Hu Yanyan, Wang Qian, Wang Zengmin, Wang Fengxue, Guo Xiaobo, Li Guimei
Department of Pediatrics, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
Department of Gastrointestinal Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
Int J Mol Med. 2015 Feb;35(2):358-66. doi: 10.3892/ijmm.2014.2016. Epub 2014 Nov 28.
Since the tissue of children with combined pituitary hormone deficiency (CPHD) is not readily accessible, a new focus in children with CPHD is the blood-based expression profiling of non-protein coding genes, such as microRNAs (miRNAs or miRs), which regulate gene expression by inhibiting the translation of mRNAs. In this study, to address this, we identified potential miRNA signatures for CPHD by comparing genome-wide miRNA expression profiles in the serum of children with CPHD vs. normal (healthy) controls. Human embryonic kidney 293T cells were transfected with miR-593 or miR-511 oligonucleotides. Potential target gene expression was validated by western blot analysis for proteins and by miR-593 or miR-511 reporter assay using PROP1 gene 3'-untranslated region (3'-UTR) reporter. The miR-593 and miR-511 levels in the serum of 103 children with CPHD were assessed using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method. We found 23 upregulated and 19 downregulated miRNAs with abnormal expression in children with CPHD compared with the normal controls using miRNA microarray analysis and RT-qPCR. miR-593 and miR-511 targeted the 3'-UTR of the PROP1 gene and attenuated the expression of PROP1. The levels of miR-593 and miR-511 in the serum of children with CPHD were increased compared with those in the control subjects. According to Youden's index, the sensitivity was 82.54 and 84.86%, and the specificity was 98.15 and 91.36% for miR-593 and miR-511, respectively. The various levels of specific miRNAs, particularly miR-593 and miR-511 whose direct target is the PROP1 gene, may serve as a non-invasive diagnostic biomarkers for children with CPHD.
由于患有联合垂体激素缺乏症（CPHD）的儿童组织不易获取，因此CPHD儿童研究的一个新重点是基于血液的非蛋白质编码基因表达谱分析，例如微小RNA（miRNA或miR），它们通过抑制mRNA的翻译来调节基因表达。在本研究中，为了解决这一问题，我们通过比较CPHD儿童与正常（健康）对照血清中的全基因组miRNA表达谱，确定了CPHD潜在的miRNA特征。将miR - 593或miR - 511寡核苷酸转染到人胚肾293T细胞中。通过蛋白质的蛋白质印迹分析以及使用PROP1基因3'非翻译区（3'-UTR）报告基因的miR - 593或miR - 511报告基因检测来验证潜在靶基因的表达。使用逆转录定量聚合酶链反应（RT-qPCR）方法评估了103例CPHD儿童血清中的miR - 593和miR - 511水平。通过miRNA微阵列分析和RT-qPCR，我们发现与正常对照相比，CPHD儿童中有23种miRNA上调，19种miRNA下调，表达异常。miR - 593和miR - 511靶向PROP1基因的3'-UTR并减弱PROP1的表达。与对照受试者相比，CPHD儿童血清中miR - 593和miR - 511的水平升高。根据约登指数，miR - 593和miR - 511的敏感性分别为82.54%和84.86%，特异性分别为98.15%和91.36%。特定miRNA的不同水平，特别是直接靶向PROP1基因的miR - 593和miR - 511，可能作为CPHD儿童的非侵入性诊断生物标志物。
