Table 1.
Primer sequences used for qRT-PCR.
Table 2.
Clinical monitoring of mice at 2 months following streptozotocin treatment.
Fig 1.
Diabetes causes alteration in expression of noncoding RNAs.
Assessment of A) microRNA-9 and B) circRNA_01216 levels showed decreased expression of miR-9 and increased expression of circRNA_012164 in male diabetic mice compared to age- and sex-matched non-diabetic controls. Female mice showed similar changes (S1 Fig). (Data are expressed as ± SEM and normalized to U6 or Actb. * = significantly different. n = 7/group).
Fig 2.
Endothelial specific microRNA-9 overexpression prevented diabetes induced cardiac abnormalities.
Echocardiographic analysis showed significant reduction of the A) E/A ratio of wild-type diabetic (B6-D) compared to wild-type non-diabetic (B6-C) mice. This reduction was prevented in endothelial specific miR-9 overexpressing transgenic mice. No phenotypic changes were observed in A) E/A ratio, B) fractional shortening, or C) ejection fraction between B6-C and either miR-9 transgenic groups. Representative echocardiography tracings are shown: (top panel) end-systolic diameter and end-diastolic diameter, (middle panel) pulsed wave Doppler recording of mitral inflow velocity, and (bottom panel) tissue Doppler imaging of the LV posterior wall of D) B6-C, E) B6-D, F) miR9-C, and G) miR9-D. Female mice showed similar changes (S2 Fig). (Data are expressed as mean ± SEM. * = significantly different from B6-C. n = 8/group).
Fig 3.
MicroRNA-9 overexpression prevented diabetes-induced cardiac fibrosis in mice.
Masson’s Trichrome staining showed cardiac fibrosis in B) wild-type diabetic (B6-D) compared to A) wild-type non-diabetic (B6-C) hearts, as indicated by arrows. Cardiac fibrosis was prevented in the D) diabetic microRNA-9 overexpressing (endothelial specific) transgenic mice (miR9-D). No differences were observed between A) B6-C and C) miR-9 transgenic non-diabetic (miR9-C) mice. mRNA expressions of E) Col1a1, F) Fn1, and G) S100a4 were upregulated in B6-D compared to B6-C hearts. These changes were prevented in miR-9 transgenic mice. mRNA expressions were normalized to Actb. Protein levels of H) COL1A1 and I) FN1 were similarly changed in B6-D and miR9-D groups. No differences were observed based on sex differences (S1 Fig). (Data are expressed as mean ± SEM. * = statistically significant. n = 6–8/group for mRNA analyses, n = 3/group for protein analyses).
Fig 4.
Knockdown of circRNA_012164 in endothelial cells prevents high glucose induced change in gene expression.
High glucose (30 mM, HG) treatment of mouse cardiac endothelial cells resulted in downregulation of A) miR-9 and upregulation of B) circRNA_012164 along with the downstream genes C) Acta2 D) Col1a1, E) S100a4, and F) Fn1. siRNA targeted to circRNA_012164 (si-circ) protected cells against HG-induced changes. Data normalized to U6 or Actb where applicable. (Data are expressed as mean ± SEM. * = p < 0.05, n = 5–6/group.) NG = normal glucose, 5 mM; scr = scrambled oligonucleotides.
Fig 5.
Knockdown of miR-9 negates the protective effects of circRNA_012164 knockdown in cardiac endothelial cells.
High glucose (30 mM, HG) treatment of mouse cardiac endothelial cells resulted in upregulation of A) Acta2 B) Col1a1, C) S100a4, and D) Fn1. Knockdown of circRNA_012164 (si-circ) prevented (HG) induced gene expression changes. Additionally inhibiting microRNA-9 (anti-m9) removed protective effect of si-circ and restored the HG phenotype. Data normalized to Actb. (Data are expressed as mean ± SEM. * = p < 0.05, n = 5–6/group.) NG = normal glucose, 5 mM; scr = scrambled oligonucleotides.