Plasma levels of miR‐29b and miR‐200b in type 2 diabetic retinopathy

Abstract MicroRNAs (miRNAs/miRs) are involved in the pathogenesis of diabetes mellitus and its chronic complications, and their circulating levels have emerged as potential biomarkers for the development and progression of diabetes. However, few studies have examined the expression of miRNAs in diabetic retinopathy (DR) in humans. This case‐control study aimed to investigate whether the plasma levels of miR‐29b and miR‐200b are associated with DR in 186 South Brazilians with type 2 diabetes (91 without DR, 46 with non‐proliferative DR and 49 with proliferative DR). We also included 20 healthy blood donors to determine the miRNA expression in the general population. Plasma levels of miR‐29b and miR‐200b were quantified by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Proliferative DR was inversely associated with plasma levels of miR‐29b (unadjusted OR = 0.694, 95% CI: 0.535‐0.900, P = 0.006) and miR‐200b (unadjusted OR = 0.797, 95% CI: 0.637‐0.997, P = 0.047). However, these associations were lost after controlling for demographic and clinical covariates. In addition, patients with type 2 diabetes had lower miR‐200b levels than blood donors. Our findings reinforce the importance of addressing the role of circulating miRNAs, including miR‐29 and miR‐200b, in DR.


| INTRODUCTION
Diabetic retinopathy (DR) is a neurovascular disorder and the leading cause of visual impairment and blindness among working-age adults. 1,2 In addition to vision loss, DR is associated with chronic kidney disease and mortality from cardiovascular disease in diabetes. 1 Although longer diabetes duration, poor glycemic control and high blood pressure are the major risk factors of DR, 1-3 epidemiological data support the hypothesis of differential genetic susceptibility to this chronic complication. 1,4 Epigenetic mechanisms, such as DNA methylation, histone post-translational modifications in chromatin and non-coding RNAs, mediate the interplay between genetic and environmental risk factors. Persistence of epigenetic changes might contribute to the metabolic memory phenomenon and to the oxidative stress, inflammation and extracellular matrix accumulation, 5,6 all of which lead to the development of DR. 5 In this context, several studies have reported abnormal expression of microRNAs (miRNAs/miRs) in retinal cells under hyperglycemic conditions and in murine models of DR. [7][8][9] MiRNAs are small endogenous non-coding RNAs that in general silence gene expression at the posttranscriptional level by pairing to specific sequences in the 3'-untranslated region of their target mRNAs, thereby repressing protein synthesis. MiRNAs regulate cell development and function by modulating the acquisition and maintenance of beta-cell identity, 10 cell growth, proliferation, differentiation, apoptosis and metabolism. 11 Given their high stability and omnipresence in body fluids, several studies have identified the potential of circulating miRNAs as biomarkers of diagnosis, prognosis and management of type 2 diabetes and its vascular complications. 8,[12][13][14] Despite this, the profile of circulating miRNAs in DR has been evaluated only recently. [15][16][17][18][19][20][21][22][23][24][25] MiR-29b and miR-200b were one of the first miRNAs found to be dysregulated in high glucose-exposed retinal cells and diabetic murines, thereby suggesting their involvement in the development of DR. MiR-29b exerts anti-apoptotic and antifibrotic effects on retinal cells 12,26 and contributes to endothelial function by increasing nitric oxide, 27 while miR-200b protects against vascular permeability and angiogenesis. Down-regulation of miR-29b results in increased matrix deposition and retinal fibrosis, whereas down-regulation of miR-200b results in increased inflammation. MiR-29b and miR-200b are down-regulated in diabetes resulting in increased angiogenesis and neovascularization in retina. 12 However, to the best of our knowledge, no study to date has assessed the circulating levels of these two miRNAs in type 2 diabetic patients with DR (cases) and without this complication (controls). Therefore, we investigated the association of plasma levels of miR-29b and miR-200b with DR in South Brazilians with type 2 diabetes mellitus.  28 and inclusion criteria in the study were age at diagnosis of diabetes ≥30 years, no need of permanent insulin treatment within the first year of diagnosis and no previous episodes of ketoacidosis. Patients underwent a clinical and biochemical evaluation consisting of physical examination and routine laboratory exams, such as glycated hemoglobin, serum creatinine, lipid profile and urinary albumin excretion, which were measured using standard methods. 29 Glomerular filtration rate was estimated (eGFR) with the CKD-EPI equation. 30  We also included 20 healthy blood donors from the Haemotherapy Division of HCPA to determine the plasma levels of miR-29b and miR-200b in our general population. They were enrolled between August and December 2017 and data regarding age, gender and skin colour/ethnicity were collected. Blood donors with a known personal and/or first-degree family history of diabetes were not included in the study, and no additional clinical or laboratory data were collected from them. This study was approved by the Research Ethics Committees of HCPA (CAAE number 35065914.9.0000.5327) and ULBRA (CAAE number 35065914.9.3001.5349), and was carried out in accordance with the World Medical Association Declaration of Helsinki. 33 All subjects provided written informed consent prior to the data and blood collection. Skin colour/ethnicity was self-reported and categorized as white or non-white (pardo or black).

| MiRNA isolation and quantification
Peripheral blood samples were collected in EDTA-containing tubes and centrifuged at 2500 g for 15 minutes at 4°C within 3 hours DANTAS DA COSTA E SILVA ET AL. Relative expression levels were estimated using the comparative method (2 -ΔΔCq ) 34 considering the spiked-in cel-miR-39 as the reference gene and a pool of cDNA samples obtained from nine subjects with type 2 diabetes randomly selected (four without DR, three with NPDR and two with PDR) as the reference sample (calibrator). Foldchange values were then log2-transformed for statistical analysis (see Data S1 for expression data).

| Statistical analysis
Categorical data are shown as absolute frequency (percentage) and continuous variables are expressed as mean ± SD or as median (25th and 75th percentiles). Data normality was assessed using the Shapiro-Wilk test. Categorical data were compared between the groups of subjects by the chi-square test, and continuous data were compared by independent Student t test, Mann-Whitney U, one-way ANOVA or Kruskal-Wallis, followed by the Tukey or Dunn post hoc analysis, as appropriate. Correlation between both miRNAs was evaluated using the Pearson (r) or the Spearman (r s ) correlation coefficient, as indicated by the normality test. Association of the plasma levels of miR-29b and miR-200b with PDR was evaluated by logistic regression analysis. Statistical analyses were done using the SPSS statistical package version 18.0 (SPSS Inc, Chicago, USA), and two-tailed P < 0.05 were considered as statistically significant.
As DR was the primary outcome of our study, power calculations indicated that the number of samples required for detecting an 1.5fold change with a statistical power of 90% at a significance level of 0.05 was 96 (48 cases and 48 controls). We used the value of 1.5 based on the assumption that this difference in miRNA expression might have a biological significance. 26,35 Power calculations were estimated using the WinPEPI (version 11.43) statistical software. 36

| Demographic and clinical profile of study subjects
The demographic and clinical characteristics of the patients with type 2 diabetes are shown in Table 1. Briefly, patients with PDR were older and more often male, had a longer duration of diabetes and lower body mass index (BMI), were less often hypertensive, had higher levels of serum creatinine and lower eGFR as compared with patients without DR. Moreover, daily insulin use was more frequent among patients with DR than in those without this complication (Table 1). Although blood donors were younger (mean age 45.5 ± 7.5 years) than type 2 diabetic patients (P < 0.001), the proportion of males (45%) and non-white subjects (30%) among blood donors was virtually the same as that observed among patients with type 2 diabetes (44% and 29%, respectively; P > 0.999 for both characteristics).

| Comparison of miRNA levels between study groups
Plasma levels of miR-29b and miR-200b in blood donors and patients with type 2 diabetes are shown in Table 2. Expression of miR-29b was detected in all study subjects, whereas miR-200b was undetectable in seven of 186 (3.8%) type 2 diabetic patients. Patients with type 2 diabetes without DR had approximately twofold lower levels of miR-200b in comparison to blood donors, while the levels of miR-29b were not significantly different between them.
With regard to retinopathy, the mean levels of miR-29b were 40% lower in patients with PDR as compared to those without DR.
The same trend was observed for the miR-200b, but the difference between the three groups of diabetic patients did not reach formal statistical significance (Table 2 and Figure 1).

| Association analysis of miRNA levels with diabetic retinopathy
Univariate regression analysis showed that increased plasma levels of miR-29b and miR-200b were associated with a decreased risk of PDR ( Table 3). As these two miRNAs were strongly correlated with each other in the group of subjects with type 2 diabetes (r s = 0.61, P < 0.001), we also used the logistic regression to test for their interaction (miR-29b*miR-200b). In this model, miR-29 was associ- was associated with PDR. However, miR-29b levels did not remain associated with PDR after adjusting for the demographic and clinical variables that were also associated with this outcome in the univariate analyses (Table 3).

| DISCUSSION
This case-control study assessed the association of the plasma levels of miR-29b and miR-200b with DR in type 2 diabetic outpatients from a tertiary university hospital in Southern Brazil. We reported, for the first time to our knowledge, that patients with PDR have lower plasma levels of miR-29b as compared to those without DR.
However, this association was lost after controlling for gender, age, diabetes duration, hypertension, BMI, insulin use and renal function.
Although our study was not designed to investigate the association of miRNAs with type 2 diabetes and we cannot exclude the possibility that some blood donors have prediabetes or even diabetes, we found that diabetic patients had lower plasma levels of miR-200b than blood donors.
In relation to miR-29b and type 2 diabetes, our results are in accordance with a recent meta-analysis that identified stress-related miRNA biomarkers in different tissue types and species. The authors found that miR-29b was not dysregulated in type 2 diabetes in the subgroup analyses of miRNAs in circulating blood and in human profiling studies. 14 However, these findings contrast with studies that reported either higher circulating levels of miR-29 in subjects with prediabetes 37 and type 2 diabetes 35,37 or lower miR-29b levels in those with prediabetes 38 and who developed type 2 diabetes in the prospective population-based Bruneck study. 39 It is tempting to speculate whether miR-29b is up-regulated in type 2 diabetes as a failed attempt to compensate for the endothelial dysfunction, as  27 The authors showed that miR-29 is required for normal endothelial function and can restore it in cardiometabolic disorders. 27 In addition, evidence obtained in knockout mice revealed that the miR-29 family has a protective role in beta cells and a pathogenic function in the liver. 40 Paradoxically, increased miR-29 expression was reported to lead to increased insulin resistance, and decreased insulin secretion and signaling in metabolic tissues. 12 With respect to miR-29b and DR, our findings are in line with experimental studies of cultured retinal cells and diabetic murine models that showed that high glucose (HG) induces apoptosis and down-regulates the expression of miR-29b. 41 methylation and histone demethylation. 26 In fact, histone methylation and acetylation are the two major mechanisms by which histone modification affects DR development. 46 In retina, miR-29b is expressed in the ganglion cells and the inner nuclear layer of normal and STZ-induced diabetic rats. 47 Taken together, these evidences indicate that down-regulation of miR-29b contribute to the progression of DR. In our study, levels of miR-29b were 40% lower in patients with PDR as compared to those without DR.
In accordance with previous clinical studies, 15,16 we observed that the plasma levels of miR-200b were decreased in patients with type 2 diabetes and even more reduced among those with PDR. In a previous study in Germany, plasma levels of miR-200b were decreased at least threefold in type 2 diabetic inpatients in comparison to healthy controls. 15 In a Chinese study, patients with DR (and diabetes type not reported) had about 60% lower plasma levels of miR-200b than subjects without diabetes. 16 In the retinas of non-diabetic rats and diabetic patients, miR-200b was found to be located in the vascular endothelium and in neuronal and glial elements. 48 Studies in vitro showed that miR-200b is highly expressed in normal The authors demonstrated experimentally that miR-200b might alleviate DR development by down-regulating its target gene VEGFA. 16 Similar findings were obtained in Akita mice (a genetic model of diabetes) at 3 months after intravitreal injection of miR-200b. 51 During the course of this study, other investigators attempted to identify dysregulated circulating miRNAs in DR in humans. [17][18][19][20][21][22][23] These studies were carried out in patients with type 1 diabetes, 17,18 type 2 diabetes [19][20][21][22] or diabetes type not reported, 23 using serum, 18,20,22,23 plasma 19,21 or endothelial progenitor cells cultured from isolated peripheral blood mononuclear cells. 17 They reported that angiogenic miRNAs are associated with incident DR, 18  Científico e Tecnológico (CNPq). We also thank Dr. Taís Silveira Assmann for her critical comments on the draft manuscript.

CONF LICT OF I NTEREST
The authors confirm that there are no conflicts of interest.

AUTHOR CONTRIBU TI ONS
MEDCS and KGS conceived the study design and performed the sta-