This article is cited by: Kang Jiehong, Chang Xiangyun, Zhu Yurong, Ba Tao, Sun Yuan. Relationship between associated cyclic RNA and the risk of type 2 diabetes in Han. Chinese Journal of General Practice[J], 2022, 25(15): 1863-1868 doi:10.12114/j.issn.1007-9572.2022.02.013
KANGJiehong, CHANGXiangyun, ZHUYurong, BATao, SUNYuan. Association of Cyclic RNAs with the Risk of Type 2 Diabetes Mellitus in Han Chinese. Chinese General Practice[J], 2022, 25(15): 1863-1868 doi:10.12114/j.issn.1007-9572.2022.02.013
Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia caused by absolute or relative insufficiency of insulin secretion, or in insensitivity of peripheral tissues to insulin. According to data published by the International Diabetes Federation (IDF), more than 100 million people are diagnosed with DM each year, and there are currently 463 million adult DM patients worldwide, and there will be 700 million DM patients worldwide by 2045 according to the growth trend [1,2]. Patients with type 2 diabetes mellitus (T2DM) are prone to serious complications, which bring a huge burden to individuals, families and society. The 6th Nm Epidemiological Survey of DM conducted by LI et al. [3] from 2015 to 2017 showed that there were large differences in the prevalence of DM among different ethnic groups, with the highest prevalence of DM in Han (12.8%) and the lowest (6.3%) in Hui. Qiao Xiaoling et al. [4] reviewed the effect of epigenetics on the secretion of T2DM first time phase insulin by inhibiting the exocytosis process, and the genetic predisposition of T2DM patients in China is genetically susceptible, and the genetic information carried by the genome is divided into two categories: genetic coding information and epigenetic information [5]. Epigenetics refers to changes in gene expression levels based on non-DNA sequence changes, such as chromatin remodeling, histone modification, DNA methylation, X chromosome inactivation, noncoding RNA (ncRNA) regulation, etc.[6]; ncRNA regulation is an important part of epigenetics, mainly including microRNAs (miRNAs), long-chain non-coding RNAs, and circular RNAs. circRNAs) et al. [7]. circRNAs are a special class of newly confirmed non-coding RNA molecules, which are tissue-specific and cell-specific in their expression patterns, and their biogenesis is regulated by specific cis-acting elements and trans-acting factors. CircRNAs are highly stable, specific, sensitive, and easy to detect, making them potential biological markers for diabetes diagnosis and prediction [8]. Some circRNAs also have richness and evolutionary conservatism, as well as a high ability to resist exonuclease or RNase degradation [9]. MEMCZAK et al. [10] reports show that circRNAs are closely related to the occurrence and development of T2DM, and this study expressed four candidate circRNAs (hsa_circ_0005414, hsa_circ_0139634, hsa_circ_0032491, and hsa_circ_ 0002922) Observations in Han T2DM patients to explore the variation of expression levels of four candidate circRNAs in Han T2DM patients and their potential value as biomarkers.
1 Objects and methods
1.1 Research Subjects
From September 2020 to June 2021, Han T2DM patients (T2DM group) and Han healthy patients (healthy control group) who were treated or examined in the Department of Endocrinology and Metabolism and Health Examination Center of the First Affiliated Hospital of Shihezi University School of Medicine were selected. Inclusion criteria: (1) long-term residence in Xinjiang, no intermarriage with other ethnic groups within three generations; (2) no complicated coronary heart disease, cardiomyopathy, malignant tumors, liver and kidney diseases, and acute and chronic infectious diseases, no hormonal, psychotropic drugs or steroid drugs; (3) T2DM diagnosis meets the 1999 World Health Organization diabetes diagnostic criteria [12], no use of insulin for hypoglycemic therapy; (4) T2DM group and healthy control group sex and age match. Finally, 21 cases were enrolled in the T2DM group, including 12 males and 9 females, aged 43-63 years old, with an average age (53.9±5.0 years old), and 22 cases in the healthy control group, including 11 males and 11 females, aged 44-63 years, with an average age (54.9±6.1) years. The study obtained the informed consent of all the study subjects, and the patients signed the informed consent form; approved by the Science and Technology Ethics Committee of the First Affiliated Hospital of Shihezi University School of Medicine, the ethical approval number KJ2020-134-01.
1.2 Research Methodology
Collect the general information of the research subjects, including gender, age, height, body mass, and blood pressure, and draw the research subjects to send them to the Laboratory Department of the First Affiliated Hospital of Shihezi University School of Medicine in the early morning on an empty stomach, and centrifuge at 4 000 r/min for 10 min using a DT4-1B-24-well medical level centrifuge, with a centrifugal radius of 15.6 cm, and aspirate 12 μl of supernatant in a centrifuge tube, according to BECKMAN COULTER AU5800 series automatic biochemical analyzer instructions for the determination of fasting blood glucose (FPG), fructosamine (FA), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), roche COBAS 8000 electrochemical immunoassay analyzer for fasting insulin (FINS), fasting C peptide (CpS). Calculate the insulin steady-state model index (HOMA-IR), HOMA-IR=FPG×FINS/22.5. In addition, 5 ml of peripheral venous blood was drawn, EDTA anticoagulation was extracted within 4 h, monocytes were extracted within 4 h, 1 ml of peripheral venous blood in the EDTA tube plus 3 ml of erythrocyte lysate (1:3) was placed in a centrifuge tube to mix well, let stand for 10 min at room temperature, centrifuged at 4 000 r/min for 5 min, centrifuged with a radius of 12.3 cm, a large number of white cell pellets were visible at the bottom of the tube, the supernatant was discarded, and 1 to 3 ml of erythrocyte lysate was added again to mix well, centrifuged at 4 000 r/min for 3 min, Centrifuge with a radius of 12.3 cm, discard the supernatant, and extract the total RNA of the blood monocytes according to the instructions of the TRIZOL kit (Invitrogen, USA).
The RT-PCR method was used to detect the levels of candidate circRNAs (hsa_circ_0005414, hsa_circ_0139634, hsa_circ_0032491, hsa_circ_0002922) in each group of samples, and the reaction system was 40 cycles, denatured at 95 °C for 1 s, and then the temperature was reduced to 60 °C and 31 s for extended reaction, and the housekeeper gene ACTB (β-actin) was used as an internal reference. The number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold is the CT value, the relative expression level of circRNAs is expressed in 2-ΔΔCT, all samples are repeated 3 times, taking the average, the higher the 2-ΔΔCT, the higher the circRNAs expression level.
1.3 Statistical methods
SPSS 22.0 software was used for statistical analysis. The measurement data conforming to the normal distribution were expressed as (x ±s), and the comparison between the two groups was expressed as a group t test; the nonnormal distribution measurement data was expressed as M (P25, P75), and the comparison between the two groups was expressed by Mann-Whitney U test; the analysis of counting data was χ2 test; and the relationship between circRNAs and T2DM clinical biochemical indexes was explored by multiple linear regression analysis The Logistic regression model was used to estimate the amount of change in the risk of T2DM in Han Chinese when the 2-ΔΔCT of circRNAs was changed, and the subject working characteristic (ROC) curve was plotted, and the diagnostic value of candidate circRNAs on T2DM was judged by the area under the ROC curve (AUC). The difference in P<0.05 is statistically significant.
2 Results
2.1 Comparison of general data, biochemical indicators and relative expression levels of candidate circRNAs by group
There was no statistical significance (P>0.05) in the healthy control group and the T2DM group comparing sex, age, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), FINS, CpS, HOMA-IR, LDL-C, HDL-C, and HDL-C, and the relative expression levels of FPG, FA, hsa_circ_0032491, and hsa_circ_0002922 in the T2DM group were higher than those in the healthy control group, hsa_circ_ 0005414 and hsa_circ_0139634 were lower than those in the healthy control group, and the difference was statistically significant (P<0.05), see Table 1.
Table 1 Comparison of general information，biochemical indicators and relative expression levels of four candidate circRNAs in T2DM Han Chinese and healthy Han Chinese
2.2 Multiple linear regression analysis explores the relationship between circRNAs and glucose metabolism-related indicators
Taking the relative expression of four candidate circRNAs as the dependent variable, the forward method was used to gradually introduce glucose metabolism-related indicators FPG, FA, Log2FINS, and CpS as independent variables for multiple linear regression analysis, and the results showed that hsa_circ_0005414 2-ΔΔCT=1.835-0.171×FPG, R2=0.290, and hsa_circ_0139634 2-ΔΔCT= 1.878-0.438×FA, R2=0.159; hsa_circ_0032491 2-ΔΔCT=3.808+0.876×FPG-1.023×Log2FINS, R2=0.503; hsa_circ_0002922 2-ΔΔCT=0.170+0.281×FPG, R2=0.168, see Table 2.
Table 2 Multiple linear regression analysis of the association of four candidate circRNAs with glucose metabolism related indicators in Han Chinese
2.3 ROC curve analysis of candidate circRNAs for diagnostic T2DM
The ROC curves of candidate circRNAs diagnostic T2DM were plotted, and the results showed that the AUC of hsa_circ_0005414, hsa_circ_0139634, hsa_circ_0032491, hsa_circ_0002922 diagnostic T2DM were 0, 0.199, 0.968 and 0.844, respectively, as shown in Table 3 and Figure 1.
Figure 1
Figure 1 ROC curve analysis of four candidate circRNAs in predicting T2DM in Han Chinese
Table 3 Analysis of the value of four candidate circRNAs for the diagnosis of T2DM in Han Chinese
2.4 Logistic regression analysis of the correlation between the relative expression levels of candidate circRNAs and Han Chinese T2DM
Taking whether or not to have T2DM as the dependent variable (assignment: yes = 1, no = 0), taking the relative expression of hsa_circ_0005414, hsa_circ_0139634, hsa_circ_0032491, hsa_circ_0002922 as the independent variables, the results showed that the increase in the relative expression level of hsa_circ_0139634 could reduce the risk of T2DM (P <0.05), increased relative expression levels of hsa_circ_0032491 and hsa_circ_0002922 increased the risk of T2DM (P<0.05), see Table 4. Multivariate logistic regression analysis of genes affected by univariate logistic regression analysis showed that model establishment could not be established.
Table 4 Univariate Logistic regression analysis of the association of three candidate circRNAs with T2DM in Han Chinese
3 Discussion
CircRNAs are considered a special class of non-coding RNAs, without 5′ caps and 3′ end tails, covalently closed RNA molecules produced by mRNA reverse splicing, rich in miRNA binding sites, with miRNA sponge effect, which in turn relieves the inhibition effect of miRNAs on their target genes and increases the expression level of target genes, a mechanism known as competitive endogenous RNA (ceRNA) mechanism [10,13]. Studies have shown that circRNAs can directly affect the epigenetic state of the promoter region of their host genes, such as the oncogene FLI1 in breast cancer cells, which not only metastasizes through the typical oncoin pathway, but also drives tumor metastasis through its exon-mediated epigenetic mechanisms [14]. Yang et al. [15] The mechanism of constructing lncRNA-circRNAs-miRNA-mRNA networks in T2DM suggests that individual lncRNAs and circRNAs can be associated with multiple miRNAs and then jointly regulate more mRNAs, and that lncRNAs and circRNAs play a central regulatory role in T2DM.
Although a large number of circRNAs have been discovered and studied in recent years, and many studies have shown that circRNAs are closely related to the occurrence and development of T2DM, such as ZHAO et al. [16] Differential expression profiles of peripheral blood circRNAs in T2DM patients and normal controls were found through microarray analysis, and the sensitivity and specificity of hsa_circ_0054633 were verified by large samples; Wang Sisi et al. [17] Three circRNAs related to insulin signaling pathways were screened by bioinformatics technology, and the expression levels of has_circ_0056891, has_circ_0063425, and has_circ_0071336 in the serum of T2DM patients were found to be reduced, and the statistical analysis was carried out again after adjusting multiple factors, and the results suggested that has_circ_0056891, has_circ_0063425, and has_ Decreased expression levels of circ_0071336 in T2DM are independent predictors of T2DM and may play an important role in the pathogenesis of T2DM and fasting blood glucose impairment (IFG). Xu Wenchao et al. [18] found that the expression of hsa_circ_401724 in the peripheral blood of patients with T2DM was significantly upregulated, and it was analyzed that it may participate in the occurrence and development of T2DM through the regulation of related signaling pathways by target miRNAs. LIMOGE et al. [19] studies have shown that cells with VPS13B gene defects accelerate adipocyte differentiation, resulting in increased fat accumulation, and insulin responses suggest abnormalities. Ning Lili et al. [20] discussed the expression profile of peripheral blood circRNAs in adult patients with occult immune diabetes mellitus (LADA) in mainland China, and the results showed that there were significant differences in the expression of hsa_circ_100670302 and hsa_circ_0035809 in LADA patients and healthy people, and the gene VPS13B hsa_circ_100670302 was associated with insulin resistance (IR), and compared with healthy patients, LADA patients hsa_ circ_100670302 expression is upregulated, and it is speculated that the hsa_circ_100670302 may affect the occurrence and development of diabetes by regulating the VPS13B gene, and hsa_circ_0035809 significantly downregulated in patients with T2DM and LADA. In summary, hsa_circ_100670302, hsa_circ_0035809 may become novel biological markers to distinguish LADA from healthy individuals. CircRNAs have also been shown to be closely related to diabetic cardiomyopathy, vascular endothelial dysfunction, diabetic foot, diabetic retinopathy, and diabetic comorbid depression [21,22,23,24,25,26].
The specific mechanism of circRNAs in T2DM is still not very clear, consult the relevant literature at home and abroad in recent years, the correlation between hsa_circ_0005414, hsa_circ_0139634, hsa_circ_0032491, hsa_circ_0002922 and Han T2DM has not been reported, and the hsa_circ_0005414, hsa_circ_0139634, hsa_circ_0032491. Hsa_circ_0002922 were observed in Han T2DM, and the results showed that there were significant differences in the expression levels of hsa_circ_0005414, hsa_circ_0139634, hsa_circ_0032491 and hsa_circ_0002922 in the Han T2DM group and the healthy control group, and the hsa_circ_0005414 and hsa_ circ_0139634 the expression level decreased in the Han T2DM group, the diagnostic value of the Han T2DM was analyzed, and it was found that the above two candidate genes had no obvious diagnostic value for Han T2DM, but through the one-way Logistic regression analysis, it can be seen that the increase in the relative expression level of the hsa_circ_0139634 can reduce the risk of T2DM, and it can be speculated that the hsa_circ_ Although 0139634 has no diagnostic value for Han T2DM, it is an independent predictor of Han T2DM, and the risk of T2DM in Han can be predicted through the change of gene expression level, so as to achieve early prevention, lifestyle intervention, and improve the early diagnosis rate of Han T2DM; while hsa_circ_0005414 has no diagnostic value for Han T2DM, nor is it correlated with its disease risk, but it is expressed at a low level in Han T2DM patients. It is needed to be further studied by scholars; the expression level of hsa_circ_0032491 and hsa_circ_0002922 in Han T2DM patients was significantly upregulated, with hsa_circ_0032491 being the most significant, and the diagnostic value of upregulated genes on Han T2DM was analyzed, and it could be used as a better biological marker for early diagnosis of Han T2DM according to AUC. At the same time, the univariate Logistic regression analysis can be seen that the increase in its relative expression level can increase the risk of T2DM in Han, which can also be used as a predictor, and hsa_circ_0032491 the relative expression level of genes can be initially assessed according to the clinical detection of FPG and FINS levels, so as to conduct early disease risk assessment for people with potential T2DM in Han, and early lifestyle intervention can slow down the progress of T2DM to a certain extent. Whether there is a correlation between hsa_circ_0032491, hsa_circ_0002922 and miRNAs and how they act in Han T2DM will be the main direction of follow-up research.
There are also deficiencies in this study, the selected Han T2DM patients did not distinguish whether it was the first diagnosis, and also lacked the study of people with abnormal glucose tolerance, and the number of selected cases was too small, and the subsequent sample size observation could be further expanded; in the study results, there was no statistical difference between the FINS and CpS of the T2DM group and the healthy control group, considering that the patients had not yet used insulin hypoglycemic therapy when the T2DM group was selected, and the study subjects only had a decrease in the ability of insulin to regulate glucose metabolism. It has not progressed to islet β decreased insulin secretion due to defects in cell function. In addition, this study is a single-center cross-sectional study, the geographical location of the study subjects is relatively concentrated, whether the population in other regions also has a similar gene expression profile, it is not very clear, with the prolongation of the course of the disease, whether there is a difference in gene expression level is not very clear. Therefore, the results of this study require further observation and follow-up in a larger sample and a more diverse population. It is believed that elucidating the specific mechanism of action of circRNAs in Han T2DM will bring the diagnosis and treatment of DM into a new field.
There is no conflict of interest in this article.
The table in this article is omitted
Bibliography
A study on the relationship between related cyclic RNA and the risk of developing type 2 diabetes in Han Chinese