MiR-21-5p Promotes Osteogenic Differentiation and Calcification of Valvular Interstitial Cells by Targeting TGFBI in Calcific Aortic Valve Disease

Figures

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  Fig. 1:. The expression of miR-21-5p is upregulated in calcified AVs. The expression of miR-21-5p in tissue samples of calcific aortic valves (n=15) and their pair-matched adjacent normal tissues (n=10) was detected by RT-qPCR. Data are presented as mean±standard deviation and compared using the unpaired Student’s t test (P<0.0001)

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  Fig. 2:. The level of miR-21-5p was possibly correlated with primary VICs differentiation and calcification. (A) MiR-21-5p levels were detected by RT-qPCR after primary VICs were transfected with miR-21-5p mimic, miR-21-5p inhibitor and their corresponding negative controls (miR-NC mimic and inhibitor). (B,C) Representive western blotting images of the expression of BMP2 and osteocalcin in transfected VICs and the grey values of the bands were anaylzed for quantification. (D) Alizarin red staining (200X) of transfected VICs for calcium nudes dectection indicated the undergoing osteogenic transition. (E) ALP staining (200X) of transfected VICs for ALP acvtivites. All the representive images were from three independent experiments for each assay. ***P<0.001. Scale bar=50 μm

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  Fig. 3:. The expression of TGFBI was deregulated in calcified AV compared with paired normal tissues. (A) Representive images of western blotting for TGFBI after primary VICs were transfected with miR-21-5p mimic, miR-21-5p inhibitor and their corresponding negative controls (miR-NC mimic and inhibitor). GAPDH was used as loading control. (B) The band grey values were anaylzed by Image J. *P<0.05,**P<0.01. (C) Relative mRNA expression levels of TGFBI in VICs transfected with different miRNAs as above. *P<0.05,**P<0.01. (D) The sequences of binding site of TGFBI 3’-UTR with miR-21-5p based on the prediction of TargetScan 7.0. (E) Duel-Luciferase reporter assay were performed to verify correlation between miR-21-5p and TGFBI. The results were expressed as mean±SD based on three independent experiments. **P<0.01, ***P<0.001. WT, wild-type; MUT, mutant

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  Fig. 4:. The osteoblastic differentiation and calcification degree of VICs were promoted by miR-21-5p via suppressing TGFBI expression. Primary VICs were transfected with miR-NC mimic + oe-NC plasmid; miR-NC mimic+oe-TGFBI plasmid; miR-21-5p mimic+oe-NC plasmid; and miR-21-5p mimic+oe-TGFBI plasmid, respectively. (A,B) The biomarker protein levels of osterblatic differentiation (BMP2 and osteocalcin) with different treatment were analyzed by western bloting assay. (C) and (D) Representative staining images for calcium nudes formation and ALP activities were obtained from transfected VICs as above. Data were shown as mean±SD from at least three independent experiments. *P <0.05, **P<0.01, ***P<0.001. Scale bar=50 μm