Study On Expression Of PTHR1and AC-cAMP-PKA/Ca2+-PKC Signal Transduction Of GRK Mediated By GRK In Meduuary Thyroid Carcinoma Cell

Background:Medullary thyroid carcinoma originated from thyroid parafollicular cell (C cells) that secreted calcitonins. It accounts for about5-10%of all of thyroid cancer. The degree of malignancy between the differentiated thyroid carcinoma and anaplastic thyroid carcinoma, the prognosis was relatively poor, the mortality rate of MTC accounts for about13.4%of all thyroid cancer-related deaths. Due to C cells do not express the TSH receptor without uptake iodine so that the traditional thyroid-stimulating hormone suppressive therapy and131I radionuclide therapy were ineffective for MTC. Meanwhile it showed poor effect to systemic chemotherapy and radiotherapy, the radical surgery has become the unique option for the most patients with MTC. The efficacy and prognosis of the patients was restricted by the single and lacked treatment. It remains unclear for the C cell surface receptor distribution and regulatory mechanism of endocrine hormones in MTC. As one of the G protein-coupled receptor, it has not yet been reported that parathyroid hormone receptors in C cell surface expression, receptors internalization and interaction in the regulation of its signaling pathway and the incidence of MTC regulatory mechanism.Objectives:To clear the thyroid C cell surface receptor distribution and PTHR1expression in medullary thyroid carcinoma tissue and explore the PTHR1signaling pathway and GRK-mediated AC-cAMP-PKA signaling system and Ca2+/PKC signaling system signaling pathway in the regulation mechanism and interaction of MTC. The project was trying to provide theoretical support and a new target for the treatment of medullary thyroid carcinoma.Methods:(1) Using immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction and protein immunoblot method to detect PTHR1, CTR and GRK2, GRK5, CaM, PKC, and PKA in MTC tissue expression, respectively. From the organization, protein and nucleic acid level to study the expression of GPCRs and GRK, AC-cAMP-PKA signaling system, Ca2+/PKC signaling system signaling pathway.(2) Medullary thyroid carcinoma TT cell cultured in vitro by exogenous interventions add of PTH1-34and PTHR McAb. The expression of PTHR1CTR, GRK2, GRK5and CaM in MTC TT cells were detected by the real-time fluorescence quantitative polymerase chain reaction and protein immunoblot method before and after dosing. The PTH and CT level of the cell culture supernatant were detected by electrochemical luminescence, the intracellular cAMP concentration was detected by radioimmunoassay competitive binding assay and the activity of PKC and PKC were detected by liquid scintillation counting γ-32P radioactivity assay during MTC TT cell cultured in F-12culture medium. We try to explore the effect of medullary thyroid carcinoma TT cells, PTHR1expression and the AC-cAMP-PKA and Ca2+/PKC signaling pathway added of PTH1-34.Results:(1) The Immunohistochemical staining showed PTHR1, CTR weak-medium expressed in normal thyroid, nodular goiter and thyroid papillary carcinoma and medullary thyroid carcinoma tissue. There was no significant difference between different tissue type group of thyroid tumors on PTHR1medium expression (p>0.05) and there was significant difference between different tissue type group of thyroid tumors on PTHR1strong positive expression (p<0.05).Expression of GRK2, GRK5was no difference between the normal thyroid and nodular goiter groups (p>0.05). GRK2expression was strong positive in papillary thyroid carcinoma and was weakly positive in medullary thyroid carcinoma tissue (p<0.05), GRK5expression were both weakly positive expression in papillary thyroid carcinoma and medullary thyroid carcinoma tissue. There was no significant difference between the two groups (p>0.05).Real-time fluorescence quantitative polymerase chain reaction showed there was no significant difference in the expression of the PTHR1mRNA CTR mRNA between the normal thyroid, nodular goiter and papillary thyroid carcinoma (P>0.05) but there was significant difference in the expression of the PTHR1mRNA CTR mRNA between the normal thyroid and medullary thyroid carcinoma (p<0.05). The expression of GRK2mRNA and GRK5mRNA was no significant difference between normal thyroid group and nodular goiter group (p>0.05). The expression of GRK2mRNA and GRK5mRNA in papillary thyroid carcinoma was significantly higher than normal thyroid group (p<0.05). GRK2mRNA expression was significantly higher than normal thyroid group and GRK5. mRNA expression was significantly lower than normal thyroid group in medullary thyroid carcinoma group which both showed the significant difference (p<0.05).Between normal thyroid, nodular goiter, thyroid papillary carcinoma of PKA mRNA and PKC mRNA expression was no significant difference (p>0.05); compared with normal thyroid group, the expression of PKA mRNA of PKC mRNA were significantly higher (p<0.05).There was no significant difference of expression CaM mRNA between normal thyroid, nodular goiter, thyroid papillary carcinoma (p>0.05).The expression CaM mRNA was higher in medullary thyroid carcinoma than normal thyroid that there was significant difference between two groups (p<0.05). Western blot showed same results as real-time fluorescence quantitative polymerase chain reaction.(2) Medullary thyroid carcinoma TT cells culture in F-12K medium showed long spindle, polygon or irregular shape to stick wall growth. It showed adherent growth of about80%of the48-to72hours after TT cells adherent fusion, give PTH1-34role significantly reduce cell number and some cells the cell body shrinkage and round cell gap is further widened, some cells showed growth delay, suspension cells and debris increased; given PTHR McAb to the role of cell number did not significantly reduce the Some cells showed a growth delay, cell morphology and vitality was no significant change. Add PTH1-34TT cell supernatant supernatant PTH levels significantly higher (p <0.05) and CT levels were significantly lower (p<0.05) between the different doses of PTH1-34TT supernatant level no significant difference (p>0.05).Conclusions:(1) Thyroid C cell surface express PTHR1and CTR. Due to the high expression of the second messenger-regulated kinase (PKA and PKC) and CaM in medullary thyroid carcinoma tissue, G protein-coupled receptor kinase-GRK2and GRK5expression differently that GRK2activity enhance and GRK5activity weakened. As different phosphorylation of GRK2and GRK5on the thyroid C cell surface GPCRs, the result showed increase expression of PTHR1and CTR in medullary thyroid carcinoma tissue.(2) Add through the exogenous PTH1-34and the PTHR McAb, observe PTH1-34in vitro effects of medullary thyroid carcinoma TT cell line, cell culture supernatant showed that PTH levels were significantly increased CT levels; of cAMP concentration increased at the same time PKA rise in the PKC lower. At the same time CaM decreased expression of GRK2reduced expression of GRK5expression was elevated, and ultimately activate the AC-cAMP-PKA signaling system and inhibition of Ca2+/PKC signaling system signaling pathway conduction, resulting in medullary thyroid carcinoma TT cell growth inhibition, the study further revealed the PTHR1signaling pathway conduction growth of malignant cells, this study suggests that the PTHR1could become one of the targets of targeted therapy of medullary thyroid carcinoma, and opens up new perspectives and ideas for the treatment of medullary thyroid carcinoma.