Analysis Of The Relationship Between Thyroid Hormone Receptor And MicroRNA Splicing Protein Drosha

Thyroid Hormone(TH)plays an important role in the growth,differentiation,development and maintenance of metabolic balance.It has been shown that this effect is achieved by binding the thyroid hormone receptor(THR)to regulate microRNAs and thus affect the transcription of downstream target genes.In order to explore the mechanism of thyroid hormone regulation of micro RNAs,this study explored the mechanism of interaction between Drosha enzyme and thyroid hormone receptor,which explored the maturation of microRNAs.In this study,immunoprecipitation(Co-IP)method was used to verify the relationship between thyroid hormone receptor and Drosha protein by Western Blot and mass spectrometry.On the other hand,the effect of thyroid hormone receptor ? / ? Gene overexpression and gene silencing,to study the effects of thyroid hormone receptors on microRNAs.The main results are as follows:1.Analysis of the relationship between thyroid hormone receptor and Drosha proteinIn this study,HEK293 cells were used to study the relationship between HEK293 cells by immunoprecipitation method and Western Blot.The experimental results were as follows: The total protein(IgG as the control antibody)in KEK293 cells was immunoprecipitated by anti-mouse THR polyclonal antibody and anti-rabbit Drosha monoclonal antibody.The THR and Drosha protein immune complexes were obtained and separated by SDS-PAGE gel electrophoresis THR and Drosha immunoprecipitated complexes were then analyzed by Western Blot with anti-rabbit Drosha monoclonal antibody and anti-mouse THR polyclonal antibody.The results showed that the presence of Drosha protein was detected by immunoprecipitation complexes with THR antibody,whereas the presence of THR was detected in Drosha immunoprecipitation complex.It has been shown that there is a correlation between thyroid hormone receptors and Drosha proteins.In order to further analyze the relationship between the two,the THR and Drosha antibody immunoprecipitated complexes were analyzed by mass spectrometry.The results showed that there were 263 proteins with high THR activity,including Drosha protein There is a 249 protein with a high degree of confidence in the relationship with the Drosha protein,which contains thyroid hormone.Analysis of mass spectrometry data revealed that 49 proteins were interrelated with both.Distribution and analysis of these proteins are mainly cell membrane and nuclear components,not only involved in thyroid hormone receptor and Drosha protein biological role,but also involved in cell migration,cell biosynthesis,apoptosis,protein metabolism and other molecular functions.Analysis of KEGG metabolic pathway showed that 49 proteins were mainly involved in glycolysis,glycerol metabolism,glutathione metabolism,arginine and proline metabolism,calcium ion signaling channel,protein assignment,amino acid biosynthesis Metabolic pathway.The results indicate that the thyroid hormone receptor and Drosha protein are indeed interrelated,and is combined with other proteins to form a protein complex.2.Analysis of microRNA expression by overexpression or silencing of thyroid hormone receptorIn this study,the thyroid hormone receptor ? / ? eukaryotic expression vector was constructed and gene overexpression and gene silencing were performed in HEK293 cells.First,the constructed thyroid hormone receptor ? / ? eukaryotic expression vector was transfected into HEK293 cells,and the expression of microRNA was detected by fluorescence quantitative PCR.The RNA interference method was used to detect the expression of thyroid hormone receptor ? / ? gene HEK293 cells were silenced,and then the expression of microRNA was detected by fluorescence quantitative PCR.The results showed that:(1)There were significant differences in the relative expression of pre-miRNA-21 between the transfected group and the untransfected group after overexpression of thyroid hormone receptor ? / ? gene,and there was no significant difference between the remaining mi RNAs The(2)The relative expression of pre-miRNA-182 was not significantly different between the untransfected group and the transfection group after THR? / ? silencing was confirmed by the siRNA interference sequence of THR? / ? gene.The rest of the pre-miRNA expression No significant difference.Thus,there is interaction between the thyroid hormone receptor and Drosha,and the thyroid hormone receptor can directly affect the expression of some of the Pre-miRNAs.As to how the thyroid hormone receptor interacts with Drosha,further research is needed.CONCLUSIONS: Thyroid hormone receptors are interrelated with Drosha proteins and are combined with other 49 proteins to form a protein complex,and thyroid hormone receptors play a direct role in the regulation of some microRNAs.