Skip to main content
. Author manuscript; available in PMC: 2019 Jul 17.
Published in final edited form as: Circulation. 2018 Feb 5;138(3):287–304. doi: 10.1161/CIRCULATIONAHA.117.031258

Fig. 5. Increasing expression of miR-34a-3p downregulates MiD49 and MiD51 in PAH PASMC; administering anti-miR-34a-3p upregulates MiD49 and MiD51 in normal PASMC.

Fig. 5

(A) Overexpression of miR-34a-3p downregulates MiD49 and MiD51. Representative images of immunoblots and densitometries showing the expressions of MiD49 and MiD51 in PAH PASMC transfected with miR-34a-3p. Cells were transfected with miR-34a-3p for 72h. β-actin was used as the loading control (*P < 0.05; n=3–4/group).

(B) Anti-miR-34a-3p treatment upregulates MiD49 and MiD51 in normal PASMC. Representative images of immunoblots and densitometries showing the expressions of MiD49 and MiD51 in normal human PASMC transfected with anti-miR-34a-3p for 72h. β-actin was used as the loading control (*P < 0.05; n=4/group).

(C–H) Overexpression of miR-34a-3p inhibits mitochondrial fission, cell proliferation and induces apoptosis in PAH PASMC.

(C) Representative images of mitochondrial networks of PAH PASMC transfected with miR-34a-3p mimic. The cells were also infected with Adv-mNeon Green and imaged 48h following infection. Mitochondria were color coded by their morphology: red: punctate; green: intermediate; purple: filamentous. Scale bar: 10μm.

(D) Mitochondrial fragmentation was quantified by mitochondrial fragmentation count (MFC) and percentage of area of punctate, intermediate and filamentous mitochondria (***P < 0.001; n=16–20/group).

(E) Mitochondrial network is restored in PAH PASMC by augmenting miR-34a-3p. Representative images of the photoactivation experiments confirmed the increase in mitochondrial network in PAH PASMCs co-transfected with miR-34a-3p and mitochondrial matrix targeted green fluorescent protein (mito-PAGFP) plasmid for 48 h. The cells were also loaded with the potentiometric dye TMRM (red). Scale bar: 10μm.

(F) Quantification of mitochondrial network in PAH PASMC by augmenting miR-34a-3p. Mitochondrial network is quantified by determining mitochondrial networking factor (MNF) which is increased in PAH PAMC following transfection with miR-34a-3p mimic. (*P < 0.05; n=6/group; AU: arbitrary unit).

(G) Augmenting miR-34a-3p inhibits proliferation of PAH PASMC. Cell proliferation was analyzed 72h following miR-34a-3p mimic transfection in PAH PASMC (*P < 0.05; n=3/group).

(H) Augmenting miR-34a-3p induces apoptosis of PAH PASMC. PAH PASMC transfected with miR-34a-3p mimic. Apoptosis was assessed by measuring the activity of caspase3/7 48h following transfection with miR-34a-3p mimic (n=2 IPAH PASMC lines/group).