基于测序的肝细胞癌血浆外泌体微小RNA-1248的差异表达及功能分析

Differential expression and functional analysis of microRNA-1248 in plasma exosomes of hepatocellular carcinoma based on sequencing

目的筛选血浆外泌体微小核糖核苷酸(microRNA)作为肝细胞癌(HCC)的诊断标志物,并分析候选miRNA在HCC进展中可能发挥的作用。方法 2018年10月至2019年2月收集12例于广州市第一人民医院初诊为"HCC"的患者及12例健康志愿者血浆样本,利用超速离心法收集血浆外泌体,经动态光散射法及蛋白质免疫印迹法鉴定后,提取RNA进行测序。通过定量聚合酶链反应(qPCR)验证候选外泌体miRNA差异表达量,使用t检验分析各miRNA表达量差异。通过生物信息学方法预测候选miRNA的靶基因及可能调控的通路,分析其在HCC进展过程中发挥的作用,组间比较采用t检验。结果以差异表达倍数2倍以上(Fold change>2.0或<0.5,且P<0.05)作为标准,分别比较HCC患者术前、术后及健康志愿者血浆外泌体miRNA的差异表达结果,结合两次结果,筛选表达同时上/下调miRNA共12个。其中,表达上调6个(miR-2115-3p、miR-214-3p、miR-511-5p、miR-1299、miR-10a-5p、miR-375-3p),表达下调6个(miR-219a-2-3p、miR-127-3p、miR-9-5p、miR-383-5p、miR-212-5p、miR-1248)。qPCR验证候选miRNA在HCC患者及健康志愿者血浆外泌体中的表达差异,HCC组miR-1248表达低于健康志愿者组(9.046 ΔCt比7.597 ΔCt,t=2.849,P<0.05)。结论血浆外泌体miR-1248有作为HCC的诊断标志物的潜力。血浆外泌体miR-1248可通过调控受体细胞的细胞外基质受体相互作用、介导代谢重编程抑制HCC进展。

Objective To screen plasma exosomal micro-ribonucleotide(miRNA)as diagnostic biomarker for hepatocellular carcinoma(HCC)and investigate the possible mechanisms of selected miRNAs in HCC progression.Methods From October 2018 to February 2019,plasma samples were collected from 12 patients initially diagnosed with"HCC"in Guangzhou First People′s Hospital and 12 healthy donators.Exosomes were validated by dynamic light scattering and Western blotting after hypervelocity centrifugation,then the exosomal RNA was extracted for sequencing.After evaluating the miRNA expression profiles,miRNA candidates were validated by quantitative polymerase chain reaction(qPCR),and different expression of them was analyzed by Student′s test.The possible role of candidate miRNAs in the progression of HCC was analyzed by bioinformatics methods.Results Arule(fold change>2 or<0.5,and P<0.05)was applied to compare the differential expression results of plasma exosomal miRNAs in HCC patients before and after surgery and in healthy donators.Combined with the two comparisons,12 miRNAs were filtrated eventually,of which 6 were up-regulated(miR-2115-3p,miR-214-3p,miR-511-5p,miR-1299,miR-10a-5p,miR-375-3p),and 6 were down-regulated(miR-219A-2-3p,miR-127-3p,miR-9-5p,miR-383-5p,miR-212-5p,miR-1248).The qPCR revealed that the expression of miR-1248 in HCC plasma exosome was significantly reduced(t=2.849,P<0.05).Conclusion Plasma exosomal miR-1248 has the potential as a diagnostic biomarker for HCC,and may influence the progression of HCC by regulating the interaction of extracellular matrix receptors of receptor cells,and mediate tumor metabolic reprogramming.