Elsevier

Life Sciences

Volume 208, 1 September 2018, Pages 201-207
Life Sciences

Plasma miR-17, miR-20a, miR-20b and miR-122 as potential biomarkers for diagnosis of NAFLD in type 2 diabetes mellitus patients

https://doi.org/10.1016/j.lfs.2018.07.029Get rights and content

Abstract

Aims

Type 2 diabetes mellitus (T2DM), with non-alcoholic fatty liver disease (NAFLD) complication, may aggravate the disturbance of metabolism, increase the risk of non-alcoholic steatohepatitis, and promote the progress of liver fibrosis. Therefore, early detection of NAFLD in T2DM patients is critical in avoiding the adverse effects of the complication. This study aimed to identify circulating miRNAs for early diagnosis of the complication.

Materials and methods

Plasma miRNA expression profiles of T2DM patients complicated with or without NAFLD were examined by miRNA array analysis and then were validated by qRT-PCR. A new index for prediction the presence of NAFLD was developed based on the result of multivariate logistic regression analysis. STZ and high fat diet were used for construction a rat model of T2DM complicated with NAFLD.

Key findings

Plasma miR-17, miR-20a, miR-20b, and miR-122 were up-regulated in T2DM patients with NAFLD complicated compared in those without NAFLD (P < 0.05). Moreover, the data from the rat model further showed that the above miRNAs were more sensitive than traditional serological markers for predicting the complication. Meanwhile, in order to improve the diagnostic accuracy, we try to construct an AUC by using the new index, 24.852 × WHR-1.121 × miR122 + 1.988 × LDL-21.838, which was significantly higher than a chance assignment (asymptotic significance P < 0.001) for predicting the presence of NAFLD.

Significance

Plasma miRNAs and the new index involving WHR, LDL, and miR-122 are potential novel tools for the early diagnosis and risk estimation of NAFLD in T2DM patients.

Introduction

Non-alcoholic fatty liver disease (NAFLD), which is a disorder characterized by excess fat in the liver from non-alcoholic causes, is a hepatic manifestation of the metabolism syndrome and strongly associated with type 2 diabetes mellitus (T2DM) and the atherosclerotic vascular disease, and has been confirmed as an independent predictive factor of these diseases [1]. The high incidence of NAFLD in patients with T2DM leads to increased mortality risk and further complications, such as non-alcoholic steatohepatitis (NASH), liver fibrosis, end-stage liver disease (ESLD), and other T2DM-associated complications. Accordingly, the presence and progression of NAFLD in patients with T2DM should be accurately determined to avoid the development of ESLD and improve the outcome of these patients [2]. The histopathological evaluation of biopsy specimens remains the gold standard for the diagnosis of NAFLD, but liver biopsy is seldom performed due to a perceived risk of complications. Ultrasound, which is the most common non-invasive tool for detecting liver diseases, can recognize NAFLD only if hepatocyte steatosis involves 1/3 of the liver [3]. In addition, early-stage NAFLD is difficult to diagnose through mild clinical manifestation. Therefore, non-invasive diagnostic methods should be urgently developed for the early diagnosis of NAFLD in T2DM patients.

MiRNAs are short, non-coding RNA molecules composed of 18 to 25 nucleotides that play an important role in regulating gene expressions. They are important regulators in a wide spectrum of biological processes and metabolic homeostasis, including protein secretion and fatty acid metabolism [4, 5]. Circulating miRNAs are extremely stable and can be protected from RNAase degradation; consequently, they have emerged as attractive candidate biomarkers for the early diagnosis of diseases and the monitoring of disease progression [6].

Many studies have addressed the role of serum/plasma miRNAs as T2DM or NAFLD biomarkers. The first study that highlighted the usefulness of circulating miRNAs as early predictors of T2DM and its vascular complications was conducted in 2010, using plasma samples collected from randomly selected individuals from the Bruneck population. Zampetaki et al. revealed a plasma miRNA signature for T2DM that includes loss of endothelial miR-126 and three other miRNAs, namely, miR-15a, miR-29b, and miR-223 [7]. Succeeding studies showed that several other miRNAs, such as miR-146, miR-130b, miR-375, miR-144, miR-192, miR-30d, miR-320a, and miR-423, can be used as prognostic biomarkers for T2DM [8]. In addition, several studies also showed a potential application of miRNAs as non-invasive biomarkers for NAFLD. The serum levels of four miRNAs, namely, miR-21, miR-34a, miR-122, and miR-451, which are involved in the regulation of hepatic fat accumulation and metabolism, were reportedly higher in NAFLD patients than in those without NAFLD [9]. Moreover, a serum miRNA panel that contains miR-27b, miR-122, miR-192, and miR-1290 has been identified as a more sensitive and specific biomarker for NAFLD diagnosis and evaluation than ALT and the fibrosis 4 (FIB4) score [10].

In the present study, we investigated whether T2DM patients with and without NAFLD have distinct circulating miRNA expression profiles. Identifying the disease progression-driven alterations in circulating miRNAs could be a novel non-invasive means for the risk estimation of NAFLD in T2DM patients.

Section snippets

Clinical samples

All T2DM patients were recruited from the Department of Endocrinology in the First Affiliated Hospital of Zhejiang University and their plasma were collected. Individuals with any other systemic inflammatory diseases, acute respiratory infection, viral hepatitis, autoimmune hepatitis, alcoholic hepatitis, or cancers were excluded. The study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University. Written informed consent was obtained from all participants.

Comparison between groups based on plasma miRNA profiling

The plasma levels of miRNAs were compared between the T2DM patients with and without NAFLD complication. Fig. 1A shows the heat map of altered miRNA expression in plasma samples. The expression profiles of 43 miRNAs were found to be significantly different (fold change > 2) between the two groups. Of these 43 miRNAs, 11 were upregulated and 32 were downregulated in the plasma of T2DM patients with NAFLD complication compared to those in T2DM patients without NAFLD complication. The dysregulated

Discussion

Metabolic abnormalities, including dysregulated insulin signaling and action, and nutritional and genetic factors are well-established contributors to the onset and progression of NAFLD [20]. Indeed, patients with T2DM have a 4- to 11-fold increased risk of developing NAFLD [21]. However, NAFLD patients usually have no apparent symptoms at the early stage, and hepatic steatosis cannot be detected by ultrasonography until it covers >1/3 of the liver tissue [22]. Therefore, developing

Conclusion

In this study, we identified significant differences in the plasma miRNA profiles of NAFLD patients in the glucose and lipid dysmetabolism status of T2DM. These miRNAs could potentially serve as novel biological markers for the early diagnosis and risk estimation of NAFLD in T2DM patients, reducing the need for liver biopsy. In addition, the new index, involving WHR, LDL, and miR-122, is more effective than other non-invasive score, HIS, and ZJU index for predicting the presence of NAFLD based

Author's contributions

YD and ZT performed the research and drafted the manuscript. LG carried out the rat model and performed the in vivo studies. XW, DF, and CG participated in case collection and performed the statistical analysis. LY conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

The work was supported by the National Natural Science Fund (81500616), the Key R & D projects of Zhejiang province (2018C03019), and Zhejiang Provincial Natural Science Foundation (LQ16H070002).

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