miR-376a inhibits glioma proliferation and angiogenesis by regulating YAP1/VEGF signalling via targeting of SIRT1

Abstract

Background: Glioma is the most common cancer in the central nervous system. Previous studies have revealed that the miR-376 family is crucial in tumour development; however, its detailed mechanism in glioma is not clear.

Methods: Cellular mRNA or protein levels of miR-376a, SIRT1, VEGF and YAP1 were detected via qRT-PCR or Western blotting. We analysed the proliferation, angiogenesis and migration abilities of glioma cell lines using colony formation, tube formation and Transwell assays. A luciferase assay was performed to determine whether miR-376a could recognize SIRT1 mRNA. Xenograft experiments were performed to analyse the tumorigenesis capacity of glioma cell lines in nude mice. The angiogenesis marker CD31 in xenograft tumours was detected via immunohistochemistry (IHC).

Results: miR-376a expression was lower in glioma cells than in normal astrocytes. miR-376a mimic inhibited SIRT1, YAP1, and VEGF expression and suppressed the proliferation, migration and angiogenesis abilities of the glioma cell lines LN229 and A172, whereas miR-376a inhibitor exerted the opposite functions. In a luciferase assay, miR-376a inhibited the luciferase activity of WT-SIRT1. SIRT1 overexpression upregulated YAP1 and VEGF in glioma cells and promoted proliferation, migration and angiogenesis. Xenografts with ectopic miR-376a expression exhibited lower volumes and weights and a slower growth curve. Overexpression of miR-376a inhibited YAP1/VEGF signalling and angiogenesis by inhibiting SIRT1 in xenograft tissues.

Conclusion: miR-376a directly targets and inhibits SIRT1 in glioma cells. Downregulation of SIRT1 resulted in decreased YAP1 and VEGF signalling, which led to suppression of glioma cell proliferation, migration and angiogenesis.

Keywords: Angiogenesis; Glioma; SIRT1; YAP/VEGF signalling; miR-376a.

Fig. 1

miR-376a regulated the proliferation, migration and angiogenesis of glioma cells. (A) The expression level of miR-376a was detected in NHA, SVGP12, U373, U251, LN229 and A172 cells. (B) The expression level of miR-376a was detected via qRT–PCR in LN229 and A172 cells transfected with a miR-376a mimic or inhibitor. (C, D and E) The glioma cell lines A172 and LN229 were transfected with miR-376a mimic or inhibitor as indicated and subjected to a clone formation assay (C), Transwell assay to assess cell migration (D) and angiogenesis assay (E). The statistical analysis is displayed in the columns. *P< 0.05, **P< 0.01, and ***P< 0.001.

Fig. 2

miR-376a directly regulated the expression of SIRT1 in glioma cells. (A) Schematic showing the binding sequence between miR-376a and the 3′-UTR of SIRT1. (B) The glioma cell lines A172 and LN229 were transfected with a SIRT1-WT or SIRT1-MUT dual-luciferase reporter system and subjected to a dual-luciferase reporter assay. (C) The mRNA level of SIRT1 was detected via qRT–PCR in the glioma cell lines A172 and LN229 after transfection with miR-376a mimic or inhibitor. (D) The protein level of SIRT1 was detected via WB in the glioma cell lines A172 and LN229 transfected with miR-376a mimic or inhibitor. *P< 0.05, **P< 0.01, and ***P< 0.001.

Fig. 3

Overexpression of SIRT1 antagonized the effects of miR-376a in glioma cells. (A) The mRNA level of SIRT1 was analysed via qRT–PCR in the glioma cell lines A172 and LN229 transfected with SIRT1 overexpression vector. (B) The protein level of SIRT1 was analysed via Western blotting in LN229 and A172 cells transfected with SIRT1 overexpression vector. (C-E) The glioma cell lines A172 and LN229 were transfected with miR-376a mimic or a SIRT1 overexpression vector as indicated. Clone formation assays (C), Transwell assays (D) and angiogenesis assays (E) were then performed in these cells. *P< 0.05, **P< 0.01, and ***P< 0.001.

Fig. 4

miR-376a regulated the YAP1/VEGF signalling axis by inhibiting SIRT1 in glioma cells. (A) The mRNA levels of YAP1 and VEGF were detected by qRT–PCR in the glioma cell lines A172 and LN229 transfected with miR-376a mimic. (B) The mRNA levels of YAP1 and VEGF were detected by qRT–PCR in the glioma cell lines A172 and LN229 transfected with miR-376a inhibitor. (C) The protein levels of YAP1 and VEGF were detected by Western blotting in the glioma cell lines A172 and LN229 transfected with miR-376a mimic or inhibitor. The mRNA (D) and protein (E) levels of YAP1 and VEGF were detected by qRT–PCR (D) and Western blotting (E) in the glioma cell lines A172 and LN229 transfected with SIRT1 overexpression vector. *P< 0.05, **P< 0.01, and ***P< 0.001.

Fig. 5

miR-376a inhibited tumorigenesis and angiogenesis in nude mice by regulating the SIRT1/YAP1/VEGF signalling axis. (A) Representative images of tumours from nude mice after subcutaneous injection of A172 cells stably expressing miR-376a mimic or mimic NC (5 mice per group). (B) Quantifications of tumour volume. (C) Quantification of tumour weight. (D) The expression levels of miR-376a, SIRT1, YAP1 and VEGF in tumour tissues were detected by qRT–PCR (n = 5). (E) The protein levels of SIRT1, YAP1 and VEGF in tumour tissues were detected by Western blotting (n = 5). (F) Immunohistochemistry analysis of CD31 levels in tumour tissues from mice in different groups. *P< 0.05, **P< 0.01, and ***P< 0.001.