Long noncoding RNA SNHG1 alleviates high glucose-induced vascular smooth muscle cells calcification/senescence by post-transcriptionally regulating Bhlhe40 and autophagy via Atg10

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  Fig. 1. LncRNA SNHG1 is downregulated in HG-induced HA-VSMCs. A Microarray heatmaps of DEGs between HG-induced cells from NG-induced cells. The color “green” and “red” denote low and high expression, respectively. B Venn diagrams of the overlapping DEGs. Three lncRNAs were downregulated in HG-induced HA-VSMCs as compared to that in NG group. C Relative expression levels of lncRNAs SNHG1, CYP1B1-AS1 and LINC01356 in HG and NG groups were quantified by qRT-PCR. D–G The effects of these three lncRNAs on HA-VSMCs calcification/senescence and contractile phenotype. HA-VSMCs were treated with NG, NG with si-CYP1B1-AS1, NG with si-LINC01356, and NG with si-SNHG1, respectively. D The protein levels of p16, p21, Runx2, and ALP were determined by Western blot in the above four groups; GAPDH was loading control. Representative images of (E) SA-β-gal staining and (F) Alizarin Red S staining in HA-VSMCs under conditions of NG with si-NC or NG with si-SNHG1. Calcium content was extracted with cetylpyridinium chloride and quantified by spectrophotometry; the red area indicated by the arrow is the calcium accumulation. Semiquantitative analysis of SA-β-gal-positive cells was performed using Image J (200…

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  Fig. 2. SNHG1 positively regulates Bhlhe40 expression in HA-VSMCs. A The overexpression (SNHG1) and knockdown (si-SNHG1) efficiencies of SNHG1 were assessed by qRT-PCR. B Volcano plot of DEGs between SNHG1-overexpressed HA-VSMCs from control group. C GO analysis of the DEGs. D Bhlhe40 mRNA levels were evaluated in indicated transfectants using qRT-PCR. E Cell extracts underwent Western blot for determination of Bhlhe40 protein expression; GAPDH was loading control. Results shown are means ± SD from triplicate experiments. **, P < 0.01; ***, P < 0.001

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  Fig. 3. SNHG1 stabilizes Bhlhe40 mRNA via RNA-RNA interaction. A Subcellular localization of SNHG1 was detected by RNA-FISH assay in HA-VSMCs; SNHG1 probes are red; Bhlhe40 probes are green (200 × magnification), scale bar = 100 µm. B Nuclear and cytoplasmic fractions of HA-VSMCs were subjected to qRT-PCR. U1 is the nuclear (Nul) positive control; β-actin is the cytoplasmic (Cyt) positive control. C Schematic diagram of the predicted interaction sequences between SNHG1 and Bhlhe40 mRNA by IntaRNA. D The RNA-RNA interaction between Bhlhe40 mRNA and SNHG1 was detected by RNA pull-down assay using in vitro transcribed biotin-labeled SNHG1. The retrieved RNA was quantified by qRT-PCR and displayed as percentage of input RNA. E, F After transfecting SNHG1 overexpression plasmid (E) or specific si-SNHG1 (F) into HA-VSMCs, the stability of Bhlhe40 mRNA over time was measured by qRT-PCR relative to time 0 after blocking new RNA synthesis with ActD (5 μg/ml) and normalized to 18S rRNA. Results shown are means ± SD from triplicate experiments. **, P < 0.01; ***, P < 0.001

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  Fig. 4. SNHG1 effectively promotes Bhlhe40 protein nuclear translocation by enhancing the SUMOylation of Bhlhe40 at K279. A Immunofluorescence assay of the localization of Bhlhe40 protein (green) in HA-VSMCs transfected with empty vector (Con) or pcDNA3.1-SNHG1 (SNHG1, 200 × magnification), scale bar = 100 µm. B The expression of Bhlhe40 protein in nucleus (Nul) and cytoplasm (Cyt) of SNHG1-overexpressing HA-VSMCs were shown by Western blot. Nuclear segregation is assayed by Histone H3; cytoplasmic segregation is assayed by β-actin. C Immunoblot analysis was performed to detect the modification of Bhlhe40 by SUMO1 after SNHG1 overexpression. Cells lysates were subjected to immunoprecipitation by anti-GFP followed by immunoblot with anti-Flag or anti-His. D Immunoblot analysis of the SUMOylation of Bhlhe40 in HA-VSMCs transfected with Bhlhe40-WT or point mutants (K159R, K279R). Cells lysates were subjected to immunoprecipitation by anti-GFP followed by immunoblot with anti-Flag or anti-His. E Immunofluorescence assay was used to detect the location of Bhlhe40 protein in HA-VSMCs (200 × magnification), scale bar = 100 µm. The cells were transfected with pcDNA3.1-SNHG1, Bhlhe40-WT or point…

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  Fig. 5. SNHG1 functions as a scaffold for Bhlhe40/PIAS3 to regulate Bhlhe40 SUMOylation. A Western blot analysis of the proteins retrieved from the SNHG1 pull-down assay using anti-Bhlhe40 and anti-PIAS3. B RIP assays were performed using anti-Bhlhe40 or anti-PIAS3 antibodies in HA-VSMCs. qRT-PCR was used to measure the enrichment of SNHG1. C Online-predicted binding sites between SNHG1 and Bhlhe40 protein on CatRAPID database. D RNA pull-down assays were used to examine the interaction between Bhlhe40 and the different mutants of SNHG1. Results shown are means ± SD from triplicate experiments

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  Fig. 6. SNHG1 regulates Bhlhe40 mediated Atg10 transcription inhibition. A Western blot was performed to detect the levels of autophagy markers in HA-VSMCs after transfection with pcDNA3.1-SNHG1. B mRFP-GFP-LC3 distribution in HA-VSMCs transfected with mRFP-GFP-LC3 and pcDNA3.1-SNHG1 were analyzed by confocal microscopy (200 × magnification), scale bar = 100 µm. C The protein levels of Atg3, Atg5, Atg7 and Atg10 were measured in HA-VSMCs after transfection of si-SNHG1 by Western blot. D The binding sites of Bhlhe40 on Atg10 promoter region and the binding sequence was obtained using the JASPAR website. E ChIP assays were performed to verify the reliability of the binding sequence of Bhlhe40 on Atg10 promoter region. F HA-VSMCs were transfected with luciferase reporter carrying WT-pGL3-Atg10 or Mut-pGL3-Atg10 and cotransfected with the adenovirus vector expressing Bhlhe40 (Bhlhe40) or not (Con). Firefly luciferase values, normalized for Renilla luciferase, are presented. G qRT-PCR analysis of Atg10 in HA-VSMCs transfected with the adenovirus vector expressing Bhlhe40 or si-Bhlhe40. H HA-VSMCs were transfected with Atg10 overexpression plasmid (Atg10) or negative control (Con),…

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  Fig. 7. A model for the regulatory mechanisms of SNHG1 in HG-induced HA-VSMCs calcification/senescence