LPS Promoted The Invasion And Metastasis Of Osteosarcoma And The Mechanism Of EMT Through The LncRNA HOTAIR/ERK1/2 Pathway

Introduction: osteosarcoma is a primary malignant bone tumor that often occurs in the metaphysis of teenagers.Due to its high degree of malignancy,simple surgical excision is often insufficient.Although significant progress has been made in the diagnosis and treatment of osteosarcoma in recent decades,the overall survival rate of patients with osteosarcoma has not changed significantly.The metastasis of primary osteosarcoma to adjacent tissues and organs has been the main issue that limits treatment success and survival of osteosarcoma patients.Determining the mechanisms involved in osteosarcoma metastasis has recently become an important topic of research.Tumor invasion and metastasis are complex processes involving cell adhesion,migration,proliferation,extracellular matrix degradation,tumor angiogenesis and invasion and metastasis.Increasing evidence suggests that many components of the tumor microenvironment are involved in tumor invasion and metastasis,including inflammatory factors.Their role in tumor invasion is,therefore,worthy of further study.Lipopolysaccharide(LPS),is a component of the outer membrane of Gram-negative bacteria,as well as an important factor in the tumor microenvironment.It affects tumor invasion by activating Toll-like receptors on the cell surface.In addition,LPS participates in tumor invasion and metastasis by activating the epithelial-mesenchymal transition(EMT)process.EMT is the process by which epithelial cells transform to mesenchymal cells under specific physiological and pathological conditions.It is the molecular basis for the invasion and metastatic abilities of tumor cells.The down-regulation or deletion of E-cadherin in the epithelial phenotype of tumor cells is an important indicator of EMT,as is as the up-regulation of mesenchymal markers such as ?-SMA,N-cadherin,and vimintin.Therefore,EMT can be identified through detection of the specific markers of these two cell types.An increasing amount of evidence suggests that EMT is the basis for tumor invasion and metastasis.Studies have found that many factors are involved in EMT,including non-coding RNAs(ncRNAs).Discovered in recent years,ncRNAs play an important role in regulating EMT and tumor invasion and metastasis.The ncRNAs include long non-coding RNA(lncRNA,>200 nucleotides in length)and microRNA(miRNA,are around nucleotides in length).Increasingly,ncRNAs have been shown to play an important role in physiological and pathological processes.Studies have shown that lncRNAs are not simply a by-product of transcription,but can play an important role in disease progression through regulation of gene expression.In this study,we examine the role of a lncRNA in tumor processes.The lncRNAs are lacking open reading frames(ORFs)that are unlike any class of known non-coding transcripts.HOX transcript antisense RNA(HOTAIR)is transcribed from the antisense strand of the HOXC gene cluster and was the first trans-acting lncRNA to be identified.It plays an essential regulatory role in many tumors.Ding and colleagues demonstrated that HOTAIR promotes the invasion and metastasis of liver cancer cells.Xu et al.found that the deletion of HOTAIR in gastric cancer inhibits tumor invasion.LPS affects the invasion and metastatic ability of osteosarcoma cells;however,the relationship between the effects of LPS and the role of HOTAIR has not yet been examined.Since 2007,HOTAIR has played an important regulatory role in a variety of tumors.Ding et al.proved that HOTAIR can promote the invasion and migration of liver cancer cells.Xu ZY et al.found that the absence of HOTAIR in gastric cancer can inhibit the invasion of gastric cancer.Chang et al.confirmed that high expression of HOTAIR was suggested in clinical samples of primary breast cancer and invasive breast cancer.In vitro experiments,it was found that overexpression of HOTAIR would enhance the invasiveness of tumor cells,and silence of HOTAIR would inhibit the growth of tumor cells.This is the first study to link HOTAIR with malignant tumor phenotypes.However,the specific mechanism by which HOTAIR is involved in tumorigenesis and development is still unclear.Studies on HOTAIR and Osteosarcoma have rarely been reported.As of January 2018,the search on PUBMED for the heading of HOTAIR and Osteosarcoma showed only five research papers and literature reviews,with the exception of one study on LPS promoting invasion and migration of Osteosarcoma through TLR4/HOTAIR,and no other study on the mechanism of LPS on Osteosarcoma.In the study of osteosarcoma,LPS has an effect on the invasion and metastasis of osteosarcoma,and the relationship between this effect and HOTAIR is worth further study.Previous studies have shown that lncRNA is abnormally expressed in tumors and further participates in tumor cell invasion and metastasis through different signaling pathways.Further research on tumor-related signaling pathways will not only help us understand the occurrence and development of tumors,but also interfere with this pathway through targeted therapy,so as to provide more effective treatment for clinical patients.The research methods of MAPK pathway are mainly expression,dominant inactivation and gene silencing.There are four main types of MAPK signaling pathways.Named MAPK for its core,it is involved in the regulation of a variety of physiological and pathological activities,and can integrate and cross pathways.The strength and activation degree of MAPK pathway signals determine the future fate of cells.MAPK signal transduction pathway transforms extracellular signals through cascade phosphorylation response effects.Therefore,strict regulation of these signaling pathways is essential for normal cell function to be deregulated leading to a variety of diseases including tumors.Three major MAPK families have been found to be associated with cancer: ERK(erk1/2),p38 MAPK,and Jun amino-terminal kinase(JNK).In non-transformed cells,erk1/2 mainly involves themitogenesis reaction,while p38 MAPK and JNK mediate cellular stress and inflammatory reaction.Whether LncRNA HOTAIR plays a role through the MAPK signaling pathway deserves further study.This study intends to observe the effect of LPS on the invasion and metastasis of osteosarcoma through the addition of LPS in the cell culture process and the relevant clinical and pathological experiments,and to explore whether LPS can achieve the invasion and metastasis of osteosarcoma through HOTAIR,so as to provide more theoretical basis for the treatment of osteosarcoma.Objective: In this study,osteosarcoma cells MG63 and saos-2 were taken as the research objects,and the mechanism of LPS promoting bone tumor invasion and metastasis was clarified through the induction of LPS,so as to further explore the mechanism of LPS and verify its effect through TLR4/HOTAIR,in order to provide theoretical basis for the treatment of osteosarcoma.Methods: First,LPS was added in the process of osteosarcoma cell culture,and then thegrowth of osteosarcoma cells was detected by MTT to find the optimal time and concentration of LPS on the growth of osteosarcoma cell lines.The expression of epithelial phenotype marker e-cad,interstitial phenotype marker n-cad,VIM and a-sma were detected by Western blot and real-time rt-pcr.TLR4 is the receptor of LPS.In order to further study the mechanism of LPS,TLR4 was inhibited by RNA interference,and the phosphorylation of protein and cell growth were detected by Western blot.Results:1.LPS promotes the invasion and metastasis of osteosarcoma cellsLPS plays an important role in promoting the invasion and metastasis of numerous tumors,however,its effects in osteosarcoma remain unclear.We studied the effects of LPS(10 ?g/mL)on invasion and metastasis of two osteosarcoma cell lines,MG63 and Saos-2,using Transwell assays.The results show that the invasion and metastatic abilities of the osteosarcoma cell lines increase significantly 24 h after LPS treatment,suggesting that LPS promotes invasion and metastasis of osteosarcoma cells.2.LPS promotes EMT in osteosarcoma cellsWe used established EMT markers to determine whether LPS promotes EMT of osteosarcoma cells.Real-time PCR revealed that the gene expression of the epithelial marker E-cadherin is significantly decreased 24 h after LPS treatment(10 ?g/mL)compared with the control group,while the mesenchymal markers,N-cadherin,Vimentin,and ?-SMA,show clear increases in expressio.We next detected the expression of E-cadherin,N-cadherin,vimentin and ?-SMA proteins by western blot.The results show that the expression of E-cadherin in osteosarcoma cells is significantly decreased 48 h after LPS treatment,while the expression of N-cadherin,vimentin,and ?-SMA shows clear increases.The results consistently show decreased epithelial markers and increased mesenchymal markers following LPS treatment,indicating the occurrence of EMT in the osteosarcoma cells.3.LPS induces EMT in osteosarcoma cells along with increased expression of TLR4We next determined whether TLR4,the receptor for LPS,participates in LPS-induced EMT in osteosarcoma cells.We analyzed TLR4 expression by RT-PCR and western blot.The results show that TLR4 mRNA and protein expression are significantlyincreased compared to the control group after LPS treatment,indicating that TLR4 participates in the process of LPS-induced EMT in osteosarcoma cells.4.LPS induces EMT in osteosarcoma cells along with increased expression of HOTAIRPrevious studies have confirmed the important regulatory role of HOTAIR in tumor invasion and metastasis.Li et al demonstrated that HOTAIR promotes the invasion and metastasis of osteosarcoma cells.Wang and colleagues found that overexpression of HOTAIR promotes the invasion and metastasis of osteosarcoma cells.In addition,HOTAIR has essential roles in the EMT process.Therefore,we hypothesized that HOTAIR takes part in the process of LPS-induced EMT in osteosarcoma cells.RT-PCR results indicated that HOTAIR expression increases following LPS treatment of osteosarcoma cells.5.Effects of HOTAIR on EMT of osteosarcoma cellsPrevious studies have confirmed the essential role of HOTAIR in tumor cell EMT,but whether HOTAIR promotes the occurrence of EMT in osteosarcoma is still unclear.We overexpressed HOTAIR in osteosarcoma cells to verify the effects of HOTAIR on EMT in osteosarcoma.Our results show that the invasion and metastatic abilities of osteosarcoma cells significantly increase after overexpressing HOTAIR.In addition,osteosarcoma cells undergo EMT following overexpression of HOTAIR.6.LPS regulates EMT in osteosarcoma through TLR4/HOTAIRIn order to clarify whether LPS promotes EMT in osteosarcoma via TLR4/HOTAIR,we knocked down TLR4 expression in osteosarcoma cells.In the TLR4 knockdown cells,the effects of LPS on tumor invasion and metastasis decreased,as did the effects on HOTAIR.Next,we treated HOTAIR knockdown osteosarcoma cells with LPS to verify the relationship between TLR4 and HOTAIR.The results reveal that LPS-induced tumor invasion and metastasis decrease significantly.These results indicate that LPS promotes EMT,invasion and metastasis of osteosarcoma cells through TLR4/HOTAIR.7.LPS inhibits the EMT of osteosarcoma through the p-erk1/2 signaling pathwayIn order to investigate whether the MAPK pathway is involved in the EMT effect of mg-63 and saos-2 in osteosarcoma cells,the total protein of osteosarcoma cells was collected and extracted,and the phosphorylation of erk1/2,p38 MAPK and JNK in theMAPK pathway was detected and analyzed.Our results confirm that LPS is accompanied by the activation of MAPK erk1/2 in the process of promoting EMT,but LPS does not affect the expression of P38 and JNK.It was also found that the expression level of p-erk1/2 in osteosarcoma tissues was higher than that in benign bone lesions.8.LPS activates the p-erk1/2 signaling pathway through lncRNA HOTAIRIn order to verify whether LncRNA HOTAIR plays a role through the MAPK signaling pathway which is closely related to inflammation and tumorigenesis,LPS was added after the expression of HOTAIR was silenced in osteosarcoma cells,and the protein expression of p-erk 1/2 was also verified.To further prove the role of LPS through the p-erk1/2 pathway,we added PD98059 in the cell culture process and then gave LPSinduction,and detected the changes in HOTAIR expression We found that LPS could still promote the expression of HOTAIR after erk1/2 activity was inhibited.This result suggested that LPS promoted the invasion and metastasis of osteosarcoma cells through HOTAIR/erk1/2.Conclusion: Our study reports the effects of LPS on EMT in osteosarcoma,and establishes that the mechanism is mediated via the TLR4/HOTAIR pathway.Our results reveal the effects of LPS in the tumor microenvironment on the invasion and metastasis of osteosarcoma and propose a mechanism through which these effects are mediated,providing experimental support for the further treatment of osteosarcoma.