Project description:Objective: Trastuzumab has been used for the treatment of HER2-positive breast cancer (BC). However, a subset of BC patients exhibited resistance to trastuzumab therapy. Thus, clarifying the molecular mechanism of trastuzumab treatment will be beneficial to improve the treatment of HER2-positive BC patients. In this study, we identified trastuzumab-responsive microRNAs that are involved in the therapeutic effects of trastuzumab. Methods and results: RNA samples were obtained from HER2-positive (SKBR3 and BT474) and HER2-negetive (MCF7 and MDA-MB-231) cells with and without trastuzumab treatment for 6 days. Next, we conducted a microRNA profiling analysis using these samples to screen those microRNAs that were up- or downregulated only in HER2-positive cells. This analysis identified miR-26a and miR-30b as trastuzumab-inducible microRNAs. Transfecting miR-26a and miR-30b induced cell growth suppression in the BC cells by 40% and 32%, respectively. A cell cycle analysis showed that these microRNAs induced G1 arrest in HER2-positive BC cells as trastuzumab did. An Annexin-V assay revealed that miR-26a but not miR-30b induced apoptosis in HER2-positive BC cells. Using the prediction algorithms for microRNA targets, we identified cyclin E2 (CCNE2) as a target gene of miR-30b. A luciferase-based reporter assay demonstrated that miR-30b post-transcriptionally reduced 27% (p=0.005) of the gene expression by interacting with two binding sites in the 3’-UTR of CCNE2. Conclusion: In BC cells, trastuzumab modulated the expression of a subset of microRNAs, including miR-26a and miR-30b. The upregulation of miR-30b by trastuzumab may play a biological role in trastuzumab-induced cell growth inhibition by targeting CCNE2. We obtained microRNA expression profiles of breast cancer cell lines, MCF7, MDA-MB-231, SKBR3, and BT474, with or without trastuzumab (4microgram/mL) treatment.
Project description:Objective: Trastuzumab has been used for the treatment of HER2-positive breast cancer (BC). However, a subset of BC patients exhibited resistance to trastuzumab therapy. Thus, clarifying the molecular mechanism of trastuzumab treatment will be beneficial to improve the treatment of HER2-positive BC patients. In this study, we identified trastuzumab-responsive microRNAs that are involved in the therapeutic effects of trastuzumab. Methods and results: RNA samples were obtained from HER2-positive (SKBR3 and BT474) and HER2-negetive (MCF7 and MDA-MB-231) cells with and without trastuzumab treatment for 6 days. Next, we conducted a microRNA profiling analysis using these samples to screen those microRNAs that were up- or downregulated only in HER2-positive cells. This analysis identified miR-26a and miR-30b as trastuzumab-inducible microRNAs. Transfecting miR-26a and miR-30b induced cell growth suppression in the BC cells by 40% and 32%, respectively. A cell cycle analysis showed that these microRNAs induced G1 arrest in HER2-positive BC cells as trastuzumab did. An Annexin-V assay revealed that miR-26a but not miR-30b induced apoptosis in HER2-positive BC cells. Using the prediction algorithms for microRNA targets, we identified cyclin E2 (CCNE2) as a target gene of miR-30b. A luciferase-based reporter assay demonstrated that miR-30b post-transcriptionally reduced 27% (p=0.005) of the gene expression by interacting with two binding sites in the 3’-UTR of CCNE2. Conclusion: In BC cells, trastuzumab modulated the expression of a subset of microRNAs, including miR-26a and miR-30b. The upregulation of miR-30b by trastuzumab may play a biological role in trastuzumab-induced cell growth inhibition by targeting CCNE2.
Project description:RNA sequencing technology has been carried out in order to evaluate mRNA expression changes after manipulation of miR-26a in both MCF-7 and MDA-MB-231 breast cancer cell lines. To evaluate the entire set of genes modulated by miR-26a in breast cancer, we performed RNA-seq after ectopic manipulation of this miRNA. We over-expressed miR-26a in MCF-7 epithelial cancer cell lines and also reduced its activity by stably transfecting MDA-MB-231 mesenchymal-like cancer cell lines with a specific sponge vector. GO terms and pathway enriched analysis of the transcripts that significantly change upon miR-26 ectopic manipulation implicates miR-26ab in cell cycle, apoptosis, cell spreading and cell adhesion in breast cancer
Project description:To reveal the potential regulation target genes of miR-26a and miR-23a/b clusters in articular chondrocytes, we performed a multi-omics analysis of LC-MSMS and RNA-seq using cultured chondrocytes samples, which were primarily isolated from 3-week-old wild-type, miR-26a -/- (with or without miR-26a mimic transfection afterwards) or miR-23a/b cluster flox/flox;Col2a1-cre mice. For LC-MSMS, protein from TRIZOL reagent was extracted, nanoLC-MSMS was performed. An expression list was made to further explore the regulation targets of miR-26a and miR-23a/b clusters.