Alternative Reading Frame Protein Induces EMT Through Wnt/β-catenin Signaling Pathway To Promote The Invasive And Metastasis Of Papillary Thyroid Carcinoma

Objective: PTC(Papillary Thyroid Carcinoma)is the most prevalent type of thyroid cancer.The incidence of PTC has been rapidly increasing in recent years.One of the characteristics of PTC is prone to cervical lymph node metastasis.Cervical lymph node metastasis of PTC was a significant risk factor of local recurrence and metastasis.Therefore,finding biomarkers associated with lymph node metastasis in patients with PTC to enable an early diagnosis is crucially importance.ARF(Alternative Reading Frame)protein is encoded by the alternative reading frame of the CDKN2 A loci.ARF is a tumor suppressor that regulates cell cycle progression and cell apoptosis through both p53-dependent and p53-independent mechanisms.However,ARF role as classic tumor suppressor has been currently challenged by recent studies.ARF over-expression has been reported in a range of cancers,and associated with a poor prognosis,but its role in invasive and metastasis of PTC remains unelucidated.This study aims to clarify the role and potential molecular mechanisms of ARF in the invasion and metastasis of PTC.Methods: 1.RNA-seq data along with clinical information on PTC were retrieved from TCGA(The Cancer Genome Atlas)and GEO(Gene Expression Omnibus).The m RNA expression levels of CDKN2 A in PTC and normal thyroid tissues were analyzed.And the correlation of CDKN2 A expression with clinical features and prognosis of PTC patients was analyzed.The GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analyses were performed on CDKN2 A related genes to gain insight into CDKN2 A mediated molecular pathways in PTC.2.A total of 156 cases of primary papillary thyroid cancer tissues and 116 cases of paracancerous tissues were collected from the Department of Vascular and Thyroid Surgery of the First Affiliated Hospital of China Medical University.Expression of ARF protein in PTC tissues and paracancerous tissues were detected by IHC(Immunohistochemistry),and the correlation of ARF expression with clinical features of PTC patients was analyzed.3.Three human PTC cell lines(BCPAP,K1,and TPC1)and one normal human thyroid epithelial cell line(Nthyorid3-1)were used.Gene silence or overexpression were performed by transfecting with si RNA(small interfering RNA)or plasmids.CCK8 and clone formation assay were utilized to observe the effects of ARF on the proliferation and colony formation of PTC cells.The cell scratch experiment was used to test the migration ability of PTC cells.The cell invasion capacity was examined using Transwell invasion assay.Cell apoptosis was analyzed by Annexin V-APC/7-AAD staining.PI(Propidium Iodide)staining assay was used to detect the effect of ARF on PTC cell cycle.4.Expression of EMT markers,N-cadherin、E-cadherin、MMP9、Vimentin and Claudin1 was determined by Western blot analysis.The effect of ARF on the Wnt signaling pathway protein,β-catenin,MMP7,c-Myc,Cyclin D1,p-GSK3β,GSK3βwas detected by Western blot.5.Immunoprecipitation combined with immunofluorescence was used to investigate whether ARF binds directly to β-catenin.After CHX(cycloheximide)were added to block protein synthesis while ARF was down-regulated,the expression changes of ARF and β-catenin proteins were detected by Western blot.After the proteasome inhibitor MG132 were added to block protein degradation while ARF was down-regulated in PTC cells,the expression changes of β-catenin were detected by Western blot.Immunoprecipitation experiment was used to observe the effect of upregulation of ARF on the ubiquitination level of β-catenin.6.We then investigated if ARF promoted the migration and invasive capability of PTC cells,activated Wnt signaling and promoted EMT progression of PTC cells by the rescue experiment.Results: 1.We found that the mRNA level of CDKN2 A was higher in PTC tissues than in adjacent tissues in TCGA and GEO dataset(P<0.05).CDKN2 A expression level was positively correlated with T classification,N classification and clinical stage(P<0.05),while it was not connected with age,sex and M classification(P>0.05).KEGG analyses showed that ARF is related to Wnt signaling pathway.2.IHC revealed that ARF was upregulated in PTC tissues compared to adjacent thyroid tissues.Correlation analysis between ARF expression level and clinicopathological features revealed that high expression of ARF in PTC was closely associated with lymph-node metastasis(P<0.05).However,no correlations with age,gender,T classification or clinical stage were observed(P>0.05).3.PTC cells were divided into the siARF group(transfected with ARF silence si RNA vector),si NC group(transfected with empty si RNA vector),ARF group(transfected with ARF overexpression plasmid vector)and NC group(transfected with empty plasmid vector).The results of CCK8 assays indicated that the OD(Optical Density)values on the fifth day of si ARF group was significantly lower than those of si NC group(P<0.05).In addition,the OD values on the fifth day of ARF group was significantly higher than those of NC group(P<0.05).The results of plate clone formation experiment showed that the colony numbers of si ARF group were significantly fewer than those of si NC group(P<0.05).And the colony numbers of ARF group were significantly more than those of NC group(P<0.05).The results of the scratch experiment showed that the migration area ratio of si ARF group was significantly lower than that of si NC group(P<0.05).And the migration area ratio of ARF group was significantly higher than that of NC group(P<0.05).The Transwell chamber invasion results showed that the number of cells passed through Transwell chamber was significantly decreased in si ARF group compared with si NC group(P<0.05).In addition,the number of cells passed through Transwell chamber was significantly increased in ARF group compared with NC group(P<0.05).Flow cytometry revealed that the rate of cell apoptosis was not significantly different between groups(P>0.05).Cell cycle distribution was not significantly different among the groups(P>0.05).4.Results of Western blot demonstrated no difference of GSK3β and p-GSK3βexpression after altering expression level of ARF in PTC cells or Nthyroid3-1 cell line.ARF overexpressing cells showed higher β-catenin,MMP7,c-Myc and Cyclin D1 protein level as compared with control group.Meanwhile,expression level of the above indices was all significantly decreased in si ARF group(P<0.05).Protein expression levels of N-cadherin and MMP9 were significantly higher in ARF group than in NC group(P<0.05),and significantly lower in siARF group than in siNC group(P<0.05).Protein expression levels of E-cadherin,Vimentin and Claudin1 were significantly lower in ARF group than in NC group(P<0.05),and significantly higher in si ARF group than in si NC group(P<0.05).5.Cell immunofluorescence and immunoprecipitation experiments found that ARF and β-catenin were directly combined in PTC cells.When CHX was added to block protein synthesis,while ARF was silenced in TPC1 cells,the degradation rate ofβ-catenin was increased in si ARF group.And after the proteasome inhibitor MG132 were added to block protein degradation while ARF was silenced in TPC1 cells,there was no obvious change in the expression of β-catenin protein,indicating that ARF participated in the degradation process of β-catenin protein.Finally,after silencing the expression of ARF in TPC1 cells,we found that the ubiquitination level of β-catenin protein increased significantly,which indicated that ARF affected the expression of β-catenin protein by affecting the ubiquitination level of β-catenin.6.To know whether ARF-β-catenin interaction is important for ARF function on PTC progression,we silenced β-catenin in ARF-overexpressing cell lines.β-catenin silence in ARF-overexpression PTC cells led to EMT reversion,Wnt signaling pathway reversion and the facilitatory effect of ARF in regulating PTC cell invasion and migration.Conclusions: In conclusion,CDKN2 A and ARF is highly expressed in PTC tissues,and is closely related to clinical features such as tumor lymph node metastasis.This study revealed the specific mechanism of ARF activating the Wnt signaling pathway by reducing the ubiquitination level of β-catenin and stabilizing β-catenin expression,inducing EMT of PTC cells,and enhancing the proliferation,migration and invasion capabilities of PTC cells.Our research provided a scientific theoretical basis for the prognosis assessment of PTC and the discovery of new therapeutic targets.