| 【Purpose】 To investigate the effects of miR-30 a on epithelial-mesenchymal transition and stem cell-like properties of breast cancer,and explore the underlying mechanism.【Methods】 1.Basic expression of miR-30 a in breast cancer cell lines was screened by q RT-PCR.Forced expression of miR-30 a in MDA-MB-231 and MCF-7 cells.Morphological changes of cells were observed by light microscopy.Immunofluorescence and western blot were used to detect the expression of EMT-related markers.The self-renewal ability of cancer cells was detected by the mammosphere formation assay.The proportion of CD44high/CD24 low cell subsets and ALDH activity were detected by flow cytometry.2.The potential target genes of miR-30 a were predicted by bioinformatics analysis and dual luciferase reporter assay verified whether SOX4 was the direct target gene of miR-30 a.Correlation analysis between miR-30 a and SOX4 in TCGA.q RT-PCR to detect the intracellular changes of miR-30 a and SOX4.3.Basic expression of SOX4 in breast cancer cell lines by q RT-PCR.We established a stable SOX4 knockdown cell line,and the effects of SOX4 on EMT and CSC phenotypes were observed in vitro experiment.4.Overexpressed the full-length SOX4 c DNA,lacking the 3’UTR,in the stably expressing miR-30 a cells to observe whether the up-regulated SOX4 weakened the inhibitory effects of miR-30 a on EMT and CSC phenotypes.5.We detected the expression of phosphorylated SMAD2/3 and TGFBR1 by Western blot.In order to varify the relationship among them,we conducted bioinformatics analysis such as GSVA and correlation analysis in TCGA.6.Subcutaneous xenograft nude mouse model was utilized and we detected the expression of related markers through IHC experiment in xenograft tissues.7.Basic expression and survival analysis of miR-30 a were analyzed in TCGA.IHC staining of SOX4 was performed on breast cancer tissues,and we analyzed the correlation between expression of SOX4 and the clinicopathological parameters.Survival analysis of SOX4 was also performed.8.We explored whether Disulfiram could upregulate the expression of miR-30 a via q RT-PCR in breast cancer cell lines in vitro,so as to play the role of miR-30 a tumor suppressor.【Results】 1.Basic expression of miR-30 a was significantly downregulated in breast cancer cell lines compared with normal breast cell lines.Found that overexpression of miR-30 a in breast cancer cells could prevent EMT morphological change induced by TGF-β and retained their cobblestone-like epithelial morphology with tight cell–cell adhesion.Forced expression of miR-30 a could upregulate E-cadherin and downregulate Vimentin,ZEB1 and Snail1.Overexpression of miR-30 a could also inhibit the ability of mammospheres formation,and reduce the proportion of CD44high/CD24 low subpopulations and the activity of ALDH.2.We analyzed the prediction results of microRNA targets,and SOX4 was selected as the candidate target gene of miR-30 a.Dual luciferase report assay confirmed that SOX4 is a direct target of miR-30 a.The expression of miR-30 a and SOX4 was negatively correlated in TCGA.When we upregulated the expression of SOX4,the expression of miR-30 a was significantly decreased and vice versa.3.The expression of SOX4 was higher in cancer cells than that in normal cells.Knockdown of SOX4 could resist morphology changes during induction of TGF-β and reversed EMT program as miR-30 a did.The knockdown of SOX4 also suppressed mammospheres formation ability and decreased the proportion of CD44high/CD24 low subpopulations and ALDH activity.4.Overexpressed the full-length SOX4 c DNA,lacking the 3’UTR,in the stably expressing miR-30 a cells and we found E-cadherin was downregulated and Vimentin,ZEB1,Snail1 were upregulated.Mammospheres formation ability,proportion of CD44high/CD24 low subpopulations and activity of ALDH were also rescued by SOX4 reintroduction.5.Overexpression of miR-30 a or knockdown of SOX4 could inhibit the expression of phosphorylated SMAD2/3 and TGFBR1.The suppression of TGF-β pathway regulated by miR-30 a could be reversed by the reintroduction of SOX4.The expression levels of EMT,CSC and TGF-β pathway related moleculars were negatively correlated with the expression levels of miR-30 a.6.Mi R-30 a could inhibit tumor growth as compared to the control group in vivo.IHC staining revealed that miR-30 a reversed EMT markers,inhibited CSC related markers and suppressed the expressions of SOX4 and TGF-β pathway(phosphorylated SMAD2/3 and TGFBR1)in vivo,as it did in vitro.7.The expression of miR-30 a was higher in normal breast tissues than in breast cancer tissues and was associated with good prognosis in TCGA.IHC results showed that the SOX4 positive rate were 52% and 56% in 42 clinical breast cancer cases and 133 cases tissue microarray,respectively.The positivity of SOX4 was significantly correlated with triple negative breast cancer.Survival analysis showed that SOX4 was a poor prognostic factor for breast cancer.8.The expression level of miR-30 a could be upregulated with the treatment of Disulfiram for 48 h at appropriate concentrations.【Conclusions】 1.MiR-30 a could inhibit the EMT and CSC phenotypes in breast cancer in vivo or in vitro.2.SOX4 is a direct target of miR-30 a and formed a double negative feedback loop with miR-30 a.3.Suppression of SOX4 is required for miR-30 a mediated inhibition of EMT and CSC phenotypes in breast cancer 4.Mi R-30a/SOX4 inhibits EMT and CSC phenotypes through suppression of TGF-β pathway in breast cancer 5.Mi R-30 a was associated with good prognosis and SOX4 was a poor prognostic factor in breast cancer.6.Disulfiram could upregulate the expression of miR-30 a so as to play the role of miR-30 a tumor suppressor. |
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