Effects Of Circ RNA＿0004435 On The Function Of Breast Cancer Cells And Exploration Of Potential CeRNA Networks

Objective: The latest cancer report data in 2020 shows that breast cancer has become the cancer with the highest incidence over the world and it is of great significance to explore the biological markers related to breast cancer.With the application of high-throughput sequencing and the development of bioinformatics technology,a large number of non-coding RNAs have been discovered by researchers,among which circRNAs,as a member of the emerging non-coding family,have attracted much attention.CircRNAs can competitively bind to miRNAs and participate in the regulation of the occurrence and development of tumors.In this study,we hope to screen out an unreported differentially expressed circRNA in breast cancer through bioinformatics analysis,and then to explore its function.We also predicted the competitively bound miRNA and downstream target genes,and the possible pathways of the circRNAs were explored to construct a circRNAs ceRNA regulatory network.Methods: This study can be divided into two parts.The first part is bioinformatics analysis: this study downloaded the data set of breast cancer patients GSE101123 from GEO database and the data set of breast cancer patients from TCGA.Based on the GSE101123 dataset,circRNAs differentially expressed in breast cancer were screened and used as the main research objective of this study.Then we used Miranda,Starbase and Target Scan software to predict the downstream targeting miRNA and mRNA of the target circRNA,and the ceRNA regulatory network of the circRNA was constructed.Then,KEGG enrichment analysis was performed to explore the signaling pathways that this network might play a role in and explore its prognostic value.The second part is the experimental part,which is the verification and exploration of the previous analysis results.Firstly,the differential expression of the circRNA was verified at tissue and cell levels by real-time quantitative PCR.The correlation between this index and the clinicopathological data of patients was analyzed by Pearson.CCK8,transwell and plate cloning experiments were used to explore the effects of the circRNA on the proliferation,invasion and migration functions of breast cancer cells.Finally,Western blotting was used to verify the expression levels of target genes in the previously predicted regulatory network and key proteins in the pathway.Results: GSE101123 was used to screen the circular RNA＿0004435 with low expression in luminal type and triple negative breast cancer.The downstream miRNA was predicted: miR-196a-5p,and TCGA data verified that miR-196a-5p was highly expressed in breast cancer(P=6.9e-20).The enrichment analysis of downstream target genes of miR-196a-5p showed that the most genes were enriched in the MAPK pathway,and the low expression mRNA of miR-196a-5p(P=1.2e-48)and prognostic significance(P=0.028)--CACNA2D1 was selected as the target gene.The expression of CACNA2D1 in breast cancer was negatively correlated with miR-196a-5p(P =7.49);The expression level of circular RNA＿0004435 in breast cancer tissues was significantly lower than that in paracancerous tissues(P < 0.0001);The expression level of circRNA in the high Ki-67 group was lower than that in the low Ki-67group(P=0.0311).The expression of cyclic RNA＿0004435 in MCF-7 and MDA-MB-231 was lower than that in MCF-10A(P < 0.001);CCK8,plate cloning assay showed that the proliferation of MCF-7 and MDA-MB-231 cells in the OE group was lower than that in the NC group(P=0.0006;P=0.0253),the proliferation ability of MCF-7 and MDA-MB-231 in SI group was higher than that in NC group(P=0.0196;P=0.0098);Transwell assay results showed that the invasion and migration ability of MCF-7 and MDA-MB-231 cells in the OE group were lower than those in the NC group(P=0.0039;P=0.0318),the ability of MCF-7 and MDA-MB-231 cells in SI group was higher than that in NC group(P<0.0001);Western blot results showed that the contents of Ca CNA2D1 and ERK1/2 in MCF-7 and MDA-MB-231 cells in the OE group were significantly higher than those in the NC group.The contents of Ca CNA2D1 and ERK1/2 in MCF-7 and MDA-MB-231 cells in the SI group were lower than those in the NC group.Conclusion: The expression of circular RNA＿0004435 is significantly decreased in breast cancer,and its low expression is related to ki-67.circular RNA＿0004435 can inhibit the proliferation,invasion and migration of breast cancer cells.The ceRNA regulatory network related to the MAPK signaling pathway of circular RNA＿0004435--miR-196a-5p-mRNA was constructed.Among them,it was preliminarily verified that annular RNA＿0004435--miR-196a-5p-CACNA2D1 may affect the proliferation,invasion and migration of breast cancer cells by affecting the MAPK pathway.