继发性甲状旁腺功能亢进疾病相关分子的筛选与验证

Screening and validation of molecules associated with secondary hyperparathyroidism

背景与目的:继发性甲状旁腺功能亢进(SHPT)是慢性肾脏疾病(CKD)最棘手的并发症之一,伴随着一系列的钙磷代谢紊乱、骨软化症与小肠吸收不良等症状,严重影响了患者的生存质量。目前临床上治疗效果不佳,尚未发现理想的靶点分子。因此,本研究旨在寻找SHPT的疾病相关分子,从而为其治疗提供新的靶点。方法:收集2017-2020年中南大学湘雅医院病理科5例新鲜正常甲状旁腺组织,15例SHPT患者甲状旁腺组织,其中2例正常甲状旁腺组织及5例SHPT患者甲状旁腺组织用于RNA转录组测序,获取差异表达基因,并通过功能富集分析、构建PPI网络和拓扑算法识别出核心基因;分别用qRT-PCR法与Western blot法验证核心基因在其余正常甲状旁腺组织与SHPT甲状旁腺组织中的表达。采用免疫组化法检测核心基因在36例SHPT甲状旁腺组织石蜡标本与16例甲状腺手术误切的正常甲状旁腺石蜡标本中的表达。结果:RNA测序发现1 323个差异基因;构建PPI网络和拓扑算法,最终筛选出10个关键基因;其中,二肽基肽酶4 (DPP4)因与糖尿病密切相关,而被用于进一步的验证。qRT-PCR结果显示,SHPT患者甲状旁腺组织中DPP4 mRNA表达水平明显高于正常甲状旁腺组织(P=0.005 1);Western blot结果显示,SHPT患者甲状旁腺组织中DPP4蛋白表达水平明显高于正常甲状旁腺组织(P=0.006 0);免疫组化结果显示,DPP4在SHPT患者甲状旁腺组织中的阳性率明显高于正常甲状旁腺组织(P=0.006 9)。结论:SHPT患者的甲状旁腺组织中,DPP4的表达水平明显上调,因此,DPP4可能参与SHPT的发生发展,或有望作为SHPT的药物治疗的潜在靶点。

Background and Aims:Secondary hyperparathyroidism(SHPT)is one of the most challenging complications of chronic kidney disease(CKD),characterized by a series of calcium and phosphorus metabolism disorders,osteomalacia,and malabsorption,which severely affects the quality of life of patients.Currently,clinical treatment is unsatisfactory,and no ideal target molecules have been discovered.This study was conducted to identify candidate target molecules for SHPT,so as to provide new targets for its treatment.Methods:The specimens of 5 fresh normal parathyroid tissues and 15 SHPT patient parathyroid tissues were collected from the Department of Pathology of Xiangya Hospital between 2017 and 2020.Among them,2 normal parathyroid tissues and 5 SHPT patient parathyroid tissues were used for RNA transcriptome sequencing to obtain the differentially expressed genes,and the core genes were identified through functional enrichment analysis,PPI network construction and topology algorithms.The expressions of the core genes in the remaining normal parathyroid tissues and SHPT patient parathyroid tissues were validated using qRT-PCR and Western blot,respectively.Immunohistochemical staining was performed to detect the expressions of the core genes in the paraffin sections of 36 SHPT patient parathyroid tissues,and 16 normal parathyroid tissues that were inadvertently resected during thyroid surgery.Results:A total of 1323 differentially expressed genes were identified by RNA sequencing,and 10 key genes were screened through construction of PPI network and topological algorithm.Among them,dipeptidyl peptidase 4(DPP4)was further validated due to its close association with diabetes.Results of qRT-PCR showed that the mRNA expression level of DPP4 in the parathyroid tissue of SHPT patients was significantly higher than that in normal parathyroid tissue(P=0.0051);results of Western blot showed that the protein expression level of DPP4 in the parathyroid tissue of SHPT patients was significantly higher than that in normal parathyroid tissue(P=0.0060);results of immunohistochemical staining showed that the positive rate of DPP4 in the parathyroid tissue of SHPT patients was significantly higher than that in normal parathyroid tissue(P=0.0069).Conclusion:The expression level of DPP4 is significantly upregulated in the parathyroid tissue of patients with SHPT,indicating that DPP4 may be involved in the occurrence and development of SHPT,and may be a potential therapeutic target for drug treatment of SHPT.