Study Of SOX5 Promotes Epithelial-mesenchymal Transition In Osteosarcoma Via Regulation Of Snail

Background:Osteosarcoma(OS)is the most common primary malignancy of bone and patients with metastatic disease or recurrences continue to have very poor outcomes.Although multiple associations have been made between the development of osteosarcoma and race,gender,age,various genomic alterations,and exposure situations among others,the etiology remains unclear and controversial.Thus,we can not completely understand and treat osteosarcoma from the perspective of etiology.Studies have shown that tumor metastasis is often the leading cause of death in cancer patients,and in osteosarcoma,the death caused by cancer metastasis is more than 90%,but the mechanism of metastasis is still poorly understood.Therefore,studying and understanding the mechanism of osteosarcoma metastasis,predicting and earlydetecting osteosarcoma metastasis,interventing and treating osteosarcoma patients with metastatic tendency,which is very important for improving the quality of life of patients with osteosarcoma and reducing mortality.EMT is a developmental process,in which epithelial cells lose polarity and develop a cell-cell adhesion.EMT is involved in the initiation of metastasis.Cancer cells with EMT may develop migratory and invasive properties,and ultimately develop cancer stem cell(CSC)properties.Sex determining region Y-box protein 5(SOX5),a transcription factor,serves important role in the regulation of embryonic development and in the determination of the cell fate.SOX5 was determined to be involved in human glioma,seminoma,and nasopharyngeal carcinomas.In recent years,more and more studies have shown that the expression of SOX5 is associated with tumor metastasis.However,to the best of our knowledge,the role of SOX5 in osteosarcoma metastasis and its molecular mechanism has not been reported.In addition,our pre-trial results show that SOX5 expression is significantly increased in osteosarcoma cell lines with high metastatic potential compared to normal cell lines,suggesting that SOX5 may play an important role in the metastasis of osteosarcoma,and may become a potential target for osteosarcoma metastasis and treatment.Objective:To analyze the relationship between SOX5 expression level and OS clinical factors and metastasis,and evaluate the predictive value of SOX5 expression level on OS metastasis by dectecting the expression of SOX5 in OS solid tumor tissue and OS cell line.In addition,the regulation of SOX5 on EMT-related gene expression and its corresponding mechanism in OS was studied by cell biology experiments in vitro.Methods:1.qRT-PCR was used to detect the expression of SOX5 mRNA in the specimens of OS and adjacent non-tumorous tissues,and analyzing the expression of SOX5 mRNA and the clinicopathological factors and metastasis of OS.2.qRT-PCR and western blotting was used respectively to detect to expression of SOX5 mRNA and protein in OS cell lines with metastatic potential and normal cells.And then the relationship between SOX5 expression and metastatic potential of OS cells was analyzed.3.Transwell invasion and migration experiments was conducted to detect the effects of SOX5 on biological behavior of OS cells in vitro after pcDNA3.1-SOX5 eukaryotic plasmids,which increases the expression of SOX5 in cells,and shRNA,which decreases the expression of SOX5 in cells,were transfected.Then the results were used to analyze the role of SOX5 in OS metastasis.4.Western blotting was used to detect the expression change of EMT-related gene in OS cells with high expression and low expression of SOX5,such as epithelial markers E-cadherin,mesenchymal marker N-cadherin and Vimentin,and the expression of EMT-associated transcription factor Snail and the expression of related signaling pathways such as p-Rafl,Rafl,p-ERK1/2 and ERK1/2.Results:1.The expression level of SOX5 mRNA in OS tissues was significantly higher than that in adjacent normal tissues(P<0.01);the expression of SOX5 mRNA was related to pulmonary metastasis.The expression of SOX5 mRNA in patients with pulmonary metastasis was significantly higher than that in patients without pulmonary metastasis(P<0.01);the expression of SOX5 mRNA in the low age group(<20 years old)was higher than that in the high age group(≥20years old),the difference was statistically significant(P = 0.024);there was no significant correlation between SOX5 mRNA expression level and sex;compared with the control group hFOB1.19 cell line,SOX5 mRNA and protein levels were significantly increased in four OScell lines with metastatic potential.2.The ability of migration and invasion of OScells in vitro was significantly enhanced after SOX5 expression increased;and the ability of migration and invasion of OS cells in vitro was significantly reduced after SOX5 expression decreased.3.After SOX5 expression increased,the expression of E-cadherin in OS cells was down-regulated,while the expression of N-cadherin and Vimetin were up-regulated,and the expression of Snail and Rafl was up-regulated;whereas after SOX5 expression reduced,the expression of E-cadherin in OS cells was up-regulated,while the expression of N-cadherin and Vimetin were down-regulated,and the expression of Snail and Rafl was down-regulated.Conclusion:SOX5 is a molecular marker,which suggests OS with high SOX5 expressionis more prone to metastasis.Furthermore,SOX5 regulates the invasion and metastasis of osteosarcoma by activating the Raf-ERK signaling pathway to regulate the expression of Snail and then induce the EMT process.