The Function And Mechanism Study Of TBX3 Protein In Papillary Thyroid Carcinoma Development

Objectives:Thyroid cancer is the most common cancer of endocrine system.The incidence of thyroid cancer has been increasing in past few decades.Although the role of BRAF^V600E mutation and RET/PTC rearrangement of chromatin in TC cases have been more clear,but other genetic events are poorly understood.Therefore,it is very important to find new molecular targets to guide early diagnosis and targeted therapy.As an important gene of the T-BOX family,TBX3 is critical for development and has emerged as an important player in the oncogenic process.It usually plays the role of oncogene and participates in various biological behaviors of tumorigenesis.Especially,it was used to promote cancer in p53 dependent and non-dependent manner by inhibiting p53 activator p14^ARF or CDK kinase inhibitor p21^WAF1/CIP1,however,the function of TBX3 in thyroid papillary carcinoma has not been reported.The main purpose of this topic is to study how TBX3 affects the occurrence and development of papillary thyroid carcinoma（PTC）and to explore the possible molecular mechanism of the abnormal expression of TBX3 in PTC.Methods:Part Ⅰ:To study the expression of TBX3 in clinical specimens of papillary thyroid carcinoma and to explore the correlation between TBX3 expression and clinicopathologic features.This part of the research is mainly described in three aspects.Firstly,we collected 98 PTC samples,71 of them with adjacent normal tissues and performed immunohistochemical staining.Secondly,qRT-PCR and western blotting were used to detect the mRNA and protein expression of TBX3 in 15pairs of thyroid cancer tissues and corresponding paracancerous tissues.At the same time,Cell lysates of five thyroid cancer cell lines and one normal thyroid epithelial cell were extracted,and the expression level of TBX3 was identified by immunoblotting technique.Finally,thec² test was used to analyze the correlation between the expression level of TBX3 in human PTC and clinicopathologic features.Part Ⅱ:To investigate the effect of TBX3 expression on proliferation of thyroid papillary carcinoma cells.On the one hand,RNAi technology was used to reduce the expression of TBX3 in PTC cells,and the proliferation phenotypes were analyzed by CCK8,clone formation experiment and flow cytometry.On the other hand,K1 cells with stable knockdown of TBX3 were inoculated on both sides of the dorsal skin of nude mice.Tumor volumes were monitored regularly and the expression changes of TBX3 and Ki-67 were detected by immunehistochemistry.In order to explore the molecular mechanism of the effect of TBX3 on proliferation,we investigated the transcriptomes in wild-type and TBX3-depleted K1 cells by high-throughput RNA sequencing（RNA-seq）.qRT-PCR and immunoblotting were used to verify the target gene and to observe the effect of TBX3 on p57^KIP2IP2 expression.After that,we transfected siRNA against CDKN1C into K1 cells with stable knockdown of TBX3and determine whether the changes of proliferation phenotypes could be rescued.Part Ⅲ:Firstly,Bioinformatics methods were used to predict the potential binding sites of TBX3 in the CDKN1C promoter region of p57^KIP2-encoding gene.After that,HEK293T cells were cotransfected with different promoter reporter constructs and TBX3 expression plasmid or corresponding vector control.Cell culture media were collected and subjected to luciferase activity assays 48h post-transfection.Secondly,the precise site of CDKN1C promoter was determined by using the endogenous ChIP experiment and DNA-pull down assays.Furthermore,CoIP technology detects the interaction between TBX3 and PRC2 complex and HDAC1/2physically.Finally,the protein expression of p57^KIP2 and cell proliferation phenotype were detected by adding the inhibitor of S-adenosylhomocysteine hydrolase and HDAC1/2 in K1 cells expressing TBX3.Results:Part Ⅰ:Compared with the adjacent tissues,TBX3 showed high expression at mRNA and protein levels in human thyroid papillary carcinoma.Further analysis revealed that the expression level of TBX3 was directly proportional to lymph node metastasis and TNM staging（p<0.001）,regardless of age,sex and tumor size.Interestingly,TBX3 highly expressed in K1 and TPC-1 cells,but it hardly express in normal thyroid epithelial cells.Part Ⅱ:TBX3 affected proliferation phenotype of PTC cell lines.In vitro experiments,the cell growth slowed down and cell cycle arrest occured during G1/S phase when the expression of TBX3 was transiently or stably reduced in PTC cells.Consistently,knockdown the expression of the TBX3 resulted in a dramatic reduction of tumor volume and weight.High throughput transcriptome sequencing analysis found a new CKI molecule associated with the proliferation changes,p57^KIP2.The expression level of p57^KIP2 was increased or decreased in the cells after the reduction or overexpression of TBX3 respectively.Of course,FACS analysis revealed that p57^KIP2 knockdown in TBX3-depleted cells resulted in a significant decrease in the percentage of cells at the G1 phase.Part Ⅲ:TBX3 directly transcriptionally regulated the expression of p57^KIP2.Luciferase activity assay and ChIP assays accurately determined that TBX3 binds to the-1297 and-800 sites of the CDKN1C promoter.Secondly,the endogenous and exogenous CoIP experiments demonstrated that TBX3 and PRC2 complexes and HDAC1/2 could physically bind to each other.Finally,it was found that the phenotype of cell growth was restored after inhibiting the role of H3K27me3 and HDAC1/2 by rescue experiments.Taken together,these experiments showed that TBX3 required PRC2 complex and HDAC1/2 to function in PTC.Conclusion:Down-regulation of TBX3 expression in thyroid papillary carcinoma cell lines can delay the G1/S phase transition,reduce cell proliferation in vitro and inhibit tumorigenesis in vivo.p57^KIP2 is a new downstream target gene of TBX3 that controls the proliferation of PTC cells.Reduction of TBX3 increased the expression of p57^KIP2,followed by knockdown of p57^KIP2 to restore the phenotype of cell cycle arrest.In the clinical samples of PTC,the expression of TBX3 was closely related to tumor classification,but negatively correlated with the expression of p57^KIP2.Mechanism studies showed that TBX3 directly binds to the promoter region of CDKN1C and inhibited its transcription.In addition,TBX3 recruited the core components of the PRC2 complex and the histone deacetylase HDAC1/2 to share transcriptional repression.In conclusion,our study showed that the TBX3-p57^KIP2 axis was involved in new functions and mechanisms of cell cycle regulation and also indicated the role of PRC2 complex and HDAC1/2 in this process.