Fig 2: piR-2158 suppressed IL11 expression and inactivated JAK-STAT signaling in breast cancer. A: Volcano plot showing the different expression genes (DEGs) regulated by piR-2158 in MDA-MB-231 cells, in which IL11 was significantly downregulated. B: Overexpression of piR-2158 in MDA-MB-231 cells in 3 independent experiments. C: QRT-PCR analysis validated downregulation of IL11 by piR-2158 in MDA-MB-231 cells in B. D: ELISA analysis showing decreased level of secretory IL-11 in the supernatants of piR-2158-overexpressing MDA-MB-231 cells. E: A negative correlation between the expression levels of piR-2158 and IL11 in tumor tissues from breast cancer patients (n = 32). F: TCGA database showing upregulation of IL11 in human breast cancer tumors (n = 1,083). G: Negative correlations between the IL11 expression levels and overall survival, disease specific survival and progression free survival in breast cancer (n = 1,083). H: KEGG pathway analysis showing the top 5 signaling pathways regulated by piR-2158. I and J: Western blot analysis showing downregulation of IL-11 and p-STAT3 by piR-2158 in the mammary tumors from mice. Total STAT3 did not show change. ß-actin served as a loading control. Data are presented as the mean ± SEM (n = 3 unless otherwise indicated). * p < 0.05, ** p < 0.01.

Fig 3: piR-2158 inhibited IL11 expression by competing with transcriptional factor FOSL1 to bind the promoter of IL11. A: BLAST analysis identified two potential binding sites (b1 and b2) of piR-2158 in the promoter region of IL11, in which the first binding site b1 also belongs to the binding site of FOSL1 (indicated with red color of font). B: ChIP-seq analyses of FOSL1 in two breast cancer cell lines (MDA-MB-231 and BT549) both indicated a binding peak in the b1 region of IL11 promoter. C: Luciferase reporter assays confirmed suppression of IL11 promoter by piR-2158, while activation of IL11 promoter by FOSL1. piR-2158 overexpression attenuated FOSL1-induced IL11 expression. D: Schematic representation of the two truncated luciferase reporters of IL11 promoter carrying with b1 (F1) or both b1 and b2 (F2). E: Luciferase reporter assays showing suppression of IL11 promoter activity by piR-2158 through both b1 and b2 sites. Data are presented as the mean ± SEM (n = 3). ** p < 0.01.

Fig 4: Tumor-targeted therapy of breast cancer with nanotechnology-based piR-2158. A: Schematic representation of treatment of mammary tumor-burden mice with hyaluronic acid (HA)-coated, piR-2158-preloaded magnetic nanoparticles (MNPs). B: Tumor growth curves showing decreased tumor growth rate in the mice treated with piR-2158-MNPs (n = 10). C: Tumor Images from the mice in A. D: Quantitative analysis of the tumor weight in C (n = 10). E, F: QRT-PCR analysis demonstrating upregulation of piR-2158 (E) and downregulation of IL11 (F) in mammary tumors from the mice treated with piR-2158-MNPs, compared to NC group (n = 6). G: Immunohistochemical staining of ki67 in mammary tumors showed inhibition of cell proliferation by piR-2158-MNPs in vivo (n = 6). H: Immunohistochemical staining of CD31 in the mammary tumors showed decreased angiogenesis upon treatment with piR-2158-MNPs (n = 3). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.

Fig 5: Schematic representation of the working model in which piR-2158 competed with AP-1 transcription factor subunit FOSL1 to suppress IL11 expression and IL-11 secretion, thereby inactivating JAK-STAT3 signaling to inhibit tumorigenesis in breast cancer. Meanwhile, piR-2158-IL11 targeted endothelial cells to inhibited angiogenesis as well.