The Functions Of FOXE1Gene In The Process Of Epithelial-mesenchymal Transition In Thyroid Cancer Cells

Forkhead box E1(FOXE1) is a member of the forkhead box family. Theforkhead family belongs to a large one of transcription factor family,and many genes in this family have important biological functions,including regulating embryonic development, cell proliferation anddifferentiation as well as tumorigeness, which have the functions of bothoncogene and tumor suppressor when their expression disordering, and playan important role in tumor development, invasion and metastasis. FOXE1gene is one of the specific thyroid transcription factors, which play animportant role in development, cell proliferation and differentiation ofthyroid gland. In recent years, the role of FOXE1in tumor oncogenesiswas concerned. Some researches have shown that FOXEl gene is related tooccurring of certain tumors, such as pancreatic carcinoma, cutaneoussquamous cell carcinoma and thyroid neoplasm. In2009, by the genome-wideassociation study, FOXE1gene was screened as a genetic risk gene ofpapillary thyroid carcinoma in the European population.Epithelial-mesenchymal transition (EMT) refers the epithelial cellslose epithelial characteristics under certain physiological andpathological conditions, and transform into mesenchymal cells. EMT playsan important role in embryonic development, tissue and organdifferentiation, as well as wound healing, tissue regeneration and organfibrosis, and other physiological and pathological processes. Recentstudies suggest that EMT is also an important molecular mechanisminvolving in invasion and metastasis of malignant tumors, and a requisitefor cancer going across the basement membrane and entering the adjacenttissues. Studies have shown that EMT may be a common phenomenon in invasivepapillary thyroid carcinoma, and may also play a role on transitionprocess of thyroid cancer from differentiation to undifferentiation.In this study, we investigated the functions of transcription factor FOXE1in epithelial to mesenchymal transition of thyroid cancer cell lines.We First discovered the mRNA and protein expression of FOXE1was relatedto the epithelial and mesenchymal characteristics in papillary thyroidcancer cell TPC-1and poorly differentiated thyroid cancer cell line SW579.The migration and invasion potential of cancer cells were enhanced afterthe expression of FOXE1gene was specifically silenced by lentivirusmediated RNA interference,which maybe pass through EMT. The downstreamgenes with expression changing in thyroid cancer cells after FOXE1geneinterferencing were further screened by microarray experiment.Throughfunctions analysis and expression verification of the relevant genes, weconfirm that FOXE1affect EMT process of thyroid cancer cell maybe byregulating platelet-derived growth factor A(PDGFA), and furthermoreinfluence invasion and metastasis of tumor, which provides theexperimental basis for clinical searching for molecular target in thetreatment of thyroid cancer. In this study, the experiment was listed asthe following three parts:Part OneObjectives To detect the relationship between FOXE1expression andepithelial and mesenchymal markers in thyroid cancer cells with differentdifferentiation, then explore the effects of FOXE1gene on epithelial tomesenchymal transition (EMT) in thyroid cancer cells.Methods qRT-PCR and Western-blotting were used to detect mRNA andprotein expression of FOXE1gene and epithelial and mesenchymal markers,such as E-cadherin and Vimentin in differentiated thyroid cancer celllines TPC-1, K1and poorly differentiated thyroid cancer cell line SW579,and analyzed the correlation between them.Results FOXE1mRNA expression was0.568,0.516and0.098respectivelyin TPC-1, K1and SW579cell line, there was no difference in FOXE1mRNA expression between the TPC-1and K1cell line (P>0.05), whereas thedifference was statistically significant in FOXE1mRNA expression betweenthe TPC-1,K1and SW579cell line (P<0.01). The E-cadherin mRNA expressionwas0.064,0.082and0.023respectively in TPC-1, K1and SW579cell line,while the mRNA expression of Vimentin was0.343,0.428and1.397in thatthree cell lines, there was no difference in the expression between TPC-1and K1cell line (P>0.05), whereas there was statistically significantdifference in the expression between TPC-1,K1and SW579cell line (P<0.01).The results of FOXE1、E-cadherin and Vimentin protein expression wereconsistent with the mRNA expression changes.Conclusions Compared with differentiated thyroid cancer cells, theexpressions of FOXE1gene and epithelial marker E-cadherin were lowwhereas the expression of mesenchymal marker Vimentin was high in poorlydifferentiated thyroid cancer cells. That suggested the expression ofFOXE1gene may be related to the epithelial and mesenchymalcharacteristics of thyroid cancer cells. The FOXE1gene could take partin the EMT process and play a negative role on EMT process in thyroid cancercells.Part TwoObjectives To investigate the effects of interfering FOXE1gene on EMTof TPC-1cell, and study the role of FOXE1in invasion and migration ofTPC-1cells.Methods The lentiviral expression vector for RNA interference of FOXE1gene was constructed to silence the expression of FOXE1. Real-timepolymerase chain reaction(RT-PCR) and western-blotting were used todetect the expressions of such EMT markers as E-cadherin, N-cadherin andVimentin. Transwell assay and scratch assay were used to analyze themigration and invasion of TPC-1cells. Results After RNA interference of FOXE1, the morphology of TPC-1cellsindicated a transformation of EMT, the expression of epithelial phenotypemarker E-cadherin decreased remarkably while mesenchymal marker Vimentinwas significantly up-regulated (P<0.01). Compared with control group, theexpression of Vimentin mRNA in the experimental group was2.24timeshigher. Meantime, the migration and invasion ability of TPC-1cellsincreased significantly, and the number of cells in transwell was2.11times of the control，s.Conclusions The silence of FOXE1gene probably increases the invasionpotential of human PTC cells through EMT.Part ThreeObjectives The process of EMT is regulated by various signalingmolecules, and it is unknown to how FOXE1gene regulates the EMT process.To further explore the molecular mechanisms of FOXE1gene regulating EMTprocess in thyroid cancer cells, the genes with expression changing inTPC-1cells with FOXE1-shRNA were screened and identified in function.Methods Thyroid cancer cell line TPC-1with FOXE1-shRNA and its controlgroup (NS-Top10) were selected. The downstream genes were screened byusing PrimeViewTM(Affymetrix) gene chip, the experimental group and thecontrol group each was repeated for three times. Then the screened geneswere analyzed on expression and function, and the mRNA expressions of agroup of genes with higher correlation were validated by using theqRT-PCR.Results The boundary was defined as1.5times for upregulation ordownregulation. As a result,115up-regulated genes and441down-regulated genes were screened by the microarray experiments, whichfunctions involve in cell proliferation, apoptosis, differentiation, andcell transport, positioning, secretion and so on. Through further function analysis, the upregulated growth factor PDGFA was found toprobably take part in the process of FOXE1regulating EMT in tumor cells.Conclusions The results show that growth factor PDGFA is the downstreamgene of FOXE1in thyroid cancer cells. The FOXE1gene could regulatethe EMT process in thyroid cancer cell through mediating PDGFA, therebyinfluencing the invasive and metastatic potential of thyroid cancer cell,which provides the experimental basis for clinical further searching formolecular target in the treatment of thyroid carcinoma.