MiR-301a-5p调节继发性甲状旁腺功能亢进症中甲状旁腺激素的分泌:基于调控钙敏感受体的表达

MiR-301a-5p modulates parathyroid hormone secretion in secondary hyperparathyroidism possibly by regulating calcium-sensing receptor

目的探索继发性甲状旁腺功能亢进症(SHPT)中引起钙敏感受体(CaSR)低表达的miRNA及其对甲状旁腺激素(PTH)分泌的影响。方法对6例正常甲状旁腺组织标本和11例SHPT的甲状旁腺组织标本进行了全转录组测序,结合网站的预测,初步筛选了7个有可能调控CaSR的miRNAs,通过双荧光素酶实验,选定出一个最可能调控CaSR的miRNA。采用qRT-PCR检测正常甲状旁腺组织与SHPT甲状旁腺组织中miR-301a-5p和CaSR信使RNA表达的差异,并分析其表达量与患者血清PTH水平的相关性。采用Western blot检测SHPT甲状旁腺原代细胞转染miR-301a-5p mimics、inhibitors及各自对照之后CaSR蛋白表达的情况,同时取其上清液测定PTH的分泌情况。结果初步选定了7个有可能调控CaSR的miRNAs,分别是:miR-15a-5p、miR-15b-5p、miR-16-5p、miR-221-3p、miR-222-3p、miR-301a-5p和miR-503-5p。与正常甲状旁腺组织相比,miR-301a-5p在SHPT中表达上调(P<0.05),其表达量与患者PTH水平呈正相关关系,但结果无统计学意义(P>0.05);而CaSR信使RNA表达下调(P<0.05),其表达量与患者PTH水平呈负相关关系,但结果无统计学意义(P>0.05)。在细胞实验中,当miR-301a-5p过表达时,SHPT甲状旁腺原代细胞中CaSR蛋白的表达较对照组减少(P<0.05);反之,当miR-301a-5p的表达被抑制时,CaSR蛋白的表达较对照组增加(P<0.05)。过表达miR-301a-5p后,PTH的分泌与对照组的差异无统计学意义(P>0.05),而拮抗miR-301a-5p的表达后,PTH的分泌较对照组增加(P<0.05)。结论miR-301a-5p可能通过调控SHPT中CaSR的表达而影响PTH的分泌。

Objective To explore the miRNAs that down-regulate calcium-sensing receptor(CaSR)in secondary hyperparathyroidism(SHPT)and their effects on parathyroid hormone(PTH)secretion.Methods Whole transcriptome sequencing was performed for 6 normal parathyroid tissue samples and 11 SHPT parathyroid tissue samples.Based on bioinformatic prediction,we screened out 7 candidate miRNAs that regulate CaSR,among which the most likely miRNA for CaSR regulation was identified by double luciferase test.We detected the differential expression of miR-301a-5p and CaSR mRNA in SHPT and normal parathyroid tissue using qRT-PCR,and analyzed the correlation between their expressions and serum PTH levels of the patients.Western blotting was used to detect the expression of CaSR protein in primary SHPT parathyroid cells transfected with miR-301a-5p mimics or inhibitors,and the level of PTH in the supernatant of the cell culture was determined.Results Among the preliminarily selected 7 miRNAs that potentially regulate CaSR(miR-15a-5p,miR-15b-5p,miR 16-5p,miR 221-3p,miR 222-3p,miR 301a 5p and miR 503-5p),miR 301a-5p was significantly upregulated in SHPT compared with normal parathyroid tissue(P<0.05),and its expression appeared to be positively correlated with PTH level,but this correlation was not statistically significant(P>0.05);The expression of CaSR mRNA was significantly downregulated in SHPT(P<0.05),and its expression tended to inversely correlate with the patient's PTH level,but the correlation was not statistically significant(P>0.05).In primary culture of SHPT parathyroid cells,miR-301a-5p overexpression caused a significant decrease of CaSR protein expression(P<0.05),and conversely,inhibition of miR-301a-5p expression increased the expression of CaSR protein(P<0.05).Although miR-301a-5p overexpression did not significantly affect PTH secretion of the cells(P>0.05),inhibition of iR 301a 5p expression strongly increased the secretion of PTH(P<0.05).Conclusion MiR-301a 5p affects PTH secretion in SHPT possibly by regulating the expression of CaSR.