2.1 Study Design
This was a single center cross-sectional study with a single time point data collection. The study was conducted at The Third Hospital of Yunnan Province, China after receiving approval from the Ethics Committee of the hospital and was conducted in compliance to the Declaration of Helsinki. The written informed consent was obtained from all subjects prior to their enrollment.
2.2 Research subjects
The study population consisted of Han Chinese individuals who received medical health check-ups from January 2018 to September 2018 in the Outpatient Department of Endocrinology, The Third Hospital of Yunnan Province, China. Individuals with malignant tumor, cardiovascular disease, nephropathy or other chronic diseases which pose latent effect on miRNAs expression and who had previously been diagnosed with diabetes mellitus or had any history of medication for 6 months prior to the study were excluded.
All the diagnoses were confirmed by Oral Glucose Tolerance Test on the basis of fasting plasma glucose (FPG) and plasma glucose (PG) levels at 30 minutes, 1, 2, and, 3 hours Among 56 individuals enrolled, 21 were healthy individuals with normal glucose tolerance (NGT), 10 were diagnosed with T2DM, first degree relatives of T2DM patients with normal glucose tolerance (FD-NGT) were 13 in number and 12 individuals were first degree relatives of T2DM patients with T2DM (FD-T2DM).
2.3 Laboratory analyses
OGTT (Oral Glucose Tolerance Test)
A 3-hour OGTT (75 g of glucose) was performed in the laboratory department of The Third Hospital of Yunnan Province, China. The samples for plasma glucose (PG) were drawn at 0, 30 minutes, 1, 2 and, 3 hours Diagnosis was based on OGTT recommended by American Diabetes Association and evaluated as follows：Patients with (FPG<5.6 mmol/L and 2-hour PG<7.8 mmol/L) were considered as normal glucose tolerance (NGT) and those with (FPG 5.6-6.9 mmol/L and 2-hour PG 7.8-11.0 mmol/L) were considered as T2DM.(17)
The Connecting peptide (C-peptide) is produced in equal amounts to insulin and is considered a measure of endogenous insulin secretion.(18) Hence, C-peptide levels were analyzed using the automated Roche diagnostics (Manheim, Germany) E170 immuno-analyser (limit of detection 3.3 pmol/l, inter- and intra-assay coefficients of variation < 4.5% and < 3.3%, respectively). Fasting C –peptide level was: 1.1 to 4.4, 1 hour after meal it was5 to 10 times the fasting level and 3 hours after meal it was back to the level of fasting.
Insulin was measured by chemiluminescent immunometric assay (Siemens Healthcare Diagnostics B.V., Breda, the Netherlands). The intra-assay variation was 6% at 47 pmol/L and 3% at 609 pmol/L. The inter-assay variation was 4% at 91 pmol/L and 6% at 120 pmol/L. The detection limit was 15 pmol/L. The fasting insulin level was 2.6 to 24.9 mIU/L, 1 hour after meal it was 5 to 10 times (13 to 249 mIU/L) the fasting level and 3 hours after meal it was back to the level of fasting.
2.4 RNA isolation and characterization
Peripheral blood was obtained by venipuncture and then coagulated at room temperature for 0.5-2 h. Following centrifugation at 3000g for 5 min, serum was collected and centrifuged again at 12,000g for 15 min to completely remove cell debris. It was then aliquoted and stored at -80℃ until miRNA detection. Total RNA containing small RNA was extracted from 500 μl of serum using mirVana isolation kit (Ambion, Austin, USA) according to the manufacturer’s protocol. The final elution volume was 100 μl. The concentration of all RNA samples was quantified by NanoDrop 1000 (Nanodrop, Wilmington, USA), and 20 ng of serum RNA containing miRNA was reverse transcribed to cDNA using TaqMan MicroRNA Reverse Transcription Kit (Applied BioSystems, Foster, USA) and miRNA-specific primers provided by the manufacturer in an Applied BioSystems 9300 Thermocycler (Applied Biosystems, Foster, USA). All Complementary DNAs (cDNAs) were stored at -20℃ until Quantitative real-time polymerase chain reaction (qRT-PCR) analysis. After 1:2 dilution, 4.5 μl was used as template in a 10 μl qPCR.
2.5 Quantitative real-time PCR (qRT-PCR)
Quantitative real-time RT-PCR (qRT-PCR) was performed to assess the levels of miR-375. Essential MicroRNA-specific data are presented in Table 1. Each reaction was performed in a final volume of 10μl containing 4.5μl cDNA, 5μl TaqMan 2 × Universal PCR Master Mix (No AmpErase) and 0.5μl TaqMan miRNA Assay (Applied BioSystems). The thermal cycle was set as start with 10 min template denaturation at 95℃, 40 cycles of denaturation at 95℃ for 15 s and combined primer annealing/elongation at 60℃ for 1 min. Each sample was run in triplicate for analysis. As the internal control gene, non-coding small RNA RNU6B was used according to the Applied Biosystems Application Note. RNU6B has demonstrated both stable and abundant expression in different human tissues and organs. It is regarded as one of the control genes with the least variability for miRNAs assays and has been widely used in different fields including diabetic research
2.6 Statistical Analysis
Data were presented as means ± standard error. For qRT-PCR data, the difference of threshold cycle (Ct) between miR-375 and RNU6B (△Ct) which was equivalent to the ratio of log2-transformed absolute copy numbers was employed to show the relative expression levels of miR-375. The one-way ANOVA followed by a post hoc multiple comparison test was used to compare the concentration of miR-375 among the four individual groups Statistical analysis was performed by using SPSS software. P value less than 0.05 was considered statistically significant.