【摘要】 目的 探讨靶向维生素D受体（VDR）基因的miRNA及其对继发性甲状旁腺功能亢进甲状旁腺激素（PTH）分泌的影响。方法 用胶原酶消化法提取、分离和培养出继发性甲状旁腺功能亢进的甲状旁腺组织的原代细胞；运用生物信息学方法及全转录组测序的方法筛选得到靶向VDR的miRNA，双荧光素酶报告基因实验验证筛选出的miRNA与VDR的靶向关系；qRT-PCR及Western blotting检测过表达或抑制miRNA后的对VDR mRNA水平及蛋白水平和对PTH分泌的影响；qRT-PCR和免疫组织化学分别验证部分miRNA、VDR mRNA和蛋白水平的表达差异。结果 成功培养得到甲状旁腺原代细胞；筛选并验证了hsamiR-149-5p、hsa-miR-221-5p、hsa-miR-222-3p、hsa-miR-29a-5p、hsa-miR-301a-5p、hsa-miR-873-5p、hsa-miR-93-3p均与VDR存在靶向关系；筛选并验证的7个miR中，过表达hsa-miR-149-5p、hsa-miR-301a-5p PTH分泌增加。hsa-miR-149-5p在继发性甲状旁腺功能亢进中高表达（P=0.046）,VDR mRNA(P=0.0267)和蛋白水平表达均下降。结论 hsa-miR-149-5p、hsa-miR-301a-5p可能通过下调VDR基因的表达促进继发性甲状旁腺功能亢进患者PTH的分泌。更多还原

【Abstract】 Objective To identify the miRNAs targeting vitamin D receptor(VDR) gene and their effect on parathyroid hormone(PTH) secretion in secondary hyperparathyroidism. Methods Primary parathyroid cells with secondary hyperparathyroidism were isolated by collagenase digestion and cultured. The miRNAs targeting VDR were screened by bioinformatics methods and full transcriptome sequencing, and dual-luciferase reporter assay was used to verify the targeting relationship between VDR and the screened miRNA. The effects of overexpression or inhibition of the candidate miRNA on VDR mRNA and protein expressions and PTH secretion were evaluated using qRT-PCR and Western blotting. The expression levels of the candidate miRNAs and VDR mRNA in clinical specimens of parathyroid tissues were verified by qRT-PCR, and the expression of VDR protein was detected by immunohistochemistry. Results We successfully isolated primary parathyroid cells.Dual-luciferase reporter assay verified the targeting relationship of hsa-miR-149-5p, hsa-miR-221-5p, hsa-miR-222-3p,hsa-miR-29a-5p, hsa-miR-301a-5p, hsa-miR-873-5p, hsa-miR-93-3p with VDR, and among them, the overexpression of hsa-miR-149-5p and hsa-miR-301a-5p significantly increased PTH secretion in the parathyroid cells. In patients with secondary hyperparathyroidism, hsa-miR-149-5p was highly expressed in the parathyroid tissues(P=0.046), where the expressions of VDR mRNA(P=0.0267) and protein were both decreased. Conclusion The two miRNAs, hsa-miR-149-5p and hsa-miR-301a-5p,may promote the secretion of PTH in patients with secondary hyperparathyroidism by down-regulating the expression of VDR gene.更多还原