MiR-340 Inhibits Triple-negative Breast Cancer Progression By Reversing EZH2 Mediated MiRNAs Dysregulated Expressions

Background: Triple negative breast cancer(TNBC)is a subtype with a high degree of malignancy.Micro RNA-340 is a non-coding single-stranded RNA molecules encoded by endogenous genes that have potent transcriptional gene expression regulation.It regulates many physiological and pathological processes.EZH2 is a potential target for miR-340,which catalyzes the trimethylation of the 27 th lysine on histone H3(H3K27me3),thus silencing gene expression.Through the study of clinical pathological tissue,in vivo and in vitro experiments,we intend to reveal a miRNAs network pathway that contributes to the molecular mechanism of TNBC by targeting EZH2.Methods: 1.FISH and immunohistochemistry were used respectively to detect the differential expression of miR-340 and EZH2 in breast cancer tissues and their adjacent tissues.Compare and grade the expression of miR-340 and EZH2,and analyze the relationship between them.2.The expression of miR-340 and EZH2 in different breast cancer cell lines were detected by RT-PCR and Western-blotting respectively.3.The potential target of miR-340 was obtained by bioinformatics prediction software.Western-blot and luciferase assay was used to confirm the result.4.We use MTT and plate cloning assay to observe the changes of cell growth ability and flow cytometry to detect the change of cell cycle and the percentage of apoptotic cells.Immunofluorescence was used to observe the expression of EMT markers.Transwell was applied to observe cell invasion.5.We construct nude mouse transplantation tumor model to detect the effect of miR-340 on breast cancer in vivo and detect the biomarkes' change in the tumor.6.We use RT-PCR to detect the changes of miR-21 and miR-200a/b in downstream pathway of EZH2 and use Western-blotting and immunohistochemistry to detect the expression of DNMT1,H3K27me3 and p-STAT3 in these pathways.Results: 1.Expression state of miR-340 was low in cancer tissues compared with adjacent tissues and is negatively correlated with the expression of EZH2.2.MDA-MB-231 and MDA-MB-468 cell lines were significantly different from other cell lines in terms of expression levels of miR-340 and EZH2.3.Mi R-340 has a conserved sequence that can be combined with EZH2.The luciferase assay showed a significant decrease of luciferase activity in miR-340-mim cell lines and an opposite result in miR-340-Inh cell line cell lines.4.In miR-340-mim cell lines,cell proliferation capability,invasion and metastasis ability and EMT mesenchymal biomarker ?-catenin was significantly decreased.G0 / G1 phase ratio and the expression level of results are just the opposite.5.In vivo studies,the ability of tumorigenicity was significantly enhanced after the injection of miR-340-mim than the injection of miR-340-Inh.Meanwhile,the expression level of EZH2 ? Ki-67 ? MMP9 was significantly decreased in miR-340-mim transplated tumor than in that of miR-340-Inh.6.In miR-340-mim cell lines,the expression of DNMT1?H3K27me3?p-STAT3 was decreased,of miR-21 was decreased and of miR-200a/b was increased.Conclusion: 1.Clinical data show that miR-340 is down-regulated in breast cancer and is negatively correlated with EZH2 expression.2.EZH2 is a new target of miR-340.3.Mi R-340 plays a role as tumor suppressor gene in TNBC.It can inhibit the tumorigenicity of TNBC through the regulation of cell growth,invasion and metastasis,apoptosis and cell cycle.It also showed an anti-tumor effect in in vivo studies.4.Mi R-340 reverses EZH2-mediated miR-21 transcriptional activation and miR-200a/b expressional silencing.