Serum miR‐195‐5p is upregulated in gestational diabetes mellitus

Abstract Background Gestational diabetes mellitus (GDM) is defined as varying degrees of glucose intolerance with an onset or first recognition during pregnancy in women without previously diagnosed diabetes. Accumulating evidence indicates that miRNAs exert crucial roles in the pathogenesis and development of diabetes, including GDM. In the present study, we aimed to determine the clinical performance of miR‐195‐5p in GDM. Methods First, the miR‐195‐5p expressions in serum samples from healthy pregnant women and women with GDM at 25 weeks pregnancy were detected using real‐time polymerase chain reaction (RT‐qPCR). Then, receive characteristic (ROC) curve was used to determine the diagnostic value of miR‐195‐5p in GDM. Finally, the correlation analysis of miR‐195‐5p expression with related clinicopathological factors was carried out to determine the clinical value of miR‐195‐5p in GDM. Results In this study, we found that miR‐195‐5p expression was significantly increased in serum samples from GDM patients as compared with that in healthy pregnancies. Furthermore, miR‐195‐5p might be a putative biomarker for GDM diagnosis with an area under the curve (AUC) of 0.8451; the cutoff value was 1.598, sensitivity was 73.69%, specificity was 96.85%, accuracy was 81.26%, and Youden index was 70.54%. Expression of miR‐195‐5p was positively associated with fasting plasma glucose, one‐hour plasma glucose, and two‐hour plasma glucose. Conclusion miR‐195‐5p might function as a putative diagnostic biomarker for GDM and contribute to identifying at‐risk mothers in pregnancy.

aberrant miRNA expression has been demonstrated in various diseases, including diabetes and GDM. 5,6 Meanwhile, several miRNAs have been reported to be related to insulin secretion and insulin resistance, which are key factors in diabetes diagnosis and treatment. 7,8 A previous study noted that miR-195-5p was upregulated in GDM patients. 9 However, the relationship between miR-195-5p and GDM remains unclear.
In the present study, we determined the expression level of miR-195-5p in GDM patients. Additionally, we tested the clinical roles of miR-195-5p in GDM to contribute to clinical diagnosis and treatment. Participants fasted overnight before tests under standardized conditions. Fasting blood samples were taken from participants and collected in evacuated silicon-coated tubes containing gel for the separation of sera from the blood clot. After the coagulation of the blood, serum samples were separated by centrifugation at 10 000 g and kept at −80°C until use. Basic clinicopathological factors in 204 samples are displayed in Table 1. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University, and written informed consent was obtained from all participants.

| Patient samples
All the participants meet the following criteria: abstinence from smoking and alcohol at least 4 days before the study, willingness to sign the defined protocol over the whole study period, and continuance of usual medication and insulin administration, if necessary.

| Laboratory parameters
Under the guidelines of the German Medical Association, 10 glucose concentrations were detected using continuous glucose monitoring systems (CGMS), glucometer system Calla Premium, and glucose oxidase strips (Wellion).

| Reverse transcription-quantitative real-time PCR (RT-qPCR) assay
Total RNA was extracted from 500 μL serum samples using TRIzol LS reagent (Life Technologies) following the manufacturer's instructions. RNA samples were quantified in a NanoDrop One spectrophotometer (Thermo Fisher Scientific), and the quality and integrity were checked in a 2100 Bioanalyzer (Agilent). cDNA

| Statistical analysis
All data analyses were performed using the SPSS version 17.0 statistical software and presented as mean ± standard deviation (SD).
Receiver operating characteristic curve (ROC) was constructed to determine the clinical diagnostic value of miR-195-5p in GDM, followed by the post hoc Tukey's test was used to distinguish the differences between the two groups. The Pearson correlation analysis was used to verify the correlation between miR-195-5p and related factors. A P value <.05 was considered statistically significant.

| miR-195-5p is upregulated in the serum of GDM patients
As demonstrated in Figure 1, the expression of miR-195-5p was significantly increased in the serum of GDM patients as compared to those in healthy pregnant women ( ** P < .01).

| Clinicopathological factors of healthy pregnancies and GDM patients
As demonstrated in Table 1, there are significant differences in body mass index (BMI), fasting plasma glucose, one-hour plasma glucose, and two-hour plasma glucose between the two groups, while there are no significant differences in age, gestational age, and fetal weight. As demonstrated in Figure 2A-D, fasting plasma glucose, 1-hour plasma glucose, two-hour plasma glucose, and BMI were significantly higher in the serum of GDM patients as compared with healthy volunteers ( *** P < .0001).

| The diagnosis value of miR-195-5p in GDM
In the present study, the ROC curve was used to measure the diagnostic value of miR-195-5p in GDM. The area under the curve (AUC) value was 0.8451, while the 95% confidence interval ranged from 0.7916 to 0.8985, and the cutoff value was 1.598, sensitivity was 73.69%, specificity was 96.85%, accuracy was 81.26%, and Youden index was 70.54%, which suggested a remarkable performance for miR-195-5p in GDM diagnosis ( Figure 3 and Table 2).

| miR-195-5p expression is positively correlated with fasting plasma glucose, one-hour plasma glucose, two-hour plasma glucose, and BMI
The expression of miR-195-5p was positively correlated with fasting plasma glucose (r = .2667, P = .0067), one-hour plasma glucose (r = 0.2781, P = .0047), two-hour plasma glucose (r = 0.3237, P = .0009), and BMI (r = 0.4359, P < .0001; Figure 4A-D).   11 Meanwhile, previous studies also reported that specific miRNAs could also control insulin expression, secretion, and processing. 12 According to a former study, miR-195-5p was also involved in regulating insulin resistance, which was a key contributor in the pathogenesis of GDM. 13 Meanwhile, a previous study noted that miR-195-5p expression was remarkably increased in women with gestational diabetes as compared to healthy volunteers, 9 which was consistent with our study.