Circulating MicroRNAs in Elderly Type 2 Diabetic Patients

Giuseppina Catanzaro, Zein Mersini Besharat, Martina Chiacchiarini, Luana Abballe, Claudia Sabato, Alessandra Vacca, Paola Borgiani, Francesco Dotta, Manfredi Tesauro, Agnese Po, Elisabetta Ferretti, Giuseppina Catanzaro, Zein Mersini Besharat, Martina Chiacchiarini, Luana Abballe, Claudia Sabato, Alessandra Vacca, Paola Borgiani, Francesco Dotta, Manfredi Tesauro, Agnese Po, Elisabetta Ferretti

Abstract

The circulating microRNAs (miRNAs) associated with type 2 diabetes (T2D) in elderly patients are still being defined. To identify novel miRNA biomarker candidates for monitoring responses to sitagliptin in such patients, we prospectively studied 40 T2D patients (age > 65) with HbA1c levels of 7.5-9.0% on metformin. After collection of baseline blood samples (t0), the dipeptidyl peptidase-IV (DPP-IV) inhibitor (DPP-IVi) sitagliptin was added to the metformin regimen, and patients were followed for 15 months. Patients with HbA1c < 7.5% or HbA1c reduction > 0.5% after 3 and 15 months of therapy were classified as "responders" (group R, n = 34); all others were classified as "nonresponders" (group NR, n = 6). Circulating miRNA profiling was performed on plasma collected in each group before and after 15 months of therapy (t0 and t15). Intra- and intergroup comparison of miRNA profiles pinpointed three miRNAs that correlated with responses to sitagliptin: miR-378, which is a candidate biomarker of resistance to this DPP-IVi, and miR-126-3p and miR-223, which are associated with positive responses to the drug. The translational implications are as immediate as evident, with the possibility to develop noninvasive diagnostic tools to predict drug response and development of chronic complications.

Figures

Figure 1
Figure 1
Experimental design for the identification of circulating miRNAs in T2D patients. The duration of the study was 2 years. The blue horizontal arrow indicates the duration of the different phases of the project. The red vertical arrows indicate the main study periods. In year 1, patients were enrolled and started on metformin + sitagliptin (see Materials and Methods for details). After 3 and 15 months of treatment (t3 and t15), HbA1c values were reassessed and patients were classified as nonresponders (NR) or responders (R). Plasma miRNA levels at baseline (t0) and t15 from groups R and NR were compared as indicated. Comparison of plasma pools: (1) NR-t0 versus R-t0, (2) R-t15 versus R-t0, (3) NR-t15 versus NR-t0, and (4) NR-t15 versus R-t15.
Figure 2
Figure 2
Glycemic control statuses of the patients at baseline and 3 and 15 months after initiation of metformin + sitagliptin. All 40 patients in poor metabolic control were enrolled. HbA1c levels were evaluated after 3 and 15 months from the addition of sitagliptin. On the basis of HbA1c values, patients were divided into responders and nonresponders. All patients showed an initial metabolic response to therapy (t3), whereas after 15 months (t15), 34/40 were responders. Based on this information, patients were divided into five groups: (1) t0 responder samples (R-t0), (2) t0 nonresponder samples (NR-t0), (3) t3 responder samples (R-t3), (4) t15 responder samples (R-t15), and (5) t15 nonresponder samples (NR-t15). miRNA profiling was performed at baseline and after 15 months of sitagliptin addition.
Figure 3
Figure 3
Venn diagram of circulating miRNAs detected in the elderly T2D cohort and in the R and NR subcohorts. Patients were classified as responders (R) or nonresponders (NR) based on their glycemic control status after 15 months of treatment with metformin + sitagliptin (t15). Venn diagrams show the number of miRNAs detected in NR and R plasma samples collected (a) before the start of combined therapy (baseline, t0) and (b) at t15 and (c) at both t0 and t15.
Figure 4
Figure 4
Heat maps showing circulating microRNAs that were differentially expressed in plasma samples from (a) R and NR patients at baseline (t0), prior to the addition of sitagliptin to the maximum-dose metformin regimen; (b) R at t0 and after 15 months of sitagliptin (t15); (c) NR patients at t15 and t0; and (d) R and NR patients at t15.

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