Demographic Data of patients:
Twenty-five children with PAH due to congenital left to right shunts were enrolled in the study. They were all symptomatic and under medication for heart failure. They were scheduled for surgery and till surgery, they continue to have anti-congestive treatment. Eight of these patients have an AVSD, twelve patients have a VSD and five patients have PDA. We have followed up on these patients at regular intervals. On each visit, we checked their signs and symptoms with physical examination, electrocardiogram, and echocardiography. Fifteen patients were female and ten were male. The median age of patients was 9.79 ± 8.21 months. The median age of the control group was 5,35 ± 3,64. Angiocardiography was performed before surgery and mPAP was measured. Fifteen patients had a mean of PAP > 40mmHg, and the rest 10 of them was between 25–40 mmHg. Tissue samples were taken from the pulmonary artery of all patients during the surgical correction.
Comparison of miRNA profiles of plasma samples preoperative patients with Control Group:
When blood miRNA expression profiles in blood samples before the operation were compared with the control group, it was determined that the expression level of 93 miRNAs increased compared to the control group, while the expression levels of 12 miRNAs decreased compared to the control group. In blood samples taken before surgery, it was determined that the expression levels of 12 miRNAs (miR-124-3p, miR-149-5p, miR-184, miR-200a-3p, miR-203a, miR-204-5p, miR-205-5p, miR-208b, miR-211-5p, miR-488-3p, miR-498, miR-885-5p) among the detected miRNAs increased more than 100 times compared to the control group. Among these miRNAs, miR-200a-3p, which increased 804 times preoperatively compared to the control group, draws attention (Fig. 1).
We have also compared the blood miRNA profiles of the patients whose PAP < 40mmHg and PAP > 40mmHg with the control group separately.
When the blood miRNA expression profile taken preoperatively from patients with PHT less than 40 mmHg was compared with the control group; It was determined that the expression level of 90 miRNAs increased in patients with PHT less than 40 mmHg, and the expression level of 16 miRNAs decreased compared to the control group. Among these 90 miRNAs, it was determined that the blood expression level of 13 (miR-124-3p, miR-149-5p, miR-184, miR-200a-3p, miR-203a, miR-204-5p, miR-205-5p, miR-208b, miR-211-5p, miR-488-3p, miR-498, miR-499a-5p, miR-885-5p) of them increased more than 100 times (p < 0.05). Among these miRNAs, miR-200a-3p was found to increase 1035 times more in blood of patients with PHT less than 40 mmHg compared to the control group (Fig. 2).
miRNA expression profile of preoperative blood samples from patients with PHT higher than 40 mmHg compared to the control group.
We have compared the blood miRNA profile of pulmonary hypertensive patients with mean PAP > 40mmHg preoperatively with the control group. We have found that the expression levels of 92 miRNA increased and 16 miRNA decreased according to the control group.
Compared to the control group, in blood, 13 miRNAs (miR-124-3p, miR-149-5p, miR-184, miR-200a-3p, miR-203a, miR-204-5p, miR-205-5p, miR-208b, miR-211-5p, miR-488-3p, miR-498, miR-656, miR-885-5p) increased more than 100 times (p < 0.05). Among these miRNAs, it was determined that miR-200a-3p increased 670 times more in the blood of patients with PHT higher than 40 mmHg compared to the control group.
When the data obtained pre and post-operative were evaluated in total, it was determined that 12 miRNAs showed a dynamic change in both the preoperative and postoperative groups in the blood (Fig. 4). However, in addition to these 12 miRNAs, miR-499a-5p increased 109 times in patients with pulmonary hypertension less than 40mmHg, while miR-656 increased 121 times in patients with pulmonary hypertension higher than 40mmHg.
miRNA expression profile of postoperative blood samples compared to the control group
When the miRNA expression profile of the postoperative blood samples was compared with the control group; It was determined that the expression of 9 miRNAs decreased and the expression of 1 miRNA increased compared to the control group (Fig. 5A).
When the miRNA expression profiles of the postoperative blood samples taken from patients with a PHT of less than 40 mmHg were compared with the control group, it was found that the expression of 8 miRNAs increased and the expression of 2 miRNAs decreased compared to the control group (Fig. 5B).
When the miRNA expression profiles of the postoperative blood samples taken from patients with PHT higher than 40 mmHg were compared with the control group; It was determined that the expression of 10 miRNAs was decreased compared to the control group (Fig. 5C) (p < 0.05).
The comparison of miRNA expression profiles of heart tissue samples from patients with a PHT > 40 mmHg compared to tissue samples from patients with a PHT < 40 mmHg.
When the miRNA expression profiles of heart tissue samples taken from patients with PHT > 40 mmHg compared to tissue samples taken from patients with PHT < 40 mmHg, were compared each other; it was determined that only the expression level of miR-218-5p decreased, and the levels of miR146b-5p, miR-144-3p also increased.
Pathway and cluster analyses of 12 miRNAs with dynamic changes in blood samples taken from patients pre and postoperative
In conclusion, we determined that expression levels of 12 miRNA showed dynamic changes in the pre-and post-operative blood samples of the patients. As a result of our analysis to determine the pathways associated with these miRNAs, we determined that these 12 miRNAs are associated with Hippo signaling, ECM (Extra Cellular Matrix) receptor interaction, Adherens junction, Fatty acid elongation, fatty acid metabolism, and fatty acid biosynthesis pathways (Fig. 7).
According to cluster analysis, 12 miRNAs identified were divided into 2 clusters in the upper class, in the first cluster, miR-200a-3p, miR-203a, miR-885-5p, miR-204-5p, miR-205-5p, miR-208b (miR-208b-5p, miR-208b-3p), miR-203a (miR-203a-3p, miR-203a-5p), miR-211-5p, miR-149-5p, miR-184 and miR-498, while the second cluster includes miR-124-3p and miR-488-3p was found to be involved. The first cluster is divided into 3 sub-clusters in itself. The first cluster includes miR-200a-3p, miR-885-5p, miR-204-5p, and miR-205-5p. The second cluster includes miR-208b (miR-208b-5p, miR-208b-3p) and miR- miR-203a (miR-203a-3p, miR-203a-5p). The third cluster includes miR-211-5p, miR-149-5p, miR-184 and miR-498 (Fig. 8).