Expression and Significance of Plasma miR-1181 in Patients with Type 2 Diabetes Mellitus
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摘要:
目的 miRNA可作为预测糖尿病发生和发展的生物标志物，探索miR-1181在2型糖尿病（Type 2 Diabetes Mellitus，T2DM)患者的表达情况。 方法 运用Agilent miRNA芯片对T2DM患者（n = 5）和正常对照组（n = 5）血浆样本进行差异筛选，荧光定量PCR技术（quantitative Real Time PCR，qRT-PCR）验证出差异表达的miRNA；通过生物信息学方法预测其靶基因并绘制miRNA-靶基因-代谢通路关系网络图，扩大样本检测血浆中差异表达的miRNA：miR-1181和靶基因MAP2K2、MAPK12表达水平。 结果 miRNA芯片筛选和qRT-PCR验证出与T2DM相关的差异表达miRNA：miR-1181，生物信息学分析得出miR-1181靶基因有CCND1、PI3KR2、MAP2K2和MAPK12；T2DM患者血浆中 miR-1181的表达水平明显下降（P < 0.001），而靶基因MAPK12 mRNA的表达水平明显升高（P < 0.01）。 结论 2型糖尿病患者血浆miR-1181水平降低，可能与其抑制靶基因MAPK12对MAPK通路影响有关。 Abstract:Objective To verify that miRNA can be used as biomarkers to predict the occurrence and development of diabetes mellitus （DM）, and to explore the expression of miR-1181 in patients with type 2 diabetes mellitus （Type 2 Diabetes Mellitus）. Methods Agilent miRNA chip was used to screen plasma samples from patients with T2DM （n = 5） and normal control group （n = 5）. The differentially expressed miRNA were verified by fluorescence quantitative PCR （qRT-PCR）. The target genes were predicted by bioinformatics methods, and the relationship network diagram of miRNA - target gene - metabolic pathway was drawn. The differentially expressed miRNA: miR-1181, MAP2K2 and MAPK12 in plasma were detected by expanding samples. Results miRNA chip screening and qRT-PCR verified the differentially expressed miRNA associated with T2DM: miR-1181.Bioinformatics analysis showed that the target genes of miR-1181 were CCND1, PI3KR2, MAP2K2 and MAPK12. The expression of miR-1181 in the plasma of T2DM was significantly decreased （P < 0.001）. While expression of MAPK12 mRNA was increased （P < 0.01）. Conclusion The decrease of plasma miR-1181 in T2DM patients may be related to inhibiting effect of target gene MAPK12 on MAPK pathway. -
Key words:
- miR-1181 /
- Type 2 Diabetes /
- Target Genes /
- MAPK12
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图 1 T2DM组与正常对照组的Agilent miRNA芯片结果
Figure 1. Agilent miRNA microarray results of T2DM group and normal control group
图 2 miR-1181在T2DM组和正常对照组表达水平
A：以cel-miR-39为miR-1181外源性内参的表达水平；B：以U6为miR-1181内源性内参的表达水平。
Figure 2. The expression levels of miR-1181 in T2DM group and normal control group
图 3 miR-1181在T2DM组与和正常对照组ROC 曲线
Figure 3. ROC Curve of miR-1181 in T2DM group and normal control group
图 4 KEGG富集miRNA-靶基因-代谢通路关系网络
注：黄色V代表miRNA，红色方块代表代谢通路，蓝色圆点代表基因。基因和代谢通路越大，表示连接度越高，值得重点关注。miR-1181的靶向作用关系用红线着重标出。
Figure 4. The miRNA Target Gene-Metabolic Pathway Network enriched by KEGG
图 5 miR-1181靶基因MAP2K2预测序列互补图
Figure 5. Complementary map of MAP2K2 prediction sequence of miR-1181 Target Gene
图 6 miR-1181靶基因MAPK12预测序列互补图
Figure 6. Complementary map of MAPK12 prediction sequence of miR-1181 Target Gene
图 7 MAP2K2和MAPK12 mRNA在T2DM组和正常对照组表达
Figure 7. MAP2K2 and MAPK12 mRNA Expressions in T2DM group and normal control group
表 1 qRT-PCR 引物及退火温度列表
Table 1. qRT-PCR Primer and annealing temperature list
引物名称 序列（5′to3′） 退火温度Tm（℃） U6-F CTCGCTTCGGCAGCACATATACT 58 U6 -R ACGCTTCACGAATTTGCGTGTC cel-miR-39-5p-RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTATTAC cel-miR-39-5p-F TGGGAGCTGATTTCGTCTTG 59.7 hsa-mir-1181-RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCGGCTC hsa-mir-1181-F AATAACCGTCGCCGCCACCCG 72.5 GAPDH-F: GGTTGTCTCCTGCGACTTCA 58.2 GAPDH-R TGGTCCAGGGTTTCTTACTCC 58.2 MAP2K2-F: CACTATCAACCCTACCATCGC 57.3 MAP2K2-R: CGCTTCCTTTGCTGCTCAT 58.8 MAPK12-F TCTGTCCTGACCAACGCAA 57.6 MAPK12-R AAGGGACTCAAAGTATGGATGG 57.6 
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表 2 患者和对照组临床资料比较[（
$\bar x \pm s$ ），M（P25，P75）]Table 2. Clinical data of patients between two groups
项目 对照组（n = 65） T2DM组（n = 65） 性别（男/女） 34/31 36/29 年龄（岁） 33.5 ± 11.69 56.27 ± 12.28▲ BMI（kg/m2） 22.09 ± 3.18 23.80 ± 3.58▲ FPG（mmol/L） 5.0（4.72，5.44） 7.05（5.45，9.7） ▲ TC（mmol/L） 4.47 ± 0.52 4.21 ± 1.0▲ TG（mmol/L） 1.1（0.79，1.64） 1.61 （1.3，2.4） ▲ LDL（mmol/L） 2.48 ± 0.44 2.88 （2.41，3.11） ▲ HDL（mmol/L） 1.37（1.21，1.68） 0.98 （0.83，1.12） ▲ UA（μmol/L） 344.01（300.25，382.5） 320.3（284.4，354.15） Cr（μmol/L） 76.0（62.5，85.25） 62.75 （52.03，70.7） eGFR（ml/min*1.73m2） 106.76 ± 17.04 108.97 ± 28.15 UACR（mg/g） - 26.8（12.34，50.86） 注：正态性分布资料以（${{\bar x} } \pm s$）表示，非正态性资料以M（P25，P75）表示，与对照组比较，▲P < 0.05。BMI：体重指数；FPG：空腹血糖；TC：总胆固醇；TG：甘油三酯；LDL：低密度脂蛋白；HDL：高密度脂蛋白；UA：尿酸；Cr：肌酐；eGFR：估计肾小球滤过率；UACR：尿白蛋白肌酐比。
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表 3 miR-1181靶基因预测结果
Table 3. Prediction Results of miR-1181 Target Genes
miRNA Transcript Total score Total energy Max score Max energy Strand miRNA
lengthTranscript
lengthTranscript
PostionmiR-1181 ZBTB4 150 −18.25 150 −18.25 3 21 5961 5584 miR-1181 MAP2K2 157 −25.76 157 −25.76 1 21 1759 1645 miR-1181 MAPK12 156 −15.92 143 −15.92 2 21 1778 1644
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[1] 叶莺,刘小华,严延生. 2型糖尿病患者血浆microRNA差异表达及生物信息学分析[J]. 中华医学遗传学杂志,2021,38(6):536-540. [2] Ying S Y,Chang D C,Lin S L. The microRNA[J]. Methods MolBiol,2018,1733(10):1-25. [3] Zhang N. Protocols for the analysis of microRNA expression,biogenesis,and function in immune cells[J]. Curr Protoc Immunol,2019,126(1):e78. [4] 周建萍,毛振彪,吴信华. 微RNA-9对胃癌细胞内CDX2基因表达的影响[J]. 国际消化病杂志,2018,38(5):314-319. doi: 10.3969/j.issn.1673-534X.2018.05.009 [5] 郭青松,王鹏,黄龑,等. 微小RNA-30b对胰腺癌干细胞迁移和侵袭的调控作用[J]. 中华医学杂志,2019,99(38):3019-3023. doi: 10.3760/cma.j.issn.0376-2491.2019.38.011 [6] Hua X,Fan K C. Down-regulation of miR-1181 indicates a dismal prognosis for nasopharyngeal carcinoma and promoted cell proliferation and metastasis by modulating Wnt/beta-catenin signaling[J]. Eur Rev Med Pharmacol Sci,2019,23(3):1077-1086. [7] Ruan L. Serum miR-1181 and miR-4314 associated with ovarian cancer:MiRNA microarray data analysis for a pilot study[J]. Eur J Obstet Gynecol Reprod Biol,2018,222(1):31-38. [8] Jiang J. MiR-1181 inhibits stem cell-like phenotypes and suppresses SOX2 and STAT3 in human pancreatic cancer[J]. Cancer Lett,2015,356(2PtB):962-70. [9] Wang J. miR-1181 inhibits invasion and proliferation via STAT3 in pancreatic cancer[J]. World J Gastroenterol,2017,23(9):1594-1601. doi: 10.3748/wjg.v23.i9.1594 [10] Nam E J. MicroRNA profiling of a CD133(+) spheroid-forming subpopulation of the OVCAR3 human ovarian cancer cell line[J]. BMC Med Genomics,2012,5(18):1-9. [11] Ruan L. Serum miR-1181 and miR-4314 associated with ovarian cancer:MiRNA microarray data analysis for a pilot study[J]. Eur J Obstet Gynecol Reprod Biol,2018,222(10):31-38. [12] 王杰. miRNA-1181通过抑制STAT3影响人胰腺癌细胞的侵袭转移能力[J]. 贵州医科大学学报,2016,41(12):1387-1392. [13] Van Allen EM,Wagle N,Sucker A,et al. The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma[J]. Cancer Discov,2014,4(1):94-109. doi: 10.1158/2159-8290.CD-13-0617 [14] Zhong W,Zhu H,Sheng F,et al. Activation of the MAPK11/12/13/14 (p38 MAPK) pathway regulates the transcription of autophagy genes in response to oxidative stress induced by a novel copper complex in HeLa cells[J]. Autophagy,2014,10(7):1285-1300. doi: 10.4161/auto.28789 [15] 窦艳,邱鹏,王丽. NEK2及ERK2在不同病理类型乳腺癌中的表达及意义[J]. 临床合理用药杂志,2019,12(14):120-121. -
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