Highly-Expressed MiR-221-3p Distinctly Increases the Incidence of Diabetic Retinopathy in Patients With Type 2 Diabetes Mellitus

Abstract

Objective: Diabetic retinopathy (DR) is the leading cause of blindness in patients with diabetes mellitus (DM). MiR-221-3p is implicated in microvascular dysfunction in DR, and we explored their relationship.

Methods: Patients with type 2 diabetes mellitus (T2DM) were allocated to the non-DR (NDR)/nonproliferative DR (NPDR)/proliferative DR (PDR) groups, with their clinical baseline and pathological data collected. The miR-221-3p and VEGF levels were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and ELISA, respectively. Peripheral blood endothelial progenitor cell (EPC) and endothelial cell (EC) ratios were ascertained by flow cytometry. The correlations between miR-221-3p levels and VEGF/EPCs/ECs, the predictive value of serum miR-221-3p levels in DR, and the independent risk factors for DR occurrence in T2DM were analyzed by Pearson's correlation analysis, receiver operating characteristic (ROC) curve, and multifactorial logistic regression analysis.

Results: Serum miR-221-3p was highly expressed in DR. Clinical severity of DR was positively correlated with miR-221-3p levels. Endothelial function was impaired in DR. Serum miR-221-3p levels in DR were favorably correlated with VEGF and ECs and negatively associated with EPCs. The area under the curve of serum miR-221-3p in evaluating DR occurrence in patients with T2DM was 0.8178 (1.235 cutoff value, 69.62% sensitivity, and 82.35% specificity). High expression of miR-221-3p increased DR incidence in patients with T2DM. Diabetes course, VEGF, EPCs, ECs, and miR-221-3p levels were independent risk factors for DR development in patients with T2DM.

Conclusions: Serum miR-221-3p levels in patients with DR were positively correlated with VEGF and ECs and negatively linked with EPCs. Highly expressed miR-221-3p distinctly increased DR incidence in patients with T2DM and was an independent risk factor for DR development in patients with T2DM.

Translational Relevance: This study assessed serum miR-221-3p level and endothelial function indicators (VEGF, EPCs, and ECs) in patients with DR and analyzed the correlation between each indicator. We found that high serum miR-221-3p expression prominently increased the incidence of DR in patients with T2DM and was an independent risk factor for the development of DR in patients with T2DM. This study provided a scientific basis for further clarification of the pathogenesis of DR, and also provided new ideas for clinical prediction and management of DR.

Introduction

Diabetes mellitus (DM) is a disease carrying high incidence, serious complications, and far-reaching clinical impact.¹ The prevalence of it is about 1 in 10 among people aged 20 to 79 years worldwide in 2021, and its prevalence will further elevate in 2045.² Symptoms of DM consist of weight loss, polyphagia, and decreased vision, among others.³ Meanwhile, devastating microvascular complications (including diabetic retinopathy [DR] and neuropathy, and diabetic kidney disease) and macrovascular complications (cardiovascular disease) cause increased kidney failure, blindness, mortality, and an overall decreased life quality in patients with diabetes.⁴ Among these, as one of the most prevalent microvascular complications of DM, DR is the main factor of blindness in the working population of rich countries.⁵ DR is considered a microvascular disease and the detection of microvascular lesions plays a pivotal role in the diagnosis of DR.⁶ Arising as the consequence of chronic hyperglycemia, DR is characterized by retinal ischemia, leaky retinal vasculature, angiogenesis, retinal inflammation, and neovascularization.⁷ The number of people globally with DR and visual impairment is increasing as the incidence of diabetes elevates and the life expectancy of people with diabetes increases.⁸ According to the degree of retinopathy, DR can be further divided into nonproliferative DR (NPDR) and proliferative DR (PDR).⁹ By 2030, the number of patients with DR will grow to 191 million.¹⁰ However, the exact mechanism of DR development is not clear so far, and there is no effective treatment for DR microvascular dysfunction. 

MicroRNA (miRNA) is a class of small non-coding RNAs participating in the regulation of different biological processes that are critical in cancer development, such as cell proliferation, apoptosis, and differentiation.^11,12 There is evidence that miRNAs implicate the pathogenesis of DM and its macrovascular complications and microvascular complications.¹³ The miRNAs may provide a novel strategy for the treatment and diagnosis of DR.^14,15 MiR-221-3p plays a vital role in metabolic diseases.¹⁶ In terms of vascular function, miR-221-3p facilitates diabetic wound healing.^17,18 A recent study demonstrated that miR-221-3p possesses the ability to ameliorate retinal vascular leakage and repress high-glucose-induced endothelial cell (EC) proliferation and migration in DM rats.¹⁹ However, the relationship between serum miR-221-3p expression and microvascular damage in DR is currently unclear. As a consequence, in this study, we determined the serum miR-221-3p levels in patients with DR and analyzed their relationship with microvascular damage in patients with DR, thereby providing a scientific reference for further clarifying the pathogenesis of DR and providing new ideas for clinical prediction and management of DR. 

Materials and Methods

Ethics Statement

This study was reviewed and approved by the Academic Ethics Committee of The Second Affiliated Hospital of Shandong First Medical University and complied with the Declaration of Helsinki. All patients were informed of the study purpose and signed an informed consent. 

Research Subjects

Among 300 patients with type 2 diabetes mellitus (T2DM) who attended The Second Affiliated Hospital of Shandong First Medical University from January 2021 to December 2022, 34 patients did not meet the inclusion criteria and 6 patients refused to participate in the study, and 260 patients with T2DM were finally included as the subjects of this study. According to the clinical diagnostic criteria for DR, patients with T2DM were allotted to the non-DR (NDR) group (N = 102), the NPDR group (N = 87), and the PDR group (N = 71). 

Inclusion and Exclusion Criteria

The inclusion criteria were as follows: in line with the American Diabetes Association T2DM diagnostic criteria,²⁰ no systemic disease, voluntary signing of the informed consent, not within the exclusion criteria, and with complete information. 

The exclusion criteria were as below: with T1DM and other special types of DM; complicated with microangiopathy or macroangiopathy other than DR; complicated with optic nerve disease, retinal vein obstruction, cataract, glaucoma, lens disease, refractive changes and other eye diseases; congenital amblyopia; acute complications of diabetic ketoacidosis, diabetic hyperosmolar coma; complicated with hyperthyroidism, cortisolism, and other diseases affecting glucose metabolism; complicated with serious dysfunctions of the liver, kidneys, and heart; complicated with malignant tumors; complicated with acute and chronic infectious diseases; complicated with immune system or hematological system diseases; in pregnancy or lactation; and with incomplete laboratory and auxiliary examination data. 

On the basis of International Clinical DR Disease Severity Scale, patients with DR are classified into five levels, the higher the severity, the higher the level.²¹ Of these, no DR manifests level 0, mild NPDR manifests level 1 (dot hemorrhages and/or microaneurysms only), moderate NPDR manifests level 2 (more than dot hemorrhages and/or microaneurysms only but less than severe NPDR), severe NPDR manifests level 3 (obvious intraretinal microvascular abnormalities observed in ≥ 1 quadrant or > 20 intraretinal hemorrhages in each of the 4 quadrants, and no PDR), and PDR manifests level 4 (preretinal/vitreous hemorrhage or neovascularization [treated or active]).²² 

Specimen Collection and Processing

Fasting venous blood (4 mL) was collected from all subjects at the time of admission, of which 2 mL was used for the examination of fasting blood glucose (FBG) and the ratios of peripheral blood endothelial progenitor cells (EPCs) and ECs. The remaining 2 mL venous blood without anticoagulation was centrifuged at 3000 r/min for 5 minutes at 4°C within 30 minutes, and the supernatant was collected for the determinations of serum miR-221-3p and vascular endothelial-derived growth factor (VEGF) levels. 

Detections of Clinicopathological Characteristics Indexes

Baseline clinical data, such as age, sex, body mass index (BMI), and duration of DM were recorded at enrollment. FBG levels were measured by glucose oxidase method using a fully automatic biochemical analyzer (P800; Roche Diagnostics, Indianapolis, IN, USA). Serum VEGF levels were determined using the ELISA kit (PV963; Beyotime, Shanghai, China). The ratios of peripheral blood EPCs and ECs was assessed using a flow cytometer (CytoFLEX LX; Beckman Coulter, Chaska, MN, USA). All procedures were performed in strict accordance with the instructions. 

Reverse Transcription Quantitative Polymerase Chain Reaction

Serum miR-221-3p expression was determined using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as per the instructions of the manufacturer and then transcribed into complementary DNA using the PrimeScript RT kit (Takara Bio, Otsu, Shiga, Japan). Afterward, qPCR was performed on an ABI 7900HT Fast PCR Real-Time System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq II (Takara). Reaction conditions were pre-denaturation at 95°C for 10 minutes, 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 34 seconds. U6 served as the internal reference. Primers were synthesized by Sangon Biotech (Shanghai, China). The primers are as follows: miR-221-3p, forward, 5′-CGGCTACATTGTCTGCCTG-3′, reverse, 5′-CAGTGCGTGTCGTGGAGT-3′; U6 forward, 5′-CGCTTCGGCAGCACATATAC-3′, and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The relative expression of serum miR-221-3p was calculated using the 2^−ΔΔCt method. 

Statistical Analysis

Statistical analysis and graphing were performed using SPSS 21.0 (IBM, Armonk, NY, USA) and GraphPad Prism version 8.01 software (GraphPad Software, San Diego, CA, USA). The Shapiro-Wilk test was adopted for normal distribution, and normally distributed data were expressed as mean ± standard deviation. Comparisons among multiple groups were made using 1-way analysis of variance (ANOVA), and post hoc analysis was conducted using Tukey's test. Correlations between markers were assessed by Pearson's correlation analysis. Non-normally distributed measures were expressed as median values (minimal to maximal values). The Kruskal-Wallis test was utilized for comparisons among multiple groups. Categorical variables were analyzed using the Chi-square test. The receiver operating characteristic (ROC) curve was proposed to analyze the predictive value of serum miR-221-3p levels for the development of DR in patients with T2DM. Independent risk factors for the development of DR in patients with T2DM were analyzed by multifactorial logistic regression. The P value was a two-sided test. Any P < 0.05 indicated that the difference was statistically significant. 

Results

Comparisons of Clinical Baseline Data

The clinical baseline characteristics of all patients are shown in Table 1. There were no significant differences in age, sex, and BMI among the three groups (P > 0.05), whereas significant differences existed in the course of DM and FBG (P < 0.001). 

Table 1.

 

Comparisons of Clinical Baseline Information

 

Serum MiR-221-3p was Highly Expressed in Patients With DR

As reflected by the results of RT-qPCR, serum miR-221-3p levels were significantly upregulated in patients with NPDR compared with patients with NDR, and further upregulated in patients with PDR versus patients with NPDR (Fig. 1; P < 0.001). The result indicated that serum miR-221-3p was highly expressed in patients with DR and that serum miR-221-3p levels were distinctly higher in patients with PDR than in patients with NPDR. 

Figure 1.

 

MiR-221-3p was highly expressed in patients with DR. RT-qPCR was performed to assess serum miR-221-3p levels. Data were expressed as mean ± standard deviation and analyzed using 1-way ANOVA with post hoc test using Tukey's multiple comparisons test. ***P < 0.001.

Comparisons of Endothelial Function Indexes

Serum VEGF levels and the ratios of peripheral blood EPCs and ECs were measured by ELISA and flow cytometry, and endothelial function was compared among the groups. The results revealed that serum VEGF levels and the proportion of peripheral blood ECs were prominently higher in the DR group than in the NDR group, and serum VEGF levels and EC ratio were remarkably elevated in the PDR group relative to the NPDR group (Figs. 2A–C; P < 0.001). Compared with patients with NDR, the proportion of peripheral blood EPCs was considerably decreased in the NPDR group, and further reduced in the PDR group in contrast to the NPDR group (see Fig. 2B; P < 0.001). The above results unveiled that endothelial function was impaired in the patients with DR compared with patients with NDR and further impaired in the patients with PDR relative to the patients with NPDR. 

Figure 2.

 

Comparisons of endothelial function indexes among groups. (A) ELISA was used to determine VEGF level. (B, C) Flow cytometry was implemented to determine the proportion of EPCs and ECs. Data were expressed as mean ± standard deviation using 1-way ANOVA. Post hoc test was performed using Tukey's multiple comparisons test. ***P < 0.001.

Relationship Between Serum MiR-221-3p Levels and Microvascular Damage in Patients With DR

There were considerable differences in serum miR-221-3p levels in patients with DR with different clinical severity levels, with patients with DR of higher clinical severity level manifesting a higher serum miR-221-3p level (Table 2; P < 0.001). The correlations between serum miR-221-3p levels and endothelial function markers (VEGF, EPCs, and ECs) in patients with DR were analyzed by Pearson's correlation analysis. Serum miR-221-3p levels were memorably positively correlated with VEGF (Fig. 3A; r = 0.5848, P < 0.001) and ECs (Fig. 3C; r = 0.3213, P < 0.001), and negatively correlated with EPCs (Fig. 3B; r = –0.3183, P < 0.001) in patients with DR. 

Table 2.

 

Comparisons of Serum miR-221-3p Levels in Patients With DR With Different Clinical Severity

 

Figure 3.

 

Correlation between serum miR-221-3p levels and microvascular injury in patients with DR. (A, B, C) Pearson's correlation analysis was conducted to analyze the correlation between serum miR-221-3p levels and endothelial function markers (VEGF, EPCs, and ECs). R was the correlation coefficient. Differences were considered to be significant at P < 0.05.

High Serum MiR-221-3p Expression Significantly Increased the DR Incidence in Patients With T2DM

Based on the previous results, the ROC curve was plotted to analyze the predictive value of the serum miR-221-3p level for DR occurrence in patients with T2DM. The area under the ROC curve of serum miR-221-3p was 0.8178, and the cutoff value was 1.235 (sensitivity = 69.62% and specificity = 82.35%; Fig. 4). Subsequently, patients with T2DM were arranged into the miR-221-3p high expression group (N = 128) and the miR-221-3p low expression group (N = 132) according to the ROC cutoff value, and the incidence of DR of the 2 groups was compared. The results unraveled that the incidence of DR in the miR-221-3p high expression group (85.94%) was conspicuously higher than that in the miR-221-3p low expression group (36.36%; see Table 3; P < 0.001). The above results suggested that highly expressed miR-221-3p significantly boosted the incidence of DR in patients with T2DM. 

Figure 4.

 

Highly-expressed serum miR-221-3p observably elevated the incidence of DR in patients with T2DM. ROC curve was utilized for the analyses of the predictive value of serum miR-221-3p levels for the incidence of DR in patients with T2DM.

Table 3.

 

Incidence of DR in Patients With T2DM Different miR-221-3p Levels

 

Serum MiR-221-3p Level was an Independent Risk Factor for the Development of DR in Patients With T2DM

Finally, in attempt to exclude confounding factors and more accurately evaluate the impact of serum miR-221-3p levels on the occurrence of DR in patients with T2DM, the course of diabetes, FBG, VEGF, EPCs, ECs, and serum miR-221-3p levels were included into the multifactorial logistic regression analysis, which indicated that the course of DM, VEGF, EPCs, ECs, and serum miR-221-3p levels were independent risk factors for the development of DR in patients with T2DM (Table 4; all P < 0.05). 

Table 4.

 

Serum miR-221-3p Level was an Independent Risk Factor for the Development of DR in Patients With T2DM

 

Discussion

As a microangiopathy of the retina, DR occurs in almost all patients with diabetes.²³ Currently, DR is the major cause of the blindness around the world, and the number of patients with DR is increasing but the treatment is limited.⁷ Evidence has shown that miRNAs are involved in the retinal cell neovascularization and changes in miRNA levels are closely associated with vascular changes in patients with DR.²⁴ As a result, our study was designed to estimate the relationship between serum miR-221-3p expression and microvascular damage in patients with DR. The experiment results unraveled that the highly expressed serum miR-221-3p observably increased the incidence of DR in patients with T2DM. 

Reasons for the development of DM into DR include prolonged duration of diabetes, poor long-term control of blood glucose, and hypertension, among others.²⁵ Our findings exhibited marked differences in duration of diabetes and FBG levels among the patients with NDR, patients with NPDR, and patients with PDR. Ulteriorly, a recent study has found that the pathogenesis of various diseases, such as DR and cancer, is related to the dysfunction of angiogenesis, and miRNAs can modulate several stages of angiogenesis.²⁶ What is more, miR-221-3p promotes viability, migrating property, and angiogenesis of human umbilical vein ECs.¹⁷ Our results found that serum miR-221-3p was highly expressed in patients with DR, and miR-221-3p was significantly higher in the PDR and NPDR groups than in the NDR group. Consistent with our results, miR-221-3p is highly expressed in DM rats and high glucose-treated human retinal microvascular ECs, and suppression of miR-221-3p ameliorates retinal vascular leakage and represses high glucose-induced EC proliferation and migration in vitro.¹⁹ To conclude, miR-221-3p is involved in the progression of DR, and the high expression of miR-221-3p significantly increases the incidence of DR in patients with T2DM. 

ECs is one of the major cellular constituents of the retinal microvasculature, and the interaction between ECs and pericytes is essential for the development of the microvasculature.²⁷ Microvascular ECs are the main target of high-glucose induced injury, and intracellular hyperglycemia does harm to the vascular endothelium via varieties of pathophysiological processes.²⁸ Healthy EPCs are critical for retinal microvasculature, and increasing EPC number and function may be a potential target for DM.²⁹ On the other hand, VEGF is known as a main angiogenic factor and the therapeutic target of DR.³⁰ Our results confirmed elevated VEGF levels and peripheral blood EC proportion, and reduced EPC proportion in the DR, PDR, and NPDR groups compared with the NDR group, with VEGF levels and peripheral blood EC proportion higher, and EPC proportion lower in the patients with PDR than the patients with NPDR. One of the studies, conducted by Caprnda et al. revealed that VEGF is highly expressed in the retina, leading to EC proliferation, neovascularization, thereby resulting in microaneurysms, fluid leakage, and other abnormalities in some patients with DM.³¹ Moreover, it has been documented that high levels of EPCs is an important risk factor for neovascularization, and EPCs may play a role in pathological neovascularization of DR.³² Altogether, endothelial function is impaired in DR and further impaired in PDR. 

MiRNAs regulate DM-induced vascular dysfunction both in large blood vessels and microvasculature.³³ For example, vascular dysfunction in the DR can be facilitated by regulating miR-15b-5p and COL12A1, which provides new potential therapeutic targets for DR.³⁴ However, the relationship between miR-221-3p levels and endothelial function indicators remains elusive. In terms of this, our findings manifested for the first time that miR-221-3p levels were related to microvascular injury in patients with DR, which were positively related with VEGF and ECs and negatively with EPCs. Circulating miRNAs have emerged as the new biomarkers of DM, such as 2 miRNAs previously documented to be implicated in angiogenesis, miR-320a and miR-27b, are related to the incidence and progression of retinopathy.³⁵ Innovatively, our findings suggested that highly expressed miR-221-3p conspicuously increased the incidence of DR in patients with T2DM, and further elicited that disease duration, VEGF, EPCs, ECs, and serum miR-221-3p levels were independent risk factors for the development of DR in patients with T2DM. In a similar light, duration of T2DM, BMI, systolic blood pressure, and total triglycerides are independent risk factors for the development of DR in T2DM,³⁶ which prooved our findings. 

All in all, our experiments unveiled that serum miR-221-3p levels in patients with DR were remarkably positively correlated with VEGF and ECs and negatively linked with EPCs. Highly expressed miR-221-3p conspicuously increased the incidence of DR in patients with T2DM and was an independent risk factor for the development of DR in patients with T2DM. Our findings provided a reference for further clarification of the DR pathogenesis, and also provided new ideas for clinical prediction and management of DR. Nevertheless, there are also limitations. First, the number of cases included in the analysis of this study was small, and further expansion of the sample size and multicenter studies were essential to increase the credibility of the results. Second, we only explored the relationship between serum miR-221-3p expression and DR microvascular damage, whereas the specific mechanism of how miR-221-3p was involved in the regulation of DR was currently unclear. In the future, we will use the database to screen the target genes and also design animal and cellular experiments to further explore the mechanisms of miR-221-3p involvement in DR regulation. 

Acknowledgments

Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. 

Authors' Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Lili Zhao and Qingmin Pan. The first draft of the manuscript was written by Lili Zhao. All authors read and approved the final manuscript. 

Disclosure: L. Zhao, None; Q. Pan, None