The Molecular Mechanism Of ERα Regulating The Proliferation And Invasion Of Thyroid Cancer Cells

Thyroid cancer is an endocrine-related malignant tumor.In recent years,the incidence rate of thyroid cancer has continued to rise around the world,and its age of onset is getting younger.Papillary thyroid cancer(PTC)accounts for more than 80% of all types of thyroid cancer.The incidence rate of PTC between men and women is about 1:3.The incidence rate of women rises rapidly after puberty,and falls rapidly after menopause.PTC’s epidemiological preference for age-appropriate women is consistent with the changes in endogenous estrogen secretion levels,suggesting the important influence of the estrogen signaling pathway on its occurrence and development.Estrogen receptor α(ERα)is not only the regulatory hub of the secondary sexual characteristics of human development,but also an important regulator of the occurrence and progression of human endocrine related malignancy.Co-activator is an important regulatory mechanism of ERα activity and an important intervention of the ERα targeted pathway.Endocrine disrupting chemical(EDC)is a class of chemical pollutants that can cause adverse effects on the endocrine system.Part of the EDC has estrogen-like effect and can be combined with ERα,so it is seen as a potential causative factor of PTC.Aminophenol is a chemical intermediate mainly used in the manufacture of drugs,dyes and imaging agents.Aminophenol is classified as EDC because it can inhibit the release of insulin.In order to clarify the relationship between ERα and PTC and further clarify the molecular mechanisms of the development of PTC happen,this article will explain the following two parts.Part I:The Effect of Aminophenol on the Proliferation of Thyroid PapillaryCarcinoma CellsBackground and Objective:Estrogen can bind to ERα and ERβ receptors in nucleus,and regulate the expression of target genes in an estrogen response element(ERE)dependent or independent manner.In addition,estrogen can also bind to GPR30 receptor on the cell membrane,leading to kinase activation and rapidly activating related signal pathways.Aminophenol is an endocrine disrupting chemical(EDC).It has an estrogen-like effect and can bind to ERα.It can increase the expression levels of ERα,ERβ and GPR30 in human papillary thyroid carcinoma cells BHP10-3.It also can promote proliferation by rapidly activating the PI3K-m TOR signaling pathway.However,the current research between EDC and thyroid cancer is still at the initial stage.There are only a few cell and animal experimental results and there is no data to support epidemiology and clinical evidence.The purpose of this study is to explore the effects of EDC on the occurrence and development of human papillary thyroid cancer from the levels of genes,proteins,cells and animals.This study also explores the significance of avoiding EDC exposure in the prevention of thyroid cancer,and explores new methods for the treatment of thyroid cancer.Methods: At the gene level,luciferase reporter gene and chromatin immunoprecipitation technology are used to analyze the ability of aminophenol to bind to ERE and analyze the effects of ERα in the promoter region of CTSD gene(ERα target gene,encoding cathepsin D).At the protein level,this study uses SDS-PAGE and Western blot to detect the effect of aminophenol on the expression level of Cyclin D1.At the cellular and animal level,this study reveals the effect of aminophenol on the proliferation of human PTC cell BHP10-3 in vivo.Results:This study concludes that aminophenol can increase the transcription factor activity of ERα in a dose-dependent manner in BHP10-3 cells: induces ERα to bind to ERE,increases the recruitment of ERα in the CTSD gene promoter region,and up-regulates the expression level of Cyclin D1.Finally,it promotes the proliferation of BHP10-3 cells.The hormone-like potency of aminophenol can be significantly inhibited by the block or knockdown of ERα.Conclusion:The results of this study initially expand the understanding of the role of EDC in the development of PTC,explore the mechanism of aminophenol in the development of PTC,suggest the necessity of avoiding exposure to aminophenol,and indicate that Erαmay be a potential target for PTC clinical treatment.Part II:Mi R-3652/PES1 Pathway Can Regulate the Proliferation of ThyroidPapillary Carcinoma CellsBackground and Objective:PES1 is a nucleolar protein with transcription factor activity.It contains breast cancer-associated gene 1(BRCA1)and BRCA1 C-terminal domain(BRCT).Micro RNA(mi Rs)is an important type of non-coding RNA,which is transcribed by RNA polymerase II(RNA Pol II)and can identify the mi RNA targeting sites in the mi RNA 3’ Un-translation regions(3’UTR)through sequence specificity.After mi Rs and m RNA recognize each other,they can induce Dicer to degrade the m RNA of the mi Rs target gene.This process is Post-transcriptional Gene Silencing(PTGS).Using bioinformatics tools can predict mi RNAs that potentially act on specific genes.Cloning mi R into the corresponding expression vector and packaging it into virus particles,transfecting malignant tumor cells can inhibit pro-oncogenes and oncogenes to achieve anti-tumor effects.Based on the function and mechanism of PES1,an important co-activator of ERα,this study aims to reveal the role of mi R-3652/PES1 in regulating ERα in thyroid cancer cells and its influence on the proliferation of thyroid cancer cells.Methods:Using luciferase reporter gene detection technology to detect the effect of PES1 on the activity of ERα transcription factor.Using quantitative PCR and western blot to detect the expression of ERα,PES1 and Cyclin D1 downstream of ERα in cells and tissues.Using Immunoprecipitation(IP)technology to determine the interaction between PES1 and Erα.Using Chromatin Immunoprecipitation(Ch IP)to detect the recruitment of ERα in the promoter region of its downstream genes;I collect tissue specimens derived from thyroid,isolate patient-derived tumor cells(PDCs)from the specimens and culture the existing thyroid cancer cell line BHP10-3.Using the online bioinformatics tool mi RNA data base(mi RDB)to predict the mi RNA(mi R-3652)acting on PES1.Using eukaryotic cell transfection experiment,western blot and luciferase reporter gene system to confirm the target of mi R-3652 on PES1.Using luciferase reporter gene system,quantitative PCR,western blot and Ch IP to detect the effect of mi R-3652 on the ERα signaling pathway,and constructing a mutant of PES1 to determine the specificity of mi R-3652.Using cell colony formation(plate cloning experiment),Transwell experiment and nude mouse tumor formation experiment to determine the effect of mi R-3652 on the invitro proliferation and the in vivo proliferation of thyroid cancer cells.In this study,SPSS software is used for statistical correlation analysis.Results:(1)PES1 can interact with ERα,increase the transcription factor activity of ERα and promote the recruitment of ERα in its downstream gene promoter region;(2)PES1overexpressed in BHP10-3 cells can promote cell proliferation and promote cell transfer and invasion in vitro;(3)mi R-3652 can inhibit the proliferation of thyroid cancer cells by acting on the 3’UTR region of PES1;(4)In thyroid-derived tissues,the expression level of PES1 is positively correlated with the expression of Cyclin D1,a downstream gene of Erα.The expression level of mi R-3652 is negatively correlated with the expression of PES1,and negatively correlated with the expression of Cyclin D1.Conclusion:This study is based on ERα,a new intervention target and regulatory hub of thyroid cancer,and focuses on the interaction between PES1 and ERα in thyroid cancer cells.mi R-3652,a potential mi RNA for Pes1,is screened and its target is confirmed.This study not only clarifies the clinical significance of the expression of related molecules in clinical specimens,but also carries out relevant research on multiple research techniques,including biochemical and molecular biology technology,cell biology technology,animal experiment,and finally determines the interaction between PES1 and ERα in thyroid cancer cells.PES1 can improve the transcription factor activity of Erα and promote the recruitment of Erα in the promoter region of its downstream gene in thyroid cancer cells.We find that mi R-3652 is a potential micro RNA for PES1,and confirm its role in thyroid cancer cells.mi R-3652 can down regulate the activity of Erα pathway in thyroid cancer cells by acting on PES1,inhibit the proliferation of thyroid cancer cells,and ultimately play an anti-tumor activity.