miR-22-3p/PGC1 β Suppresses Breast Cancer Cell Tumorigenesis via PPAR γ

Abstract

In this study, we found that miR-22-3p expression was decreased in breast cancer (BC) cell lines and tissues. Overexpression of miR-22-3p inhibited the proliferation and migration of BC cells in vitro and in vivo, while depletion of miR-22-3p exhibited the opposite effect. Importantly, miR-22-3p could directly target PGC1β and finally regulate the PPARγ pathway in BC. In conclusion, miR-22-3p/PGC1β suppresses BC cell tumorigenesis via PPARγ, which may become a potential biomarker and therapeutic target.

Figure 1

miR-22-3p was decreased in BC cell lines and tissues. (a, b) miR-22-3p had low expression in BC tissues compared with adjacent normal tissues. (c) miR-22-3p had low expression in BC cell lines. (d) Detection of colocalization of miR-22-3p in cytoplasm by RNA FISH assay (magnification, ×400). Red, miR-200a-3p; blue, DAPI. ^∗∗p < 0.1; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 2

miR-22-3p suppressed cell proliferation of BC cells. (a, b) Expression of miR-22-3p was confirmed by RT-qPCR in MDA-MB-231 and MCF-7 cells. (c, d) Effect of miR-22-3p on proliferation in MDA-MB-231 and MCF-7 cells by MTT assay. (e, f) Effect of miR-22-3p on proliferation in MDA-MB-231 and MCF-7 cells by colony formation assay. (g, h) Effect of miR-22-3p on proliferation in MDA-MB-231 and MCF-7 cells by western blotting. ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 3

miR-22-3p suppressed cell migration of BC cells. (a–c) Wound healing assays were performed in MDA-MB-231 cell line treated with miR-22-3p mimics or miR-22-3p inhibitor (miR-NC as negative control). (d–f) Cell migration assays were performed in MDA-MB-231 cell line treated with miR-22-3p mimics or miR-22-3p inhibitor (miR-NC as negative control). ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 4

PGC1β is a direct target of miR-22-3p. (a, c) Putative complementary sites within miR-22-3p and PGC1β predicted by bioinformatics analysis (TargetScan). (b, d) Dual-luciferase reporter assays demonstrated that PGC1β is a direct target of miR-22-3p. (e) PGC1β mRNA level was determined by RT-PCR in MDA-MB-231 and MCF-7 cells with different treatment. (f–h) Representative western blots and quantification of PGC1β and PPARγ in MDA-MB-231 and MCF-7 cells with different treatment. β-Actin was used as an internal control. ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 5

miR-22-3p suppressed the proliferation and migration of BC cells via PGC1β. (a–d) Knockdown of PGC1β partially reversed miR-22-3p inhibitor-induced promotion of proliferation in MDA-MB-231 and MCF-7 cells determined by MTT assay and colony assay. (e, f) Knockdown of PGC1β partially reversed miR-22-3p inhibitor-induced promotion of migration in MDA-MB-231 and MCF-7 cells determined by transwell assay. (g–i) Western blotting analysis for PGC1β/PPARγ protein level in MDA-MB-231 and MCF-7 cells. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 6

Inhibition of PPARγ attenuates suppression of miR-22-3p on BC cells. (a) Upregulated miR-22-3p increased the expression of PPARγ and decreased the expression of PGC1β, C-myc, NF-κB, CyclinD1, cyclin E, MMP2, and MMP9. (b) Downregulation of C-myc, NF-κB, CyclinD1, cyclin E, MMP2, and MMP9 induced by miR-22-3p was inverted by PPARγ inhibition (GW9662).

Figure 7

miR-22-3p suppressed BC tumor growth in vivo. (a) Overexpression of miR-22-3p in MDA-MB-231 cells was verified by RT-qPCR. (b) Representative images of xenograft tumors in nude mice. (c) The growth curves of xenografts. (d) Extract protein from tumors and measuring the expression of PGC1β/PPARγ by western blotting. (e, f) Immunohistochemistry (IHC) staining of PGC1β/PPARγ in xenografts. (g) The mechanism diagram was generated to illustrate the mechanism of miR-22-3p-PGC1β-PPARγ in BC. ^∗p < 0.05; ^∗∗∗∗p < 0.0001.