Study On The Mechanism Of METTL3-Mediated SNHG1 Methylation Modification Promoting The Proliferation,Invasion And Metastasis Of Osteosarcoma

Introduction Adolescents are most likely to suffer from osteosarcoma(OS),which accounts for2.4% of all malignant tumors in children.Osteosarcoma is characterized by a high degree of malignancy,and can rapidly destroy surrounding tissues for metastasis.Relevant research shows that about 10-20% of patients with osteosarcoma have developed metastatic disease at the time of diagnosis.The prognosis of osteosarcoma patients is often poor due to the early metastasis of the disease.The survival rate of osteosarcoma patients,despite the enhancement of surgical resection in conjunction with systemic chemotherapy or radiotherapy,remains very low.Patients with localized osteosarcoma have a 5-year event-free survival rate of approximately 70%,whereas those with metastatic osteosarcoma have a survival rate of between 15% and30%.Roughly 80% of those who have undergone surgical treatment for osteosarcoma relapse,drastically impacting their outlook.Therefore,it is necessary to further study the molecular carcinogenesis of osteosarcoma and find new targets for the treatment of osteosarcoma.The most frequent internal chemical alteration of m RNA,N6-methyladenosine(m6A),is a post-transcriptional alteration of RNA.M6 A can be divided into three subtypes according to its functions: "writers","readers" and "erasers".The modification of m6 A was promoted by writers and removed by erasers;In addition,it can also recruit specific reader proteins.M6A-related regulatory factors participate in various physiological and pathological processes of the body by regulating RNA stability,m RNA splicing and translation,and micro RNA processing.The catalyzing of mammalian m6 A modification is mainly methyltransferase-like 3(METTL3)and methyltransferase-like 14(METTL14),which is then reversed by ALKBH5 and FTO,a dynamic process.METTL3 is the first m6 A "writers" reported.It is widely involved in all stages of RNA life cycle,including precursor m RNA splicing,3 ’-terminal processing,translation regulation and mi RNA processing.Studies conducted recently have demonstrated that METTL3 is a critical factor in the advancement of colon,bladder,and ovarian cancer.Peng W et al.For example,in bladder cancer,METTL3 is significantly overexpressed and is associated with proliferation,invasion and tumorigenicity in vivo.METTL3 modifies AFF4 and NF through m6A-κ B m RNA promotes tumor progression and activates MYC transcription.In hepatoblastoma,the abnormally high expression of METTL3 leads to a significant increase in the level of m6 A in the tumor.m6 A is enriched not only near the m RNA termination codon,but also in the coding sequence(CDS)region.Due to its m6 A modification,the stability of CTNNB1 is improved,resulting in Wnt/ β-The significant activation of the catenin signal pathway promotes the proliferation of tumor cells.Sometimes METTL3 also acts as a tumor inhibitor.It has been reported that the deletion of METTL3 changes the enrichment of m6 A on ADAM19 and promotes the carcinogenesis of glioblastoma stem cells.It has also been confirmed that METTL3 mediates the m6 A modification of FBXW7 and subsequently inhibits the development of lung adenocarcinoma.Therefore,METTL3 regulates the fate of these RNAs through m6 A modification on key transcripts,which in turn affects the development of many cancers,including hematological malignancies and solid tumors.Small nucleolar RNA host gene 1(SNHG1)is the host of 8 sno RNAs,located in the 11q12.3 region of chromosome,and has 11 exons.The carcinogenic lnc RNA SNHG1 has been extensively studied,and its up-regulation in a variety of cancer types,such as ovarian,neuroblastoma,glioma,gastric,laryngeal squamous cell carcinoma,cervical,renal cell carcinoma,colorectal,hepatocellular,lung,prostate,esophageal,nasopharyngeal,and osteosarcoma,is well-documented.The abnormal expression of SNHG1 is significantly related to important clinical features such as vascular invasion,size and stage of advanced tumor,TNM stage and total survival period.SNHG1’s molecular and cellular mechanisms,which mediate its action,are intricate and intertwined with numerous elements.Moreover,SNHG1 is involved in cell activities such as proliferation,migration,invasion,and apoptosis.At present,there are few studies on the upstream regulation mechanism of SNHG1.This study seeks to investigate METTL3’s regulatory role in the migration of osteosarcoma cells,initially elucidating the mechanism of action,based on the SNHG1-mediated proliferation,invasion and metastasis of osteosarcoma cells,with m6 A alteration influencing the amount of long-chain non-coding RNA as the starting point.The objective of this research is to create a novel objective for the molecular and gene-based treatment of osteosarcoma.Objective This research seeks to explore the influence of METTL3 on the proliferation,invasion,and metastasis of osteosarcoma cells,as well as to determine if it can modify SNHG1 expression and thus influence the proliferation,invasion,and metastasis of osteosarcoma cells through m6 A alteration.Methods(1)A Bioinformatics examination of the varying expression of METTL3 in The Cancer Genome Atlas(TCGA)and GSE12865 and GSE42352,osteosarcoma-related data sets from the GEO database;(2)The tissue samples of 60 patients with osteosarcoma were collected,and the differential expression of METTL3 in osteosarcoma tissues and corresponding adjacent tissues was detected by immunohistochemistry,western blot and real-time fluorescence quantitative reverse transcriptase polymerase chain reaction(RT-q PCR);(3)RT-q PCR and western blot were employed to discern the varying expression of METTL3 in human osteoblast and osteosarcoma cell lines.;(4)Models of osteosarcoma cells with varying METTL3 expression levels were fashioned.To ascertain if the model was constructed successfully,Western blot and q RT-PCR were employed to measure METTL3 expression;additionally,transwell and clonogenic assays were utilized to assess the influence of varying METTL3 expression levels on the proliferation,invasion,and metastasis of HOS and U2 OS osteosarcoma cells;(5)Bioinformatics was employed to investigate the association between METTL3 and SNHG1 expression in sarcoma tissues in the TCGA database;(6)Bioinformatics analysis of the correlation between METTL3 and SNHG1 in the osteosarcoma related data sets GSE87437 and GSE33458 in the GEO database;(7)RT-q PCR and western blotting were employed to discern the dissimilar expression of SNHG1 in human osteoblast and osteosarcoma cell lines;(8)Osteosarcoma cell models with different SNHG1 To ascertain the success of the model,Western blot and RT-q PCR were employed to ascertain SNHG1 expression levels;additionally,transwell and clonogenic assay were utilized to assess the influence of varying SNHG1 expression levels on the proliferation,invasion,and metastasis of HOS and U2 OS osteosarcoma cells;(9)RT-q PCR was employed to ascertain the influence of varying METTL3 concentrations on SNHG1 expression in osteosarcoma cell models,which were constructed with varying METTL3 expression levels;(10)Me RIP-q PCR was employed to detect the influence of varying METTL3 expression levels on SNHG1 m6 A levels in osteosarcoma cell models;(11)Actinomycin test revealed the influence of METTL3 on SNHG1’s steadiness;(12)Experiments involving transwelling and cloning were conducted to explore the influence of SNHG1 at various expression levels on the proliferation,invasion,and metastasis of osteosarcoma cells HOS and U2 OS,which is mediated by METTL3.Results(1)In osteosarcoma related data sets GSE12865 and GSE42352,METTL3 was significantly higher in 103 osteosarcoma samples than in non-osteosarcoma samples(15 cases);(2)In the TCGA database,a significantly higher METTL3 expression level was observed in 206 sarcoma tissue samples than in two normal tissue samples;(3)METTL3 expression in the osteosarcoma tissue samples gathered was markedly greater than that of the neighboring tissues;(4)Compared with osteoblast h FOB 1.19,METTL3 expression was increased in osteosarcoma cell lines;(5)Transfection of METTL3 specific small interfering RNA(sh METTL3-1 and sh METTL3-2)can The expression of METTL3 in HOS and U2 OS cells is significantly diminished,thus hindering their migration capacity;however,transfection of the METTL3 overexpression vector plasmid(oe METTL3)can significantly raise the expression of METTL3 in HOS and U2 OS cells.improve the migration ability of HOS and U2 OS cells;(6)In the TCGA database,the expression of SNHG1 and METTL3 was significantly positively correlated;(7)In osteosarcoma related data sets GSE87437 and GSE33458,SNHG1 is positively correlated with METTL3 in both databases.(8)The expression of SNHG1 in the osteosarcoma tissues and cell lines collected was markedly greater than that in the neighboring tissues;(9)The expression level of SNHG1 in HOS and U2 OS cells can be significantly decreased by transfection of SNHG1-1 and sh SNHG1-2,while the expression level of SNHG1 overexpression vector plasmid(oe SNHG1)can be significantly increased,thus enhancing the migration ability of HOS and U2 OS cells;(10)After down-regulation of METTL3,the expression level of SNHG1 in HOS and U2 OS cells was inhibited;After METTL3 was up-regulated,the expression level of SNHG1 in HOS and U2 OS cells increased.(11)After down-regulation of METTL3,the level of SNHG1 m6 A in HOS and U2 OS cells was inhibited;After METTL3 was up-regulated,the level of SNHG1 m6 A in HOS and U2 OS cells increased.(12)After down-regulation of METTL3,the m6 A modification level of SNHG1 in MG-63 and U2 OS cells was inhibited,and the half-life of SNHG1 was significantly shortened;(13)Down-regulating the expression of SNHG1 in METTL3 overexpressed cells can weaken the promotion of METTL3 on the proliferation,invasion and metastasis of osteosarcoma cells,and enhancing the expression of SNHG1 in METTL3 silenced cells can enhance the promotion of METTL3 on the proliferation,invasion and metastasis of osteosarcoma cells.Conclusion Osteosarcoma tissues and cells displayed a heightened expression of METTL3 and SNHG1.and there was a positive correlation between them;METTL3 and SNHG1 can promote the proliferation,invasion and metastasis of osteosarcoma cells HOS and U2OS;SNHG1 has a theoretical m6 A modification site;METTL3 can affect the m6 A modification level of SNHG1 and affect its stability;METTL3 can promote the proliferation,invasion and metastasis of osteosarcoma cells MG-63 and U2 OS by regulating SNHG1.