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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.

Objective: To explore the genetic basis for a child featuring idiopathic epilepsy and autism., Methods: Peripheral blood samples of the child and his parents were collected with informed consent for the extraction of genome DNA. Whole exome sequencing was carried out for the family trio. Candidate variants were verified by Sanger sequencing and bioinformatic analysis., Results: The proband was found to harbor a heterozygous nonsense c.3025C>T (p.Arg1009Ter) variant in exon 7 of the CASR gene exon 7, which may produce a truncated protein. Based on the guidelines of the American College of Medical Genetics and Genomics, the variant was predicted to be deleterious and classified as possibly pathogenic (PVS1+PM2)., Conclusion: The c.3025C>T (p.Arg1009Ter) variant of the CASR gene probably underlay the disease in this child.

Objective: To establish an cell model of hyperparathyroidism by isolation, in vitro culture, and identification of parathyroid cells from patients with secondary hyperparathyroidism (SHPT)., Methods: The parathyroid gland tissues obtained from 10 patients with SHPT were dissociated by collagenase digestion for primary culture of the parathyroid cells. Morphological changes and growth characteristics of the cells were assessed by microscopic imaging and cell counting. The mRNA and protein expression levels of parathyroid hormone (PTH), calcium-sensing receptor (CaSR), and glial cells missing 2 (GCM2) in the primary and passaged cells were determined by immunofluorescence, qRT-PCR, and Western blotting., Results: Primary cultures of parathyroid cells were successfully obtained. The cells exhibited a high expression of PTH shown by immunofluorescence assay and had a population doubling time of approximately 71.61 h. PTH secretion in the second-passage (P2) cells was significantly lower than that in the primary (P0) and first-passage (P1) cells ( P < 0.001). Despite a significant downregulation of CaSR mRNA ( P =0.017) and protein ( P =0.006) in P1 cells as compared with P0 cells, no significant differences were found in mRNA and protein expressions of PTH or GCM2 between the two cell generations., Conclusion: Primary cultures of parathyroid cells isolated from SHPT patients by collagenase digestion show similar biological properties to the cells in vivo .

Objective: To explore the genetic basis for a child with neonatal severe hyperparathyroidism., Methods: Genomic DNA was extracted from peripheral blood samples from the patient and her parents. Whole exome sequencing was carried out to screen potential mutations. Suspected mutation was verified by Sanger sequencing., Results: The proband was found to carry compound heterozygous variants c.179G>A (p.Cys60Tyr) and c.1525G>A (p.Gly509Arg) of the CaSR gene. The c.179G>A variant was derived from her mother and was unreported previously. The c.1525G>A variant was derived from her father and known to be pathogenic., Conclusion: The compound heterozygous variants of c.179G>A and c.1525G>A of the CaSR gene probably underlie the disease in the patient. The results of genetic testing has enabled diagnosis and genetic counseling for her family.

The study was designed to investigate the effects and mechanism of a calcium-sensing receptor (CaSR) polymorphism at E942K on the proliferation of gastric cancer cells. Single nucleotide polymorphisms (SNPs) were detected between gastric cancers group and normal controls group by DNA sequence analysis. The cell model was constructed by transfection of E942K mutant plasmid and wild-type (WT) plasmid into SGC-7901 and HEK-293 cells. The effect of E942K mutation on cell proliferation ability was detected by CCK8 and cell clone formation experiments. The effect of E942K mutation on calcium signaling was detected by calcium imaging. Western blot experiments were used to detect changes in phosphorylation levels of key proteins ERK1/2 and β-catenin in downstream signaling pathways after E942K mutation. The results showed that the mutation rate of E942K in gastric cancer group was significantly higher than that in normal control group (P < 0.05). CCK8 and cell clone formation experiments showed that E942K mutation significantly improved the proliferation ability of SGC-7901 gastric cancer cells and HEK-293 cells. E942K mutation enhanced calcium signaling in SGC-7901 and HEK-293 cells. E942K mutation enhanced ERK1/2 phosphorylation without affecting β-catenin phosphorylation. The results suggest that E942K mutation in CaSR may ultimately promote the proliferation of gastric cancer cells by enhancing intracellular calcium signaling and ERK1/2 phosphorylation. These results have potential clinical implications for the diagnosis and targeted therapy of gastric cancer.

Objective: To study the effect of calcium-sensing receptor (CaSR) agonists and antagonists on the expression of CaSR in neonatal mice with persistent pulmonary hypertension (PPHN), and to clarify the role of CaSR in neonatal mice with PPHN., Methods: Forty-nine neonatal mice were randomly divided into four groups: control (n=10), hypoxia (PPHN; n=11), agonist (n=13), and antagonist (n=15). The mice in the PPHN, agonist, and antagonist groups were exposed to an oxygen concentration of 12%, and those in the control group were exposed to the air. The mice in the agonist and antagonist groups were intraperitoneally injected with gadolinium chloride (16 mg/kg) and NPS2390 (1 mg/kg) respectively once daily. Those in the PPHN and the control groups were given normal saline daily. All the mice were treated for 14 consecutive days. Hematoxylin and eosin staining and immunohistochemistry were used to observe the changes in pulmonary vessels. Laser confocal microscopy was used to observe the site of CaSR expression and measure its content in lung tissues. qRT-PCR and Western blot were used to measure the mRNA and protein expression of CaSR in lung tissues., Results: Compared with the control group, the PPHN group had significant increases in the pulmonary small artery wall thickness and the ratio of right to left ventricular wall thickness (P<0.05), which suggested that the model was successfully prepared. Compared with the control group, the PPHN group had a significant increase in the mRNA and protein expression of CaSR (P<0.05), and the agonist group had a significantly greater increase (P<0.05); the antagonist group had a significant reduction in the mRNA and protein expression of CaSR (P<0.05)., Conclusions: CaSR may play an important role in the development of PPHN induced by hypoxia in neonatal mice.

To investigate the effect of Ca(2+)-sensing receptor (CaR) on Spermine-induced extracellular Ca(2+) influx and NO generation in human umbilical vein endothelial cells (HUVEC), the small interference RNA (siRNA) specifically targeting CaR gene was designed, synthesized and transfected into HUVEC according to the cDNA sequence of human CaR gene in GenBank. The transfection efficiency and the interference efficiency of CaR protein were determined by laser scanning confocal microscopy and Western blot, respectively. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by Fura-2/AM loading. The production of NO and the activity of endothelial nitric oxide synthase (eNOS) were determined by the DAF-FM diacetate (DAF-FM DA). Western blot results demonstrated that siRNA targeting the CaR specifically decreased the expression of CaR protein in CaR siRNA group 48 h after transfection (P < 0.05). At the same time, the Spermine-induced [Ca(2+)](i), eNOS activity and NO generation were also significantly reduced (P < 0.05) in CaR siRNA group compared with those in the untransfected or negative siRNA transfected group. In conclusion, the present study suggests that the CaR plays an important role in the Spermine-evoked process of extracellular Ca(2+) influx and NO generation in HUVEC.

Objective: To investigate the effect of interleukin-6 (IL-6) and calcium sensing receptor (CASR) gene polymorphisms on bone mass accrual in Chinese adolescent girls., Methods: A total of 228 premenarche Chinese girls (9-11.5 years) were recruited for a 2-year study. Bone mineral densities (BMD) and Bone mineral contents (BMC) in the total body, total left hip including femoral neck, trochanter, intertrochanter and Ward's triangle area, and lumbar spine (L1-L4) were measured by DEXA. The -634C/G polymorphism of IL-6 gene was detected by PCR-restriction fragment length polymorphism and A986S polymorphism of CASR gene was detected by allele-special mutagenically separated amplication-PCR., Results: There were 176 available subjects when the 2-year study was completed. The -634C/G polymorphism of IL-6 gene and A986S polymorphism of CASR gene were significantly associated with bone mass accrual after adjusting the potential confounding factors. Girls with CC genotype of IL-6 -634G/C gene had higher percentage of increase in BMD of total body (P= 0.027) and femoral trochanter (P= 0.028) than those with CG+ GG genotypes. Girls with AA genotype of CASR gene had greater percentage of increase in L1-L4 lumbar spine BMC (P= 0.022) and Ward's triangle area BMD (P= 0.049) than their AS+ SS counterparts. Subjects with G allele of IL-6 gene in combination with S allele of CASR gene had less increase in BMC of femoral neck and L1-L4 lumbar spine., Conclusion: Adolescent girls carrying the G allele of IL-6 gene at -634C/G and S allele of CASR gene at A986S could potentially be the risk population of lower bone mass accrual.

Objective: To investigate the distribution of calcium-sensing receptor (CASR) gene polymorphisms in healthy young women of Han nationality in Beijing area and to explore the association of CASR genotypes and serum calcium, parathormone (PTH) level in healthy women., Methods: 202 healthy young women aged (27 +/- 5) years of Han nationality in Beijing area were recruited in this study. Whole blood genome DNA was extracted with QIAGEN DNA extraction kit. A986S and G990R genotypes were determined with mutagenically separated polymerase chain reaction (MS-PCR) and PCR-restriction fragment length polymorphisms (RFLP), respectively. Serum calcium, albumin, phosphorus, PTH were determined., Results: (1) There were A986S and G990R polymorphisms in healthy young women of Han nationality in Beijing. The frequencies of genotypes were as follows: SS absent, AA 95.0% and AS 5.0% for A986S, RR21.3%, GR51.0% and GG27.7% for G990R. Hardy-Weinberg equilibrium was evident. (2) In subjects carrying AA and AS genotype, serum Ca was (2.46 +/- 0.09) mmol/L vs (2.45 +/- 0.08) mmol/L, calcium adjusted by albumin was (2.31 +/- 0.10) mmol/L vs (2.30 +/- 0.09) mmol/L, serum P was (1.23 +/- 0.14) mmol/L vs (1.24 +/- 0.11) mmol/L, serum PTH was (41.6 +/- 18.6) ng/L vs (50.0 +/- 25.1) ng/L. There were no significant differences among them. (3) In subjects carrying GG, GR and RR genotype, serum Ca was (2.44 +/- 0.10), (2.46 +/- 0.08) and (2.48 +/- 0.08) mmol/L; calcium adjusted by albumin was (2.30 +/- 0.10), (2.32 +/- 0.09) and (2.32 +/- 0.10) mmol/L; serum P was (1.22 +/- 0.13), (1.24 +/- 0.15) and (1.20 +/- 0.15) mmol/L, serum PTH was (37.6 +/- 16.0), (42.1 +/- 20.2) and (45.9 +/- 18.1) ng/L. There were significant difference in serum Ca, calcium adjusted by albumin and PTH (P = 0.042, 0.020 and 0.014, respectively)., Conclusions: (1) There are A986S and G990R polymorphisms in CASR gene in Han nationality in Beijing area. The frequencies of genotypes are as follows: SS absent, AA 95.0% and AS 5.0% for A986S, RR21.3%, GR51.0% and GG27.7% for G990R. Hardy-Weinberg equilibrium is evident. A and G alleles are more common. It is indicated that the distribution of CASR gene polymorphisms in Chinese is different from that in Caucasians. (2) G990R polymorphism is associated with serum calcium and PTH levels in healthy women. Subjects with R allele have higher levels of serum calcium and PTH. There is no correlation between A986S polymorphism and serum calcium or PTH.

Objective: To investigate the relationship between calcium-sensing receptor protein (CaSR) expression and rat cardiomyocyte apoptosis and related signal transduction pathways., Methods: The CaSR, BCl2, Caspase3 protein and ERK1/2 phosphorylation or non-phosphorylation were detected by Western blot. Cardiomyocyte apoptosis was detected by flow cytometry and immunofluorescence., Results: CaSR protein was detected in rat cardiac tissue and CaSR activator gadolinium (GdCl3) induced cardiomyocyte apoptosis and increased ERK1/2 phosphorylation and expression of BCl2 and activated Caspase3. The selective mitogen-activated protein kinase (MAPK) inhibitor PD98059 abolished gadolinium -induced ERK1/2 activation and BCl2 expression, further increased the activation of Caspase3 and cardiomyocyte apoptosis., Conclusion: Our results demonstrate the CaSR existence in cardiomyocytes and CaSR activation by gadolinium can induce myocyte apoptosis by activating Caspase3 and tyrosine protein kinase pathway.

Objective: To investigate the association of a calcium-sensing receptor (CaSR) gene missense polymorphism, 986Ala/Ser (A986S), with bone mineral density (BMD) and bone size in healthy Chinese premenopausal women., Methods: A total of 285 healthy Chinese premenopausal women (20.0 to 41.9 years of age) of Han nationality in the urban area of Shanghai were recruited for this study. The BMD and bone size of the spine and hip were measured by dual-energy X-ray absorptiometry. All the subjects were genotyped at the CaSR A986S site in exon 7 with polymerase chain reaction followed by restriction enzyme BsaHI digestion. The presence of the restriction fragment site was represented by alanine (A), while its absence by serine (S), rendering the genotypes AA, AS, and SS., Results: The genotype AS was rare and SS absent in these Chinese women, and no significant differences in the BMD or bone size of either the spine or hip were found between the two genotypes., Conclusion: Given the important role of the CaSR in calcium metabolism, further studies with useful genetic markers may have better chances to define the association of the CaSR gene with bone phenotype variations.