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. 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. 4. miR-34a-3p is decreased in PAH and is a negative regulator of MiD49 and MiD51.

Fig. 4

(A) miRNA expression profiling in PAH and normal PASMCs. Volcano plot showing expression change of miRNAs in PAH relative to control samples. Each dot represents one probe set. Red: reduction; green: increase; n=3 for normal PASMC and n=6 for PAH PASMC.

(B) In silico prediction of miR-34a-3p targeting MiD49 and MiD51. (i) The putative binding site of miR-34a-3p on the 3′-UTR of MiD49 as predicted by nucleotide BLAST. (ii) Nucleotide sequences of MiD49 3′-UTR and miR-34a-3p. The predicted binding site is highlighted in red. Nucleotide positions are indicated in parentheses. (iii) The two putative binding sites of miR-34a-3p on the 3′-UTR of MiD51. (iv) Nucleotide sequences of MiD51 3′-UTR and miR-34a-3p. Predicted target sites are highlighted in blue. Nucleotide positions are indicated in parentheses. The expect value is calculated by BLAST to describe the number of hits one can “expect” to see by chance. The lower the expect value, the more significant the match.

(C) miR-34a-3p is decreased in PAH PASMC. Quantification of miR-34a-3p was performed by qRT-PCR (**P < 0.01; n=4 for normal PASMC and n=6 for PAH PASMC).

(D) miR-34a-3p binds to 3′-UTRs of MiD49 and MiD51. miR-34a-3p was found to repress the activity of luciferase reporter, indicating its binding to the 3′-UTRs of MiD49 and MiD51 genes (**P < 0.01; n=5 and 8 for MiD49 and MiD51, respectively).

(E) miR-34a-3p is decreased in whole blood and plasma from IPAH patients. miR-34a-3p expression from whole blood and plasma from two cohorts were normalized to U6 and miR-23-3p, respectively, and analyzed by qRT-PCR (*P < 0.05, ***P < 0.001; n=11–29 for healthy volunteer and n=14–39 for IPAH patient).

(F) miR-34a-3p identified patients with IPAH. Receiver operating characteristic (ROC) curves showing sensitivity and specificity of whole blood and plasma miR-34a-3p for differentiating patients with IPAH from healthy volunteers at the time of diagnosis (Sheffield whole blood: AUC=0.7857, P = 0.0160; Beijing whole blood: AUC=0.807, P = 0.0002; Sheffield plasma: AUC=0.7573, P = 0.0010; Beijing plasma: AUC=0.8846, P < 0.0001).