The Role Of Long Non-coding RNA HOTAIR On Proliferation And Migration Of Osteosarcoma Cells And Its Related Mechanism

BackgroundOsteosarcoma is a malignant bone tumor derived from mesenchymal tissue.Although the incidence is low,the prognosis of osteosarcoma patients with lung metastases is poor.The standard treatment for osteosarcoma consist of extensive surgical resection,chemotherapy,and radiotherapy.However,despite these aggressive interventions,patients,outcomes have not significantly improved for decades,the five-year overall survival rate for patients with osteosarcoma is only 60-70%.Relapse rates also remain high at approximately 35%.Moreover patients,with metastatic disease have an even worse prognosis,with five-year overall survival rates of approximately 10–30%.Therefore,It is urgent to explore the pathogenesis of osteosarcoma to provide a theoretical basis for designing a new and effective treatment plan for osteosarcoma.HOX transcript antisense RNA(HOTAIR)is localized to the mammalian HOXC gene cluster;it can not only interact with polycomb repressive complex 2 and the lysine-specific histone demethylase/ CoREST/REST complex and manipulate the expression of various genes.HOTAIR promotes invasion and metastasis of tumor by silencing tumor suppressors and activating oncogenes and signaling pathways.HOTAIR overexpression in many human cancers has been reported and plays an important role in the development and progression of various tumors,including osteosarcoma,but its role in osteosarcoma is unclear.ObjectivesTo explore the role of long non-coding RNA HOTAIR on proliferation and migration of osteosarcoma cells and related mechanisms.Methods1.Real-time quantitative PCR was used to detect the expression level of HOTAIR mRNA in two human osteosarcoma cell lines MG63,U2 OS and normal human osteoblast cell line h FOB1.19.2.Construction of sh-HOTAIR lentiviral vector(sh-HOTAIR#1;sh-HOTAIR#2;sh-HOTAIR#3)based on sequence information of HOTAIR gene,and sh-HOTAIR lentivirus was packaged to infect MG63 cell line.The transfection efficiency was detected by flow cytometry and the stable cell line of knockdown HOTAIR was selected by puromycin.Real-time PCR was used to detect HOTAIR knockdown efficiency,and the cells with the highest knockdown efficiency were used for subsequent cell function experiments and whole transcriptome sequencing.The effects of knockdown HOTAIR on proliferation and migration of osteosarcoma cells were detected by CCK8 proliferation assay,scratch assay,transwell assay and cell cycle assay.3.The whole transcriptome sequencing technology screened the differentially expressed genes before and after HOTAIR knockdown and carried out gene ontology(GO)and Kyoto Gene and Genomic Encyclopedia(KEGG)enrichment analysis for differentially expressed genes.Result1.The expression level of HOTAIR mRNA in MG63 and U2 OS was significantly higher than that in human osteoblast cell line h FOB 1.19(P<0.01).2.According to the obtained three sh-HOTAIR,it was found by RT-PCR that sh-HOTAIR#3 was the highest expression efficiency.Knockdown of HOTAIR inhibits MG63 cell proliferation,reduces migration capacity,and induces G1 arrest.3.A total of 119 differentially expressed genes were screened by whole transcriptome sequencing,including 87 differential expression up-regulation and 32 differential expression down-regulation.A total of 54 significantly enriched signaling pathways were obtained from the KEGG pathway enrichment analysis of differentially expressed genes.Among them,chemokines and chemokine receptors were most abundant in the signaling pathway.Conclusion1.HOTAIR is highly expressed in osteosarcoma cells compared to human osteoblasts.2.Knockdown of HOTAIR inhibits os cell proliferation,migration,and induce G1 arrest.3.AMOT and SPHK1 may interact with HOTAIR as a carcinogenic factor to regulate the proliferation and migration of osteosarcoma cells.HOTAIR may participate in the development and progression of osteosarcoma through downstream chemokines and chemokine receptors.