Circular Rna Hsa_circ₀052112 Promotes Cell Migration And Invasion By Acting As Sponge For MiR-125a-5p In Breast Cancer

Breast cancer has become one of the most common malignancies threatening female’s health and life in worldwide.According to Global Cancer Statistics estimates,about 1.7 million new cases were diagnosed with breast cancer in 2012,accounting for 25 percent of all cancer cases among females,and 521,900 deaths,accounting for 15 percent of female cancer deaths.At present,the main treatment of breast cancer is surgery,combined with chemotherapy,radiotherapy,endocrine therapy and targeted therapy,etc.Although the comprehensive therapy of breast cancer can greatly improve the prognosis and survival rate of breast cancer patients,it will eventually lead to the metastasis of cancer cells.According to the research,about 10-15% of breast cancer patients will develop distant metastases in the first three years after cancer treatment.Therefore,it is urgent to explore the mechanism of invasion and migration of breast cancer.Circular RNAs（CircRNAs）are a class of novel,long,endogenous non-coding RNA molecules,which are involved in the regulation of gene expression at transcriptional and posttranscriptional level.CircRNA has a relatively stable structure and highly conserved sequence,and has a highly expressed tissue specificity in eukaryotic transcripts.Recent studies have found circRNAs harbor abundant miRNAs binding sites,inhibiting the activity of miRNAs by competing with miRNAs,thus deregulating the target genes and mediating the biological function of cells.At present,circRNAs have become a research hotspot.However,the role of circRNAs in the regulation of invasion and migration in breast cancer is not clear,so in this experiment,we will study the relationship between circRNA and breast cancer.PART Ⅰ Screening of differentially expressed circRNAs in breast cancer cells using microarray Objective: To identify the differentially expressed circRNAs in breast cancer MCF-7 cells and MDA-MB-231 cells by microarray.The circRNA with significant difference was further selected and the relevant characteristics was verified,laying a foundation for the subsequent study of function and mechanism.Methods: The circular RNA expression profile was performed using microarray based on the highly invasive breast cancer cells（MDA-MB-231）and low invasive cells（MCF-7）;according to the circBase,the circRNA sequences were obtained,and the divergent primers were designed according to the principle of back-splicing;the total RNA of MCF-7 cells and MDA-MB-231 cells was extracted and digested by RNase R,and the differentially expressed circRNAs in MCF-7 and MDA-MB-231 cells were further verified by real-time quantitative PCR（RT-qPCR）,then one circRNA with large difference was screened out;the circRNA was identified by agarose gel electrophoresis;the backsplice junction of circRNA was verified by Sanger sequencing;Fluorescence in situ hybridization（FISH）was used to identify the localization of circRNA in cells.Results: It was found that there were 5842 differentially expressed circRNAs（Fold change ≥4.0,p < 0.05）by microarray analysis of MDA-MB-231 cells and MCF-7 cells,and based on the expression level of circRNA in MCF-7 cells,2598 circRNAs were up-regulated and 3244 circRNA were down-regulated in MDA-MB-231 cells.The circRNAs with large difference were further verified by real time quantitative PCR,and then the circular RNA hsa_circ₀052112 was screened out;it was found that the RT-qPCR products amplified with cDNA as template could show bands by agarose gel electrophoresis,while gDNA as template did not show the target bands,suggesting the divergent primers could specifically amplify hsa_circ₀052112.The back-splice junction of hsa_circ₀052112 was confirmed by the Sanger sequencing experiment,consistent with that in circBase.Fluorescence in situ hybridization showed that hsa_circ₀052112 was mainly located in the cytoplasm.Conclusions: A large number of differentially expressed circRNAs were screened based on microarray,which may be closely correlated with the invasion and migration of breast cancer.Moreover,the expression of hsa_circ₀052112 was significantly up-regulated in MDA-MB-231 cells,suggesting that it might be a potential oncogene in breast cancer.PART Ⅱ Hsa_circ₀052112 promotes cell migration and invasion by sponge for mi R-125a-5pObjective: The first part of the study confirmed that hsa_circ₀052112 was significantly up-regulated in MDA-MB-231 cells,and hsa_circ₀052112 was mainly located in the cytoplasm.Meanwhile the hsa_circ₀052112/mi RNAs interaction was predicted using bioinformatics software.Therefore,we further study the relationship between hsa_circ₀052112/mi RNA and elucidate the mechanism of hsa_circ₀052112 regulating the migration and invasion of breast cancer.Methods: Hsa_circ₀052112 over-expression vector or blank vector was constructed,the sequences of vector construction were identified by Sanger sequencing experiment,and then transfected into MCF-7 cells by electroporation,si-hsa_circ₀052112 and negative control were electroporated into MDA-MB-231 cells.After transfection successful,wound-healing and transwell assay were used to detect the ability of cell migration and invasion;hsa_circ₀052112/mi RNAs interaction network was predicted by using the mi Randa software and RNAhybrid software;the target genes of hsa_mi RNA-125a-5p were predicted by four databases（Target Scan,Pic Tar,Micro Cosm and mi RDB）,and the circ RNA-mi RNA-m RNA interaction network was established;the wild-type and mutant sequences of hsa_circ₀052112 were constructed,and were identified by Sanger sequencing experiment,and the dual luciferase activity assay was used to verify whether hsa_circ₀052112 could target mi R-125a-5p;mi R-125a-5p mimics and negative control were transfected into MDA-MB-231 cells by electroporation,the expression of hsa_circ₀052112 was detected by RT-q PCR after transfection;the mixture of mi R-125a-5p mimics and hsa_circ₀052112 overexpression vector,mi R-125a-5p mimics and negative control were transfected into MDA-MB-231 cells by electroporation,respectively,and the migration and invasion of MDA-MB-231 cells were detected by transwell assay;the relationship between mi R-125a-5p and MMP11 was studied by Western blot.Level 3 mi RNA-seq isoform quantification,RNA-Seq data（HTSeq-Counts）and clinical data of breast cancer patients were downloaded from the Cancer Genome Atlas（TCGA）,and were analyzed by bioinformatics.Results: After successful transfection,the promotion on cell migration by hsa_circ₀052112 was further confirmed by wound-healing assay,and overexpression of hsa_circ₀052112 promoted breast cancer cell migration and invasion by transwell assay;Furthermore,down-regulation of hsa_circ₀052112 inhibited the cell migration and invasion.It was found that hsa_circ₀052112 could bind to 39 mi RNAs according to the conserved seed sequence matches through mi Randa software and RNAhybrid software,and a total of 3722 target genes of hsa_mi RNA-125a-5p were predicted by Target Scan,Pic Tar,Micro Cosm and mi RDB,and 51 target genes of which could be predicted by the above mentioned three softwares simultaneously.Hsa_circ₀052112 could combine with mi R-125a-5p by the dual luciferase activity assay,but the expression of hsa_circ₀052112 did not change significantly after mi R-125a-5p mimics was transfected into MDA-MB-231 cells.Mi R-125a-5p significantly inhibited the migration and invasion of breast cancer cells by transwell assay.However,overexpression of hsa_circ₀052112 partially abrogated the effect of mi R-125a-5p mediated inhibition of migration and invasion in MDA-MB-231 cells.Western blot and RT-q PCR showed that mi R-125a-5p inhibited MMP11 expression,but co-transfected mi R-125a-5p mimics and hsa_circ₀052112 overexpression vector,the inhibitory effect of MMP11 was partially hindered.In addition,the expression of mi R-125a-5p was significantly down-regulated in breast cancer tissues by analyzing dataset from TCGA.However,the high or low expression of mi R-125a-5p had no significant difference in overall survival rate（OS）of breast cancer patients;the expression of MMP11 was significantly up-regulated in breast cancer tissues,and the high expression of MMP11 was associated with the poor relapse free survival（RFS）and disease-free survival rate（DFS）of breast cancer patients by Kaplan-Meier plotter survival analysis.Conclusions: Hsa_circ₀052112 regulates the migration and invasion of breast cancer cells by acting as sponge for mi R-125a-5p,which increases the expression of MMP11.PART Ⅲ Preliminary study on the relationship between hsa_circ₀052112 and host gene ZNF83Objective: The circ RNA hsa_circ₀052112 is originated from exon 3–4 of ZNF83 gene.Therefore,it is further explored whether hsa_circ₀052112 can affect the expression of the host gene ZNF83.Methods: Real-time quantitative PCR was used to detect the expression of ZNF83 in MCF-7 and MDA-MB-231 cells;the expression of ZNF83 was detected again by real-time quantitative PCR after hsa_circ₀052112 overexpression vector and blank vector were transfected into MCF-7 cells;RNA-Seq data（HTSeq-Counts）and clinical data of breast cancer patients were downloaded from the Cancer Genome Atlas（TCGA）,and were analyzed by bioinformatics.Results: The expression of ZNF83 in MDA-MB-231 cells was higher than that in MCF-7 cells;Moreover,the expression of ZNF83 in MCF-7 cells transfected with hsa_circ₀052112 over-expression vector was significantly higher than that in negative control cells.The expression of ZNF83 was significantly up-regulated in breast cancer tissues,and the high expression of ZNF83 was closely correlated with the poor RFS of breast cancer patients by Kaplan-Meier plotter survival analysis.Conclusions: Overexpression of hsa_circ₀052112 promotes the expression of ZNF83 acted as its host gene.