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Basic Research
miR-32-5p regulates the biological behaviors of breast cancer MDA-MB-231 cells by targeting the expression of Dickkopf-related protein 3
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Abstract:
Objective: To screen the key differentially expressed miRNAs and their target genes in breast cancer using bioinformatics tools, and to observe the effect of interfering their expression on the function of breast cancer cells. Methods: The GEO database was used to screen the differentially expressed miRNAs in breast cancer, and the ENCOR1 database was further used to verify the expression of screened miRNAs to identify the most differentially expressed miRNA as the research target. Starbase, miRDB and miRWalk databases were used to predict the target genes of miR-32-5p. GO analysis and KEGG analysis of target genes were done by using DAVID database. PPI network analysis and screening of hub genes were performed using String database and Cytoscape3.6.2 software. The Dickkopf-related protein 3 (DKK3) gene with the most significant degree was selected from the hub genes for follow[1]up experiments. qPCR was used to detect the expression of miR-32-5p in human normal breast MCF10A cells and human breast cancer cells (MCF7, MDA-MB-231 and MDA-MB-453 cells). MiR-32-5p mimics, miR-32-5p inhibitors and their control (NC) sequences were transfected into MDA-MB-231 cells. The effects of over-expression or inhibition of miR-32-5p on cell proliferation,apoptosis and invasion were detected by CCK-8 method, flow cytometry, and Transwell experiment, respectively. Results: Two differentially expressed miRNAs were identified from the two datasets in GEO database. ENCORI database was adopted to verify the differentially expressed miRNAs and showed that the expression level of miR-32-5p was consistent with the result in the GEO database, so it was selected for subsequent research. 198 potential target genes of miR-32-5p were predicted and 10 hub genes (DKK3,WNT2B, SFRP5, SFRP2, SFRP1, LRP6, WNT6, KREMEN1, NEDD4L and TRIP12) were identified, among which DKK3 showed the most significant degree. So, miR-32-5p/DKK3 axis was selected for the subsequent research. The expression of miR-32-5p in three breast cancer was significantly higher than that in normal mammary gland cells (all P<0.01), with the highest expression in MDA[1]MB-231 cells. Dual-luciferase reporter gene assay verified cell lines that miR-32-5p tageted and down-regulated. After transfection with miR-32-5p mimics or miR-32-5p inhibitors, the expression of miR-32-5p in MDA-MB-231 cells was successfully increased or inhibited. Compared with the control group, overexpression of miR-32-5p could inhibit the apoptosis of MDA-MB-231 cells and promote cell proliferation and invasion (P<0.05 or P<0.01), while knocking down miR-32-5p played an opposite role (all P<0.01).Conclusion: miR-32-5p/DKK3 axis may be a key pathway affecting the development of breast cancer. Over-expression of miR-32-5p can inhibit the apoptosis but promote cell proliferation and invasion of breast cancer MDA-MB-231 cells.
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Project supported by the Youth Program of Natural Science Foundation of Hainan Province (No. 818QN314)
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