Figure 1
Clinicopathological parameters involved in E, D, ED, and N groups. (A) The comparation of age distribution among E (essential hypertension), D (type 2 diabetes mellitus), ED (patients with essential hypertension and concomitant type 2 diabetes mellitus), and N (healthy donors) groups. (B-J) Comparations of clinicopathological parameters among N, E, D, and ED groups, including BMI (B), FBG (C), LDL-C (D), HDL-C (E), ALT (F), ALB (G), AST (H), TC (I), and TG (J). All data are shown as Mean±SEM. *, p<0.05; **, p<0.01; NS, not significant.
Figure 2
Comparation of plasma miRNA expression profile among patients and healthy donors. A Heatmap assay of the differentially expressed miRNAs in plasma samples of healthy donors (N-1, N-2, N-3, N-4, N-5), patients with essential hypertension (E-1, E-2, E-3, E-4, E-5), type 2 diabetes (D-1, D-2, D-3, D-4, D-5), patients with essential hypertension and type 2 diabetes mellitus (ED-1, ED-2, ED-3, ED-4, ED-5). B Hierarchical cluster analysis of plasma miRNA expression profile among patients (E, D, ED) and healthy donors (N). C-E The scatter plots illustration of differentially expressed miRNAs between the D and N groups (C), E and N groups (D), and ED and N groups (E). F-G Venn Map analysis of the distributions of the differentially downregulated (F) or upregulated (G) miRNAs in plasma samples among the indicated groups (N, E, D, and ED).
Figure 3
Plasma miR-195-5p expression was downregulated in the patients. A-D Validation of plasma levels of significantly downregulated miRNAs, including miR-197-5p (A), miR-130a-5p (B), miR-27a-5p (C), and miR-195-5p (D) by miRNA chip (A) and qRT-PCR. U6 was used as an internal control. All data are shown as the mean±SEM (n=5), *p<0.05; NS, not significant.
Figure 4
DRD1 functions as the direct inhibitory target of miR-195-5p. A Validation of plasma levels of DRD1 by qRT-PCR. B Fold change of DRD1 mRNA expression in HEK-293T cells after transfection of miR-195-5p mimic or miR-195-5p inhibitor for 48h. Control mimics and inhibitor control mimics served as negative controls of miR-195-5p mimics and miR-195-5p inhibitors, respectively. C-D Expression levels of DRD1 proteins in the aforementioned four groups were quantified by western-blot assay (C), and gray-scale scanning with ImageJ software (D). GAPDH was used as an internal control. All data are shown as the mean±SEM (n=3), *p<0.05. E Putative wildtype (WT) and mutant type (MUT) sequences of miR-195-5p binding sites in the DRD1 3′ UTR. F Luciferase reporter assay of the inhibitory activity of miR-195-5p upon transfection with DRD1 3′-UTR and DRD1-mut 3'-UTR and/or miR-195-5p mimic or control mimic in in HEK-293T cells for 24h. All data are shown as the mean±SEM (n=3), *p<0.05; NS, not significant.
Figure 5
ED patients exhibited miR-195-5p binding SNP locus mutation in the 3′-UTR of DRD1. A Two miR-195-5p binding SNP locus mutations (231T->A, 233C->G) were identified in the 3′-UTR of DRD1 in patients with essential hypertension and type 2 diabetes mellitus (ED group). B Number of patients with SNP locus mutation in the ED and N groups.
Figure 6
Relevance of miR-195-5p with multiple clinicopathological parameters. A-B The relevance of miR-195-5p with positively related ED-associated clinicopathological parameters, including the Cr (A) and ALP (B). C Relevance of miR-195-5p with age. D-H MiR-195-5p showed no significant difference with other ED-associated clinicopathological parameters, including LDL-C (D), BUN (E), AST (F), FBG (G), and TC (H). I ROC analysis of miR-195-5p between N and ED groups. Cut-off value=4.020, Sensitivity=0.846, Specificity=0.769, AUC=0.772, p=0.018.
Figure S1
Clinicopathological parameters involved in E, D ED, and healthy donor (N) groups. A-F ED-associated clinical parameters in E, D, ED, and N groups, including ALP (A), BUN (B), Cr (C), r-GGT (D), TP (E), and UA (F). All data are shown as the mean±SEM. NS, not significant.
Figure S2
Comparation of plasma miRNA expression profiling among patients and healthy donors. A-C Scatter plot illustration of serum miRNAs between the D and N groups (A), E and N groups (B), and ED and N groups (C). D-F Scatter plot illustration of serum miRNAs between the D and E groups (D), E and ED groups (E), and D and ED groups (F).
Figure S3
Identification of differentially expressed miRNAs among patients and healthy donors. A-B Amplification plot (A) and melting curve plot (B) of ACTIN, miR-197-3p, miR-130a-3p, miR-27-3p, and miR-195-5p.
Figure S4
Luciferase reporter assay to investigate the binding of miR-195-5p to the DRD1 3′-UTR. A Luciferase reporter activity indicating the inhibitory effect of miR-195-5p on DRD1 in HEK-293T cells transfected with DRD1-3′-UTR and DRD1-mut 3'-UTR (MUT DRD1-3′ UTR) and/or miR-195-5p mimic or control mimic for 24h. All values are shown as firefly/renilla ratio. All data are shown as the mean±SEM (n=3), *p<0.05; NS, not significant.
Figure S5
miRNA-binding SNP locus mutation analyses in the 3′-UTR of DRD1 in the ED group. A Analyses of miRNA-binding SNPs in other regions of the 3′-UTR of DRD1 in healthy donors (N-1, N-2) and ED groups with essential hypertension and type 2 diabetes mellitus (DM-1, DM-2, DM-3, DM-4), such as 971-1014 (A), 1016-1058 (B), 1104-1146, (C) and 1667-1708 (D).
Figure S6
The relevance of miR-195-5p with multiple clinicopathological parameters. A-H MiR-195-5p showed no significant difference with other ED-associated clinicopathological parameters, including the TP (A), ALB (B), ALT (C), UA (D), BMI (E), TG (F), r-GGT (G), and HDL-C (H) values.
Table S1
General information of representative groups (E, D, ED) and healthy donors (N).
Table S2
Primer sequences for the amplification of candidate miRNAs.
Table S3
Ten significant upregulated miRNAs in patients with EH and T2DM.
Table S4
Ten significant downregulated miRNAs in patients with EH and T2DM.
Table S5
SNP in the miR-195-5p-binding region of DRD1 3′-UTR.
Table S6
Relative miRNA expression levels.
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