| Purpose:1.To explore the effect of lncRNA FOXD2-AS1 on the biological function of osteosarcoma.2.To study the molecular mechanism of lncRNA FOXD2-AS1 regulating the progression of osteosarcoma.3.To investigate lncRNA FOXD2-AS1,EZH2 and HIF-1α relationship with clinical features and overall survival in patients with osteosarcoma.Method:This study is divided into four parts:Part Ⅰ: To study the expression and biological function of lncRNA FOXD2-AS1 in high-grade conventional intramedullary osteosarcoma tissues and cells.The expression of lncRNA FOXD2-AS1 in osteosarcoma tissues and different cell lines was detected by real-time fluorescence quantitative PCR(RT-PCR).The expression of lncRNA FOXD2-AS1 in osteosarcoma cells was specifically knocked down by plasmid or lentivirus transfection.The effects of knockdown of lncRNA FOXD2-AS1 on tumor cell proliferation,apoptosis,tumor invasion and migration were detected by CCK-8,annexin V-FITC / PI double staining and Transwell invasion experiment,The expression of metastasis related proteins was detected by Western blot,and through the construction of nude mouse tumor-bearing model,in vitro experimental verification,to detect tumor growth.Part Ⅱ: To study the molecular mechanism of lncRNA FOXD2-AS1 regulating the progression of osteosarcoma.Through bioinformatics analysis and literature review,we speculate that lncRNA FOXD2-AS1 may interact with EZH2 to catalyze lysine trimethylation at position 27 of histone H3,inhibit the expression of gene p21(CDKN1A),and promote the occurrence and development of osteosarcoma.We prepared the plasmid of lncRNA FOXD2-AS1 and EZH2 or lentivirus interference to transfect osteosarcoma cells.The expression,malignant biological characteristics,growth and metastasis of osteosarcoma cells after knockdown of lncRNA FOXD2-AS1 and EZH2 were detected in vitro and in vivo;The distribution of lncRNA FOXD2-AS1 in cytoplasm / nucleus was determined by bioinformatics analysis and cytoplasmic nuclear subcellular localization experiment.The regulation of EZH2 by lncRNA FOXD2-AS1 was analyzed by RNA immunocoprecipitation experiment;The effect of lncRNA FOXD2-AS1 on EZH2 expression was detected by RT-PCR and Western blot.In addition,we also used RT-PCR,Western blot and chip experiments to clarify that histone modification is involved in the regulation of cyclin dependent kinase inhibitor related gene p21(CDKN1A)expression by lncRNA FOXD2-AS1.Part Ⅲ: To explore the molecular mechanism of the up-regulation of lncRNA FOXD2-AS1 expression in osteosarcoma.We predict that there may be some transcription factor regulating the transcription of lncRNA FOXD2-AS1.Through bioinformatics analysis,we found that HIF-1α may have a binding site to the promoter region of lncRNA FOXD2-AS1;further verify that HIF-1α is an upstream regulator of lncRNA FOXD2-AS1 through dual luciferase report experiments and Ch IP,and The binding site of HIF-1α and lncRNA FOXD2-AS1 was determined;finally,after the HIF-1α overexpression plasmid was introduced into osteosarcoma cells(MG63),the changes in the expression level of lncRNA FOXD2-AS1 were detected by RT-PCR.Part Ⅳ: Analyze the relationship between the expression of lncRNA FOXD2-AS1,EZH2 and HIF-1α and clinicopathological characteristics.We used RT-PCR experiments to detect the expression of lncRNA FOXD2-AS1 in osteosarcoma tissues and adjacent tissues,and verified the relationship between lncRNA FOXD2-AS1 and clinicopathological characteristics in groups,and then studied the use of lncRNA FOXD2-AS1 through bioinformatics.The impact of AS1,EZH2 and HIF-1α on the overall survival rate,and Kaplan-Meier should be used to analyze the impact of the expression of lncRNA FOXD2-AS1 on the overall survival of patients with osteosarcoma.Result:1.The expression of lncRNA FOXD2-AS1 in osteosarcoma tissues was higher than that in normal adjacent tissues,and the expression in osteosarcoma cell lines was higher than that in human osteoblast cell lines,especially in MG63 and Saos-2 cell lines.In vitro cell experiments showed that knockdown of lncRNA FOXD2-AS1 could inhibit the malignant biological behavior of osteosarcoma cells,including reducing proliferation,promoting apoptosis and inhibiting invasion and migration.In vivo animal experiments showed that knockdown of lncRNA FOXD2-AS1 could inhibit the increase of tumor volume and weight,that is,inhibit tumor growth.2.Lnc RNA FOXD2-AS1 is mainly distributed in the nucleus of osteosarcoma cells.By interacting with EZH2,it specifically binds to H3K27me3 in the promoter region of the target gene and silences the expression of p21(CDKN1A),thereby promoting the progression of osteosarcoma.3.The process of Lnc RNA FOXD2-AS1 promoting the progression of osteosarcoma can be regulated by the upstream transcription factor HIF-1α.The binding site of transcription factor HIF-1α is located in the precursor promoter region of lncRNA FOXD2-AS1;exogenous expression of HIF-1α can promote the expression of lncRNA FOXD2-AS1,and there is a positive correlation between them.4.The expression of lncRNA FOXD2-AS1 in osteosarcoma tissue is higher than that in normal adjacent tissues.Its expression has nothing to do with the patient’s age and gender,but is related to tumor size and clinical stage;its high expression is more common in large tumors,Patients with high stage,suggesting that there is a significant positive correlation with poor clinicopathological characteristics and poor prognosis.The overall survival rate of osteosarcoma patients with high expression of lncRNA FOXD2-AS1 is reduced.Conclusion:lncRNA FOXD2-AS1 is highly expressed in osteosarcoma tissues and cells,and promotes the progression of osteosarcoma.The mechanism is that lncRNA FOXD2-AS1 interacts with EZH2 to enhance PRC2 activity,catalyze H3K27me3 in the promoter region of target gene p21(CDKN1A),and silence p21 expression.This regulatory process can be activated by the upstream transcription factor HIF-1α.In addition,in clinical practice,the high expression of lncRNA FOXD2-AS1 is common in patients with large tumors and high stages,and it is associated with poor prognosis and reduced overall survival.This study clarified the function of lncRNA FOXD2-AS1 and specifically inhibited HIF-1α/ lncRNA FOXD2-AS1 / EZH2 signaling pathway may become a potential means to inhibit the progression of osteosarcoma.At the same time,it also provides a scientific basis for screening biomarkers and prognosis evaluation in clinic. |
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