Han population is six times as likely as Kazak population to present with type 2 diabetes mellitus (T2DM) in China. We hypothesize that differential expression and CpG methylation of miR-375 may be an ethnic-related factor that influences the incidence of T2DM. The expression level of miR-375 was examined using real-time PCR on Kazak and Han T2DM plasma samples. Furthermore, the methylation levels of CpG sites of miR-375 promoter were determined by MassARRAY Spectrometry in these samples. The relative expression levels of plasma miR-375 in Kazak T2DM samples are 1, and the relative expression levels of plasma miR-375 in Han T2DM samples are 3. The mean level of miR-375 methylation, calculated from the methylation levels of the CpG sites, was 8.47% for the Kazak T2DM group and 10.38% for the Han T2DM group. Further, five CpG units showed a statistically significant difference between Kazak and Han T2DM samples, and, among them, four were hypomethylated and only one CpG unit showed hypermethylation in Kazak T2DM samples. These findings indicate that the expression levels of plasma miR-375 and its CpG methylation in the promoter region are ethnically different, which may contribute to the different incidence of diabetes observed in Kazak and Han populations.
Type 2 diabetes mellitus (T2DM) is commonly associated with obesity and results from defects in insulin secretion and/or diminished sensitivity of target tissues to insulin action. MicroRNAs (miRNAs) are endogenous about 22 nucleotides noncoding RNAs. They participate in posttranscriptional regulation of gene expression, therefore, to directly control the expression of a large portion of the human genome [
miRNAs are also important regulators of specialized
In addition to classical genetic abnormalities in the pathogenesis of T2DM, epigenetic modifications have emerged as a central driving force for the development of T2DM. Epigenetic modifications, especially DNA hypermethylation, are believed to play an important role in the regulation of essential protective genes for T2DM. Found in the tumor area, epigenetic regulations of miRNA genes within or near CpG islands in their promoter regions can result in aberrant expressions of a number of miRNAs.
In China, Kazak population is significantly overweight and has insulin resistance, hypertension, smoking, and other risk factors for T2DM, but its T2DM prevalence rate is much lower than the Han population in the same region [
This study was prospectively performed and approved by the Institutional Ethics Committees of the First Affiliated Hospital of Shihezi University School of Medicine in China and conducted in accordance with the ethical guidelines of the Declaration of Helsinki. One hundred patients with T2DM from Kazak population and another 100 T2DM from Han population were recruited from the Departments of Endocrinology and Metabolism of First Affiliated Hospital of Shihezi University School of Medicine in China from 2010 to 2011. Written informed consents were obtained from all patients before they entered the study. Diagnosis of type 2 diabetes was based on the World Health Organization criteria: fasting glucose ≥ 7 mmol/L (126 mg/dL) or the 2-hour oral glucose tolerance test glucose level ≥ 11.1 mmol/L (200 mg/dL) or clinical diagnosis of the disease. All subjects were recruited when they were hospitalized for treatment of poor glycemic control and any patients suspected of infectious disease or autoimmune disease were excluded from current study.
RNAs were isolated from plasma samples of Kazak and Han T2DM patients using the miRNeasy Serum/Plasma Kit (Qiagen, Germany) and genomic DNA was isolated from blood cells using the DNeasy Blood and Tissue Kit (Qiagen, Germany) according to manufacturer’s instruction. The nucleic acid samples were quantified by measuring their absorption at 260 nm.
Real-time reverse transcription PCR (RT-PCR) was performed using an ABI Prism 7500 Fast Real-time PCR System with Taqman Universal PCR Master Mix, Taqman Reverse Transcription kit, Taqman MicroRNA Assays, and Human Panel Early Access kit from Applied Biosystems (Foster City, CA) according to the manufacturer’s instructions. Expression levels of miRNA genes were determined by normalizing the amount of the target message to that of the control microRNA-16 transcript.
To quantify methylation levels of the miR-375 CpG islands in the clinical samples, the high-throughput MassARRAY platform (Sequenom, San Diego, USA) was carried out as described previously [
The associations between categorical variables were assessed using the
To find out if there is difference in miR-375 expression levels between Kazak and Han populations, the expression levels of miR-375 in Chinese Kazak T2DM samples and corresponding Han T2DM samples were determined by quantitative real-time PCRs. Figure
The levels of
In order to know whether CpG methylation of miR-375 occurs in Kazak and Han populations, methylation patterns of miR-375 in 100 samples from each population were quantitatively analyzed using MassARRAY Spectrometry. Hierarchical clustering showed substantial differences in the quantitative methylation profiling of Kazak T2DM cases compared with Han T2DM cases (Figure
Hierarchical clustering of miR-375 methylation profiles in Kazak and Han T2DM samples. MassARRAY analysis quantified DNA methylation status of miR-375 gene promoter region in Kazak and Han T2DM samples. Each row represented a sample and each column represented a CpG unit, which was a single CpG site or a combination of CpG sites. Color coding reflected the degree of methylation with yellow being 100% red being 0%, and gray being no data.
The promoter region of miR-375 was mapped by UCSC Genome Browser’s CpG island annotations and miRbase release 13.0. As illustrated in Figure
Illustrative map of
The DNA methylation levels of
We noted heterogeneity among individual CpG units, so eight CpG units of the total 17 analyzed CpG units were further analyzed and they span 267 bp on the promoter region of
Comparison of specific
To further explore the role of methylation, we analyzed the correlation analysis between CpG methylation levels of miR-375 promoter and plasma levels of miR375. In Kazak and Han T2DM patients, we found an inverse correlation for clear trend towards a negative correlation between DNA methylation levels and plasma levels of miR-375 (Figure
The correlation analysis between CpG methylation levels of miR-375 promoter and plasma levels of miR-375. An inverse correlation for clear trend towards a negative correlation between DNA methylation levels and plasma levels of miR-375 is shown for each of the two sample sets: (a) Kazak T2DM and (b) Han T2DM. Spearman correlation coefficients are reported.
Han population is six times as likely as Kazak population to present with T2DM in China [
Some recent studies show that DNA methylation contributes to downregulation of miR-375 during tumorigenesis [
In this study, we found that the plasma level of miR-375 was significantly lower in Kazak T2DM samples compared to Han T2DM samples (Figure
Epigenetic modification of DNA, such as methylation, is thought to play a key role in T2DM progression and miRNAs are epigenetically regulated by DNA methylation [
We further evaluated the methylation status of the CpG units to test the possibility to use them to predict the progression of Kazak T2DM. Our results showed statistically significant differences in the frequency of methylation at individual CpG units between Kazak and Han T2DM samples. And more than half of the studied individual CpG units were hypomethylated in Kazak T2DM patients, CpG 1.2, CpG 3.4, CpG 5.6, and CpG 21.22.23.34, as compared with Hans (
In summary, we demonstrated, in this study, the presence of ethnic differences in the expressions of miR-375 and CpG methylation of miR-375 promoter regions, and this difference may contribute to the different patterns of T2DM frequency observed in Kazak and Han populations. Meanwhile, miR-375 methylation patterns could be added to the known risk factors in predicting the progression of Kazak T2DM.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Xiangyun Chang, Siyuan Li, and Jun Li contributed equally to this work.
This work was supported by the National Natural Science Foundation of China (81060065/H0711).