强直性脊柱炎患者血清中长链非编码RNA差异性表达的初步筛查

Differential expression of lncRNA in the serum of ankylosing spondylitis patients

目的初步筛查强直性脊柱炎(ankylosing spondylitis,AS)患者血清中差异性表达的长链非编码核糖核酸(long noncoding RNA,lncRNA),为AS早期诊断及药物靶向治疗提供潜在新型分子标志物。方法收集2017年1月至9月于南京鼓楼医院脊柱外科就诊的AS患者及同时段体检的健康对照血清样本各19例,AS组及对照组年龄分别为(38.74±7.42)岁和(37.00±6.86)岁。选取年龄相近的男性AS患者及健康对照血清样本各3例行高通量lncRNA测序,并根据表达差异的P值及倍数大小从测序结果中筛选出待验证的lncRNA。于AS组及对照组剩余各16例中利用TRizol试剂快速提取法提取RNA,逆转录为cDNA后针对待验证lncRNA行RT-qPCR验证测序所得结果。结果高通量lncRNA测序发现AS组与对照组血清中差异性表达的lncRNA共43个,其中41个表达上调,2个表达下调。根据表达差异的P值排序后,从测序结果选取差异倍数>2的lncRNA作为待验证的目标基因,共4个:ENST00000365494.1(P=2.62×10^-277,2.05倍);ENST00000364938.1(P=2.49×10^-77,2.19倍);ENST00000363046.1(P=2.67×10^-29,2.51倍);ENST00000384756.1(P=6.17×10^-21,2.28倍)。RT-qPCR结果示:ENST00000365494.1的相对表达量为1.80±0.22(P=0.304),ENST00000364938.1的相对表达量为0.78±0.07(P=0.417),ENST00000363046.1的相对表达量为1.28±0.24(P=0.793),ENST00000384756.1的相对表达量为1.52±0.25(P=0.611)。其中3个lncRNA呈上调趋势,符合测序所得结果,但其相对表达量的差异均未达统计学意义。结论验证测序所得AS患者血清中lncRNA的表达差异阴性结果,提示差异性表达的lncRNA可能未存在于外周血中,未来相关研究可偏向于病灶组织中差异性表达lncRNA的鉴定与功能探究。

Objective To investigate the differential expression of lncRNA in the serum of ankylosing spondylitis (AS) patients, with the goal of findingnew potential biomarkers for the diagnosis and targeted treatment of AS. Methods A total of 19 AS patients and 19 age-matched healthy controls treated at Nanjing Drum Tower Hospitalfrom January 2017 to September 2017 were recruited. Average age were 38.74±7.42 (range, 25-51) and 37.00±6.86 (range, 26-50). High-throughput lncRNA sequencing technology was used to detect differently expressed lncRNAs in the serum of 3 AS patients and 3 healthy controls. Target lncRNAs for further validation were selected according to the P values and fold-changes. In the rest of the serum samples (16 AS patients and 16 healthy controls), Trizol-based technique was used to extract total RNA, and after reverse transcription to obtain cDNA, RT-qPCR was preformed to confirm the sequencing results. Results Using high-throughput lncRNA sequencing, a total of 41 up-regulated and 2 down-regulated lncRNAs were detected in the serum of AS patients. After sorted by the P values, 4 lncRNAswith a fold-change larger than 2 were chosen as the target genes for RT-qPCR (ENST00000365494.1, P=2.6×10^-277, fold-change: 2.05;ENST00000364938.1, P=2.49×10^-77, fold-change: 2.19;ENST 00000363046.1, P=2.67×10^-29, fold-change: 2.51;ENST00000384756.1, P=6.17×10^-21, fold-change: 2.28). RT-qPCR results showed the relative expression of lncRNA ENST00000365494.1 was 1.80±0.22 (P=0.304), lncRNA ENST00000364938.1was 0.78±0.07 (P=0.417), lncRNA ENST00000363046.1was 1.28±0.24 (P=0.793), lncRNA ENST00000384756.1 was 1.52±0.25 (P=0.611)and tendency of up-regulation was found in 3 of them, which was consistent with the sequencing results. However, the difference did not achieve statistical significance. Conclusion Sequencing result could not be confirmed by RT-qPCR with a larger sample size, which implied the differential expression of lncRNA might not exist in the peripheral blood of AS patients, and further studies regarding lncRNA in AS could focus more on its differential expression and function in the focal tissue.