MiR-5047在乳腺癌细胞增殖迁移中的作用及表达调控

The Role and Expression Regulation of MiR-5047 in the Proliferation and Migration of Breast Cancer Cells

探讨miR-5047在乳腺癌细胞中的表达及其在乳腺癌细胞增殖和迁移中的作用,并明确地西他滨在miR-5047表达调控中的作用。通过实时荧光定量PCR(qRT-PCR)检测人乳腺癌细胞系和正常乳腺上皮细胞MCF10A中miR-5047的表达水平;将miR-5047模拟物(mimic),阴性对照(NC)分别转染至MDA-MB-231和MCF7细胞,经平板克隆实验、MTT实验、划痕愈合实验检测乳腺癌细胞的增殖和迁移能力,通过qRT-PCR和Western blot检测相关基因表达及蛋白水平。使用浓度5μmol/L和10μmol/L的地西他滨分别处理MDA-MB-231和MCF-7细胞,经qRT-PCR检测不同浓度和处理时间条件下地西他滨对miR-5047表达的影响。同时,通过形态观察和Western blot检测地西他滨对乳腺癌细胞上皮间质转化的影响。与正常乳腺上皮细胞MCF-10A相比,miR-5047在乳腺癌细胞中表达均显著下调。miR-5047过表达可显著抑制乳腺癌细胞的增殖和迁移,促进上皮细胞标志物E-cadherin的表达,抑制间质细胞标志物Vimentin的表达。不同浓度地西他滨处理MDA-MB-231和MCF7细胞后,miR-5047表达均增强,且10μmol/L作用48 h效果最显著。地西他滨可诱导MDA-MB-231细胞向上皮样转变。miR-5047在乳腺癌细胞系中表达显著下调,过表达miR-5047可抑制乳腺癌细胞的增殖和迁移,地西他滨可促进乳腺癌细胞中miR-5047的表达,并诱导细胞向上皮样转变。

To explore the expression of miR-5047 in breast cancer cells and its role in breast cancer cell proliferation and migration,and to clarify the role of decitabine(DAC)in the regulation of miR-5047 expression.The expression level of miR-5047 in human breast cancer cell lines and normal breast epithelial cells MCF10 A was detected by real-time quantitative PCR(qRT-PCR).Transfect miR-5047 mimic and negative control mimic NC into MDA-MB-231 and MCF7 cells,respectively,and verify the transfection efficiency by qRTPCR.Plate cloning experiments,MTT experiments,and scratch healing experiments were used to detect the proliferation and migration ability of breast cancer cells,and qRT-PCR and Western blot methods were used to detect the expression of related genes and proteins after over-expression of miR-5047.MDA-MB-231 and MCF-7 cells were treated with DAC at final concentrations of 5μmol/L and 10μmol/L,and qRT-PCR was used to detect the effect of DAC on miR-5047 expression under different concentrations and treatment time.At the same time,the effect of DAC on the epithelial mesenchymal transition(EMT)of breast cancer cells was detected by morphological observation and Western blot.Compared with normal breast epithelial cells MCF-10 A,the expression of miR-5047 in breast cancer cells was significantly down-regulated.Overexpression of miR-5047 can significantly inhibit the proliferation and migration of breast cancer cells,promote the expression of epithelial cell marker E-cadherin,and inhibit the expression of mesenchymal cell marker Vimentin.The expression of miR-5047 can promote the expression of epithelial cell marker E-cadherin and inhibit the expression of mesenchymal cell marker Vimentin.After treating MDA-MB-231 and MCF7 cells with different concentrations of DAC,the expression of miR-5047 was enhanced,and the effect was most significant when 10μmol/L DAC was used for48 h.DAC can induce epithelial transformation of MDA-MB-231 cells.The expression of miR-5047 is significantly down-regulated in breast cancer cell lines.Overexpression of miR-5047 can inhibit the proliferation and migration of breast cancer cells.The low expression of miR-5047 in breast cancer cells can inhibit the proliferation and migration of breast cancer cells.DAC treatment can enhance the expression of miR-5047 in breast cancer cells and induce epithelial transformation of the cells.