| ObjectiveThe occurrence and development of tumor is a complex procedure related multi-genes changes and experienced multi-stage and multi-step progress. The activation of proto-oncogene and mutation or inactivation of tumor suppressor gene is often involved in this procedure. In recent study, microRNA (miRNA) plays an essential role in genesis and progression of tumor. MiRNA, as a hot field of tumor molecular orientation research lately, can explain the pathogenesis of cancer and can supply a new treatment strategy to tumor. Heterogeneity of breast cancer is very high. And different miRNA function in different stage in breast cancer. But, so far, there is a little study in the stage of precancerous lesion of breast cancer about miRNA. In our research, we study the expression level of miRNA in ductal carcinoma in situ (DCIS) as precancerous lesion by the technology of miRNA microarray and real-time PCR. We hope find some new miRNA playing a specific role in the early stage of breast cancer and explore their function in the development of breast cancer, through which can offer efficient tumor markers for finding breast cancer in the early stage and evidence-based clues for treatment of breast cancer.Method21paired fresh frozen tissues come from Tianjin Medical University Cancer Institute and Hospital, obtained by mammary surgery during2007years and2012years. Every paired tissue includes one DCIS tissue and correspondingly adjacent normal tissue (ANT), which do not any preoperative treatment including chemotherapy and radiotherapy. One paired tissue is detected by miRNA microarray analysis. According to the result, we screen out some miRNA which expression level occur obvious change for validation of the follow-up work. By the above research work, we screen out miRNA-132which expression level has a significant deregulation in DCIS tissues compared to ANTs, for functional studies in vitro. We choose three kind breast cancer cell lines, including MDA-MB-231, MCF-7and BT-549. CCK-8reagent, colony formation assay and flow cytometry (FCM) are used to study the function of miRNA-132in process of division and proliferation breast cancer cell.Results1. Compared with the ANTs, miRNA microarray detects out852miRNA expression levels happen change in DCIS tissue. However, there is only155 miRNA accord with demand when we put FoldChange≥3as screening criteria. We can distinguish easily the two different kind tissues by this differential miRNA expression profiling.2. Based on the data from miRNA microarray, we select some miRNA which expression level happen obviously changes to validate by real-time PCR in21paired tissues. The statistic results show that the expression deregulation of miRNA-125b, miRNA-145, miRNA-10b, miRNA-132, miRNA-409, miRNA-154and miRNA-382have significant statistical significance. And all that miRNA are downregulated in DCIS tissues.3. With the help of a variety of functional experiment in vitro, we study the effect of miRNA-132in breast cancer. MiRNA-132can inhibit breast cancer cell proliferation in MDA-MB-231, MCF-7and BT-549cell line by CCK-8reagent. It can suppress colony-forming efficiency in MDA-MB-231and MCF-7cell line in colony formation assay. Besides, FCM results in BT-549cell line revealed that miRNA-132can retardant cell cycle at the stage of S/G2.Conclusion1. By the technology of miRNA microarray and real-time PCR, we verify miRNA-125b, miRNA-10b and miRNA-145related to invasion and metastasis of breast cancer have occurred obviously change in precancerous lesion of breast cancer. And this miRNA are downregulated in most of DCIS tissues.2. According to the result of real-time PCR, we demonstrated that miRNA-132, miRNA-409, miRNA-154and miRNA-382which are little involved in field of mammary gland are deregulated. All results are in accordance with miRNA microarray analysis results.3. MiRNA-132can inhibit breast cancer cell proliferation acting as a tumor suppressor. It plays an important part in the development of breast cancer and need to further study in function and pathogenesis. |
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