中国骨质疏松杂志

lncRNA FKBP15作为绝经后骨质疏松症肾虚血瘀证潜在生物标志物的可行性

Identification of lncRNA FKBP15 as potential biomarker for diagnosis of renal deficiency and blood stasis in postmenopausal osteoporosis

DOI：10.3969/j.issn.1006-7108.2025.04.009

中文关键词: 绝经后骨质疏松症肾虚血瘀证 lncRNA 潜在生物标志物 RNA测序

英文关键词:postmenopausal osteoporosis renal deficiency and blood stasis lncRNA potential biomarker RNA sequencing

基金项目:国家自然科学基金面上项目（82074458）；江苏省自然科学基金青年项目（BK20220470）；江苏省高等学校自然科学研究面上项目（22KJB360012）；江苏省中医退行性骨关节病临床医学创新中心资助项目(苏中医科教〔2021〕4号）；江苏省卫生健康委员会科研基金项目（Z2022059）；无锡市卫生健康委员会中青年拔尖人才支持计划（HB2023121）；无锡市中医药管理局科技项目计划立项项目（ZYYB03）

作者

单位

朱弈桦1 孙海涛2 彭晨健3 杨浩淼3 储旭东2 郭杨1.4 马勇1.4.5.6 王礼宁1.3.5*

1.南京中医药大学骨伤修复与重建新技术实验室,江苏南京 210023 2.南通大学杏林学院附属惠山医院,无锡市惠山区人民医院骨科,江苏无锡 214100 3.南京中医药大学附属南京中医院,南京市中医院骨科,江苏南京 210023 4.南京中医药大学无锡附属医院,江苏省中医退行性骨关节病临床医学创新中心,江苏无锡 214071 5.南京中医药大学中西医结合学院,江苏南京 210023 6.南京中医药大学附属盐城中医院,盐城市中医院,江苏盐城 224001

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中文摘要:

目的探讨人外周血单个核细胞（peripheral blood mononuclear cell，PBMC）中差异表达的长链非编码RNA（long noncoding RNA，lncRNA）作为绝经后骨质疏松症肾虚血瘀证（PMOP-SXXY）潜在生物标志物的可行性和临床应用价值。方法采用病例对照研究，初期纳入PMOP-SXXY和骨量正常女性患者共10例，采用密度梯度离心法获取PBMC，使用Trizol法进行RNA提取并进行文库构建，后利用RNA测序技术得到lncRNA和mRNA表达谱，筛选差异表达的lncRNAs（differential expressed lncRNA，DElncRNAs）和mRNAs（differential expressed mRNA，DEmRNAs）（筛选标准为表达倍数变化FC≥2，P＜0.05）；根据差异表达基因进行GO、KEGG富集分析，同时选择显著差异表达的10个lncRNAs与20个mRNAs进行lncRNA-mRNA相关性分析，筛选出潜在可作为生物诊断标志物的lncRNAs；采用ROC曲线分析，评估各潜在lncRNAs作为PMOP-SXXY诊断标志物的诊断价值；后再次纳入PMOP-SXXY和骨量正常女性患者共30例，行实时荧光定量PCR（quantitative real-time PCR，qPCR），对筛选结果进行外部验证。结果 ①与骨量正常组相比，PMOP-SXXY组中共2599个DElncRNAs和751个DEmRNAs。②GO富集分析中发现差异基因功能主要富集在蛋白结合上以及膜、质膜、胞外基质等细胞组分上；KEGG分析则显示差异表达的主要富集在细胞外基质受体互作（ECM-receptor interaction）、PI3K-Akt信号通路（PI3K-Akt signaling pathway）、焦点粘附（Focal adhesion）、癌症通路（Pathways in cancer）和蛋白质消化与吸收（Protein digestion and absorption）等信号通路中；lncRNA-mRNA相关性网络显示，lncRNA FKBP15具有最多的节点数，共15个，其次为lncRNA HMGCR、lncRNA SLC25A37和lncRNA IQGAP1等。③利用ROC曲线分析筛选出对PMOP-SXXY诊断具有较好预测价值的lncRNAs：lncRNA FKBP15：其AUC为1.000，95%CI：1.000～1.000；lncRNA SLC25A37：其AUC为0.900，95%CI：0.690～1.000；lncRNA HMGCR：其AUC为1.000，95%CI：1.000～1.000；lncRNA IQGAP1：其AUC为0.960，95%CI：0.843～1.000，其中lncRNA FKBP15和lncRNA HMGC的诊断价值最高。④qPCR结果显示，lncRNA FKBP15、lncRNA HMGCR、lncRNA SLC25A37和lncRNA IQGAP1与PMOP-SXXY的发生均有显著相关性（P＜0.05），而lncRNA FKBP15在PMOP-SXXY组和正常组中具有更明显的差异性表达。结论 lncRNA FKBP15在PMOP-SXXY患者外周血中高表达，与绝经后骨质疏松症的发生密切相关，可作为PMOP-SXXY的临床诊断的潜在生物标志物。

英文摘要:

Objective To explore the feasibility and clinical application value of differentially expressed lncRNA in human peripheral blood PBMC as a potential biomarker for renal deficiency and blood stasis in postmenopausal osteoporosis (PMOP-SXXY). Methods In this study, a case-control trial was conducted to collect a total of 10 samples of peripheral blood from PMOP-SXXY and from women with normal bone mass. LncRNAs and mRNAs expression profiles were detected by using RNA sequencing to screen for DElncRNA and DEmRNAs, with the criteria for screening in FC≥2 and P<0.05; GO and KEGG enrichment analyses were carried out for differential expressed genes; 10 DElncRNAs and 20 DEmRNAs with significant differential expression were selected for lncRNA-mRNA correlation analysis to screen out the lncRNAs that could be used as potential biomarkers. Then, ROC curve analysis was used to evaluate the diagnostic value of DElncRNAs as clinical potential diagnostic markers for PMOP-SXXY. Afterwards, 30 patients with PMOP-SXXY and normal bone mass were included again, and qPCR was performed to externally validate the screening lncRNAs. Results ① Compared with the group with normal bone mass, 2599 DElncRNAs and 751 DEmRNAs in PMOP-SXXY. ② Bioinformatics technology was used to analyze the differential expressed gene, the GO analysis showed that the activities of the gene products were mainly related to the Protein binding, membrane, plasma membrane and extracellular region. The results of KEGG enrichment analysis showed that it was mainly enriched in ECM-receptor interaction, PI3K-Akt signaling pathway, Focal adhesion, Pathways in cancer and Protein digestion and absorption. ③ Diagnostic efficacy of screened lncRNAs using ROC curve analysis and the results showed that lncRNA FKBP15: AUC: 1.000, 95% CI (1.000-1.000); lncRNA SLC25A37: AUC: 0.900, 95% CI (0.690-1.000); lncRNA HMGCR: AUC: 1.000，95%CI (1.000-1.000); lncRNA IQGAP1: AUC: 0.960, 95% CI (0.843 -1.000). Among them, lncRNA FKBP15 and lncRNA HMGC were the best predictors of PMOP-SXXY. ④The qPCR results showed that lncRNA FKBP15, lncRNA HMGCR, lncRNA SLC25A37 and lncRNA IQGAP1 were all significantly correlated with the occurrence of PMOP-SXXY (P<0.05). However, lncRNA FKBP15 had a more significant differential expression in the PMOP-SXXY group and the normal group. Conclusion The high expression of lncRNA FKBP15 in the peripheral blood of PMOP-SXXY suggests that it is closely related to postmenopausal osteoporosis, and it is expected to be a potential diagnostic biomarker for PMOP-SXXY.

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