LRG-1 Enhances The Metastasis Of Thyroid Cancer Cells Through Epithelial-mesenchymal Transition Via Activating MAPK/p38 Pathway

ObjectiveThyroid cancer represents the most common endocrine malignancy and its incidence is rapidly increasing worldwide year by year.Most types of thyroid cancer usually have good prognoses owing to their well-differentiation and low malignant biological behavior as well as the ability to absorb radioiodine.However,a small number of thyroid cancer patients develop radioactive iodine-resistant tumors and demonstrate aggressive behavior and/or distant metastasis,significantly reducing the survival rate.Over the last several decades,numerous genetic alterations involved in thyroid cancer development have been identified,including point mutations of BRAF and the RAS gene,as well as fusions involving the RET and NTRK1 tyrosine kinases.However,a greater understanding of the mechanisms regulating tumor malignant biological behavior in aggressive thyroid cancer is still needed.Research has demonstrated that the invasion and metastasis of malignant tumors,including thyroid cancer,are largely due to the epithelial-mesenchymal transition(EMT)process of cancer cells,and this process is considered as a critical early step in cancer progression.In normal thyroid tissue,epithelial cells show apical-basal polarity,they adhere and communicate with each other through specialized inter-cellular junctions,and they are positioned on a basement membrane that helps to define their physiology.During the process of EMT,epithelial cells lose their characteristics of cell polarity and adhesion,acquiring a motile mesenchymal phenotype,which results in enhanced mobility and invasiveness in carcinoma cells.Additionally,cells that have undergone EMT acquire resistance to senescence and apoptosis.In a study of thyroid carcinoma,EMT-related gene expression levels were significantly upregulated in anaplastic thyroid carcinoma tissues compared with those in well-differentiated thyroid cancer tissues,which indicated that EMT plays an important role in the aggressive biological behavior of thyroid cancer cells.Recently,many studies revealed different regulatory mechanisms of EMT in thyroid cancer cells.For instance,SDC4 gene silencing inhibited thyroid cancer cell EMT via the Wnt/β-catenin pathway.Puli et al showed that the transcription factor ETV5 decreased BRAFV600E-induced TWIST1 expression in thyroid cancer cells.In addition,micro RNA-150-5p affected EMT by regulating the BRAFV600 E mutation in thyroid cancer cells.Furthermore,lnc RNA BANCR promoted EMT in thyroid cancer via the Raf/MEK/ERK signaling pathway.Leucine-rich-alpha-2-glycoprotein1(LRG-1)is the founding member of the leucine-rich repeat(LRR)family,which was first isolated from human serum in 1977.Recently,LRG-1 has been revealed as a new regulator of pathogenic angiogenesis and a novel oncogene-associated protein.Reports have shown that LRG-1 is overexpressed in several types of carcinomas,such as bladder,ovarian and biliary tract cancer.Additionally,LRG-1 has an important role in glioma cell invasion and migration;in addition,LRG-1 was found to promote EMT in colorectal cancer via HIF-1α activation.However,the biological function of LRG-1 in thyroid cancer and the potential molecular mechanisms of LRG-1 are still unknown.As a result,we try to explore the role of LRG-1 in thyroid cancer and angiogenesis,at the meanwhile,explain the mechanism.Methods1.Clinical cases study: To investigate the expression level of LRG-1 in thyroid cancer tissues,we analyzed the microarray data of thyroid cancer from Gene Expression Omnibus(GEO)database of the National Center for Biotechnology Information(NCBI);In addition,by using RT-PCR and Western blot,we verified the transcription and expression of LRG-1 in thyroid cancer tissues and matched normal thyroid tissues of thyroid cancer patients who received radical surgery from 2006 to 2007 in the Affiliated Minzu Hospital of Guangxi Medical University.We analyzed the relationship between LRG-1 expression and clinicopathological feathers and survival.2.In vitro study: We established LRG-1 stably silenced cells and investigated the biological function of LRG-1 in thyroid cancer cells by performing proliferation assay,Flow cytometric analysis,wound healing assay and transwell assays.We detected the expression of EMT related factors by western blot assay.3.In vivo study: Utilizing the xenograft mice model to explore the role of LRG-1 in vivo angiogenesis.LRG-1-knochdown cells and control cells were injected subcutaneously in nude mice.We measured the volume of xenograft tumor each 3 days and on the 30 th day sacrificed the mice to get the tumor body.4.Initial study on mechanism of LRG-1 promoting EMT: To confirm that LRG-1-induced EMT via the MAPK/p38 signal pathway,we detected the expression of EMT,MAPK/p38 signal pathway related factors by WB,and identified whether LRG-1 affected thyroid cancer EMT via MAPK/p38 signal pathway with the p38 inhibitor.Results1.The transcriptong of LRG-1 was overexpressed in thyroid cancer,and high expression of LRG-1 was corelated with clinical characteristics and prognosis.By bioinformatic analysis of microarray data from three datasets GDS1732、GDS1665 and GDS5362,we found that the transcription level of LRG-1 was significantly higher in thyroid cancer tissues than that in normal tissues.And we also detected the expression of LRG-1 in 97 thyroid cancer cases by immunohistochemistry(IHC)staining assays,IHC results showed that LRG-1 was overexpressed in thyroid carcinoma tissues(p <0.001),and late tumor stage and high LRG-1 expression were significant independent prognostic factors.Patients with higher LRG-1 levels suffered significantly worse disease-free survival(p <0.01).The logistic regression model analysis found that higher LRG-1 expression was correlated with later tumor stage(HR,19.01,P<0.001)and lymph node metastasis(HR,43.94,P<0.001).2.LRG-1 promotes the malignant behavior of thyroid cancer.By establishing the HTC/C3 and SW579 cells lines with LRG-1 knockdown.In vitro study,we found that knockdown of LRG-1 inhibited cell migration and invasion,but did not affect proliferation and apoptosis in thyroid cancer cells.In vovo study,growth curve of xenograft mice model indicated that knockdown of LRG-1 significantly attenuated thyroid cancer growth in vivo.Nude mice injected with LRG-1-knockdown cells exhibited obviously decreased tumor volume at all examined time-points compared with nude mice injected with control scramble sh RNA-infected cells(p<0.001).3.LRG-1 induced the EMT of thyroid cancer cells.We found the expression of the epithelial biomarker triggered by LRG-1 were upregulated in LRG-1 knockdown thyroid cancer cells,while the mesenchymal biomarkers triggered by LRG-1 were downregulated,which indicating the role of LRG-1 induced the EMT in thryroid cancer.4.LRG-1 promoted the metastasis of thyroid cancer through MAPK/p38 pathwayWe found that LRG-1 markedly increased p38 phosphorylation in a dose-dependent manner.And the upregulation of the epithelial biomarker and the downregulation of the mesenchymal biomarkers triggered by LRG-1 were reversed in the presence of a p38 inhibitor.Additionally,inhibition of the p38 pathway by SB203580 also diminished the expression of LRG-1-induced key transcription factors.Furthermore,when p38 phosphorylation was blocked in the wound healing and Transwell assays,the enhanced migration and invasion abilities of the thyroid cancer cells induced by LRG-1 were almost completely abolished.Conclusion1.LRG-1 was overexpressed in thyroid carcinoma tissues,and high LRG-1 expression predicted poor patient survival and late tumor stage.2.LRG-1 knockdown inhibited cell migration and invasion,but did not affect proliferation and apoptosis in thyroid cancer cells.3.LRG-1 induced epithelial-mesenchymal transition(EMT)in thyroid cancer cells.4.LRG-1 promoted the metastasis of thyroid cancer cells via MAPK/p38 pathway.Overall,LRG-1 may be a candidate oncogene in thyroid cancer.