Abnormal Expression Of MicroRNA In Serum Of Breast Cancer Patients And Preliminary Functional Study Of Mir-205

Background:MicroRNAs(miRNAs)are a new class of non-protein-coding, endogenous, small RNAs and of about 20-24 nucleotides in length, which are cleaved from the 70-80 nt partially duplexed precursor by the RNase III Dicer. Mature miRNAs are known to negatively regulate gene expression or destroy the stability of genes via incomplete or complete matching with the 3'UTR of their target genes at the post-transcriptional level. They are involved in processes such as the regulation of organismal development, nerve differentiation, cell proliferation, apoptosis and fat metabolism. Many miRNAs have relatively conservative sequences among species. Though the functions of these miRNAs are not clear, it is sure that they have different express patterns, which can regulate many physiological and pathological processes and abnormal expression of miRNAs may result in some human diseases including tumors. miRNAs are recognized as oncogenes or anti-oncogenes and they have specific express profiles in different tumor tissues. It has been demonstrated that circulating nucleic acid (CNA) which exists in body fluids including serum and blood plasma is closely related to cell metabolism in physiological and pathological condition. The detection and gene diagnosis of CNA has played a very important role in the diagnosis of malignant tumors. Recently circulating miRNAs are found in serum and blood plasma. Thus, it is possible that circulating miRNAs become a new class of clinical biomarkers in the early detection of malignant tumors.Objective:1. To demonstrate that the extraction of RNA and subsequent identification of miRNA from serum is feasible and to detect the expression of mir-21, mir-200c and mir-205 in serum between breast cancer patients and healthy controls.2. To investigate the effect of mir-205 on cell growth and apoptosis in breast cancer cell lines.3. To predict the target genes of mir-205 using bioinformatics methods and provide theoretical support and experimental basis for further research on mechanism of mir-205 in breast cancer development. 4. In summary, this study hopes to find new molecular targets in treatment of breast cancer and new biomarkers for diagnosis and prognosis.Methods:1.Real-time quantitative PCR is used to detect the abnormal expression of mir-21, mir-200c and mir-205 in the serum of breast cancer patients compared with healthy controls.2.mir-205 mimics is transfected into MCF-7 cells and fluorescent dyes cy3 linked at the oligonucleotide was observed by fluorescence microscope and qRT-PCR of mir-205 was chosen to observe the changes of miRNA expression. MTT was tested to evaluate the changes in cell growth. Cell cycle was observed by assessment of flow cytometry PI staining and apoptosis was observed by assessment of FITC-AnnexinV/PI two-color flow cytometry before and after transfection.3.Different bioinformatic ways are used to predict the target genes of miR-205 which is down-regulated in breast cancer cell lines MCF-7.Results:1.mir-21, mir-200c and mir-205 were detected in serum of 10 breast cancer patients and 3 healthy controls with real-time quantitative PCR. Results show that mir-200c is up-regulated while mir-205 is down-regulated in breast cancer patients.2.Observation of fluorescent dyes cy3 in cells and qRT-PCR of mir-205 after transfection confirmed the efficiency of transfection. Up-regulation of mir-205 would decrease the cell activity while increasing apoptosis but has no effect on cell cycles.3.There are 113 target genes predicted by at least two bioinformatic ways, most of the genes are involved in cell cycles, cell proliferation, differentiation, apoptosis, signal transduction and so on.Conclusions:1. miRNA could be detected in serum and some miRNA abnormally expressed in serum of breast cancer patients compared with healthy controls. Circulating miRNAs may become a novel class of biomarkers for early diagnosis of breast cancer.2. mir-205 could decrease cell proliferation activity while increasing apoptosis, but has no effect on cell cycles. 3. Bioinformatic analysis that predicts miR-205 might be involved in origin and progression of breast cancer as target gene regulators.