Fig 1: scRNA-seq revealed that miR-874 downregulates PMVK and upregulates p53 target genes in MCF-7 cells. MCF-7 cells were transfected with control (cont) miRNA or miR-874 for 36 h or control siRNA or siPMVK for 48 h. The viable population of MCF-7 cells was sorted, and each of the four groups was profiled by droplet-based scRNA-seq. (a) scRNA-seq data (n = 46,130) across all four groups of treated MCF-7 cells are shown as nonlinear representations of the top 50 principal components. Cells are coloured according to UMAP-based clusters. (b,c) Proportions of each cluster from miRNA (b) or siRNA transfections (c). Clusters increased by miR-874 (b) or siPMVK transfection (c) are marked with red frames. (d) Cells are coloured according to UMAP-based clusters of cell phase, according to treatment. (e,f) Proportions of the cell phase within each group (e) or cluster (f). (g) Venn diagram showing 349 common differentially expressed genes (DEGs) among 9695 genes identified by bulk RNA-seq and 437 identified by scRNA-seq between cells transfected with cont miRNA and miR-874. (h) The table shows multiple pathways enriched in 349 common DEGs analysed by Metascape. (i) The table shows the prediction of upstream transcription factors among the DEGs by TRRUST analysis in Metascape. (j) Volcano plots showing DEGs between cont miRNA and miR-874. (k) Dot plot showing p53 target genes. (l) The table shows the prediction of upstream transcription factors from the DEGs between control siRNA and siPMVK by TRRUST analysis in Metascape. (m,n) Violin plot of the miR-874 targets PMVK (m) and PPP1CA (n) in each group (****p < 0.0001; Welch’s t test).
Fig 2: Supplementation with GGPP partially restores apoptosis induced by miR-874. (a) MCF-7 cells were transfected with control (cont) miRNA or miR-874, treated with mevalonate (MVA, 1 mM), mevalonate-5-phosphate (MVP, 0.5 mM), mevalonate-5-pyrophosphate (MVA-5PP, 0.5 mM), isopentenyl pyrophosphate (IPP, 15 µM), farnesyl pyrophosphate (FPP, 10 µM), or geranyl pyrophosphate (GGPP, 10 µM) for 72 h and subjected to the Alamar Blue assay. For mock samples, 70% methanol was added. Data are presented as the mean ± SD. ****p < 0.0001 (one-way ANOVA). (b) MCF-7 cells were transfected with cont miRNA or miR-874, treated with GGPP for 48 h, and subjected to annexin V-633/PI double staining with flow cytometry analysis. For the mock sample, 70% methanol was added. Representative images are shown. (c) The percentages of annexin V-633-negative/PI-negative cells (lower left, live cells), annexin V-633-positive/PI-negative cells (lower right, early apoptosis), and annexin V-633-positive/PI-positive cells (upper right, late apoptosis) were quantified. n = 4 per group. Data are presented as the mean ± SD. ****p < 0.0001 (one-way ANOVA). (d) MCF-7 cells were transfected with cont miRNA or miR-874 and treated with GGPP for 40 h. Western blotting was performed to analyse the effects of miR-874 transfection and GGPP treatment on c-Myc and p53 in MCF-7 cells. *indicates nonspecific bands, and triangles show bands for SREBP2. The numbers below the bands are the relative values of expression. (e) qRT-PCR analysis of PMVK, SREBF2, BAX, and PUMA in MCF-7 cells transfected with control or miR-874 for 48 h. Data are presented as the mean ± SD (n = 6, *p < 0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA).
Fig 3: Identification of PMVK as a novel miR-874 target in breast cancer cells. (a,b) Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays were used to analyse the effects of miR-874 on PMVK mRNA and SREBF2 mRNA in MCF-7 and MDA-MB-231 cells. Data are presented as the mean ± SD. ***p < 0.001, ****p < 0.0001 (one-way ANOVA). (c,d) Western blotting for PMVK, SREBP2, and actin in MCF-7 and MDA-MB-231 cells transfected with cont miRNA or miR-874 for 72 h. Triangles indicate specific bands, and asterisks indicate nonspecific bands. (e) Bioinformatic analysis using TargetScan predicted that miR-874 might target both the PMVK 3'-UTR and the SREBF2 3’-UTR. (f) Putative miR-874 sites in the 3'-UTR of PMVK, as determined by the STarMir program. (g) p53/c-Myc double-knockout (DKO) MCF-7 cells were transfected with the PMVK 3'-UTR in a vector construct and miR-874 or control (cont) miRNA to analyse the effects on luciferase activity in the psiCHECK-2-PMVK-3'-UTR reporter. n = 3. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01 (Student’s t test). (h) Putative miR-874 sites in the 3'-UTR of SREBF2, as determined by the STarMir program. (i) p53/c-Myc DKO MCF-7 cells were transfected with the SREBF2 3'-UTR in a vector construct and miR-874 or cont miRNA to analyse the effects on luciferase activity in the psiCHECK-2-SREBF2-3'-UTR reporter. n = 6. Data are presented as the mean ± SD. **p < 0.01 (Student’s t test).
Fig 4: Expression of miR-874, PMVK, and SREBF2 in The Cancer Genome Atlas (TCGA) database analysis and meta-analysis. (a) Expression of miR-874 (left), PMVK (middle), and SREBF-2 (right) in breast cancer tissues compared with normal tumour-adjacent tissues in the TCGA breast cancer dataset (normal samples [n = 104] versus tumour samples [n = 1093]). The data are presented as logarithmic values. The central line represents the median gene expression. Outliers above the 95th percentile and below the 5th percentile are displayed as dot plots. (b) Correlation analysis between the expression of miR-874 and PMVK (Pearson’s r = - 0.255, p < 0.0001). (c) Cohort studies for PMVK gene expression in a breast cancer meta-analysis were combined using random-effects models. The integrated hazard ratios (HRs) of relapse-free survival and its 95% confidence interval are shown. All cohorts were divided by the median gene expression ratio. Source data are provided in Supplementary Table S3. (d) Overall survival for the high and low PMVK mRNA expression groups (upper panel) and PMVK protein expression groups (lower panel) depicted using Kaplan–Meier plotter (https://kmplot.com/analysis). (e) A schematic view of a proposed mechanism through which miR-874 induces cancer cell death via suppression of the mevalonate pathway. miR-874 suppresses the mevalonate pathway by directly targeting PMVK and SREBF2, resulting in GGPP depletion, which activates the c-Myc and p53 signalling pathways and causes apoptosis and cell cycle arrest.
Fig 5: PMVK knockdown induces p53-mediated cell proliferation. (a) qRT?PCR analysis of PMVK, p21/CDKN1A, BAX, NOXA, PUMA, and SREBF2 in MCF-7 cells transfected with control or PMVK siRNAs and treated with or without geranylgeranyl pyrophosphate (GGPP) for 60 h (n = 4 or 6, mean ± SD, *p < 0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA). (b) Western blotting for PMVK, c-Myc, p53, p21/CDKN1A, and actin in MCF-7 cells transfected with control or PMVK siRNAs for 48 h. (c) p53 WT MCF-7 cells were transfected with control or PMVK siRNAs and subjected to a cell proliferation assay. Error bars represent the SD of the mean (n = 4, *p < 0.05, ****p < 0.0001 by one-way ANOVA). (d) MCF-7 cells were transfected with control or PMVK siRNAs for 64 h. The effect of PMVK knockdown on cell apoptosis was detected via annexin V-633/PI double staining of MCF-7 cells and flow cytometry assessment. Representative images are shown. (e) The percentages of annexin V-633-positive/PI-negative cells (lower right, early apoptosis) and annexin V-633-positive/PI-positive cells (upper right, late apoptosis) were quantified. (n = 3, **p < 0.01, ***p < 0.001 by one-way ANOVA). (f) MCF-7 cells were transfected with control or PMVK siRNA, treated with geranylgeranyl pyrophosphate (GGPP) for 48 h and subjected to an EdU assay with flow cytometry analysis. For the control sample, 70% methanol was added. Representative images are shown. (g) The proportion of cells in the S phase was quantitated and plotted (n = 3). Error bars represent the SDs of the means. ****p < 0.0001 (one-way ANOVA).
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