The Regulation Of Malignant Phenotype Of Breast Cancer By MiR-106a/20b Cluster And The Underlying Mechanism

BackgroundCancer is a leading cause of death worldwide. In China, more than 1.6 million people are diagnosed with cancer each year, and 1.2 million die of it. Breast cancer is the most common malignant tumor in women in European and American countries. In China, the mortality of breast cancer ranks third among all cancers, and its mortality and morbidity is increasing each year. Therefore, it is important to explore the mechanism underlying the initiation and progression of breast cancer and to identify effective diagnosis and treatment methods to improve its prognosis. In recent years, a group of small, single-stranded, non-coding RNA molecules(microRNAs, miRNAs) have become hotspot in research. miRNAs commonly regulate several or a group of target genes with similar functions, and play an important role in regulating biological functions of cells. Research has already confirmed the important role of miRNAs in regulating genes in breast cancer.ObjectiveBased on the existing research on miRNA profiles of breast cancer, and bioinformatics analysis, this study aims to predict the key miRNAs associated with breast cancer, validate their roles in proliferation, invasion and migration in the breast cancer model, and identify the relevant target genes. In addition to elucidating the mechanism, this study is expected to offer new methods and treatment targets for the diagnosis and treatment of breast cancer, and provide rationale for improving treatment and prognosis.Methods1. We developed miRNAs-Gene-network and miRNAs-GO-network, based on the miRNA profiles of breast cancer and breast cancer stem cells by bioinformatics methods,including breast cancer-specific analysis, Gene Ontology analysis(GO-analysis), and target gene analysis. We then selected the mi RNAs that could play key roles in regulating malignant phenotype of breast cancer with high degree in the networks.2. We transfected MCF-7 and T47 D breast cancer cells with miRNA agonist(agomir)and antagonist(antagomir), and investigated the effect of miRNAs on invasion, migration and proliferation of breast cancer cells using the experiments of transwell- invasion and migration,wound healing repair assay and BrdU proliferation.3. We predicted the target genes of miRNAs using online tools TargetScan and miRanda,and validated the expression of target genes in miRNA-transfected breast cancer cells using western blotting methods.Results1. miRNAs-Gene network and miRNAs-GO-network showed that both miR-106 a and miR-20 b had a high degree of regulation, and are located on the X chromosome(Xq26.2,GRCh37) with a distance of less than 1.0 kb. They are a new miRNA cluster, and may be the key molecules that regulate the malignant phenotype of breast cancer.2. miR-106 a inhibited the invasion and migration of breast cancer cells, and functioned as an anti-oncogene; miR-20 b promoted the migration of breast cancer cells, and functioned as an oncogene.3. Based on the function of proliferation and differentiation and binding free energy, we obtained 18 candidate target genes of miR-106a/20 b. After preliminary screening by qRT-PCR assay, we chose two potential genes CCND1 and E2F1 for further validation. Using MCF-7 cells as the experimental system, we validated the target genes by dual-luciferase reporter assay and western blot. The results showed that CCND1 and E2F1 were the target genes of miR-106 a, miR-106 a directly inhibited the expressions of CCND1 and E2F1.However, CCND1 and E2F1 were not the target genes of miR-20 b, and miR-20 b indirectly promoted the expressions of CCND1 and E2F1.Conclusion1. The combination of gene microarray and GO-analysis is helpful for the screening of key miRNAs and their target genes that play important roles in regulating malignant phenotype of breast cancer.2. miR-106 a and miR-20 b, as a new miRNA cluster, function as the cancer-suppressing and cancer-promoting genes of malignant phenotype of breast cancer respectively, and play a balanced role in the initiation and progression of breast cancer.3. The cancer-suppressing role of miR-106 a is the result of its direct and negative regulation of the target genes CCND1 and E2F1; while the cancer-promoting role of miR-20 b could be the result of its indirect and positive regulation of CCND1 and E2F1.