MicroRNA-21 deficiency suppresses prostate cancer progression through downregulation of the IRS1-SREBP-1 signaling pathway

Abstract

Sterol regulatory element-binding protein 1 (SREBP-1), a master transcription factor in lipogenesis and lipid metabolism, is critical for disease progression and associated with poor outcomes in prostate cancer (PCa) patients. However, the mechanism of SREBP-1 regulation in PCa remains elusive. Here, we report that SREBP-1 is transcriptionally regulated by microRNA-21 (miR-21) in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of SREBP-1, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) in Pten/Trp53 double-null mouse embryonic fibroblasts (MEFs) and Pten/Trp53 double-null mutant mice. Strikingly, miR-21 loss significantly reduced cell proliferation and suppressed the prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, miR-21 inactivation decreased the levels of SREBP-1, FASN, and ACC in human PCa cells through downregulation of insulin receptor substrate 1 (IRS1)-mediated transcription and induction of cellular senescence. Conversely, miR-21 overexpression increased cell proliferation and migration; as well as the levels of IRS1, SREBP-1, FASN, and ACC in human PCa cells. Our findings reveal that miR-21 promotes PCa progression by activating the IRS1/SREBP-1 axis, and targeting miR-21/SREBP-1 signaling pathway can be a novel strategy for controlling PCa malignancy.

Keywords: ACC; FASN; Fatty acid signaling and mouse models; PTEN; TP53.

Fig. 1.

miR-21 knockout suppresses prostate tumorigenesis of Pten-Trp53 mutant mice. (A) Biopsies of anterior prostates (AP) from Wt, miR-21, Pten-Trp53, Pten-Trp53-miR-21 mice at 6 months of age. (B) The weight distribution of AP prostates or tumors in mice from (A). N indicates the mouse number. (C) AP weight/body weight ratio for the indicated genotypes at 6 months of age. (D) Cumulative survival of indicated genotypes of mice at specified age. (E) Representative images of H&E staining show the pathological changes of indicated genotypes of mice from (A). (Magnification: 4X and 20X). Wt represents wild type mice, miR-21 represents miR-21^−/− mice, Pten-Trp53 represents Pten^pc−/−,Trp53^pc−/− mice; Pten-Trp53-miR-21 represents Pten^pc−/−, Trp53^pc−/−, Mmu-miR-21^−/− mice. Comparison between groups was performed using two-tailed Student’s t-test. NS-not significant, *P < 0.05 and **P < 0.01, ***P < 0.005.

Fig. 2.

miR-21 inactivation impairs the IRS1/SREBP-1 signaling in vitro in MEFs. (A) Effects of miR-21 inactivation on the cell proliferation of MEFs. (B) Quantification analysis of the relative expression levels of IRS1, SREBF1, FASN, and ACACA genes from the indicated genotypes of MEFs. (C) Western blotting analysis of IRS1, SREBP-1, FASN, and ACC and β-galactosidase (β-gal) protein levels in the indicated genotypes of MEFs. (D) Quantification analysis of IRS1, SREBP-1, FASN, and ACC protein levels in the indicated genotypes of MEFs. (E) Immunofluorescence (IF) images showing the subcellular locations of SREBP-1, FASN, and ACC protein and the quantification analysis in the indicated genotypes of MEFs. (F) Upper panel: Western blotting analysis showing the differential changes of IRS1, SREBP-1, FASN, and ACC protein levels to IGF-1 stimulation in PP-m21^+/+ and PP-m21^−/− MEFs. Lower panel: Quantification of protein levels for IRS1, SREBP-1, FASN, and ACC in the upper panel. PP-m21^+/+ represents Pten^Δ/Δ-P53^{Δ/Δ _}Mmu-miR-21^+/+, PP-m21^+/− represents Pten^Δ/Δ-P53^Δ/Δ-Mmu-miR21^+/− , and PP-m21^−/− represents Pten^Δ/Δ-P53^Δ/Δ-Mmu-miR21^−/− . Error bars are representative of the SEM in triplicate. Comparison between groups was performed using two-tailed Student’s t-test. *P < 0.05 and **P < 0.01, ***P < 0.005.

Fig. 3.

miR-21 knockout reduces adipose deposition in mice by impairing IRS1/SREBP-1 signaling. (A) Body weights of Pten-Trp53, Pten-Trp53-miR-21^+/− , and Pten-Trp53-miR-21^−/− mutant mice at indicated ages (n = 10). (B) Biopsies of fat tissues accumulated in mice with indicated genotypes at 6 months of age. (C) The quantification analysis of fat tissues from (B). (D) The comparison of IRS1, SREBF1, FASN, and ACACA gene expression in liver tissues of mice at indicated genotypes (n = 4). (E) Western blotting analysis of IRS1, SREBP-1, FASN, and ACC protein levels in liver tissues of mice with indicated genotypes. Two mouse liver samples for each genotype. (F) Quantification analysis of the protein expression from the panel (E), the quantification analysis is the mean for n = 3, but 2 distinct samples were chosen as representatives. Error bars are representative of the SEM in triplicate. (G) Western blotting analysis of IRS1, SREBP-1, FASN, and ACC protein levels in prostate tissues of mice with indicated genotypes. Two mouse prostate samples for each genotype. (H) Quantification analysis of the relative expression protein levels from (G). (I) IHC staining of SREBP-1 in prostate tissues of the indicated mice as well as the quantification analysis. Comparison between groups was performed using two-tailed Student’s t-test. *P < 0.05 and **P < 0.01, ***P < 0.005.

Fig. 4.

miR-21 overexpression increases the IRS1/SREBP-1 signaling in prostate cells. (A) Relative miR-21 expression in human prostate cells. LNCaP, PC3, 22Rv-1, and C4–2B are prostate cancer cell lines, and RWPE1 is a non-tumorigenic cell line. (B) Quantitative RT-PCR analysis to show the miR-21 expression in RWPE1 cells upon miR-21 overexpression (OE). (C) Relative expression of IRS1, SREBF1, FASN, and ACACA genes in RWPE1 cells upon miR-21 OE. (D) Western blotting analysis showing the increased levels of IRS1, SREBP-1, FASN, and ACC in RWPE1 cells upon miR-21 OE. (E) Quantification analysis of the protein levels from (D). (F) Quantitative RT-PCR analysis to show the miR-21 expression in miR-21 OE LNCaP clones. (G) Effects of miR-21 OE on the cell proliferation of LNCaP cells. (H) Quantitative RT-PCR analysis to show the relative expression of IRS1, SREBF1, FASN, and ACACA genes in miR-21 OE LNCaP cells. (I) Western blotting analysis to show the levels of IRS1, SREBP-1, FASN, and ACC proteins in miR-21 OE LNCaP cells. (J) Quantification analysis of the protein levels from (I). (K) Western blotting analysis showing the differential changes of IRS1, SREBP-1, FASN, and ACC protein levels in C4–2B cells to IGF stimulation. (L) Quantification analysis of the protein levels from (K). Comparison between groups was performed using two-tailed Student’s t-test. *P < 0.05 and **P < 0.01, ***P < 0.005. @ denotes not significant.

Fig. 5.

miR-21 inhibition decreases the IRS1/SREBP-1 signaling in human prostate cancer cells. (A) Effects of miR-21 inhibition on the viability of C4–2B cells. (B) Quantification of relative miR-21 expression levels after miR-21 inhibition from (A). (C–E) Effects of miR-21 inhibition on cell invasion (C), cell migration (D), and colony formation (E) in C4–2B cells. (F) Quantitative RT-PCR analysis to show the effects of miR-21 inhibition on the expression of IRS1, SREBF1, FASN, and ACACA genes in C4–2B cells. (G) Western blotting analysis showing the effects of miR-21 inhibition on IRS1, SREBP-1, FASN, and ACC protein levels in C4–2B cells. (H) Quantification analysis of the protein levels from (G). (I) Oil red staining showing the effects of miR-21 inhibition on the levels of lipid deposits in C4–2B cells. (J) Working model of miR-21 regulation on fatty acid signaling towards tumor cell proliferation. Error bars are representative of the SEM in triplicate. Comparison between groups was performed using two-tailed Student’s t-test. *P < 0.05 and **P < 0.01, ***P < 0.005.