| Objectives As a result of the invasion and metastasis of breast cancer patients with tumor recurrence and death,the main reasons for the understanding and the understanding molecular mechanisms such as breast cancer cell invasion and metastasis of breast cancer has great significance to the reasonable treatment.miRNAs due to its powerful regulation of target genes in the organism physiological and pathological process played an important role.Recent study found that miRNAs in breast cancer and other malignant tumors may play an important role in the process of development.Current research on the relationship between miRNA-136 and the tumor is less,and there is a dispute,studies have found that miRNA-136 in non-small cell lung cancer is significantly higher than that of peritumoral tissue in the organization.And in the content of miRNA-136 in glioma significantly lower than the peritumoral tissue.And associated with the invasion and metastasis of tumor.The meaning of this research,the miRNA-136 in breast cancer tissue,a variety of expression in breast cancer cell line,and targeted RASAL2 genotype of TNBC.invasion and migration ability of breast cancerMethods(1)Extracting total RNA in tissue and cell,using the real-time quantitative PCR detection of micrornas-136 in breast cancer tissue and normal breast tissue,human normal mammary epithelial cell line MCF10 A and 3 kinds of breast cancer cell line(MCF7 ZR751,MAD-MB-231)in the expression,and analyses its significance.(2)miRNA-136 transfection,to the high invasive breast cancer cell line MAD-MB-231,wound healing and cell migration experiments using cells tested expression of micrornas-136 impact on breast cancer cell migration.At the same time using immunofluorescence,Westemblot and heterogeneous into tumor in mice model experiment method,common validated in vivo in vitro expression of micrornas-136 EMT occur in breast cancer biology;(3)The potential target genes of miRNA-136 were predicted by bioinformatics tool,and further determined the potential target genes based on their roles in cell invasion and migration,respectively.Experimental determination was performed by using transfection technique.The 3’UTR region of potential target gene was cloned into the p CMV2-myc vector which containing luciferase reporter gene,and then co-transfected with miRNA-136 mimics into breast cancer cells.Finally the target genes of miRNA-136 were validated by Luciferase Reporter Assay;(4)Extraction of total RNA and protein in the organization,and application of real-time quantitative PCR,Westemblot and immunohistochemical of40 for fresh breast cancer and matched normal tissue samples RASAL2 expression,and analyses its significance.Carrying the miRNA-136 and RASAL2 MCF10 A and MAD-MB-231 cells,using real-time quantitative PCR,Westemblot and cell migration experiment to evaluate the miRNA-136 and RASAL correlation expression in breast cancer cells and the influence of the change of cell migration.Results(1)The miRNA-136 expression in breast cancer tissue was significantly lower than normal tissue;Three kinds of breast cancer cells(MCF7 ZR751,MAD-MB-231)in the miRNA-136 expression was significantly lower than normal mammary epithelial cell MCF10A(2)The results of the flow cytometric analysis showed that the percentage of primary ICC reached 7.87%.Under inverted microscope ICC displayed spindle morphology and obvious connections between each other.Ultrastructurally,ICC were characterized by the presence of many mitochondria in the cytoplasm,circular or oval in shape with a large nucleus and many cytoplasmic processes with a dichotomous branching pattern.As the time extension and increased concentration of co-culture,ICC were seriously injured.(3)Bioinformatics analysis: online database Target Scan,mi RBase,mi RGen Targets predict the target genes of the miRNA-136 RASAL2;By Blast database,3 ’UTR region RASAL2 are associated with the Mi RNA-136-5’ end sequences against each other.Luciferase report gene results confirmed the miRNA-136 combined with RASAL2 3 ’UTR region(4)RASAL2 expression in TNBC group was significantly higher than that of normal tissue,and with TNBC is closely relative to breast cancer tissue classification.Rt-PCR,Western blot detection to the miRNA-136 in the expression of m RNA and protein level were cut RASAL2.RASAL2 high expression can reverse the miRNA-136 regulation to inhibit the action of the breast cancer cell invasion.Conclusions miRNA-136 was marked down regulated while RASAL2 was up regulated in TNBC and both of them were significantly correlated with clinical stage.Furthermore,our results suggested miRNA-136 suppressed cell migration and invasion as well as EMT process in breast cancer.Moreover,RASAL2 harbors a binding site of miRNA-136 and overexpression of miRNA-136 significantly decreased the m RNA level of RASAL2 which indicated miRNA-136 may directly regulate RASAL2 expression in the development of breast cancer.In brief,these results may validate a pathogenetic role of a miRNA-136 in TNBC and establish a potentially regulatory mechanism which involved in miRNA-136/RASAL2/EMT in TNBC. |
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