-qPCR analysis revealed circ＿0003045 m6A levels to be significantly lower in 3 pairs of tumor samples from patients with breast cancer than in adjacent tissue samples.We then analyzed the expression levels of m6A methyltransferases in breast cancer tissues using the TNM plot database.The results showed that METTL14 were the down-regulated m6A methylation transferases and ALKBH5 were up-regulated m6 demethylation transferase in breast cancer.Further investigation showed that down-regulation of m6A level induced by interference of METTL14 and ALKBH5 could down-regulate or up-regulate the circ＿0003045 expression levels.6.RNA interference results showed that the down-regulation of DHX9 and ADAR1 could upregulate the expression level of circ＿0003045,indicating that the expression level of circ＿0003045 was affected by RBP.ConclusionCirc＿0003045 was shown to be highly down-regulated in breast cancer in this study,and it’s possible that its low expression level is associated with the patients’ poor prognosis.Overexpression of circ＿0003045 decreased the proliferation,migration and invasion ability of breast cancer cells.In terms of downstream molecular mechanisms,circ＿0003045 may influence the development of breast cancer via circ＿0003045-miR-654-3p/643/577-SRSF1Wnt pathway.In terms of upstream molecular mechanisms,we hypothesized that regulatory genes of m6A RNA methylation as well as RBP may influence the downregulation of circ＿0003045 expression in breast cancer.In this study,we explored the pathological role of circ＿0003045 in breast cancer and its potential molecular mechanisms involved in tumorigenesis,which provides a direction for us to further explore the molecular targeted therapy in breast cancer treatment.