miRNA表达谱的变化作为2型糖尿病早期诊断标志物的研究
发布时间：2019-06-11 00:34
【摘要】：目的:T2DM已经成为影响全球健康的元凶之一,目前尚无特异性T2DM早期预测标志物,因此寻找其早期预测生物标志物十分必要。本文旨在筛选不同糖耐量状态下micro RNA的表达差异,寻找T2DM潜在的生物标志物,并初步探讨其在T2DM中的可能作用,为早期预测和干预T2DM提供新的靶点。方法:1.选取健康对照者,糖调节受损者,2型糖尿病者各2例,应用micro RNA基因芯片方法筛选各组间差异表达的mi RNA,筛选标准为两组间比较有显著性差异(倍数差异2或倍数差异0.5),随后在得到的有显著性差异的mi RNA中选取表达量高,倍数差异明显的mi RNA进行进一步验证及研究。2.选取健康对照者108例,糖调节受损者92例,2型糖尿病者117例,分别抽取其清晨空腹外周静脉血,提取血清,应用实时荧光定量PCR技术检测三组中mi R-3200-5p、mi R-100-5p、mi R-574-5p、mi R-3135b、mi R-1972、mi R-133b、mi R-1273f表达量以验证芯片的准确性,分析7个mi RNA与临床参数的关系,并建立ROC曲线,评价其诊断的灵敏性和特异性。最后用靶基因预测软件和KEGG通路分析软件,分析这7个mi RNA在糖尿病中可能发挥的作用。结果：1.micro RNA基因芯片结果显示,三组共2549个mi RNA,以倍数差异2或倍数差异0.5为标准,健康对照组与糖调节受损组间有5个差异表达的mi RNA,其中上调5个,下调0个;糖调节受损组与2型糖尿病组间有4个差异表达的mi RNA,其中上调2个,下调2个;健康对照组和2型糖尿病组有26个差异表达的mi RNA,其中上调17个,下调9个。2.根据这些mi RNA的组间差异,表达量及相关的生物学分析,选取7个mi RNA(mi R-3200-5p、mi R-100-5p、mi R-574-5p、mi R-3135b、mi R-1972、mi R-133b、mi R-1273f)进行进一步验证和研究。q RT-PCR技术检测结果显示,mi R-3200-5p、mi R-574-5p、mi R-3135b、mi R-100-5p和mi R-1972在健康对照组、糖调节受损组和2型糖尿病组的表达依次降低,其中mi R-3200-5p和mi R-574-5p三组间均有显著性差异(P0.05),mi R-100-5p在健康对照组和糖调节受损组之间无统计学差异(P0.05),mi R-1972在糖调节受损组和2型糖尿病组间无统计学差异(P0.05)。mi R-133b、mi R-1273f在健康对照组、糖调节受损组和2型糖尿病组的表达先降低再上升,但mi R-133b在三组间均有统计学差异(P0.05),mi R-1273f在糖调节受损和2型糖尿病组间无统计学差异(P0.05)。其中,mi R-100-5p的PCR验证结果与基因芯片结果相反,其余6个mi RNA的PCR验证结果与基因芯片结果一致。3.对7个mi RNA不同组合来评估其在早期诊断2型糖尿病中的价值,发现mi R-574-5p、mi R-133b、mi RNA-3135b、mi R-1273f、mi R-1972联合检验时,区分健康对照组与糖调节受损组的诊断准确性最高,其曲线下面积为0.805(P0.01);mi R-3200-5p、mi R-574-5p、mi R-133b、mi R-100-5p联合检验时,区分糖调节受损组与2型糖尿病组的诊断准确性最高,其曲线下面积为0.738(P0.01);mi R-3200-5p、mi R-574-5p、mi R-1273f、mi R-100-5p联合检验时,区分健康对照组与2型糖尿病组的诊断准确性最大,其曲线下面积为0.935(P0.01)。多元相关分析显示,与健康对照组相比,糖调节受损组和2型糖尿病组的FBG、TG、Hb A1c、BMI、WHR显著升高(P0.05),HDL-C显著降低(P0.05);与糖调节受损组相比,2型糖尿病组FBG、SBP、DBP、TG、Hb A1c、WHR、BMI显著升高(P0.05),HDL-C显著降低(P0.05)。结论：1.mi RNA基因芯片结果显示,当倍数差异2或倍数差异0.5时,三组间分别有不同差异表达的mi RNA;2.筛选出7个mi RNA(分别为mi R-3200-5p、mi R-100-5p、mi R-574-5p、mi R-3135b、mi R-1972、mi R-133b、mi R-1273f)进行q RT-PCR验证,发现其中的6个mi RNA(除mi R-100-5p)在糖尿病的进程中有显著变化趋势,有望成为2型糖尿病早期生物学标志物。3.7种mi RNA与临床资料密切相关,且在不同的组合情况下的诊断价值不同,联合检测可提高诊断效率。同时,7种mi RNA的靶基因可能通过蛋白消化和吸收、TGF-β信号通路、胰岛分泌、p53信号通路、磷酸化信号系统等分子生物学作用和代谢通路发挥作用。
[Abstract]:Objective: T2DM has become one of the elements that affect the global health, and there is no specific early prediction marker of T2DM, so it is necessary to find its early prediction biomarkers. The purpose of this study is to screen the differential expression of micro RNA in different glucose tolerance states, to find the potential biomarkers of T2DM, and to explore its possible role in T2DM and to provide a new target for early prediction and intervention of T2DM. Method:1. The expression of mi-RNA in each group was selected by microRNA gene chip method, and the screening criteria were significant difference (multiple difference of 2 or multiple difference of 0.5). And then the mi RNA with high expression quantity and the multiple difference is selected to be further verified and studied in the obtained mi RNA with significant difference. 108 patients with healthy controls,92 patients with impaired glucose regulation and 117 patients with type 2 diabetes were selected, and the fasting peripheral venous blood in the morning was extracted, serum was extracted, and the mi R-3200-5p, mi R-100-5p, mi R-574-5p, mi R-3135b, mi R-1972, mi R-133b were detected by real-time fluorescence quantitative PCR. The expression of mi R-1273f was used to verify the accuracy of the chip, the relationship between the 7 mi RNA and the clinical parameters was analyzed, and the ROC curve was established to evaluate the sensitivity and specificity of the diagnosis. Finally, the target gene prediction software and the KEGG pathway analysis software were used to analyze the possible role of the seven mi-RNA in the diabetes. Results: The results of microRNA gene chip showed that there were a total of 2549 mi-RNA in the three groups, with a multiple difference of 2 or a multiple of 0.5 as the standard. There were 4 different expression of mi-RNA in the group with impaired glucose regulation group and type 2 diabetes group, of which 2 were up-regulated and 2 were down-regulated; there were 26 difference expression of mi-RNA in the healthy control group and type 2 diabetes group, including 17 up-regulation and 9 down-regulation. 7 mi-RNA (mi R-3200-5p, mi R-100-5p, mi R-574-5p, mi R-3135b, mi R-1972, mi R-133b, mi R-1273f) were selected for further verification and study according to the inter-group difference, expression and related biological analysis of these mi-RNA. The results of q-RT-PCR showed that the expression of mi R-3200-5p, mi R-574-5p, mi R-3135b, mi R-100-5p, and mi R-1972 in healthy control group, sugar-adjusted damaged group and type 2 diabetes group decreased in sequence, among which, there was a significant difference among the three groups of mi R-3200-5p and mi R-574-5p (P0.05). mi r-100-5p had no statistical difference between the healthy control group and the impaired glucose control group (P0.05). mi r-1972 no significant difference between the sugar-adjusted and 2-type diabetic group (p0.05). mi r-133b, mi r-1273f, in the healthy control group, the expression of the sugar-adjusted damaged group and the type 2 diabetes group was first reduced and then increased, But the mi R-133b had a statistical difference among the three groups (P0.05), and the mi R-1273f had no statistical difference between the sugar regulation and the type 2 diabetes group (P0.05). The results of PCR validation of mi R-100-5p were in contrast to the results of gene chip, and the results of PCR of the remaining six mi-RNA were consistent with the results of gene chip. The results showed that the diagnostic accuracy of mi R-574-5p, mi R-133b, mi-RNA-3135b, mi R-1273f, mi R-1972 was the highest in the early diagnosis of type 2 diabetes, the area of the curve was 0.805 (P0.01), mi R-3200-5p, mi R-574-5p, In the combined test of mi R-133b, mi R-100-5p, the diagnostic accuracy of the sugar-adjusted damaged group and type 2 diabetes group was the highest, the area under the curve was 0.738 (P0.01), mi R-3200-5p, mi R-574-5p, mi R-1273f, mi R-100-5p combined test, the diagnostic accuracy of the healthy control group and type 2 diabetes group was the largest, The area under the curve is 0.935 (P0.01). The multivariate correlation analysis showed that the FBG, TG, Hb, BMI, WHR of the damaged group and type 2 diabetes group were significantly higher than those in the healthy control group (P0.05), and the HDL-C was significantly lower (P0.05); and the FBG, SBP, DBP, TG, Hb, and WHR in type 2 diabetes group were compared with that of the group 2 diabetes. The BMI was significantly higher (P0.05), and the HDL-C was significantly lower (P0.05). Conclusion: The results of 1.mi RNA gene chip show that when the multiple difference is 2 or a multiple of 0.5, there are different expression of mi-RNA in the three groups. 7 mi-RNA (mi R-3200-5p, mi R-100-5p, mi R-574-5p, mi R-3135b, mi R-1972, mi R-133b, mi R-1273f) were screened for q-RT-PCR. It is expected to be an early biological marker of type 2 diabetes. 3.7 kinds of mi-RNA are closely related to clinical data, and in different combinations, the diagnostic value is different, and the combined detection can improve the diagnostic efficiency. At the same time, the target gene of seven kinds of mi-RNA can play a role in molecular biology and metabolic pathway of protein digestion and absorption, TGF-signaling pathway, pancreatic islet secretion, p53 signal pathway, and phosphorylation signal system.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
本文编号：2496856
[Abstract]:Objective: T2DM has become one of the elements that affect the global health, and there is no specific early prediction marker of T2DM, so it is necessary to find its early prediction biomarkers. The purpose of this study is to screen the differential expression of micro RNA in different glucose tolerance states, to find the potential biomarkers of T2DM, and to explore its possible role in T2DM and to provide a new target for early prediction and intervention of T2DM. Method:1. The expression of mi-RNA in each group was selected by microRNA gene chip method, and the screening criteria were significant difference (multiple difference of 2 or multiple difference of 0.5). And then the mi RNA with high expression quantity and the multiple difference is selected to be further verified and studied in the obtained mi RNA with significant difference. 108 patients with healthy controls,92 patients with impaired glucose regulation and 117 patients with type 2 diabetes were selected, and the fasting peripheral venous blood in the morning was extracted, serum was extracted, and the mi R-3200-5p, mi R-100-5p, mi R-574-5p, mi R-3135b, mi R-1972, mi R-133b were detected by real-time fluorescence quantitative PCR. The expression of mi R-1273f was used to verify the accuracy of the chip, the relationship between the 7 mi RNA and the clinical parameters was analyzed, and the ROC curve was established to evaluate the sensitivity and specificity of the diagnosis. Finally, the target gene prediction software and the KEGG pathway analysis software were used to analyze the possible role of the seven mi-RNA in the diabetes. Results: The results of microRNA gene chip showed that there were a total of 2549 mi-RNA in the three groups, with a multiple difference of 2 or a multiple of 0.5 as the standard. There were 4 different expression of mi-RNA in the group with impaired glucose regulation group and type 2 diabetes group, of which 2 were up-regulated and 2 were down-regulated; there were 26 difference expression of mi-RNA in the healthy control group and type 2 diabetes group, including 17 up-regulation and 9 down-regulation. 7 mi-RNA (mi R-3200-5p, mi R-100-5p, mi R-574-5p, mi R-3135b, mi R-1972, mi R-133b, mi R-1273f) were selected for further verification and study according to the inter-group difference, expression and related biological analysis of these mi-RNA. The results of q-RT-PCR showed that the expression of mi R-3200-5p, mi R-574-5p, mi R-3135b, mi R-100-5p, and mi R-1972 in healthy control group, sugar-adjusted damaged group and type 2 diabetes group decreased in sequence, among which, there was a significant difference among the three groups of mi R-3200-5p and mi R-574-5p (P0.05). mi r-100-5p had no statistical difference between the healthy control group and the impaired glucose control group (P0.05). mi r-1972 no significant difference between the sugar-adjusted and 2-type diabetic group (p0.05). mi r-133b, mi r-1273f, in the healthy control group, the expression of the sugar-adjusted damaged group and the type 2 diabetes group was first reduced and then increased, But the mi R-133b had a statistical difference among the three groups (P0.05), and the mi R-1273f had no statistical difference between the sugar regulation and the type 2 diabetes group (P0.05). The results of PCR validation of mi R-100-5p were in contrast to the results of gene chip, and the results of PCR of the remaining six mi-RNA were consistent with the results of gene chip. The results showed that the diagnostic accuracy of mi R-574-5p, mi R-133b, mi-RNA-3135b, mi R-1273f, mi R-1972 was the highest in the early diagnosis of type 2 diabetes, the area of the curve was 0.805 (P0.01), mi R-3200-5p, mi R-574-5p, In the combined test of mi R-133b, mi R-100-5p, the diagnostic accuracy of the sugar-adjusted damaged group and type 2 diabetes group was the highest, the area under the curve was 0.738 (P0.01), mi R-3200-5p, mi R-574-5p, mi R-1273f, mi R-100-5p combined test, the diagnostic accuracy of the healthy control group and type 2 diabetes group was the largest, The area under the curve is 0.935 (P0.01). The multivariate correlation analysis showed that the FBG, TG, Hb, BMI, WHR of the damaged group and type 2 diabetes group were significantly higher than those in the healthy control group (P0.05), and the HDL-C was significantly lower (P0.05); and the FBG, SBP, DBP, TG, Hb, and WHR in type 2 diabetes group were compared with that of the group 2 diabetes. The BMI was significantly higher (P0.05), and the HDL-C was significantly lower (P0.05). Conclusion: The results of 1.mi RNA gene chip show that when the multiple difference is 2 or a multiple of 0.5, there are different expression of mi-RNA in the three groups. 7 mi-RNA (mi R-3200-5p, mi R-100-5p, mi R-574-5p, mi R-3135b, mi R-1972, mi R-133b, mi R-1273f) were screened for q-RT-PCR. It is expected to be an early biological marker of type 2 diabetes. 3.7 kinds of mi-RNA are closely related to clinical data, and in different combinations, the diagnostic value is different, and the combined detection can improve the diagnostic efficiency. At the same time, the target gene of seven kinds of mi-RNA can play a role in molecular biology and metabolic pathway of protein digestion and absorption, TGF-signaling pathway, pancreatic islet secretion, p53 signal pathway, and phosphorylation signal system.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
【参考文献】
相关期刊论文 前1条
1 Mustafa Abdo Saif Dehwah;;MicroRNAs and Type 2 Diabetes/Obesity[J];遗传学报;2012年01期
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