Analysis Of The Serum MicroRNAs Expression Profiles In Patients With NPDR

Objective:The chip technology analyses the expression profiles of the serum microRNAs from patients of non-proliferative diabetic retinopathy (NPDR) and patients of non-diabetic retinopathy(NDR), searching for specific serum miRNA for NPDR, to identify novel biomarkers for early diagnosis of diabetic retinopathy(DR), and to provide theoretical basis for the further study of the role of miRNA in the pathogenesis of DR.Methods:A total of 60 patients with type 2 diabetes (T2DM) were selected and divided into two groups based on their fundus fluorescein angiography results:test group---T2DM with non-proliferative diabetic retinopathy(NPDR), n=30,11 males,19 females; and control group---T2DM without diabetic retinopathy(NDR), n=30,12 males, 18 females. The serum miRNAs were checked with μParaflo/TM microRNA microarray assay and ones with differential expressions were detected from two groups. Real-time quantitative PCR (RT-qPCR) was adopted to verify the results of microarray, and the purpose miRNAs were identified. The correlation between the serum expression level of the purpose miRNAs and blood glucose and blood lipid was analysed with Pearson correlation analysis. PicTar, TargetScan and miRanda were applied to comprehensively predict the target genes of the purpose miRNAs, and their intersection was regarded as the final genes.Results:1. There is no significant difference in age, gender, BMI, FBQ UAER, ABI, PWV and diabetes duration between the two groups (P>0.05). Compared with NDR group, TG、TC、HbAIC, however, were significantly higher in the NPDR group (P＜0.05).2. The gene expression profiles of serum miRNAs of NPDR patients is established. Compared with NDR patients, serum miR-19a, miR-29c, miR-4659b, miR-144 were significantly raised (respectively 2.09 times,2.34 times,5.46times,1.01 times), while miR-6837, miR-449c, miR-3139, miR-3944, hsvl-miR-H15 were significantly decreased (respectively 10.18 times,12.59 times,10.48 times,2.07 times,1.26 times) (P＜0.05) in patients with NPDR.3. MiRNAs with differential expressions from 30 serum samples of NPDR patients and 30 NDR patients were tested via RT-qPCR. MiR-19a, miR-29c, miR-4659b and miR-144 that raised in serum of patients with NPDR were verified. Result:miR-19a, miR-29c, miR-4659b were expressed significantly (Z values were-4.184,-4.45,-2.794, P<0.05), consistent with the results of the chip. More than half of cycle quantification value with miR-144 were above 35, which suggested that the seurm expression level of miR-144 was low and was excluded from follow-up experiment.4. Pearson correlation analysis showed:the relative expression level of miR-19a was positively correlated with high density lipoprotein level (r=0.302, P＜0.05) and significantly increased with the rise of HDL value. Positive correlation between the expression level of miR-29c、 miR-4659b and hemoglobin AIC level (r=0.379,0.350, P＜0.05) was observed. With the increase of HbAIC level, the relative expression level of miR-29c, miR-4659b was raised.5. ROC analysis:The areas under the receiver operating characteristic curve was 0.814 for miR-19a,0.834 for miR-29c and 0.710 for miR-4659b, all of which was more than 0.7. It indicates that the 3 miRNAs are valuable in the diagnosis of NPDR. The area under the receiver operating characteristic curve for miR-29c was the largest, which indicated the sensitivity and specificity of serum miR-29c in the diagnosis of NPDR were higher than others.6.Bioinformatics analysis:through the prediction of the target gene software, we found that miR-19a regulated PIK3CA, PAFAH2, CR2,TFPI, IL20 and were involved widely in VEGF signaling pathway, ErbB signaling pathway, lipid metabolism, apoptosis etc; miR-29c regulated AKT2, LAMA2, MDM2, PMID,CYCS, MAP4KA and were involved widely in MAPK signaling system, insulin signaling pathway, cytokine-cytokine receptor interaction, ECM-receptor interaction, p53 signaling pathway etc; miR-4659b regulated ADCY6, CXCL5, TSC1, ILII,EDA2R and were involved widely in gap junction, vascular smooth muscle contraction, insulin signaling pathway, mTOR signaling pathway, cytokine-cytokine receptor interaction etc.Conclusion:1.Serum miR-19a, miR-29c, miR-4659b were significantly upregulated in NPDR patients. That the 3 miRNAs have some value in the diagnosis of NPDR with the receiver operating characteristic curve analysis.2. Serum miR-19a expression level positively correlates with HDL level, and the expression level of miR-29c、miR-4659b positively correlates with HbAIC value.3. MiRNAs from NPDR patients may regulate glucose metabolism, lipid metabolism pathway, VEGF and various cell factors, ECM-receptor interaction, apoptosis, etc to participate in the pathogenesis of DR.4. Serum miR-19a, miR-29c, miR-4659b may have great potential to serve as novel biomarkers for early diagnosis of DR.