Differential expression of circRNA CPT1A in peripheral blood of patients with type 2 diabetes mellitus

WANG Juan, CHANG Xiang-yun, ZHU Yu-rong, BA Tao

Department of Endocrinology and Metabolism, the First Affiliated Hospital of Shihezi University School of Medicine, Shihezi 832008, China

Keywords:

ABSTRACT Objective:To investigate the clinical value of circRNA CPT1A in type 2 diabetes mellitus(T2DM) by observing the relative expression levels of circRNA CPT1A in the peripheral blood of type 2 diabetic patients.Methods:Peripheral blood, as well as general data were collected from 20 type 2 diabetic patients and 20 healthy check-ups attending the First Affiliated Hospital of Shihezi University School of Medicine, and a glucose tolerance test was perfected to detect relevant biochemical indexes in peripheral blood of diabetic and healthy control groups, and the relative expression level of circRNA CPT1A was detected by qRT-PCR technique; ROC curve was used to evaluate the diagnostic value of circRNA CPT1A in T2DM;correlation analysis was used to explore the correlation between target circRNA and glucolipid metabolic indexes; logistic regression model was used to analyze the effect of circRNA CPT1A on the risk of T2DM.Results:The relative expression levels of circRNA CPT1A were higher in the T2DM group than in the healthy control group (P＜0.001).ROC curves showed that circRNA CPT1A has a diagnostic value in T2DM.The correlation analysis showed that the expression level of circRNA CPT1A correlated with fasting glucose, 2 h post-glucose load,HOMA-IR, HOMA-β, and triglycerides (P＜0.05).logistic regression analysis showed that the relative expression level of circRNA CPT1A increased the risk of T2DM (P＜0.05).Conclusion:The expression levels of circRNA CPT1A were significantly elevated in the peripheral blood of T2DM patients, suggesting that they may be a potential diagnostic marker for T2DM.

1.Introduction

China is the largest country in the world with diabetes, and an epidemiological study conducted in 1980 in 14 provinces and cities across the country showed that the prevalence of diabetes was 0.67%[1], and a diabetes epidemiological survey conducted from 2015 to 2017 concluded that the prevalence of diabetes among people aged 18 years and older in China had reached 11.2%[2].In recent years, the prevalence of diabetes in China has been increasing year by year and has become a recognized major public health problem, causing both a great economic burden to patients and huge medical pressure on society[3] Therefore, it is urgent to prevent and treat diabetes.Therefore, the prevention and treatment of diabetes have become urgent.

Patients with type 2 diabetes mellitus (T2DM) often do not have obvious symptoms in the early stages, and often develop complications before the diagnosis is confirmed, which often involve microvascular and macrovascular organs, such as the eyes,kidneys, and feet, and can be disabling and life-threatening in severe cases.Therefore, it is important to explore the new pathogenesis and diagnostic and therapeutic means of T2DM in depth.A large number of epigenetic studies in recent years have provided new insights into the development of diabetes.Epigenetics usually refers to the absence of altered nucleotide sequences, but altered gene expression levels through DNA methylation, non-coding RNA, histone modifications, etc[4].There is increasing evidence that circRNA, a non-coding RNA, plays an important role in the development of T2DM.CircRNAs have a stable structure, high evolutionary conservation,and tissue specificity, and play an essential role in gene expression and regulation, which makes them suitable candidates for disease biomarker studies[5].In recent years, circRNAs are aberrantly expressed in a variety of diseases, such as tumors and diabetes.A deeper exploration of its structure and function can help us to better understand the onset and progression of diabetes and thus further improve the diagnosis and treatment of related diseases.It has been shown that the expression of the CPT1A gene increases the risk of metabolic diseases, and the expression level is positively correlated with fasting glucose[6].Therefore, this study aimed to explore the relationship between circRNA CPT1A and T2DM, and initially explore the diagnostic value of circRNA CPT1A for T2DM.

2.Materials and Methods

2.1 General Information

Twenty patients with T2DM who attended the First Affiliated Hospital of Shihezi University School of Medicine from September 2021 to June 2022 were included.All patients with T2DM were diagnosed by the 1999 World Health Organization diagnostic criteria for diabetes mellitus; and patients with combined hypertension,heart, liver, kidney, and tumor diseases, as well as patients with acute and chronic infectious diseases and those taking oral hormones were excluded.Another 20 healthy individuals who attended the health check-up center of the First Affiliated Hospital of Shihezi University School of Medicine during the same period were selected as the control group (NC group), whose laboratory indexes were within the normal range and had no history of other diseases.The included individuals were Han Chinese, long-term residents of Xinjiang, had not intermarried with other ethnic groups within three generations,and were sex- and age-matched.The study was approved by the ethics committee of the First Affiliated Hospital of Shihezi University School of Medicine, ethics approval number: KJX-2021-093-01, and all participants had signed the relevant informed consent forms.

2.2 Methodology

2.2.1 Collection of routine indicators

All enrolled subjects underwent general data collection, as well as a screening of glucose tolerance test, physical and laboratory examinations to collect systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FPG), 2 h post-glucose load blood glucose (2H-PG), fasting insulin (FINs) and C-peptide (CP),high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), total cholesterol (TC), triglycerides (TG) and other biochemical indices, and the islet function was assessed by insulin resistance index (HOMA-IR) and islet beta-cell function index (HOMA-β).

2.2.2 Detection of relative expression levels of circRNA CPT1A

① Primer selection: Our group performed high-throughput sequencing of peripheral blood in Xinjiang Uyghur T2DM patients in the early stage, and screened 134 differentially expressed circRNAs, among which circRNA CPT1A was more significantly up-regulated expression.[7].②Peripheral blood circRNA CPT1A was detected by real-time fluorescence quantitative PCR using the same reaction system and parameters with GADPH as the internal reference.circRNA CPT1A relative expression levels were calculated using the 2-ΔΔCtmethod.The test was repeated three times to take the average.

2.3 Statistical treatment

SPSS 23.0 and GraphPad Prism 9.0 software were used for statistical processing of the data.The t-test was used for comparison between groups of normally distributed measures; the Mann-Whitney U test was used for comparison between groups of nonnormally distributed measures; the χ2test was used for analysis of count data; the ROC curve was used to evaluate the diagnostic value of circRNA CPT1A in T2DM; Spearman correlation analysis was used for circRNA CPT1A relative expression was compared with glycolipid metabolic indexes using Spearman correlation analysis.Logistic regression model was used to analyze the effect of circRNA CPT1A on the risk of T2DM.The difference was considered statistically significant at P＜0.05.

3.Results

3.1 Comparison of general data and biochemical indicators of study subjects

The general data and biochemical indexes of the type 2 diabetes group were compared with those of the healthy control group (NC group), in which fasting glucose, 2 h post-glucose after glycemic load, triglycerides, HDL, fasting insulin, insulin resistance index,and pancreatic β-cell function index were statistically different in the T2DM and NC groups (P ＜ 0.01), while gender, age, BMI,systolic blood pressure, diastolic blood pressure, total cholesterol,low-density lipoprotein, and fasting C-peptide were not statistically different in the two groups (P ＞ 0.05), as shown in Table 1.

Tab 1 Comparison of general data, biochemical indexes in each group

3.2 Comparison of relative expression levels of circRNA CPT1A

The relative expression levels of circRNA CPT1A were elevated in patients in the T2DM group compared with the NC group, with statistically significant differences between the groups (P ＜ 0.001).See Table 2 and Figure 1.

Tab 2 Comparison of circRNA CPT1A gene relative expression levels

Fig 1 Relative expression levels of circRNA CPT1A quantified by qRT-PCR

3.3 Diagnostic value of circRNA in T2DM

ROC curves were plotted with grouping as the state variable and relative expression level2-ΔΔCt values as the test variable, and the results showed that the AUC of circRNA CPT1A for the diagnosis of T2DM was 0.835 (95% CI: 0.711-0.959; P＜0.001; sensitivity=80%;specificity=80%), as shown in Table 3 and Figure 2.

Tab 3 ROC curve analysis of circRNA CPT1A for the diagnosis of T2DM

Fig 2 ROC curve of circRNA CPT1A in the diagnosis of type 2 diabetes mellitus

3.4 Correlation of circRNA CPT1A with metabolic indexes

Spearman′s correlation analysis of the relative expression levels of circRNA CTP1A2-ΔΔCtand metabolic indexes showed that the relative expression levels of circRNA CTP1A in the group were positively correlated with fasting glucose, 2 h post-glucose load glucose, HOMA-IR, triglycerides and negatively correlated with HOMA-β (P＜0.05).The relative expression levels of circRNA CTP1A in the T2DM group were positively correlated with fasting glucose and negatively correlated with HOMA-β (P＜0.05).The relative expression level of circRNA CTP1A was positively correlated with fasting glucose and negatively correlated with HOMA-β (P＜0.05).The relative expression level of circRNA CTP1A was positively correlated with fasting insulin and HOMA-IR in the NC group (P＜0.05).See Table 4.

3.5 Logistic regression analysis of circRNA CPT1A and

A one-way logistic regression analysis was performed with the presence or absence of T2DM as the dependent variable and the relative expression of circRNA CPT1A level as the independent variable, and the results showed that high expression of circRNA CPT1A increased the risk of T2DM (P＜0.05).After further adjustment for TG and HDL-C, the results showed that the increased expression level of circRNA CPT1A increased the risk of T2DM(P＜0.05).See Table 5.

Tab 4 Correlation between circRNA CTP1A and metabolic indexes

Tab 5 Logistic regression analysis of circRNA CTP1A and type 2 diabetes mellitus

4.Discussion

Diabetes mellitus is a metabolic disease characterized by chronically elevated blood glucose, and the prevalence of diabetes mellitus in China has shown an obvious increasing trend in recent years.In the process of the development and progression of type 2 diabetes, two links play a very critical role, which is impaired pancreatic β-cell function and insulin resistance, respectively.Numerous studies have shown that in conditions where insulin resistance is already present, the body can easily develop a hyperglycemic state if the pancreatic β-cell function fails.It has been confirmed that one of the key conditions for the development of type 2 diabetes is defective islet β-cell function[8] HaqueS[9]team study showed that circRNA is abundantly expressed in human islets and can be differentially regulated.In addition, overexpression of circPPM1F can lead to further islet damage by enhancing M1 macrophage activation[10].CAI[11] demonstrated that upregulation of circHIPK3 expression could lead to hyperglycemia and insulin resistance.[12] found that circRNA is associated with the pathogenesis of gestational diabetes.All these studies confirmed the association of circRNA with the development of T2DM.

The expression level of circRNA CPT1A has good diagnostic efficacy for T2DM.Our group has previously screened the differentially up-regulated expression of circRNA CPT1A by highthroughput sequencing, and related studies have confirmed that the CPT1A gene has a strong association with diabetes[13, 14].In order to further validate the association between the CPT1A gene and diabetes, the present study was conducted by sequencing the CPT1A gene.For further validation, this study examined the relative expression levels of circRNA CPT1A in the T2DM group and healthy controls by qRT-PCR, and found that the expression of circRNA CPT1A was upregulated in T2DM patients, and further analyzed the diagnostic value of circRNA CPT1A in T2DM by ROC curve, showing that the sensitivity of circRNA CPT1A in diagnosing T2DM was 0.8 and the specificity was 0.8.suggested that circRNA CPT1A has the potential to become a diagnostic marker for T2DM.The circRNA CPT1A may play an important role in the development of T2DM.In Spearman correlation analysis, we found that the relative expression levels of circRNA CPT1A in peripheral blood of all enrolled subjects were positively correlated with fasting glucose, 2 h post-glucose load glucose, HOMA-IR, triglycerides, and negatively correlated with HOMA-β, suggesting that the upregulation of circRNA CPT1A expression was associated with insulin resistance and islet β-cell function defects.The relative expression level of circRNA CTP1A in the T2DM group was positively correlated with fasting glucose and negatively correlated with HOMA-β, which may imply that circRNA CTP1A is involved in the pathogenesis and disease progression of T2DM.logistic regression analysis suggested that circRNA CPT1A relative expression level could increase the risk of type 2 diabetes The risk of developing type 2 diabetes can be speculated that a high expression level of circRNA CPT1A is a risk factor for T2DM (OR＞1).

In summary, circRNA CPT1A is upregulated in the peripheral blood of T2DM patients and could be a potential biological marker of type 2 diabetes and may be involved in the pathogenesis and progression of type 2 diabetes.Limitations of this study.(1) Only diabetic patients were included in this study, and pre-diabetic patients were not included for comparison.(2) This study only provides an idea for the role and mechanism of circRNA in T2DM CPT1A, which can be further verified in cellular and animal experiments at a later stage.(3) The sample size of this study is small, and its accuracy can be further verified in a large sample population at a later stage.(4) The subjects included in this study are all Han Chinese, living in Xinjiang for a long time, and it is unclear whether their gene expression is consistent in other populations.

Authors′ contributions

Wang Juan proposed the research idea, performed the experimental operation and wrote the paper; Ba Tao provided part of the experimental idea and participated in the experimental operation;Chang Xiang-Yun and Zhu Yu-Rong were responsible for and supervised the overall article.

This article does not present any conflict of interest.