The Study That MiR-301a-5p Affects Parathyroid Hormone Secretion In SHPT By Regulating CaSR

Background: Secondary hyperparathyroidism(SHPT)is often secondary to chronic renal dysfunction,seriously affecting the quality of life of patients with the disease progression.The pathogenesis is associated with the low expression of calcium-sensitive receptor(CaSR)and the abnormal expression of microRNA(miRNA),but the specific development process is not completely clear.Objective: To explore the miRNA causing low CaSR expression in SHPT and its effect on the secretion of parathyroid hormone(PTH),and to provide some new ideas for the pathogenesis and new basis for the treatments of SHPT.Methods:(1)Total transcriptomic sequencing was performed on 6normal parathyroid tissue samples and 12 parathyroid tissue samples of SHPT.Combined with the prediction of the website,7 miRNAs that may regulate calcime-sensitive receptors were initially screened.After that,a miRNA that is most likely to regulate CaSR was selected by the double-luciferase assay.(2)Real-time Quantitative Polymerase Chain Reaction(qRT-PCR)was used to detect miR-301a-5p and CaSR message RNA(mRNA)in normal parathyroid tissue and SHPT parathyroid tissue,and analyze whether the expression level was correlated with the serum PTH level of patients.(3)Western blot(WB)was used to detect the CaSR protein expression of SHPT primary parathyroid cells after transfection with mimics,inhibitors and their respective controls.Meanwhile,the secretion of PTH was measured by supernatant.Results:(1)Seven miRNAs that may regulate CaSR were initially selected,which were as follows: miR-15a-5p,miR-15b-5p,miR-16-5p,miR-221-3p,miR-222-3p,miR-301a-5p and miR-503-5p,and further experiments showed that miR-301a-5p had the strongest regulatory effect on CaSR.(2)Compared with normal parathyroid tissue,miR-301a-5p was up-regulated in SHPT(P<0.05),and the expression level of miR-301a-5p was positively correlated with PTH level of patients(P>0.05).CaSR mRNA expression was down-regulated(P<0.05),and the expression level of CaSR was negatively correlated with PTH level of patients(P>0.05).(3)In cell experiments,when miR-301a-5p was overexpressed,the expression of CaSR protein in primary parathyroid cells of SHPT decreased compared with the control group(P<0.05).Conversely,when the expression of miR-301a-5p was inhibited,the expression of CaSR protein was increased compared with the control group(P<0.05).At the same time,the overexpression of miR-301a-5p showed no significant difference in the secretion of PTH compared with the control group(P>0.05),while the antagonistic expression of miR-301a-5p significantly increased the secretion of PTH compared with the control group(P<0.05).Conclusion: miR-301a-5p may affect PTH secretion by regulating the expression of CaSR in SHPT.