| Objective: In this experiment, through the analysis plasma micro RNA(mi RNA) differential expression profiles in the normal control group, type2 diabetes mellitus(T2D) patients and patients with prediabetes, we found the differently expressed mi RNAs, and to explore the pathogenesis of T2 D from plasma mi RNA levels, so as to find the possible new biological markers in the early diagnosis of T2 D. Methods:(1) Total RNA was extracted from 9 cases of individual plasma,which divided into three groups: prediabetes group, T2 D group and healthy control group in 3 cases, respectively. We utilized the Agilent mi RNA microarray chip testing expression level of the mi RNAs. According the microarray results, we choose the significant differentially expressed plasma mi RNAs after compared in pairs for subsequent verification analysis.(2) We collected 100 cases in the Guizhou Provincial People’s Hospital of Endocrinology clinic from March 2014~August 2015, newly diagnosed T2 D patients and prediabetes in 3 cases, respectively. While those of healthy, age and sex matched with T2 D and prediabetes samples were obtained from50 normal controls. Total RNA was extracted from the samples, and verify the expression of mi RNAs by SYBR Green quantitative real time polymerase chain reaction(q RT-PCR).(3) We use bioinformatics databases—Targetscan, micro RNAorg and pita for mi RNA target gene prediction, then get the three databases are predicted mi RNA target genes and then analyze its biological function.(4) We finally using statistical analyze and further establish the receiver operting characteristic curve(ROC curve), and by calculating the area under curve(AUC) to determine the clinical diagnostic capabilities of each mi RNA. Results:(1) Compared with normal control group, fasting plasma glucose(FPG), 2-hour plasma glucose(2h PG) and hemoglobin dyslipidemia(Hb A1c) exhibit significantly higher in prediabetes and T2 D group(P<0.0001).(2) mi RNA microarray analysis results showed that hsa-mi R-1249,hsa-mi R-320 b and hsa-mi R-6069 showed a decreasing in the healthy control group,T2 D and prediabetes; and hsa-mi R-572 showed a decreasing in T2 D, prediabetes and the healthy control group.(3) In q RT-PCR validation, hsa-mi R-1249 showed a decreasing in the healthy control group, T2 D and prediabetes, hsa-mi R-320 b showed a decreasing in the healthy control group, prediabetes and T2 D, hsa-mi R-572 showed a decreasing in T2 D, prediabetes and healthy control group down, while hsa-mi R-6069 has not been verified. So q RT-PCR validation results are basically consistent with the microarray results.(4) Hsa-mi R-1249, hsa-mi R-572 and hsa-mi R-320 b forecast to a corresponding target gene 18, 29 and 915, respectively.GO and KEGG pathway analysis to get these target genes mainly related to the development of multicellular organisms, signal transduction biological function guide,cell differentiation, apoptosis, ion transport, cell metabolism and other related regulation.(5) Our results revealed that hsa-mi R-1249, hsa-mi R-320 b and hsa-mi R-572(P<0.05) were differentially expressed among the three groups, which yielded an area under the receiver operator characteristics(ROC) curve(AUC) of0.784 [95% confidence interval(CI) : 0.685~0.883], 0.946(95% CI: 0.906~0.985),and 0.843(95% CI: 0.766~ 0.920) discriminating T2 D patients from NGT control groups, respectively, while the AUC was 0.887(95% CI: 0.818~0.957), 0.635(95%CI: 0.525 ~ 0.744) and 0.69(95% CI: 0.580 ~ 0.793) discriminating prediabete patients from NGT control groups, respectively. Conclusion: In this study, we obtained differentially expressed mi RNA in T2 D and prediabetes(hsa-mi R-1249,hsa-mi R-320 b and hsa-mi R-572), which may be play an important role in the early diagnosis of T2 D, and may be involved in the development of T2 D. |
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