The Role Of Thyroid Hormone Receptor ?1 In Atypical Meningiomas And Its Mechanism

Objective:Thyroid hormone and its receptor have been reported to have a certain relationship with the onset of meningioma,and the specifie mechanism is still unclear.TRpl,one of the important thyroid hormone receptors,has been shown to be a tumor suppressor in a variety of tumors,but is rarely reported in meningiomas.To clarify the link between TR?1 and meningioma and to explore the relevant mechanisms,we designed this experiment.Firstly,the relationship between thyroid hormone receptor ?1(TR?1)and the degree of malignancy of meningioma was studied.On the basis of this,the expression of TR?1 protein in benign meningioma and atypical meningioma was detected.In addition,the obtained fresh meningioma tissue was used.A primary meningioma cell culture system was established,and the role of thyroid hormone receptor ?1 in atypical meningioma cells was investigated at the cellular level and its mechanism was further studied..Methods:The frozen meningioma tissue was ground in a liquid nitrogen atmosphere,and after total RNA was extracted,real-time PCR was performed to determine the expression of thyroid hormone receptor ?1 mRNA in meningioma tissue,and the Ki67 index corresponding to the sample(Correlation comparison was performed by the pathology department.Immunohistochemistry(IHC)staining of meningioma tissue samples and Western blot analysis of thyroid hormone receptor ?1 protein expression in benign and atypical meningioma tissues.The primary meningioma cell system was established,and the fresh meningioma tissue samples obtained were cut and digested with trypsin to culture the primary meningioma cells,and the cultured cells were identified by cell immunochemistry.Subsequent experiments were performed on primary cultured tissue cells identified as pathologically identified as atypical meningioma.The effects of thyroid hormone T3 on atypical meningioma cells were examined by MTT,siRB and EDU.First,the cells were seeded in a 96-well plate for 24 hours,and then 1 ng/ml,5 ng/ml,10 ng/ml,and 20 ng/ml of four different concentrations of T3 were added.After 48 hours,the cell viability was determined by MTT assay,and the most T3 was determined.Good concentration.Then,the cells were seeded in a 48-well plate,and after 12 hours of cell adherence stabilization,the cells were further cultured in a complete medium containing 20 ng/ml of T3,respectively,at 0 days,1 day,2 days,3 days,4 days,10 days.The siRB test was performed after the%TCA was fixed.The cells were also subjected to EDU assay 48 hours after 20 ng/ml T3 intervention.In order to further clarify the relationship between thyroid hormone receptor and atypical meningioma cells,siRNA of thyroid hormone receptor ?1was treated with cells,and the expression of thyroid hormone receptor ?1 on the cells was down-regulated by western blot.The cell viability of the thyroid hormone receptors?1,T3 and atypical meningi cell proliferation was analyzed by MTT assay using the effective siRNA band treatment and 20 ng/ml T3 intervention.Western blot was used to detect the relevant downstream pathways that affected cell proliferation after intervention with an effective siRNA band and 20 ng/ml T3.Results:1,the expression of TR?1 mRNA in 24 cases of meningiomas detected,and its corresponding Ki67 index correlation analysis found that with the increase of tissue Ki67 index,the expression of TRpl mRNA is lower.Then,IHC staining was used to detect the expression of TR?1 protein in benign meningiomas and atypical meningioma tissues.The positive staining cells in the visual field were counted,and the positive cells of TR?1 protein expression were 37.25±5.56/field in atypical meningioma.In benign meningiomas,90.75±18.73/field of view,compared with benign meningioma,the expression of TR?1 protein in atypical meningiomas was lower,and the difference was statistically significant(**,P<0.01).Western blot analysis of the two tumor tissue proteins revealed that the expression of TR?1 protein was lower in atypical meningiomas than in benign meningioma tissues.After gray scale analysis of the bands,TR?1 in atypical meningiomas could be found.The mean gray value of protein/intemal reference was 0.32,which was lower than 1.32 of benign meningioma tissue.The difference was statistically significant(*,P<0.05).2.The morphology of primary meningioma cells after culture in different time periods accorded with the growth process of typical meningioma cells.Immunochemical staining of epithelial membrane antigen(EMA)and vimentin(Vim)on cells showed that the cultured cells were all cultured.Meningioma cells.MTT assay showed that different concentrations of T3 inhibited the activity of atypical meningioma cells in a concentration-dependent manner.The difference between the drug-added group and the control group was the highest at T3 concentration of 20 ng/ml(***,P<0.001).The siRB test indicated that the number of cells in the dosing group was relatively small as compared with the control group.The EDU results showed that the average cell proliferation rate of the control group was 48.59%.The average cell proliferation rate of the drug-added(T3)group was 27.45%,which was lower than the former,and the difference was statistically significant(*,P<0.05).The expression of TRpl protein in the cells was down-regulated by 48%,Which was statistically significant(*,P<0.05).After MTT assay,the cell activity after intervention with effective siRNA band treatment and 20 ng/ml T3 was observed,and in the NC group,the cell activity after dosing(T3)was lower than that of the control group,and it was statistically significant(*,P<0.05),and in the siRNA group,there zwas no significant difference between the cell activity after dosing and the control group,and there was no statistical significance.The expression of phosphorylated ERK(P-ERK)in the NC group after T3 treatment was found in the downstream pathways that affected cell proliferation by Western blot analysis with effective siRNA band treatment and 20 ng/ml T3 intervention.Compared with the control group,it was statistically significant(*,P<0.05).In the siRNA group,the expression of phosphorylated ERK(P-ERK)in the cells after T3 treatment was slightly higher than that in the control group,but there was no statistical significance.Conclusion:In tissues,the expression of TR?1 mRNA was negatively correlated with the proliferation index of Ki67 cells in meningioma tissues,indicating that TR?1 was negatively correlated with the malignant degree of meningiomas.The expression of TR?1 protein in non-typical meningioma was lower than that in benign meningioma.Compared with benign meningioma,there is a certain loss of TRpl expression in atypical malignant meningioma,which may be one of the potential mechanisms for its high recurrence and poor prognosis.In vitro,20 ng/ml T3 was found to be effective in inhibiting the proliferation of atypical meningioma cells,and the inhibition of tumors was attenuated after down-regulation of TR?1 expression by siRNA.Phosphoric acid was also detected in the downstream pathway of proliferation.Abnormal expression of ERK(P-ERK)suggests that TR?1 may inhibit the proliferation of atypical meningioma cells by reducing the phosphorylation of ERK under the action of T3.