Chinese Journal of Tissue Engineering Research ›› 2025, Vol. 29 ›› Issue (26): 5608-5620.doi: 10.12307/2025.787
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Qiu Boyuan1, 2, Liu Fei1, Tong Siwen1, Ou Zhixue2, Wang Weiwei1
Received:
2024-10-11
Accepted:
2024-11-12
Online:
2025-09-18
Published:
2025-02-25
Contact:
Ou Zhixue, Chief physician, MD, Professor, Department of Joint and Sports Medicine, Guilin Traditional Chinese Medicine Hospital, Guilin 541000, Guangxi Zhuang Autonomous Region, China
Co-corresponding author: Wang Weiwei, Physician, PhD candidate, Graduate School of Guangxi University of Chinese Medicine, Nanning 530000, Guangxi Zhuang Autonomous Region, China
About author:
Qiu Boyuan, Master candidate, Graduate School of Guangxi University of Chinese Medicine, Nanning 530000, Guangxi Zhuang Autonomous Region, China; Department of Joint and Sports Medicine, Guilin Traditional Chinese Medicine Hospital, Guilin 541000, Guangxi Zhuang Autonomous Region, China
Supported by:
CLC Number:
Qiu Boyuan, Liu Fei, Tong Siwen, Ou Zhixue, Wang Weiwei. Bioinformatics identification and validation of aging key genes in hormonal osteonecrosis of the femoral head[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(26): 5608-5620.
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2.1 激素性股骨头坏死衰老基因的差异分析 使用 “limma “R软件包和|logFC|> 0.8和Padj < 0.05的筛选标准鉴定了858个DEGs,其中432个基因在激素性股骨头坏死中上调,426个基因下调。使用热图和火山图描述了差异特征,见图2A,B。图2C显示了激素性股骨头坏死差异基因与衰老相关基因集之间的交集情况。这些交集产生了21个DEG-衰老基因。 2.2 WGCNA分析和激素性股骨头坏死潜在衰老基因的鉴定 使用“WGCNA”R包将前25%基因的表达方差作为筛选条件,剔除波动性较低的基因,并使用4 708个基因构建了共表达网络。选取18作为最佳软阈值(R2=0.9)来构建无标度网络,见图3A。随后,利用聚类分析确定高度相似的模块,最小模块大小设为100个基因。利用动态杂交剪切获得了3个基因模块,其中一个蓝色模块(881个基因)与激素性股骨头坏死的相关性(cor)最高(cor=0.72;P < 0.001),见图3B。此外,在蓝色模块中,GS和MM之间也有很强的相关性(cor=0.73;P < 0.001),见图3D。将蓝色模块中的881个基因与衰老相关基因集取交集,得到了31个WGCNA-衰老基因,见图3E。将DEGs-衰老基因和WGCNA-衰老基因进行取并集,发现了41个激素性股骨头坏死潜在衰老基因,见图3F。GO和KEGG富集分析弦图见图3G,结果表明激素性股骨头坏死潜在衰老基因主要富集在衰老、氧化应激反应、RNA聚合酶Ⅱ转录调节复合物、转录调节复合物、DNA结合转录因子结合、抗氧化活性、FOXO信号通路及肿瘤坏死因子信号通路等方面。具体见表3,4。 2.3 激素性股骨头坏死衰老枢纽基因的鉴定结果 为了进一步明确激素性股骨头坏死衰老枢纽基因,此研究使用了LASSO和SVM-RFE两种机器学习算法来进行鉴定,见图4A-D。结合两种算法的结果(交集),共得到了5个激素性股骨头坏死衰老枢纽基因,它们分别是过氧化氢酶、结缔组织生长因子、叉头框蛋白O3、胰岛素受体底物2和丝裂原活化蛋白激酶激酶11,见图4E。其中,Student’s t检验结果显示,过氧化氢酶、结缔组织生长因子、叉头框蛋白O3 和丝裂原活化蛋白激酶激酶11 在激素性股骨头坏死中低表达,而胰岛素受体底物2在激素性股骨头坏死中高表达,见图4F。 2.4 激素性股骨头坏死衰老枢纽基因风险预测模型的构建 此研究根据激素性股骨头坏死衰老枢纽基因的表达建立了一个激素性股骨头坏死诊断nomogram图,以获得一个更适用于临床的激素性股骨头坏死诊断模型,见图5A。通过构建该模型的临床决策曲线和临床校准曲线,明显可见该模型对激素性股骨头坏死具有较高的预测能力,见图5B,C。 2.5 不同分子亚型之间的差异分析和免疫浸润分析结果 累积分布函数(Cumulative Distribution Function,CDF)图显示当k=3时波动较小,共识矩阵的热图显示了清晰明确的边界,见图6A,B。PCA图显示,a,b和c 组显著区分"
细胞、Th17细胞及Th2细胞的丰度在3个分子亚类中有显著差异,见图 6F。图6G显示了5个激素性股骨头坏死衰老中枢基因与 23个免疫细胞之间的相关性。 2.6 枢纽基因的实验验证结果 通过生物信息数据分析明确了激素性股骨头坏死衰老枢纽基因(过氧化氢酶、结缔组织生长因子、叉头框蛋白O3、胰岛素受体底物2和丝裂原活化蛋白激酶激酶11),并基于Nomogram模型,验证了衰老枢纽基因对激素性股骨头坏死诊断的预测能力。为了进一步验证上述结果,使用股骨头临床样本进行实验验证。其中股骨颈骨折需进行人工全髋关节置换患者的股骨头作为对照组,激素性股骨头坏死需进行人工全髋关节置换患者的股骨头作为激素性股骨头坏死组,见图7A。 对两组共16个样本提取RNA进行qPCR检测,每组分别随机挑选3个样本并提取蛋白进行Western blot验证。qPCR的结果显示,与对照组相比衰老枢纽基因过氧化氢酶(P < 0.01)、结缔组织生长因子(P < 0.01)、叉头框蛋白O3(P < 0.01)和丝裂原活化蛋白激酶激酶11(P < 0.01)"
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