基于生物信息学筛查CLEC3B蛋白及其在脓毒症诊断中的作用

目的:研究正常人与脓毒症患者血清差异表达蛋白中C-型凝集素域家族3成员B(CLEC3B)蛋白,探讨目标CLEC3B蛋白作为脓毒症分子标志物的可能性。方法:收集10名健康志愿者(健康组)和18例脓毒症患者(脓毒症组)外周血,利用数据独立采集法对外周血清蛋白数据进行采集,数据上传至i DEP在线平台分析脓毒症患者外周血中差异表达蛋白。并对这些差异蛋白进行生物信息学分析,筛选出脓毒症关键蛋白。酶联免疫吸附法(ELISA)验证并绘制关键蛋白受试者工作特征(ROC)曲线。结果:蛋白质组学分析筛选出138个差异表达蛋白(DEPs),其中34个蛋白显著下调,104个蛋白显著上调。DEPs主要富集在细胞过程、生物调节、生物过程调节、参与绑定、催化活化、分子功能监管、免疫系统、信号转导等。DEPs构建蛋白-蛋白相互作用网络(PPI)筛选出感兴趣的关键蛋白CLEC3B。ELISA实验表明脓毒症组患者CLEC3B蛋白浓度(297.73±22.00)ng/ml显著低于健康组(452.42±191.72)ng/ml,差异具有统计学意义(t=13.13,P=0.000)。CLEC3B蛋白的ROC曲线下面积为0.998,灵敏度为97.73%,特异度为100.0%。结论:CLEC3B蛋白在脓毒症组中显著降低,其灵敏度高,特异度高,可以作为脓毒症潜在的生物学诊断标志物。临床试验注册号中国临床试验注册中心,ChiCTR1900021261。

基于TCGA数据库筛选、分析并验证宫颈癌生物标志物

目的基于生物信息学筛选分析宫颈癌差异表达基因(differentially expressed gene,DEGs)及差异表达miRNA,并进一步对差异基因和蛋白进行验证,以期寻找潜在的生物标志物和治疗靶点.方法从肿瘤基因组图谱(the cancer genome atlas,TCGA)数据库获取宫颈癌相关数据,edgeR算法筛选DEGs和差异miRNAs.利用Cytoscape3.8.2软件构建mRNA-miRNA共表达网络.利用DAVID软件对DEGs和通过miRWalk网站预测的差异miRNA的目标基因进行GO富集分析和KEGG富集分析.利用qPCR和Western印迹技术对DEGs进行进一步验证.结果筛选出149个上调的DEGs和171个下调的DEGs,以及46个上调的差异miRNAs和64个下调的差异miRNAs.DEGs和miRNA目标基因在细胞组成上的富集具有一致性,都富集在胞质、核和核质中.但共表达网络发现DEGs和差异miRNAs之间不存在明显的调控关系.因此,后续实验重点放在了对DEGs的验证上,对差异表达性较为显著的TCEAL6、CLEC3B、LMOD1、CNN1进行了验证.qPCR显示它们在宫颈癌中表达量均显著降低,符合预期,对CNN1进行的Western印迹也显示其在宫颈癌中的低表达.结论TCEAL6、CLEC3B、LMOD1、CNN1在宫颈癌中均显著低表达,有望成为宫颈癌生物标志物.

The Expression of <i>TIMP</i>1, <i>TIMP</i>2, <i>VCAN</i>, <i>SPARC</i>, <i>CLEC</i>3<i>B</i>and <i>E</i>2<i>F</i>1 in Subcutaneous Adipose Tissue of Obese Males and Glucose Intolerance

We investigated the expression of TIMP1, TIMP2, SPARC, VCAN, and CLEC3B genes, encoded matricellular proteins with pleiotropic functions, and glucose intolerance in obese male subjects with normal and impaired glucose tolerance. The purpose of this study was to examine the association between the gene expressions and glucose intolerance in obesity. The results indicate that obesity leads to significant increase of TIMP1, TIMP2, E2F1 and CLEC3B gene expressions in subcutaneous adipose tissue, especially TIMP2 gene. However, more significant increase of the expression of TIMP1 and TIMP2 was found in adipose tissue of obese patients with glucose intolerance. No significant changes were found in the expression of VCAN and SPARC genes in adipose tissue of obese subjects with normal glucose tolerance but increased in the group of obese subjects with glucose intolerance. At the same time, the E2F1 and CLEC3B gene expressions were decreased in adipose tissue of obese patients with glucose intolerance. Results of this study provide evidence that changes in the expression of genes encoded TIMP1, TIMP2, VCAN, SPARC, E2F1 and CLEC3B in subcutaneous adipose tissue of obese individuals associate with glucose intolerance.