Actin Related Protein ACTR2 Induces EMT Through FAK/Src-ERK1/2 Pathway To Promote The Invasion And Metastasis Of Papillary Thyroid Carcinoma

Objective:Papillary thyroid carcinoma（PTC）,as the most common pathological type of thyroid cancer,has an increasing incidence rate and a higher lymph node metastasis rate.To explore the molecular mechanism of invasion and metastasis of PTC is of great significance for the diagnosis and treatment of PTC.The remodeling of actin cytoskeleton plays an important role in the process of tumor cell invasion and metastasis.Actin related protein 2（ACTR2）,as an important subunit in Arp2/3complex,is the nucleation site of new actin filaments.It stimulates actin polymerization and forms microfilaments,which is indispensable in the remodeling of actin cytoskeleton.So far,ACTR2 is considered to be closely related to many malignant tumors,but its specific impact on the invasion and metastasis of PTC is not clear.This study focused on the role of ACTR2 in the invasion and metastasis of PTC.The specific effects and related molecular regulation mechanisms were studied and discussed in detail.Methods:1.The cancerous tissues and adjacent normal thyroid tissues of 118 patients with PTC diagnosed in Sheng Jing Hospital of China medical university were collected.The different expression of mRNA and protein of ACTR2 in PTC cancer tissues and matched normal tissues were detected by real-time quantitative PCR（RT-qPCR）,western blot and immunohistochemistry（IHC）,the relationship between the abnormal expression of ACTR2 and the PTC patients’clinicopathological characteristics was analyzed.2.The expression of ACTR2 in three PTC cell lines（K1,TPC-1,BCPAP）and thyroid follicular epithelial cells（Nthy-ori3-1）was compared.PTC cell lines with ACTR2 stable knockdown(KD_ACTR2)and stable overexpression(OE_ACTR2)were constructed in PTC cells by lentivirus knockdown（KD）and plasmid overexpression（OE）ACTR2 genes,respectively.The effect of ACTR2 gene on the proliferation of PTC cells was detected by MTS experiment;The effect of ACTR2 on the migration ability of PTC cells was detected by scratch test;The effect of ACTR2 gene on the invasion ability of PTC cells was detected by Transwell test;The effect of ACTR2 gene on PTC cell cycle was detected by propidium iodide（PI）staining;Annexin v-APC/7-AAD assay was used to detect the effect of ACTR2 gene on PTC cell apoptosis.3.Collect and integrate PTC related data series in GEO（Gene Expression Omnibus）database,analyze and predict the gene Oncology（GO）and Kyoto Encyclopedia of Genes and Genomes pathway（KEGG）signaling pathway of ACTR2 in PTC through GSEA（gene set enrichment analysis）combined.The effects of ACTR2 gene on downstream signal pathway and EMT related protein expression were further detected by western blot,MTS,scratch test and Transwell test.Results:1.The results of RT-qPCR,western blot and IHC showed that the expression of ACTR2mRNA and protein in PTC was significantly higher than that in adjacent normal tissues（P<0.05）.The high expression of ACTR2 in PTC was closely related to tumor size,ETE and lymph node metastasis（P<0.05）.2.The expression of ACTR2 mRNA and protein in three PTC cell lines（K1,TPC-1,BCPAP）was significantly higher than that in Nthy-ori3-1（N3）cells（P<0.05）.MTS results showed that the optical density（OD）of the three PTC cells in KD_ACTR2group decreased significantly on the fifth day,while the OD value of OE_ACTR2group increased significantly on the fifth day（P<0.05）;The results of scratch test showed that the proportion of migration area in scratch area in KD_ACTR2group decreased significantly,while that in OE_ACTR2group increased significantly（P<0.05）.The results of invasion test showed that the number of cells in KD_ACTR2group decreased significantly,while the number of cells in OE_ACTR2group increased significantly（P<0.05）.There was no significant difference among the negative control groups（P>0.05）.Cell cycle test showed that there was no difference in the proportion of different cell cycles among the three PTC cell lines,and the results of apoptosis test showed that there was no significant difference in the rate of apoptosis among the three groups（P>0.05）.3.The GO analysis of GSEA showed that ACTR2 could promote the occurrence of biological processes such as acid secretion,amino acid transport,action potential formation and adenylate cyclase activation.KEGG pathway showed that ACTR2 could promote calcium signaling,cell adhesion molecules,chemokines,cytokine-cytokine receptor interaction,hematopoietic cell lineage pathways.Western blot showed that the expression of focal adhesion kinases（FAK）,Src tyrosine kinase（Src）and extracellular signal regulated kinases 1/2（ERK1/2）had no effect on the expression of ACTR2 in the three PTC cells（P>0.05）.The relative protein expression of p-FAK,p-Src,p-ERK1/2increased significantly.After knockdown of ACTR2,the expression of these proteins decreased significantly.E-cadherin（E-cad）decreased significantly,while N-cadherin（N-cad）,vimentin（VIM）and matrix metalloproteinase 9（MMP9）increased significantly in the overexpression group of ACTR2.In the knockdown ACTR2 group,E-cad increased significantly,while N-cad,VIM and MMP9 decreased significantly（P<0.05）.When FAK phosphorylation inhibitor Y15(OE_ACTR2+Y15)was added to OE_ACTR2group,western blot results showed that there was no difference in the expression of ACTR2,FAK,Src and ERK1/2 in OE_ACTR2+Y15 group compared with OE_ACTR2group,but p-Fak,p-Src,p-ERK1/2 decreased significantly（P<0.05）.Compared with OE_ACTR2group,E-cad increased significantly,while N-cad,VIM and MMP9 decreased significantly in OE_ACTR2+Y15 group.MTS results showed that the OD value of OE_ACTR2+Y15 group was significantly lower than OE_ACTR2group on the fifth day;The results of scratch test showed that the proportion of migration area in scratch area in OE_ACTR2+Y15 group was significantly less than that in OE_ACTR2group;The results of invasion test showed that the number of cells in OE_ACTR2+Y15 group was significantly less than that in OE_ACTR2group,and there was no significant difference among the other negative control groups（P>0.05）Conclusions:1.ACTR2 mRNA and protein are highly expressed in PTC carcinoma,and are closely related to pathological characteristics as tumor size,ETE and lymph node metastasis.2.There is a high expression of ACTR2 mRNA and protein in PTC cells,and ACTR2gene is involved in regulating the proliferation,metastasis and invasion of PTC cells,but has no influence on cell cycle and apoptosis.3.ACTR2 can induce EMT through FAK/Src-ERK1/2 signaling pathway,and then promote the invasion and metastasis of PTC cells.