Exploring The Function Of LINC01549 In Human Osteosarcoma Based On Single-cell RNA Sequencing Technology

Objective Osteosarcoma occurs most frequently in adolescents and is associated with a high degree of malignancy and poor prognosis.The current treatments for osteosarcoma are surgery combined with neoadjuvant chemotherapy.For patients with metastases and recurrences,there has been no change in overall survival for osteosarcoma over the past 30 years.This situation is mainly due to the strong heterogeneity of osteosarcoma and the complex tumor microenvironment.Osteosarcoma drug resistance,recurrence and metastasis often lead to poor prognosis.Therefore,a deeper understanding of its complex tumor microenvironment is required.The purpose of this study was to use single cell RNA sequencing(scRNA-seq)to parse the osteosarcoma profile of non-chemotherapy clinical samples,to identify the heterogeneity of various cells in the osteosarcoma microenvironment,and to identify the heterogeneity of various cells in the osteosarcoma microenvironment through novel groupings.New malignant cell populations were identified and LncRNA expression profiles were identified.Combined with traditional Bulk RNA osteosarcoma sequencing data with significance for survival and metastasis,potential targeting LncRNAs were screened to provide theoretical support for clinical prognostic indicators and drug development..Methods This study included single-cell data from 6 non-chemotherapy osteosarcoma samples,traditional Bulk RNA sequencing data from 3 pairs of osteosarcoma samples,85 osteosarcoma data from the TARGET database,and GSE21257 and GSE01549 osteosarcoma data from the GEO database.In this study,tissues were obtained from 6 non-chemotherapy osteosarcoma patient samples,followed by single-cell RNA sequencing using the 10 × Genomics platform.After the data were obtained,the main cell types were identified through quality control and dimensionality reduction clustering.The map of the main cell types were analyzed from the aspects of gene expression,cell function analysis,quasi-temporal analysis and RNA rate analysis,gene set variation analysis(GSVA),metabolic pathway analysis,drug sensitivity analysis,and cell communication analysis.Combined with survival and metastasis data in the TARGET database,a new algorithm in the Scissor package was used combined with traditional Bulk RNA sequencing data to group major cell types and screen out meaningful LncRNAs and m RNAs.Combined with Bulk RNA sequencing results,the screened high-expressing LncRNAs were verified.Combined with the database predicted mi RNAs,a network of competing endogenous RNAs was constructed.Prediction of new drugs for chemotherapy-insensitive group of osteosarcoma by calculation of drug sensitivity analysis.Finally,the biological function of the screened osteosarcoma cell LncRNAs was verified by in vitro and in vivo experiments.Results A total of 31,514 cells were obtained after quality control of the single-cell data from 6 non-chemotherapy osteosarcoma samples.A total of 6main cell types were identified by dimensionality reduction clustering: myeloid cells,osteosarcoma cells,NK/T cells,and osteoclasts.cells,endothelial cells,B cells.Copy number variation analysis showed that osteosarcoma cells had obvious copy number variation on the first and eighth chromosomes.Quasi-temporal analysis analysis suggested that the grouping method of malignant tumor groups by the Scissor package algorithm was ideal.LncRNAs related to survival were finally screened: CRNDE,LINC00667,LINC01549,DNM3 OS,MIR181A1HG,LINC00662,RASSF8-AS1,TBX2-AS1,PITPNA-AS1,FRMD6-AS1,SNHG9.The differentially expressed genes related to survival include: CCT6 A,HSPD1,MRPL.GSVA analysis found that related to survival and metastasis are: RAMASWAMY＿METASTASIS＿UP,BIDUS＿METASTASIS＿UP,WANG＿METASTASIS＿OF＿BREAST＿CANCER＿ESR1＿UP,WINNEPENNINCKX＿MELANOMA＿METASTASIS＿UP,VICENT＿METASTASIS＿UP.Metabolism-related pathways include:Mannose type O-glycan biosynthesis,Ubiquinone and other terpenoid-quinone biosynthesis,and Lysine degradation.degradation.Drug sensitivity analysis showed that the first sensitive drug related to survival and metastasis was SULPRIDE.Combined with GSE21257 data,a survival analysis model was constructed,and LINC01549 was highly expressed in the low survival group.The co-expressed genes of MRPL4,CCT6 A and HSPD1 were analyzed,and LINC01549 constructed a network of competing endogenous RNAs.The traditional Bulk RNA sequencing results showed that LINC01549 was highly expressed in the osteosarcoma tissue group.In vitro,the expression of LINC01549 in osteosarcoma cell lines was also significantly higher than that in control group.Silenced LINC01549 was significantly lower than the normal group in cell phenotype experiments such as proliferation,cloning formation,migration,invasion.In vivo experiments confirmed that silenced LINC01549 inhibited the growth of subcutaneous tumors in nude mice.Conclusion Single-cell transcriptome sequencing technology revealed that osteosarcoma has a complex microenvironment,mainly composed of myeloid cells,osteosarcoma cells,NK/T cells,osteoclasts,endothelial cells,and B cells.LncRNAs related to survival and metastasis were obtained,and LINC01549 was screened as a potential target LncRNA for osteosarcoma treatment.Three co-expressed genes,MRPL4,CCT6 A,and HSPD1,were screened,and a network diagram was constructed.These provide theoretical support for clinical treatment and prognostic indicators.