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Increased serum miR-7 is a promising biomarker for type 2 diabetes mellitus and its microvascular complications
Accepted Manuscript Increased serum miR-7 is a promising biomarker for type 2 diabetes mellitus and its microvascular complications Shujun Wan, Jun Wang, Jing Wang, Jia Wu, Jiaxi Song, Chen-Yu Zhang, Chunni Zhang, Cheng Wang, Jun-Jun Wang PII: DOI: Reference:
S0168-8227(16)31836-8 http://dx.doi.org/10.1016/j.diabres.2017.06.005 DIAB 6987
To appear in:
Diabetes Research and Clinical Practice
Received Date: Revised Date: Accepted Date:
24 December 2016 15 April 2017 6 June 2017
Please cite this article as: S. Wan, J. Wang, J. Wang, J. Wu, J. Song, C-Y. Zhang, C. Zhang, C. Wang, J-J. Wang, Increased serum miR-7 is a promising biomarker for type 2 diabetes mellitus and its microvascular complications, Diabetes Research and Clinical Practice (2017), doi: http://dx.doi.org/10.1016/j.diabres.2017.06.005
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Increased serum miR-7 is a promising biomarker for type 2 diabetes mellitus and its microvascular complications
Shujun Wana,b,†, Jun Wangc,†, Jing Wanga, Jia Wua, Jiaxi Songa, Chen-Yu Zhangb, Chunni Zhanga,b, Cheng Wanga,b,* and Jun-Jun Wanga,b,*
a
Department of Clinical Laboratory, Jinling Hospital, Nanjing University School of
Medicine, Nanjing, 210002, China.
b
State Key Laboratory of Pharmaceutical
Biotechnology, Nanjing Advanced Institute for Life Sciences, Nanjing University School of Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Nanjing University, Nanjing, 210023, China; cDepartment of Cardiology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, 210002, China.
†
These authors contributed equally to this work.
*Correspondence authors: Jun-Jun Wang ([email protected]) and/or Cheng Wang ([email protected] or [email protected]) at Department of Clinical Laboratory, Jinling Hospital, Nanjing University School of Medicine, Nanjing, 210002, China. Tel.: +86 25 80861177; Fax.: +86 25 80861177
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Abstract Aims: To investigate the alteration pattern and physiologic state of islet-specific miR-7 in the serum of patients with type 2 diabetes mellitus (T2DM) and T2DM-associated microvascular complications (T2DMC) and to evaluate its clinical significance.
Methods: The levels of serum miR-7 were firstly examined and compared in 76 T2DM patients, 76 T2DMC patients and 74 age-gender matched controls using RT-qPCR. Subsequently, the physiologic state of serum miR-7 was characterized by determining its concentrations in isolated exosomes and corresponding exosome-free samples from the same three cohorts’ samples. Moreover, statistical analyzes were performed to evaluate the associations of serum miR-7 with T2DM and T2DMC.
Results: Serum miR-7 were significantly elevated in the T2DM patients [(401.0 ± 34.37) fmol/L, P < 0.001] and in the T2DMC patients [(501.4 ± 81.69) fmol/L, P < 0.001] when compared with the controls [(175.7 ± 16.59) fmol/L]. Circulating miR-7 were mainly existed as exosome-free form rather than in membrane-bound exosomes. The concentrations of exosome-free miR-7 was markedly higher in the T2DM group [(107.2 ± 9.63) fmol/L, P < 0.001] and in the T2DMC group [(122.1 ± 10.80) fmol/L, P < 0.001] compared to the control group [(54.18 ± 2.37) fmol/L]. Logistic regression
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and ROC curve analyses revealed the serum miR-7 was significantly associated with T2DM and microvascular complications (P < 0.05).
Conclusion: Increased serum miR-7 might have the potential as a promising marker for T2DM and its microvascular complications.
Keywords: serum microRNA; miR-7; type 2 diabetes; microvascular complications; biomarkers
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1. Introduction Type 2 diabetes mellitus (T2DM) is a major global healthcare problem, especially in China [1]. According to the epidemiological survey from the International Diabetes Federation, 382 million people had diabetes in 2013 worldwide, even worse, the greatest increases in T2DM incidence in the next few decades are expected to rise to 592 million by 2035 [2]. T2DM is characterized by high blood glucose levels and insulin resistance, with or without insufficient insulin secretion for the body’s needs, worse still, persistent increased blood glucose will result in vascular complications including microvascular complications [3]. Microvascular complications are the major outcome of T2DM progression, which were broadly classified as neuropathy, nephropathy, and retinopathy. These complications can be life threatening and will greatly reduce the quality of life, if left untreated [4]. With regard to these facts, it is critical to treat T2DM early in its progression to slow down or even stop the microvascular complications. However, the main mechanism underlying the pathogenesis of T2DM and its microvascular complications remains obscure. Moreover,
non-invasive
reliable
blood
biomarkers
for
T2DM
associated
microvascular disease are currently unavailable. Thus, it is important for us to find
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novel diagnostic biomarkers and provide new insights into the pathological process in diabetic complications. MicroRNAs (miRNAs) are a class of small (~22 nucleotide), non-coding RNAs that regulate gene expression at post-transcriptional level by inhibiting translation or inducing target mRNA degradation via binding with their target sites [5]. Accumulating evidence demonstrated that miRNAs are participated in various physiological and pathophysiological process including diabetes and diabetic microvascular complications [6]. To date, more than 2,000 human miRNAs have been registered in the miRBase 22.0, which are predicted to regulate up to 60% of the human genes [7]. Several miRNAs exhibit a tissue-specific expression manner, thereby contributing to cell identity and function. Among them, miR-7 is one of the most abundant miRNA in pancreas, with predominant expression in human and mouse pancreatic islet cells such as β cells [8-10]. Emerging evidences have documented that miR-7 is evolutionarily highly conserved and highly expressed miRNA in pancreas, moreover, it has also been reported to be involved in pancreatic β-cell development as well as insulin secretion[11-13]. Additionally, several reports have identified that disturbing miR-7 expression in transgenic mouse or β cells can also lead to diabetes [14, 15]. These findings suggest that miR-7 may have the potential as novel biomarker for monitoring normal development and function of pancreatic β-cells, and dysregulated miR-7 may closely related to the occurrence and development of T2DM.
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Recent studies from our group and others demonstrated that miRNAs were also existed in circulation in a remarkably stable form that is resistant to endogenous RNase activity and several other harsh conditions treatment, and the unique pattern of circulating miRNAs may provide a useful biomarker for supplemental diagnosis for various diseases including diabetes mellitus [16].Up to date, a number of elevated or decreased circulating miRNAs have been identified in both T2DM-susceptible subjects, T2DM patients as well as T2DMC patients [17]. However, most existing studies are focused on a few specific vascular endothelial enriched miRNAs or immuno-inflammatory related miRNAs, whether pancreatic islet-specific miR-7 might act as a circulating factor in T2DM and T2DMC has barely been explored. On the other hand, intensive studies from different groups have unraveled that serum miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes
or
presented
in
the
circulation
in
a
cell-free
RNA-binding
protein-associated form, but this has not been systematically characterized for miR-7 in the peripheral blood of T2DM patients with or without diabetic microvascular complications. Therefore,
in
the
present
study,
we
performed
a
quantitative
reverse-transcriptions PCR (RT-qPCR) to examine and compare the expression levels of miR-7 in the serum of T2DM patients, T2DMC patients and non-diabetic healthy controls. We also investigated the physical state of serum miR-7 in those two patients’ groups as well as controls with the goal to determine whether miR-7 in serum are
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primarily in exosomes or circulating freely that if there has any benefit of using serum miR-7 in biomarker studies.
2. Material and methods 2.1 Study Design and Patients The present study consisted of 152 T2DM patients who were newly diagnosed or with previously diagnosed disease but during drug withdrawal more than one month in the Department of Endocrinology, Jinling Hospital, Nanjing, China from September 2013 to May 2016. In brief, T2DM patients with the drug withdrawal mean that those patients were not use any oral hypoglycemic agents (including sulfonylurea, biguanide, thiazolidinedione, alpha glucosidase inhibitors and dipeptidyl peptidase inhibitor) at least one month before the sample collection. In addition, patients being treated with insulin were also excluded. T2DM was diagnosed according to the World Health Organization criteria (WHO) and was defined as fasting plasma glucose of ≥ 7.0 mmol/L (126 mg/dl) and/or 2-h of glucose of ≥ 11.1 mmol/L (200 mg/dl) in the 75-g OGTT and/or HbA1c ≥ 6.5% [18]. Of the 152 patients, 76 were diagnosed with microvascular complications (29 with diabetic retinopathy, 28 with diabetic neuropathy, 6 with diabetic nephropathy and 2 with diabetic foot; the others had a combination of complications), and the other 76 T2DM patients were without clinical signs and symptoms of any complications. T2DM patients with microvascular complications were documented by medical records as previously described [19].
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Participants associated with other diseases such as type 1 diabetes mellitus, other types of diabetes, a severe infection, acute cerebrovascular disease, or recent surgery were excluded from this study. In the meanwhile, 74 age- and gender-matched healthy volunteers were recruited as non-diabetic controls from a large pool of individuals seeking routine health check-up at Jinling Hospital and exhibited no evidence of disease. The health checkup included a detailed history, physical examination and blood test. The study protocol was approved by the ethics committees of Jinling Hospital and was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all of the participants prior to the study.
2.2 Blood collection, sample processing and serum biochemical parameters determination Peripheral venous blood (3~5 ml) was collected in serum vacutainer tubes with gel and clot activator (Becton, Dickinson and Company, Franklin Lakes, New Jersey) from participants after at least 12 h of fasting, and serum was immediately separated by a 10-min centrifugation at 1,500 g. The remaining serum samples were stored at -80 °C until miRNA analysis. Serum glucose levels were examined by the auto-analyzer method (Hitachi 7600, Hitachi High-Technologies Corporation, Tokyo, Japan). Other clinical biochemical parameters including serum total cholesterol (TC), triglyceride (TG), low density
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lipoprotein cholesterol (LDL) and high density lipoprotein cholesterol (HDL) concentrations were measured using commercial reagents (RANDOX) on a Hitachi 7600 analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan). HbA1C were assessed using commercial reagents (TOSOH Corporation) on a HLC-723GB auto-analyzer (TOSOH Corporation, Tokyo, Japan).
2.3 Total serum exosome isolation, RNA extraction and quantitative real-time PCR analysis Total exosomes were isolated from individual serum of the two groups’ patients and controls using a commercial kit (Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008) according to the manufacturer’s instructions, which were described in supplementary materials associated with this article online. Total serum RNA was extracted from 100 µL serum using 1-step phenol/chloroform purification protocol as previously described [19]. Exosomal and exosome-free miRNAs were extracted and purified using Trizol Reagent (Invitrogen, Carlsbad, CA), per manufacturer’s instructions with minor modification as described in supplementary materials associated with this article online. Isolated miRNA was reverse-transcribed into complimentary DNA with AMV RT system (Takara) by using the TaqMan primer sets for miRs (Applied Biosystems) as previously described [19]. Quantitative real-time PCR (RT-qPCR) was performed on a Roche LightCycler® 480 Sequence Detection System (Roche Life Science of
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Roche Diagnostics Corporation, Indianapolis, Indiana, USA) by using TaqMan miRNA probes (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions with a minor modification as previously described [19]. All of the samples were run in triplicate. Since the Cq values of spiked-in MIR2911 showed quite constant among the three studied groups (Supplementary Fig. S1A), the relative expression content of serum miR-7 to the spiked-in exogenous MIR2911 was calculated using the equation 2 -∆Cq, and ∆Cq was calculated by subtracting the Cq value of MIR2911 from that of the miR-7. Nevertheless, due to the different nature of serum exosome and exosome-free samples, we found that the Cq value of the spiked-in exogenous MIR2911 displaying markedly difference between the two patients’ groups and control group as well as between those two different types sample (Supplementary Fig. S1B-D). Thus, with the purpose to effectively compare the miR-7 levels in serum, exosomes and exosome-free samples, we still constructed calibration curve with corresponding synthetic single-strand mature miR-7 oligonucleotide as previously described [20] to calculate the absolute concentration of miR-7 in exosome and corresponding exosome-free samples. The quantification calibrators were prepared by ten-fold serial dilution of synthetic miR-7 oligonucleotide (TaKaRa, Dalian, China) from 10 fmol/L to 107 fmol/L, and the level of this miR-7 oligonucleotide was assessed by RT-qPCR assay. The resulting Cq values were plotted versus the log10 of the amount of the synthetic miR-7 (Supplementary Fig. S2). Each sample and each dilution of the calibrator were run in
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triplicate for analysis.
2.4 Statistical analysis Statistical analysis was performed with SPSS 16.0 software (Chicago, USA). The concentrations of miR-7 with abnormal distribution were presented as the mean ± SEM, and the other normality or continuous variables were expressed as the mean ± SD or percent for categorical variables,
respectively.
The
nonparametric
Mann-Whitney U-test and two-sided χ2 test were used to analyze the differences in the miRNA concentrations and other variables among the groups, respectively. Pearson correlation or Spearman’s rank correlation analysis were used to measure the strength of the association between the two variables. A P-value < 0.05 was considered statistically significant.
3. Results 3.1 Demographic and baseline characteristics of studying subjects A total of 226 subjects including 76 T2DM patients, 76 T2DMC patients and 74 non-diabetic controls were recruited in this study. Table 1 summarized their ages, gender, HbA1c/hyperglycemia, BMI, lipid profile, complication, smoking history and alcohol consumption status. The mean DM duration was 1.8 ± 2.6 years for T2DM patients and 5.0 ± 5.3 years for T2DMC patients. As we expected, there were no significant differences in age distributions among the two patients’ groups and the
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control groups (Table 1). However, both T2DM and T2DMC patients showed markedly elevation in fasting glucose and HbA1c level as comparing with the non-diabetic controls (p < 0.001). Furthermore, it is notably that the T2DM patients and T2DMC patients also appeared to have a significantly disturbed lipid profile as comparing with the nondiabetic controls (Table 1). The prevalence of retinopathy, neuropathy, and nephropathy was 38.2%, 36.8%, and 7.9%, respectively, and 11 patients presented with at least one of the DM-associated microvascular complication. (Table 1).
3.2 Up-regulation of miR-7 in the serum of T2DM and T2DMC patients We then performed RT-qPCR assays to measure the serum concentrations of miR-7 in the three studied groups. We firstly calculated the absolute concentrations of serum miR-7 in the three groups using a calibrator as previously described [20], the content of serum miR-7 were obviously increased in T2DM and T2DMC groups when compared with the normal groups (Fig. 1A). However, there was no marked difference in serum miR-7 levels between T2DM and T2DMC groups (p = 0.224). Additionally, to further confirm the above findings, we also calculated the relative of serum miR-7 in the three groups using a calibrator as previously described [19], and we observed consistent increases of miR-7 levels in the two patients’ groups as compared to controls (Fig. 1B).
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3.3 Majority of serum miR-7 are presented outside exosomes and circulating freely Next, to determine whether the miR-7 in serum is contained in exosomes or free-circulating, we extracted the RNA from the exosomes pellet and from the exosome-depleted supernatant from the same three cohorts’ individual serum samples that used for miR-7 determination. The amount of miR-7 was then measured by RT-qPCR. Because our results revealed that the Cq value of the spiked-in exogenous MIR2911 which used for RNA extraction efficiency normalization displaying markedly difference between the supernatant and exosomal, we normalized the miR-7 concentration to the equal original sample volume. As shown in Figure 2, the concentration of the miR-7 was consistently higher in the exosome-free supernatant samples as compared with exosome samples in all the three studied groups. The mean content of miR-7 in the supernatant is 1.8-fold, 3.2-fold and 4.0-fold higher than in the exosomal pellet for the control group, T2DM group and T2DMC groups, respectively (Fig. 2A). The difference ranges from 0.82 ~ 18.04 folds (mean fold change = 3.28, 95% CI = 2.89 ~ 3.66) for the individual supernatant sample compared with its correspondence exosomal samples of the three cohorts (Fig. 2B-D). Taken together, these data demonstrated that the majority of miR-7 are freely circulating rather than encapsulated in exosomes in serum.
3.4 T2DM-associated abnormalities in miR-7 distribution are exhibited primarily
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in non-exosome fractions Since T2DM and T2DMC are associated with changes in circulating levels of miR-7, we next explored the potential clinical relevance of the distribution of circulating miR-7 and T2DM patients. we firstly analyzed the concentration of miR-7 in the exosome between the three studied cohorts using RT-qPCR, however, the absolute quantification of exosomal miR-7 showing no significant difference between the three groups (Fig. 2E). We then compared the miR-7 levels in exosome-free supernant, consequently, RT-qPCR results showed that concentrations of freely circulated miR-7 were markedly higher in the two groups’ patients as comparing with controls (Fig. 2F), even though no markedly difference was observed between the two patients’ groups (p = 0.329). Taken together, these results show that T2DM was associated with significant changes of circulating miR-7, and these changes were observed predominantly in the exosome-free fraction. To further explore the associations of serum miR-7 alterations and its physiologic state in T2DM, we subsequently examined the correlations between the serum miR-7 levels and the concentrations of corresponding exosome miR-7 or exosome-free miR-7 in all the studied individuals. Association analysis revealed that the serum miR-7 levels were positively correlated with exosome-free miR-7 contents but not exosomal miR-7 (r = 0.27, p < 0.001 and r = -0.10, p = 0.145, respectively) (Fig. 2G-H). These results further suggested that serum miR-7 differences in T2DM patients were mainly due to the alteration in the freely circulated miR-7 in sera.
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3.5 Increased levels of miR-7 in serum and exosome-depleted supernatant are closely associated with the presence of T2DM in patients with or without microvascular complications To evaluate the clinical usefulness of increased miR-7 in serum and exosome-depleted supernatant for T2DM patients with or without microvascular complications, we performed multivariable logistic regression analyzes to confirm that the correlation between serum miR-7 or freely circulated miR-7 and T2DM with or without complications. In the multiple model, after adjusting for age, gender, BMI, smoking status, alcohol consumption habits and blood pressure status, we observed that both serum miR-7 and exosome-free miR-7 contents were independently correlated with T2DM and T2DMC. The odds ratios (ORs) of serum miR-7 and exosome-free miR-7 for T2DM were 7.9 (95% CI, 2.4 – 25.7, P = 0.001) and 17.0 (95% CI, 5.2 – 56.0, P < 0.001), for T2DMC were 11.0 (95% CI, 3.4 – 35.5, P < 0.001) and 27.8 (95% CI, 8.9 – 87.1, P < 0.001). As our expectation, miR-7 in serum exosomes showed no significant association with T2DM (P = 0.236) and T2DMC (P = 0.679). These results revealed that both miR-7 in serum or exosome-depleted supernatant are potential independent risk factors from other traditional risk factors f or the presence of T2DM and its associated microvascular complications.
3.6 Diagnostic utility of circulating miR-7 in T2DM and T2DMC
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Receiver operating curve analyses (ROC) were used to evaluate the discriminative ability of the serum miR-7 or freely circulated miR-7 for the patients’ group and control group. As shown in Figure 3, serum miR-7 was able to discriminate T2DM and T2DMC from controls with similar areas under the receiver operator characteristics curve (AUC) of 0.76 (95% CI 0.68 – 0.83) and 0.73 (95% CI 0.64 – 0.81), respectively (Fig. 3). In those with paired serum miR-7, the AUC for freely circulated miR-7 discriminating cases and controls for T2DM was 0.75 (95% CI 0.67 – 0.83), and was superior for T2DMC (0.77, 95% CI 0.69 – 0.85), as shown in Figure 3.
3.7 Relationships of circulating miR-7 with biochemical Parameters We still analyzed the correlations of levels of miR-7 in serum or exosome-depleted supernatant with blood lipid and biochemical parameters. As shown in Table 2, the serum levels of miR-7 were positively correlated with blood glucose and TG, while negatively associated with HDL-C levels in all of the studied subjects. Interestingly, a significant positive correlation between levels of miR-7 in exosome-depleted supernatant and serum glucose, and negative correlation with HDL-C content were also found (Table 2). However, there was no significant association between exosome miR-7 levels and blood lipid profiles in the two groups’ patients and controls (Table 2). Together with the RT-qPCR results, these data suggest that the elevated levels of miR-7 in T2DM patients may be involved in T2DM pathogenesis and diabetic
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microvascular complications, and detection of miR-7 in serum as well as exosome-depleted supernatant may provide useful information for β cell dysfunction and T2DM development.
4. Discussion Circulating miRNA expression is dysregulated in a variety of diseases, and has been recognized as promising novel biomarkers for diagnosis and monitoring of almost all diseases, including T2DM. miR-7 is evolutionarily highly conserved and is considered to be a prototypical endocrine miRNA, being expressed at high levels in the pancreatic islet [8-10]. However, the dynamic changing of miR-7 expression pattern in the circulation of T2DM patients is remain unclear. Here, we provide the first evidence that miR-7, one of the most preferentially expressed miRNAs in endocrine cells of human pancreas, was significantly increased in serum of T2DM patients with or without microvascular complications when compared with control subjects. Furthermore, we also demonstrated that T2DM-associated abnormalities in serum miR-7 distribution are exhibited primarily in non-exosome fractions, and elevated levels of miR-7 in serum and exosome-free supernatant are closely associated with the presence of T2DM in patients with or without microvascular complications. By applying high-throughput approaches such as next generation sequencing or microRNA microarray technology combined with real-time quantitative RT-PCR
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technology, more than one hundred studies have now reported the miRNA profile in serum, plasma or blood cells in an attempt to discuss the usefulness of these new approaches to predict the development and progression of diabetes mellitus. These pioneering studies provided useful evidence for the potential of circulating miRNAs as novel non-invasive biomarkers for T2DM. However, the heterogeneity of the different studies obtained underscores the need for large prospective and large sample studies to identify reliable miRNA signatures for diagnosing T2DM. Accumulating evidence from in vivo and in vitro studies demonstrated that the expression of miRNAs is often tissue-specific or developmental specific, and thus miRNAs play important roles in regulation of gene expression at specific tissue or stages in various pathologic process. For instance, it has been reported that miRNAs enriched in mouse and human pancreatic islets and emerged as key regulators not only in mammalian insulin secretion but also in development of pancreatic islets [6]. Thus, it is therefore not surprising that many miRNAs have also been implicated in diabetes and its complications, and detecting those pancreatic islets specific miRNAs in circulation may provide useful information for the occurrence and development of T2DM. Actually, several pancreatic islets and β-cells enriched miRNAs which involved in inhibition of insulin secretion and compensatory β-cell proliferation were found to be dysregulated in plasma of T2DM patients [21-26]. As one of the most abundant miRNAs in human β-cells, miR-7 has been showed to targets multiple components of the mTOR signaling pathway in adult β-cells, and inhibition of miR-7 activates
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mTOR signaling and promotes adult β-cell proliferation in both mouse and human primary islets, indicating that miR-7 functions as a negative regulator of adult β-cell proliferation and dysregulated miR-7 as a therapeutic target for the treatment of diabetes [11]. This result was further confirmed by genetic knockout of miR-7 and resulted in increased glucose-stimulated insulin secretion, while transgenic overexpression of miR-7 in pancreatic β cells, occasioned chronic hyperglycemia, decreased circulating levels of insulin and impaired GSIS independent of obesity [14]. Moreover, miR-7 levels were negatively correlated with β cell function in transplanted human islets exposed to an obesogenic environment in mice [14]. These evidence from mechanistic studies fully demonstrated that increased miR-7 levels were closely associated with the occurrence and development of T2DM. In accordance with those previously reports [11, 14], we found that serum miR-7 levels were markedly higher in both T2DM and T2DMC patients, furthermore, serum miR-7 levels were also positively correlated with serum glucose, which indicated that circulating miR-7 were linked to β-cell dysfunction and impaired insulin secretion. In addition, our ROC analysis and logistic regression analysis further demonstrated that serum miR-7 concentrations were closely associated with T2DM with or without complications and might be useful biomarkers to distinguish diabetes from controls. Currently, the dominant view for the physiologic state of circulating miRNA is that miRNAs are actively secreted from cells in membrane-bound vesicles, including exosomes and shedding vesicles, which protect them from blood RNase activity [27].
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Nevertheless, in apparent contrast to this notion, two recent publications found that a significant portion of circulating miRNAs are predominantly exosome-free in cell culture media [28], and bound to Argonaut-2 protein in plasma and serum [29]. In our present study, we observed that nearly two-thirds of miR-7 were freely circulated rather than exosome-encapsulated in the serum of T2DM patients as well as in healthy subjects. Moreover, we also observed that serum miR-7 in exosome-free fraction but not in exosomes fraction displayed significant higher levels in the two patients’ groups than control group, and serum miR-7 concentrations were well correlated with freely circulated miR-7, and these results promote us to hypothesize that elevated miR-7 in serum of T2DM patients may originated from pancreatic β cells and reflect cell pathological status, since existing evidences demonstrated that circulating serum miRNAs may passively derived from broken cells after tissue injury, cell apoptosis or necrosis and chronic inflammation. Furthermore, our findings would suggest that measurement of serum miR-7 in nonexosomal fraction may be an effective strategy for improving sensitivity and miRNA biomarker performance in T2DMC patients. In the current study, we also noticed that miR-7 in circulation and exosome-depleted serum showing no significant difference between the two T2DM patients’ groups with or without microvascular complications. Previous studies have uncovered several circulating miRNAs such as miR-126 were markedly reduced in T2DM patients with diabetes-induced endothelial dysfunction, while miR-503, miR-15a and miR-16 were increased in the circulation of critical limb ischemia
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patients with T2DM [30, 31]. Our previous study also identified a panel of five serum miRNAs including miR-661, miR-571, miR-770-5p, miR-892b and miR-1303 were higher in T2DM patients with complications than in those without complications [19]. Nevertheless, those altered miRNAs have been reported to be participate in the inflammatory response, vascular endothelia damage and fibrosis processes[19, 30, 31]. Since miR-7 was demonstrated to be involved in β-cell dysfunction and insulin secretion but not the pathological processes of diabetic complications, it is reasonable that this pancreatic islet enriched miRNA showing no significant difference between T2DM patients with and without microvascular complications. However, further experimental studies are needed to examine whether miR-7 binding with specific serum protein such as Ago2 and HDL, in addition, the potential mechanisms of these protein carrier-based miRNA complexes participate the pathogenesis of T2DM were also needed to be investigated. An important question arises about the potential impact of the pharmacological treatments used in T2DM on the expression of serum miR-7. Although no report has fully addressed this issue in clinical setting currently, one pioneer study found that the expression levels of 13 plasma miRNAs showing no markedly difference between diabetic patients with or without drug treatment [30]. Nevertheless, studies for evaluating the role of hypoglycemic agents on circulating miRNA expression in T2DM and associated microvascular complications are still needed. In summary, our findings show that serum levels of miR-7 are significantly
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increased in patients with T2DM and T2DM-associated microvascular complications, and the serum miR-7 is predominantly exosome-free in circulation. Moreover, elevated levels of miR-7 in serum and exosome-free supernatant are closely associated with the T2DM presence and might has the potential as novel ancillary diagnostic marker and risk indicators for T2DM.
Supplementary materials Supplementary methods and data (Fig. S1 and S2) associated with this article can be found in the online version.
Conflict of Interest: We all have no conflicts of interests to disclose.
Acknowledgements: This study was supported by the grants from the National Natural Science Foundation of China (81401257, 81472021 and 81171661), the Natural Science Foundation of Jiangsu Province (BK20140730 and BK20140733), the Postdoctoral Scientific Foundation of China (2015M582901) and the Postdoctoral Scientific Foundation of Jiangsu Province (1501121C).
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[18] Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabetic medicine : a journal of the British Diabetic Association. 1998;15:539-53. [19] Wang C, Wan S, Yang T, Niu D, Zhang A, Yang C, et al. Increased serum microRNAs are closely associated with the presence of microvascular complications in type 2 diabetes mellitus. Scientific reports. 2016;6:20032. [20] Luo Y, Wang C, Chen X, Zhong T, Cai X, Chen S, et al. Increased serum and urinary microRNAs in children with idiopathic nephrotic syndrome. Clinical chemistry. 2013;59:658-66. [21] Seyhan AA, Nunez Lopez YO, Xie H, Yi F, Mathews C, Pasarica M, et al. Pancreas-enriched miRNAs are altered in the circulation of subjects with diabetes: a pilot cross-sectional study. Scientific reports. 2016;6:31479. [22] Delic D, Eisele C, Schmid R, Luippold G, Mayoux E, Grempler R. Characterization of Micro-RNA Changes during the Progression of Type 2 Diabetes in Zucker Diabetic Fatty Rats. International journal of molecular sciences. 2016;17. [23] Higuchi C, Nakatsuka A, Eguchi J, Teshigawara S, Kanzaki M, Katayama A, et al. Identification of circulating miR-101, miR-375 and miR-802 as biomarkers for type 2 diabetes. Metabolism: clinical and experimental. 2015;64:489-97. [24] Marchand L, Jalabert A, Meugnier E, Van den Hende K, Fabien N, Nicolino M, et al. miRNA-375 a Sensor of Glucotoxicity Is Altered in the Serum of Children with
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Newly Diagnosed Type 1 Diabetes. Journal of diabetes research. 2016;2016:1869082. [25] Erener S, Mojibian M, Fox JK, Denroche HC, Kieffer TJ. Circulating miR-375 as a biomarker of beta-cell death and diabetes in mice. Endocrinology. 2013;154:603-8. [26] Kong L, Zhu J, Han W, Jiang X, Xu M, Zhao Y, et al. Significance of serum microRNAs in pre-diabetes and newly diagnosed type 2 diabetes: a clinical study. Acta diabetologica. 2011;48:61-9. [27] Chen X, Liang H, Zhang J, Zen K, Zhang CY. Secreted microRNAs: a new form of intercellular communication. Trends in cell biology. 2012;22:125-32. [28] Turchinovich A, Weiz L, Langheinz A, Burwinkel B. Characterization of extracellular circulating microRNA. Nucleic acids research. 2011;39:7223-33. [29] Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:5003-8. [30] Zampetaki A, Kiechl S, Drozdov I, Willeit P, Mayr U, Prokopi M, et al. Plasma microRNA profiling reveals loss of endothelial miR-126 and other microRNAs in type 2 diabetes. Circulation research. 2010;107:810-7. [31] Spinetti G, Fortunato O, Caporali A, Shantikumar S, Marchetti M, Meloni M, et al. MicroRNA-15a and microRNA-16 impair human circulating proangiogenic cell functions and are increased in the proangiogenic cells and serum of patients with critical limb ischemia. Circulation research. 2013;112:335-46.
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Table 1.
Demographic and clinical features of the T2DM, T2DMC and
non-diabetic control group.a Variable Age (years)
Controls
T2DM
( n = 74 )
( n = 76 )
48.8 (15.2)
48.5 (14.5)
Female 2
BMI (kg/m )
51.1 (13.6)
50 (65.8)
55 (72.4)
33 (44.6)
26 (34.2)
21 (27.6)
23.0 (2.7)
25.2 (3.7) < 0.001 0.940
13 (17.6)
13 (17.1)
61 (82.4)
63 (82.9)
25.2 (3.9)
d
p valueb
p valuec
0.334
0.259
0.031d
0.380d
< 0.001
0.981
d
0.083d
0.754d
0.085d
0.797
0.100 22 (28.9) 54 (71.1)
0.159
Alcohol consumption-no.(%)
Ever and Current Never
( n = 76 )
41 (55.4)
Somking status- no.(%)
Ever and Current Never
0.894
T2DMC
0.193d
Sex- no.(%) Male
p valueb
d
15 (20.3)
9 (11.8)
17 (22.4)
59 (79.7)
67 (88.2)
59 (77.6)
SBP (mmHg)
120.9 (6.3)
133.1 (16.5)
< 0.001 132.4 (13.7) < 0.001
DBP (mmHg)
77.6 (4.7)
80.9 (9.2)
Diabetes duration (years)
0.006
80.7 (10.5)
1.8 (2.6)
5.0 (5.3)
0.019
0.915
Glucose (mmol/L)
4.9 (0.5)
8.9 (3.3) < 0.001
9.5 (3.7)
< 0.001
0.327
HbA1c (percent)
5.3 (0.4)
9.9 (2.9) < 0.001
10.3 (2.5)
< 0.001
0.29
Total cholesterol (mmol/L)
4.4 (0.5)
4.8 (2.1)
4.6 (1.0)
0.078
0.459
Triglyceride (mmol/L)
1.0 (0.3)
2.3 (2.3) < 0.001
2.2 (1.9)
< 0.001
0.711
LDL-C (mmol/L)
2.5 (0.6)
2.8 (1.0)
2.8 (0.9)
0.006
0.677
HDL-C (mmol/L)
1.4 (0.3)
1.0 (0.2) < 0.001
1.0 (0.2)
< 0.001
0.923
0.081 0.003
Complications Diabetic retinopathy
29 (38.2)
Diabetic neuropathy
28 (36.8)
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Diabetic nephropathy
6 (7.9)
Diabetic foot
2 (2.6)
Combined a
11 (14.5)
Data are mean (SD) or number (%).b Compared with control group.c Compared
between the two case group.d Two sides χ2 test. T2DM= type 2 diabetes. T2DMC= type 2 diabetes with microvascular complications. LDL-C= low density lipoprotein cholesterol. HDL-C= high density lipoprotein cholesterol.
Table 2.
Spearman rank correlations between serum miR-7, freely circulated
miR-7 and exosome miR-7 and sera indices in all the studied samples.a Varibles serum miR-7 (n = 226) exosome-free miR-7 (n = 226) exosome miR-7 (n = 226) a
r/p r p r p r p
Glucose 0.327 < 0.001 0.335 < 0.001 -0.005 0.934
TC 0.058 0.384 0.043 0.522 0.080 0.231
TG 0.271 < 0.001 0.224 0.001 0.000 0.999
HDL-C -0.278 < 0.001 -0.263 < 0.001 0.055 0.407
LDL-C 0.039 0.555 0.050 0.451 0.074 0.269
TC=total cholesterol. TG= Triglyceride. LDL-C= low density lipoprotein cholesterol.
HDL-C= high density lipoprotein cholesterol.
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Figure captions
Figure 1. Expression levels of serum miR-7 in the T2DM, T2DMC and non-diabetic control groups. (A) Absolute concentrations of the serum miR-7. Cq values were converted to absolute concentrations using the corresponding calibration curves. (B) The relative levels of the serum miR-7 to MIR2911. Cq values were converted to relative concentrations normalized to MIR2911 values and were calculated using the comparative Cq method (2-∆Cq). Each point represents the mean of triplicate samples. Each p-value was derived from a nonparametric Mann–Whitney U-test. **p < 0.01; ***p < 0.001.
Figure 2. The distribution of serum miR-7 in the T2DM, T2DMC and non-diabetic control groups. (A-D) Absolute concentration of miR-7 in exosome and exosome-free portions from 76 T2DM patients, 76 T2DMC patients and 74 non-diabetic control using qRT-PCR analysis. (E-F) Correlations of miR-7 expression content in serum with exosomal miR-7 (E) and with exosme-free miR-7 (F) in the 226 studied individuals, calculated by Pearson correlation analysis. ***p < 0.0001.
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Figure 3. ROC curves for the capacity of the serum miR-7 and freely circulated miR-7 to differentiate T2DM or T2DMC cases from control individuals. (A, B) ROC curves for the ability of the serum concentrations of miR-7 to differentiate the T2DM cases (n = 76) and the T2DMC cases (n = 76) from the control subjects (n = 74). (C, D) ROC curves for the ability of the concentrations of exosome-free miR-7 to differentiate the T2DM cases (n = 76) and the T2DMC cases (n = 76) from the control subjects (n = 74).
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Highlights:
A case-control study was conducted to characterize serum miR-7 signature in T2DM.
Serum miR-7 were significantly higher in the T2DM and T2DMC patients than controls.
Increased serum miR-7 is novel marker for T2DM and its microvascular complications.
S0168-8227(16)31836-8 http://dx.doi.org/10.1016/j.diabres.2017.06.005 DIAB 6987
To appear in:
Diabetes Research and Clinical Practice
Received Date: Revised Date: Accepted Date:
24 December 2016 15 April 2017 6 June 2017
Please cite this article as: S. Wan, J. Wang, J. Wang, J. Wu, J. Song, C-Y. Zhang, C. Zhang, C. Wang, J-J. Wang, Increased serum miR-7 is a promising biomarker for type 2 diabetes mellitus and its microvascular complications, Diabetes Research and Clinical Practice (2017), doi: http://dx.doi.org/10.1016/j.diabres.2017.06.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Increased serum miR-7 is a promising biomarker for type 2 diabetes mellitus and its microvascular complications
Shujun Wana,b,†, Jun Wangc,†, Jing Wanga, Jia Wua, Jiaxi Songa, Chen-Yu Zhangb, Chunni Zhanga,b, Cheng Wanga,b,* and Jun-Jun Wanga,b,*
a
Department of Clinical Laboratory, Jinling Hospital, Nanjing University School of
Medicine, Nanjing, 210002, China.
b
State Key Laboratory of Pharmaceutical
Biotechnology, Nanjing Advanced Institute for Life Sciences, Nanjing University School of Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Nanjing University, Nanjing, 210023, China; cDepartment of Cardiology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, 210002, China.
†
These authors contributed equally to this work.
*Correspondence authors: Jun-Jun Wang ([email protected]) and/or Cheng Wang ([email protected] or [email protected]) at Department of Clinical Laboratory, Jinling Hospital, Nanjing University School of Medicine, Nanjing, 210002, China. Tel.: +86 25 80861177; Fax.: +86 25 80861177
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Abstract Aims: To investigate the alteration pattern and physiologic state of islet-specific miR-7 in the serum of patients with type 2 diabetes mellitus (T2DM) and T2DM-associated microvascular complications (T2DMC) and to evaluate its clinical significance.
Methods: The levels of serum miR-7 were firstly examined and compared in 76 T2DM patients, 76 T2DMC patients and 74 age-gender matched controls using RT-qPCR. Subsequently, the physiologic state of serum miR-7 was characterized by determining its concentrations in isolated exosomes and corresponding exosome-free samples from the same three cohorts’ samples. Moreover, statistical analyzes were performed to evaluate the associations of serum miR-7 with T2DM and T2DMC.
Results: Serum miR-7 were significantly elevated in the T2DM patients [(401.0 ± 34.37) fmol/L, P < 0.001] and in the T2DMC patients [(501.4 ± 81.69) fmol/L, P < 0.001] when compared with the controls [(175.7 ± 16.59) fmol/L]. Circulating miR-7 were mainly existed as exosome-free form rather than in membrane-bound exosomes. The concentrations of exosome-free miR-7 was markedly higher in the T2DM group [(107.2 ± 9.63) fmol/L, P < 0.001] and in the T2DMC group [(122.1 ± 10.80) fmol/L, P < 0.001] compared to the control group [(54.18 ± 2.37) fmol/L]. Logistic regression
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and ROC curve analyses revealed the serum miR-7 was significantly associated with T2DM and microvascular complications (P < 0.05).
Conclusion: Increased serum miR-7 might have the potential as a promising marker for T2DM and its microvascular complications.
Keywords: serum microRNA; miR-7; type 2 diabetes; microvascular complications; biomarkers
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1. Introduction Type 2 diabetes mellitus (T2DM) is a major global healthcare problem, especially in China [1]. According to the epidemiological survey from the International Diabetes Federation, 382 million people had diabetes in 2013 worldwide, even worse, the greatest increases in T2DM incidence in the next few decades are expected to rise to 592 million by 2035 [2]. T2DM is characterized by high blood glucose levels and insulin resistance, with or without insufficient insulin secretion for the body’s needs, worse still, persistent increased blood glucose will result in vascular complications including microvascular complications [3]. Microvascular complications are the major outcome of T2DM progression, which were broadly classified as neuropathy, nephropathy, and retinopathy. These complications can be life threatening and will greatly reduce the quality of life, if left untreated [4]. With regard to these facts, it is critical to treat T2DM early in its progression to slow down or even stop the microvascular complications. However, the main mechanism underlying the pathogenesis of T2DM and its microvascular complications remains obscure. Moreover,
non-invasive
reliable
blood
biomarkers
for
T2DM
associated
microvascular disease are currently unavailable. Thus, it is important for us to find
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novel diagnostic biomarkers and provide new insights into the pathological process in diabetic complications. MicroRNAs (miRNAs) are a class of small (~22 nucleotide), non-coding RNAs that regulate gene expression at post-transcriptional level by inhibiting translation or inducing target mRNA degradation via binding with their target sites [5]. Accumulating evidence demonstrated that miRNAs are participated in various physiological and pathophysiological process including diabetes and diabetic microvascular complications [6]. To date, more than 2,000 human miRNAs have been registered in the miRBase 22.0, which are predicted to regulate up to 60% of the human genes [7]. Several miRNAs exhibit a tissue-specific expression manner, thereby contributing to cell identity and function. Among them, miR-7 is one of the most abundant miRNA in pancreas, with predominant expression in human and mouse pancreatic islet cells such as β cells [8-10]. Emerging evidences have documented that miR-7 is evolutionarily highly conserved and highly expressed miRNA in pancreas, moreover, it has also been reported to be involved in pancreatic β-cell development as well as insulin secretion[11-13]. Additionally, several reports have identified that disturbing miR-7 expression in transgenic mouse or β cells can also lead to diabetes [14, 15]. These findings suggest that miR-7 may have the potential as novel biomarker for monitoring normal development and function of pancreatic β-cells, and dysregulated miR-7 may closely related to the occurrence and development of T2DM.
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Recent studies from our group and others demonstrated that miRNAs were also existed in circulation in a remarkably stable form that is resistant to endogenous RNase activity and several other harsh conditions treatment, and the unique pattern of circulating miRNAs may provide a useful biomarker for supplemental diagnosis for various diseases including diabetes mellitus [16].Up to date, a number of elevated or decreased circulating miRNAs have been identified in both T2DM-susceptible subjects, T2DM patients as well as T2DMC patients [17]. However, most existing studies are focused on a few specific vascular endothelial enriched miRNAs or immuno-inflammatory related miRNAs, whether pancreatic islet-specific miR-7 might act as a circulating factor in T2DM and T2DMC has barely been explored. On the other hand, intensive studies from different groups have unraveled that serum miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes
or
presented
in
the
circulation
in
a
cell-free
RNA-binding
protein-associated form, but this has not been systematically characterized for miR-7 in the peripheral blood of T2DM patients with or without diabetic microvascular complications. Therefore,
in
the
present
study,
we
performed
a
quantitative
reverse-transcriptions PCR (RT-qPCR) to examine and compare the expression levels of miR-7 in the serum of T2DM patients, T2DMC patients and non-diabetic healthy controls. We also investigated the physical state of serum miR-7 in those two patients’ groups as well as controls with the goal to determine whether miR-7 in serum are
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primarily in exosomes or circulating freely that if there has any benefit of using serum miR-7 in biomarker studies.
2. Material and methods 2.1 Study Design and Patients The present study consisted of 152 T2DM patients who were newly diagnosed or with previously diagnosed disease but during drug withdrawal more than one month in the Department of Endocrinology, Jinling Hospital, Nanjing, China from September 2013 to May 2016. In brief, T2DM patients with the drug withdrawal mean that those patients were not use any oral hypoglycemic agents (including sulfonylurea, biguanide, thiazolidinedione, alpha glucosidase inhibitors and dipeptidyl peptidase inhibitor) at least one month before the sample collection. In addition, patients being treated with insulin were also excluded. T2DM was diagnosed according to the World Health Organization criteria (WHO) and was defined as fasting plasma glucose of ≥ 7.0 mmol/L (126 mg/dl) and/or 2-h of glucose of ≥ 11.1 mmol/L (200 mg/dl) in the 75-g OGTT and/or HbA1c ≥ 6.5% [18]. Of the 152 patients, 76 were diagnosed with microvascular complications (29 with diabetic retinopathy, 28 with diabetic neuropathy, 6 with diabetic nephropathy and 2 with diabetic foot; the others had a combination of complications), and the other 76 T2DM patients were without clinical signs and symptoms of any complications. T2DM patients with microvascular complications were documented by medical records as previously described [19].
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Participants associated with other diseases such as type 1 diabetes mellitus, other types of diabetes, a severe infection, acute cerebrovascular disease, or recent surgery were excluded from this study. In the meanwhile, 74 age- and gender-matched healthy volunteers were recruited as non-diabetic controls from a large pool of individuals seeking routine health check-up at Jinling Hospital and exhibited no evidence of disease. The health checkup included a detailed history, physical examination and blood test. The study protocol was approved by the ethics committees of Jinling Hospital and was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all of the participants prior to the study.
2.2 Blood collection, sample processing and serum biochemical parameters determination Peripheral venous blood (3~5 ml) was collected in serum vacutainer tubes with gel and clot activator (Becton, Dickinson and Company, Franklin Lakes, New Jersey) from participants after at least 12 h of fasting, and serum was immediately separated by a 10-min centrifugation at 1,500 g. The remaining serum samples were stored at -80 °C until miRNA analysis. Serum glucose levels were examined by the auto-analyzer method (Hitachi 7600, Hitachi High-Technologies Corporation, Tokyo, Japan). Other clinical biochemical parameters including serum total cholesterol (TC), triglyceride (TG), low density
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lipoprotein cholesterol (LDL) and high density lipoprotein cholesterol (HDL) concentrations were measured using commercial reagents (RANDOX) on a Hitachi 7600 analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan). HbA1C were assessed using commercial reagents (TOSOH Corporation) on a HLC-723GB auto-analyzer (TOSOH Corporation, Tokyo, Japan).
2.3 Total serum exosome isolation, RNA extraction and quantitative real-time PCR analysis Total exosomes were isolated from individual serum of the two groups’ patients and controls using a commercial kit (Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008) according to the manufacturer’s instructions, which were described in supplementary materials associated with this article online. Total serum RNA was extracted from 100 µL serum using 1-step phenol/chloroform purification protocol as previously described [19]. Exosomal and exosome-free miRNAs were extracted and purified using Trizol Reagent (Invitrogen, Carlsbad, CA), per manufacturer’s instructions with minor modification as described in supplementary materials associated with this article online. Isolated miRNA was reverse-transcribed into complimentary DNA with AMV RT system (Takara) by using the TaqMan primer sets for miRs (Applied Biosystems) as previously described [19]. Quantitative real-time PCR (RT-qPCR) was performed on a Roche LightCycler® 480 Sequence Detection System (Roche Life Science of
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Roche Diagnostics Corporation, Indianapolis, Indiana, USA) by using TaqMan miRNA probes (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions with a minor modification as previously described [19]. All of the samples were run in triplicate. Since the Cq values of spiked-in MIR2911 showed quite constant among the three studied groups (Supplementary Fig. S1A), the relative expression content of serum miR-7 to the spiked-in exogenous MIR2911 was calculated using the equation 2 -∆Cq, and ∆Cq was calculated by subtracting the Cq value of MIR2911 from that of the miR-7. Nevertheless, due to the different nature of serum exosome and exosome-free samples, we found that the Cq value of the spiked-in exogenous MIR2911 displaying markedly difference between the two patients’ groups and control group as well as between those two different types sample (Supplementary Fig. S1B-D). Thus, with the purpose to effectively compare the miR-7 levels in serum, exosomes and exosome-free samples, we still constructed calibration curve with corresponding synthetic single-strand mature miR-7 oligonucleotide as previously described [20] to calculate the absolute concentration of miR-7 in exosome and corresponding exosome-free samples. The quantification calibrators were prepared by ten-fold serial dilution of synthetic miR-7 oligonucleotide (TaKaRa, Dalian, China) from 10 fmol/L to 107 fmol/L, and the level of this miR-7 oligonucleotide was assessed by RT-qPCR assay. The resulting Cq values were plotted versus the log10 of the amount of the synthetic miR-7 (Supplementary Fig. S2). Each sample and each dilution of the calibrator were run in
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triplicate for analysis.
2.4 Statistical analysis Statistical analysis was performed with SPSS 16.0 software (Chicago, USA). The concentrations of miR-7 with abnormal distribution were presented as the mean ± SEM, and the other normality or continuous variables were expressed as the mean ± SD or percent for categorical variables,
respectively.
The
nonparametric
Mann-Whitney U-test and two-sided χ2 test were used to analyze the differences in the miRNA concentrations and other variables among the groups, respectively. Pearson correlation or Spearman’s rank correlation analysis were used to measure the strength of the association between the two variables. A P-value < 0.05 was considered statistically significant.
3. Results 3.1 Demographic and baseline characteristics of studying subjects A total of 226 subjects including 76 T2DM patients, 76 T2DMC patients and 74 non-diabetic controls were recruited in this study. Table 1 summarized their ages, gender, HbA1c/hyperglycemia, BMI, lipid profile, complication, smoking history and alcohol consumption status. The mean DM duration was 1.8 ± 2.6 years for T2DM patients and 5.0 ± 5.3 years for T2DMC patients. As we expected, there were no significant differences in age distributions among the two patients’ groups and the
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control groups (Table 1). However, both T2DM and T2DMC patients showed markedly elevation in fasting glucose and HbA1c level as comparing with the non-diabetic controls (p < 0.001). Furthermore, it is notably that the T2DM patients and T2DMC patients also appeared to have a significantly disturbed lipid profile as comparing with the nondiabetic controls (Table 1). The prevalence of retinopathy, neuropathy, and nephropathy was 38.2%, 36.8%, and 7.9%, respectively, and 11 patients presented with at least one of the DM-associated microvascular complication. (Table 1).
3.2 Up-regulation of miR-7 in the serum of T2DM and T2DMC patients We then performed RT-qPCR assays to measure the serum concentrations of miR-7 in the three studied groups. We firstly calculated the absolute concentrations of serum miR-7 in the three groups using a calibrator as previously described [20], the content of serum miR-7 were obviously increased in T2DM and T2DMC groups when compared with the normal groups (Fig. 1A). However, there was no marked difference in serum miR-7 levels between T2DM and T2DMC groups (p = 0.224). Additionally, to further confirm the above findings, we also calculated the relative of serum miR-7 in the three groups using a calibrator as previously described [19], and we observed consistent increases of miR-7 levels in the two patients’ groups as compared to controls (Fig. 1B).
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3.3 Majority of serum miR-7 are presented outside exosomes and circulating freely Next, to determine whether the miR-7 in serum is contained in exosomes or free-circulating, we extracted the RNA from the exosomes pellet and from the exosome-depleted supernatant from the same three cohorts’ individual serum samples that used for miR-7 determination. The amount of miR-7 was then measured by RT-qPCR. Because our results revealed that the Cq value of the spiked-in exogenous MIR2911 which used for RNA extraction efficiency normalization displaying markedly difference between the supernatant and exosomal, we normalized the miR-7 concentration to the equal original sample volume. As shown in Figure 2, the concentration of the miR-7 was consistently higher in the exosome-free supernatant samples as compared with exosome samples in all the three studied groups. The mean content of miR-7 in the supernatant is 1.8-fold, 3.2-fold and 4.0-fold higher than in the exosomal pellet for the control group, T2DM group and T2DMC groups, respectively (Fig. 2A). The difference ranges from 0.82 ~ 18.04 folds (mean fold change = 3.28, 95% CI = 2.89 ~ 3.66) for the individual supernatant sample compared with its correspondence exosomal samples of the three cohorts (Fig. 2B-D). Taken together, these data demonstrated that the majority of miR-7 are freely circulating rather than encapsulated in exosomes in serum.
3.4 T2DM-associated abnormalities in miR-7 distribution are exhibited primarily
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in non-exosome fractions Since T2DM and T2DMC are associated with changes in circulating levels of miR-7, we next explored the potential clinical relevance of the distribution of circulating miR-7 and T2DM patients. we firstly analyzed the concentration of miR-7 in the exosome between the three studied cohorts using RT-qPCR, however, the absolute quantification of exosomal miR-7 showing no significant difference between the three groups (Fig. 2E). We then compared the miR-7 levels in exosome-free supernant, consequently, RT-qPCR results showed that concentrations of freely circulated miR-7 were markedly higher in the two groups’ patients as comparing with controls (Fig. 2F), even though no markedly difference was observed between the two patients’ groups (p = 0.329). Taken together, these results show that T2DM was associated with significant changes of circulating miR-7, and these changes were observed predominantly in the exosome-free fraction. To further explore the associations of serum miR-7 alterations and its physiologic state in T2DM, we subsequently examined the correlations between the serum miR-7 levels and the concentrations of corresponding exosome miR-7 or exosome-free miR-7 in all the studied individuals. Association analysis revealed that the serum miR-7 levels were positively correlated with exosome-free miR-7 contents but not exosomal miR-7 (r = 0.27, p < 0.001 and r = -0.10, p = 0.145, respectively) (Fig. 2G-H). These results further suggested that serum miR-7 differences in T2DM patients were mainly due to the alteration in the freely circulated miR-7 in sera.
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3.5 Increased levels of miR-7 in serum and exosome-depleted supernatant are closely associated with the presence of T2DM in patients with or without microvascular complications To evaluate the clinical usefulness of increased miR-7 in serum and exosome-depleted supernatant for T2DM patients with or without microvascular complications, we performed multivariable logistic regression analyzes to confirm that the correlation between serum miR-7 or freely circulated miR-7 and T2DM with or without complications. In the multiple model, after adjusting for age, gender, BMI, smoking status, alcohol consumption habits and blood pressure status, we observed that both serum miR-7 and exosome-free miR-7 contents were independently correlated with T2DM and T2DMC. The odds ratios (ORs) of serum miR-7 and exosome-free miR-7 for T2DM were 7.9 (95% CI, 2.4 – 25.7, P = 0.001) and 17.0 (95% CI, 5.2 – 56.0, P < 0.001), for T2DMC were 11.0 (95% CI, 3.4 – 35.5, P < 0.001) and 27.8 (95% CI, 8.9 – 87.1, P < 0.001). As our expectation, miR-7 in serum exosomes showed no significant association with T2DM (P = 0.236) and T2DMC (P = 0.679). These results revealed that both miR-7 in serum or exosome-depleted supernatant are potential independent risk factors from other traditional risk factors f or the presence of T2DM and its associated microvascular complications.
3.6 Diagnostic utility of circulating miR-7 in T2DM and T2DMC
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Receiver operating curve analyses (ROC) were used to evaluate the discriminative ability of the serum miR-7 or freely circulated miR-7 for the patients’ group and control group. As shown in Figure 3, serum miR-7 was able to discriminate T2DM and T2DMC from controls with similar areas under the receiver operator characteristics curve (AUC) of 0.76 (95% CI 0.68 – 0.83) and 0.73 (95% CI 0.64 – 0.81), respectively (Fig. 3). In those with paired serum miR-7, the AUC for freely circulated miR-7 discriminating cases and controls for T2DM was 0.75 (95% CI 0.67 – 0.83), and was superior for T2DMC (0.77, 95% CI 0.69 – 0.85), as shown in Figure 3.
3.7 Relationships of circulating miR-7 with biochemical Parameters We still analyzed the correlations of levels of miR-7 in serum or exosome-depleted supernatant with blood lipid and biochemical parameters. As shown in Table 2, the serum levels of miR-7 were positively correlated with blood glucose and TG, while negatively associated with HDL-C levels in all of the studied subjects. Interestingly, a significant positive correlation between levels of miR-7 in exosome-depleted supernatant and serum glucose, and negative correlation with HDL-C content were also found (Table 2). However, there was no significant association between exosome miR-7 levels and blood lipid profiles in the two groups’ patients and controls (Table 2). Together with the RT-qPCR results, these data suggest that the elevated levels of miR-7 in T2DM patients may be involved in T2DM pathogenesis and diabetic
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microvascular complications, and detection of miR-7 in serum as well as exosome-depleted supernatant may provide useful information for β cell dysfunction and T2DM development.
4. Discussion Circulating miRNA expression is dysregulated in a variety of diseases, and has been recognized as promising novel biomarkers for diagnosis and monitoring of almost all diseases, including T2DM. miR-7 is evolutionarily highly conserved and is considered to be a prototypical endocrine miRNA, being expressed at high levels in the pancreatic islet [8-10]. However, the dynamic changing of miR-7 expression pattern in the circulation of T2DM patients is remain unclear. Here, we provide the first evidence that miR-7, one of the most preferentially expressed miRNAs in endocrine cells of human pancreas, was significantly increased in serum of T2DM patients with or without microvascular complications when compared with control subjects. Furthermore, we also demonstrated that T2DM-associated abnormalities in serum miR-7 distribution are exhibited primarily in non-exosome fractions, and elevated levels of miR-7 in serum and exosome-free supernatant are closely associated with the presence of T2DM in patients with or without microvascular complications. By applying high-throughput approaches such as next generation sequencing or microRNA microarray technology combined with real-time quantitative RT-PCR
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technology, more than one hundred studies have now reported the miRNA profile in serum, plasma or blood cells in an attempt to discuss the usefulness of these new approaches to predict the development and progression of diabetes mellitus. These pioneering studies provided useful evidence for the potential of circulating miRNAs as novel non-invasive biomarkers for T2DM. However, the heterogeneity of the different studies obtained underscores the need for large prospective and large sample studies to identify reliable miRNA signatures for diagnosing T2DM. Accumulating evidence from in vivo and in vitro studies demonstrated that the expression of miRNAs is often tissue-specific or developmental specific, and thus miRNAs play important roles in regulation of gene expression at specific tissue or stages in various pathologic process. For instance, it has been reported that miRNAs enriched in mouse and human pancreatic islets and emerged as key regulators not only in mammalian insulin secretion but also in development of pancreatic islets [6]. Thus, it is therefore not surprising that many miRNAs have also been implicated in diabetes and its complications, and detecting those pancreatic islets specific miRNAs in circulation may provide useful information for the occurrence and development of T2DM. Actually, several pancreatic islets and β-cells enriched miRNAs which involved in inhibition of insulin secretion and compensatory β-cell proliferation were found to be dysregulated in plasma of T2DM patients [21-26]. As one of the most abundant miRNAs in human β-cells, miR-7 has been showed to targets multiple components of the mTOR signaling pathway in adult β-cells, and inhibition of miR-7 activates
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mTOR signaling and promotes adult β-cell proliferation in both mouse and human primary islets, indicating that miR-7 functions as a negative regulator of adult β-cell proliferation and dysregulated miR-7 as a therapeutic target for the treatment of diabetes [11]. This result was further confirmed by genetic knockout of miR-7 and resulted in increased glucose-stimulated insulin secretion, while transgenic overexpression of miR-7 in pancreatic β cells, occasioned chronic hyperglycemia, decreased circulating levels of insulin and impaired GSIS independent of obesity [14]. Moreover, miR-7 levels were negatively correlated with β cell function in transplanted human islets exposed to an obesogenic environment in mice [14]. These evidence from mechanistic studies fully demonstrated that increased miR-7 levels were closely associated with the occurrence and development of T2DM. In accordance with those previously reports [11, 14], we found that serum miR-7 levels were markedly higher in both T2DM and T2DMC patients, furthermore, serum miR-7 levels were also positively correlated with serum glucose, which indicated that circulating miR-7 were linked to β-cell dysfunction and impaired insulin secretion. In addition, our ROC analysis and logistic regression analysis further demonstrated that serum miR-7 concentrations were closely associated with T2DM with or without complications and might be useful biomarkers to distinguish diabetes from controls. Currently, the dominant view for the physiologic state of circulating miRNA is that miRNAs are actively secreted from cells in membrane-bound vesicles, including exosomes and shedding vesicles, which protect them from blood RNase activity [27].
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Nevertheless, in apparent contrast to this notion, two recent publications found that a significant portion of circulating miRNAs are predominantly exosome-free in cell culture media [28], and bound to Argonaut-2 protein in plasma and serum [29]. In our present study, we observed that nearly two-thirds of miR-7 were freely circulated rather than exosome-encapsulated in the serum of T2DM patients as well as in healthy subjects. Moreover, we also observed that serum miR-7 in exosome-free fraction but not in exosomes fraction displayed significant higher levels in the two patients’ groups than control group, and serum miR-7 concentrations were well correlated with freely circulated miR-7, and these results promote us to hypothesize that elevated miR-7 in serum of T2DM patients may originated from pancreatic β cells and reflect cell pathological status, since existing evidences demonstrated that circulating serum miRNAs may passively derived from broken cells after tissue injury, cell apoptosis or necrosis and chronic inflammation. Furthermore, our findings would suggest that measurement of serum miR-7 in nonexosomal fraction may be an effective strategy for improving sensitivity and miRNA biomarker performance in T2DMC patients. In the current study, we also noticed that miR-7 in circulation and exosome-depleted serum showing no significant difference between the two T2DM patients’ groups with or without microvascular complications. Previous studies have uncovered several circulating miRNAs such as miR-126 were markedly reduced in T2DM patients with diabetes-induced endothelial dysfunction, while miR-503, miR-15a and miR-16 were increased in the circulation of critical limb ischemia
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patients with T2DM [30, 31]. Our previous study also identified a panel of five serum miRNAs including miR-661, miR-571, miR-770-5p, miR-892b and miR-1303 were higher in T2DM patients with complications than in those without complications [19]. Nevertheless, those altered miRNAs have been reported to be participate in the inflammatory response, vascular endothelia damage and fibrosis processes[19, 30, 31]. Since miR-7 was demonstrated to be involved in β-cell dysfunction and insulin secretion but not the pathological processes of diabetic complications, it is reasonable that this pancreatic islet enriched miRNA showing no significant difference between T2DM patients with and without microvascular complications. However, further experimental studies are needed to examine whether miR-7 binding with specific serum protein such as Ago2 and HDL, in addition, the potential mechanisms of these protein carrier-based miRNA complexes participate the pathogenesis of T2DM were also needed to be investigated. An important question arises about the potential impact of the pharmacological treatments used in T2DM on the expression of serum miR-7. Although no report has fully addressed this issue in clinical setting currently, one pioneer study found that the expression levels of 13 plasma miRNAs showing no markedly difference between diabetic patients with or without drug treatment [30]. Nevertheless, studies for evaluating the role of hypoglycemic agents on circulating miRNA expression in T2DM and associated microvascular complications are still needed. In summary, our findings show that serum levels of miR-7 are significantly
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increased in patients with T2DM and T2DM-associated microvascular complications, and the serum miR-7 is predominantly exosome-free in circulation. Moreover, elevated levels of miR-7 in serum and exosome-free supernatant are closely associated with the T2DM presence and might has the potential as novel ancillary diagnostic marker and risk indicators for T2DM.
Supplementary materials Supplementary methods and data (Fig. S1 and S2) associated with this article can be found in the online version.
Conflict of Interest: We all have no conflicts of interests to disclose.
Acknowledgements: This study was supported by the grants from the National Natural Science Foundation of China (81401257, 81472021 and 81171661), the Natural Science Foundation of Jiangsu Province (BK20140730 and BK20140733), the Postdoctoral Scientific Foundation of China (2015M582901) and the Postdoctoral Scientific Foundation of Jiangsu Province (1501121C).
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[2] Guariguata L, Whiting DR, Hambleton I, Beagley J, Linnenkamp U, Shaw JE. Global estimates of diabetes prevalence for 2013 and projections for 2035. Diabetes research and clinical practice. 2014;103:137-49. [3] Sayin N, Kara N, Pekel G. Ocular complications of diabetes mellitus. World journal of diabetes. 2015;6:92-108. [4] Sattar N, Preiss D. Diabetic microvascular complications as simple indicators of risk for cardiovascular outcomes and heart failure. The lancet Diabetes & endocrinology. 2016;4:555-6. [5] Huntzinger E, Izaurralde E. Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nature reviews Genetics. 2011;12:99-110. [6] Filios SR, Shalev A. beta-Cell MicroRNAs: Small but Powerful. Diabetes. 2015;64:3631-44. [7] Kozomara A, Griffiths-Jones S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic acids research. 2014;42:D68-73. [8] Correa-Medina M, Bravo-Egana V, Rosero S, Ricordi C, Edlund H, Diez J, et al. MicroRNA miR-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas. Gene expression patterns : GEP. 2009;9:193-9. [9] Bravo-Egana V, Rosero S, Molano RD, Pileggi A, Ricordi C, Dominguez-Bendala J, et al. Quantitative differential expression analysis reveals miR-7 as major islet microRNA. Biochemical and biophysical research communications. 2008;366:922-6. [10] Joglekar MV, Joglekar VM, Hardikar AA. Expression of islet-specific
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[18] Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabetic medicine : a journal of the British Diabetic Association. 1998;15:539-53. [19] Wang C, Wan S, Yang T, Niu D, Zhang A, Yang C, et al. Increased serum microRNAs are closely associated with the presence of microvascular complications in type 2 diabetes mellitus. Scientific reports. 2016;6:20032. [20] Luo Y, Wang C, Chen X, Zhong T, Cai X, Chen S, et al. Increased serum and urinary microRNAs in children with idiopathic nephrotic syndrome. Clinical chemistry. 2013;59:658-66. [21] Seyhan AA, Nunez Lopez YO, Xie H, Yi F, Mathews C, Pasarica M, et al. Pancreas-enriched miRNAs are altered in the circulation of subjects with diabetes: a pilot cross-sectional study. Scientific reports. 2016;6:31479. [22] Delic D, Eisele C, Schmid R, Luippold G, Mayoux E, Grempler R. Characterization of Micro-RNA Changes during the Progression of Type 2 Diabetes in Zucker Diabetic Fatty Rats. International journal of molecular sciences. 2016;17. [23] Higuchi C, Nakatsuka A, Eguchi J, Teshigawara S, Kanzaki M, Katayama A, et al. Identification of circulating miR-101, miR-375 and miR-802 as biomarkers for type 2 diabetes. Metabolism: clinical and experimental. 2015;64:489-97. [24] Marchand L, Jalabert A, Meugnier E, Van den Hende K, Fabien N, Nicolino M, et al. miRNA-375 a Sensor of Glucotoxicity Is Altered in the Serum of Children with
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Table 1.
Demographic and clinical features of the T2DM, T2DMC and
non-diabetic control group.a Variable Age (years)
Controls
T2DM
( n = 74 )
( n = 76 )
48.8 (15.2)
48.5 (14.5)
Female 2
BMI (kg/m )
51.1 (13.6)
50 (65.8)
55 (72.4)
33 (44.6)
26 (34.2)
21 (27.6)
23.0 (2.7)
25.2 (3.7) < 0.001 0.940
13 (17.6)
13 (17.1)
61 (82.4)
63 (82.9)
25.2 (3.9)
d
p valueb
p valuec
0.334
0.259
0.031d
0.380d
< 0.001
0.981
d
0.083d
0.754d
0.085d
0.797
0.100 22 (28.9) 54 (71.1)
0.159
Alcohol consumption-no.(%)
Ever and Current Never
( n = 76 )
41 (55.4)
Somking status- no.(%)
Ever and Current Never
0.894
T2DMC
0.193d
Sex- no.(%) Male
p valueb
d
15 (20.3)
9 (11.8)
17 (22.4)
59 (79.7)
67 (88.2)
59 (77.6)
SBP (mmHg)
120.9 (6.3)
133.1 (16.5)
< 0.001 132.4 (13.7) < 0.001
DBP (mmHg)
77.6 (4.7)
80.9 (9.2)
Diabetes duration (years)
0.006
80.7 (10.5)
1.8 (2.6)
5.0 (5.3)
0.019
0.915
Glucose (mmol/L)
4.9 (0.5)
8.9 (3.3) < 0.001
9.5 (3.7)
< 0.001
0.327
HbA1c (percent)
5.3 (0.4)
9.9 (2.9) < 0.001
10.3 (2.5)
< 0.001
0.29
Total cholesterol (mmol/L)
4.4 (0.5)
4.8 (2.1)
4.6 (1.0)
0.078
0.459
Triglyceride (mmol/L)
1.0 (0.3)
2.3 (2.3) < 0.001
2.2 (1.9)
< 0.001
0.711
LDL-C (mmol/L)
2.5 (0.6)
2.8 (1.0)
2.8 (0.9)
0.006
0.677
HDL-C (mmol/L)
1.4 (0.3)
1.0 (0.2) < 0.001
1.0 (0.2)
< 0.001
0.923
0.081 0.003
Complications Diabetic retinopathy
29 (38.2)
Diabetic neuropathy
28 (36.8)
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Diabetic nephropathy
6 (7.9)
Diabetic foot
2 (2.6)
Combined a
11 (14.5)
Data are mean (SD) or number (%).b Compared with control group.c Compared
between the two case group.d Two sides χ2 test. T2DM= type 2 diabetes. T2DMC= type 2 diabetes with microvascular complications. LDL-C= low density lipoprotein cholesterol. HDL-C= high density lipoprotein cholesterol.
Table 2.
Spearman rank correlations between serum miR-7, freely circulated
miR-7 and exosome miR-7 and sera indices in all the studied samples.a Varibles serum miR-7 (n = 226) exosome-free miR-7 (n = 226) exosome miR-7 (n = 226) a
r/p r p r p r p
Glucose 0.327 < 0.001 0.335 < 0.001 -0.005 0.934
TC 0.058 0.384 0.043 0.522 0.080 0.231
TG 0.271 < 0.001 0.224 0.001 0.000 0.999
HDL-C -0.278 < 0.001 -0.263 < 0.001 0.055 0.407
LDL-C 0.039 0.555 0.050 0.451 0.074 0.269
TC=total cholesterol. TG= Triglyceride. LDL-C= low density lipoprotein cholesterol.
HDL-C= high density lipoprotein cholesterol.
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Figure captions
Figure 1. Expression levels of serum miR-7 in the T2DM, T2DMC and non-diabetic control groups. (A) Absolute concentrations of the serum miR-7. Cq values were converted to absolute concentrations using the corresponding calibration curves. (B) The relative levels of the serum miR-7 to MIR2911. Cq values were converted to relative concentrations normalized to MIR2911 values and were calculated using the comparative Cq method (2-∆Cq). Each point represents the mean of triplicate samples. Each p-value was derived from a nonparametric Mann–Whitney U-test. **p < 0.01; ***p < 0.001.
Figure 2. The distribution of serum miR-7 in the T2DM, T2DMC and non-diabetic control groups. (A-D) Absolute concentration of miR-7 in exosome and exosome-free portions from 76 T2DM patients, 76 T2DMC patients and 74 non-diabetic control using qRT-PCR analysis. (E-F) Correlations of miR-7 expression content in serum with exosomal miR-7 (E) and with exosme-free miR-7 (F) in the 226 studied individuals, calculated by Pearson correlation analysis. ***p < 0.0001.
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Figure 3. ROC curves for the capacity of the serum miR-7 and freely circulated miR-7 to differentiate T2DM or T2DMC cases from control individuals. (A, B) ROC curves for the ability of the serum concentrations of miR-7 to differentiate the T2DM cases (n = 76) and the T2DMC cases (n = 76) from the control subjects (n = 74). (C, D) ROC curves for the ability of the concentrations of exosome-free miR-7 to differentiate the T2DM cases (n = 76) and the T2DMC cases (n = 76) from the control subjects (n = 74).
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Highlights:
A case-control study was conducted to characterize serum miR-7 signature in T2DM.
Serum miR-7 were significantly higher in the T2DM and T2DMC patients than controls.
Increased serum miR-7 is novel marker for T2DM and its microvascular complications.