Long Non-Coding RNA Involvement In Regulating Proliferation,Migration And Drug Resistance Of Osteosarcoma And Gastric Cancer

Background: Malignant tumors are the major chronic diseases that seriously endanger the life and health of Chinese.According to different tissue sources,malignant tumors from epithelial tissues are collectively referred to as carcinomas,while those from mesenchymal tissues are as sarcomas.Among them,osteosarcoma is the most common mesenchymal malignancy,while gastric cancer is the common adenocarcinoma.Whether osteosarcoma or gastric cancer,its pathogenesis and mechanism are still urgent problems to be solved.New therapeutic targets and biomarkers need to be found,which could provide new ideas and theoretical basis for the clinical diagnosis and treatment of malignant tumors.LncRNAs,long non-codingRNAs,involved in regulating a variety of intracellular mechanisms,including transcription,translation,protein modification and some signaling pathways,and lncRNAs often act as sponges to regulate the level of miRNAs and downstream genes.Recently,LBX2-AS1,a cancer-promoting lncRNA,was discovered in esophageal squamous carcinoma.As ceRNA,it could be as ceRNA to regulate the expression of target genes and promote the development and progression in many cancers,such as gastric cancer,liver cancer,ovarian cancer,glioma ect.However,it has not been reported in osteosarcoma.LncRNA TUG1 is highly expressed in a variety of tumors including lung cancer,ovarian cancer and pancreatic cancer,and is also involved in the regulation of cell proliferation,apoptosis and EMT signaling pathway.However,the role of TUG1 in gastric cancer remains unclear.In this study,two typical pathological types of malignant tumors,osteosarcoma and gastric cancer from different tissue sources,were selected as the research objects to explore the involvement of lncRNAs in tumorgenisis and progression of both sarcoma and carcinoma.Part one: LBX2-AS1 promotes proliferation and migration of osteosarcoma cells through LBX2-AS1/miR-5010-5p/WNT7 B axis Objective:(1)To illustrate the expression level of LBX2-AS1 in osteosarcoma cells and explore whether it is involved in the regulation of proliferation and migration in osteosarcoma.(2)To clarify whether LBX2-AS1 regulates Wnt7 b expression by competitively absorbing miR-5010-5p as ceRNA.Methods:First we cultured the human osteoblast h FOB1.19 and osteosarcoma cells Saos and 2143-b in DMEM,then throughRT-qPCR was used to detect the expression LBX2-AS1 in osteoblast and osteosarcoma cells,and then si-LBX2-AS1 was transfected in the Saos-2 and 143 b cell to construct low expression cell lines,theRT-si-LBX2 qPCR detection-AS1 transfection efficiency in cells,CCK8 experiment was used to test what is the influence of LBX2 AS1 low expression in proliferation,and the effect of LBX2-AS1 silencing in osteosarcoma cell migration was detected by wound-healing assay and transwell assay.The directly combined seed sequences was used to predict the miRNA and downstream target genes regulated by LBX2-AS1.The relationship between LBX2-AS1 and miR-5010-5p and Wnt7 b was verified by luciferase reporter assay,Weston Bolt andRT-qPCR.The effect of simultaneous silencing of LBX2-AS1 and miR-5010-5p on proliferation and migration of osteosarcoma cells was analyzed by recovery experiment.Results:(1)Fluorescent quantitative PCR results showed that compared with osteoblasts,the expression level in LBX2-AS1 osteosarcoma cells was significantly increased.(2)RT-PCR results showed that the expression level of LBX2-AS1 in the control group was three times that in the si-LBX2-AS1 osteosarcoma cells transfected with si-LBX2-AS1(P <0.05),and the cell line with low expression of LBX2-AS1 was successfully constructed.(3)The results of CCK-8,wound-healing assay and transwell assay confirmed that LBX2-AS1 promoted the proliferation and migration in osteosarcoma cells.(4)The prediction results showed that might be combined and regulated by LBX2-AS1,and Wnt7 b was downstream target gene of miR-5010-5p.The luciferase report assay results showed that the co-transfection of miR-5010-5p with LBX2-AS1(WT)and Wnt7b(WT)could affect the luciferase intensity,which proved that LBX2-AS1 could bind with miR-5010-5p,while Wnt7 b was the target of miR-5010-5p.The results of Weston Bolt andRT-qPCR confirmed that LBX2-AS1,as a role of ceRNA,adsorbed miR-5010-5p to upregulate Wnt7 b.(5)CCK-8,wound-healing,and migration assay results showed that silencing miR-5010-5p could restore the effects of LBX2-AS1 down-regulation on proliferation and migration of osteosarcoma cells.Conclusions:(1)LBX2-AS1 is highly expressed in osteosarcoma and promotes the growth and migration in osteosarcoma.(2)LBX2-AS1 is involved in the progression of osteosarcoma by regulating the miR-5010-5p/ Wnt7 b axis.Part Two: Bioinformatics analysis and preliminary verification of of TUG1 on multidrug resistance in gastric cancerObjective:(1)Analysis the expression level,correlation of clinical features and diagnostic value of TUG1 in gastric cancer.(2)Bioinformatics analysis of the potential role of TUG1 in gastric cancer.(3)The effect of TUG1 on multidrug resistance of gastric cancer was preliminarily verified.Methods:(1)Clinical data andRNASEQ data related to gastric cancer were downloaded from the TCGA database to analyze the expression of TUG1 in paired or unpaired normal tissues and cancer tissues.(2)The correlation between TUG1 expression and clinical features was analyzed based on the clinical data of gastric cancer in the TCGA database.(3)Kaplan-Meier analysis was performed to compare the OS,FP,and PPS rate between the high and lowRRM2 gene expression groups using KMplotter database.(4)The clinical data related to gastric cancer in TCGA database andRNASEQ data were used to analyze the diagnostic value of TUG1 in gastric adenocarcinoma.(5)The co-expressed genes of TUG1 were analyzed in gastric cancer in the TCGA database,followed by GO and KEGG enrichment analysis.(6)TheRNAseq data in gastric cancer in the TCGA database are grouped according to the high and low expression of TUG1,and the biological processes and signaling pathways affected by TUG1 were predicted through GSEA enrichment analysis.(7)The expression of TUG1 in gastric cancer tissue was verified by in situ hybridization assay.(8)RT-qPCR was used to verify the TUG1 expression in drug-resistant cells,and TUG1 overexpression and low-expression cell lines were constructed.(9)CCK-8,wound-healing and migration assays verified whether TUG1 was involved in drug resistance regulation by affecting the biological functions of gastric cancer cells.Results:(1)The expression level of TUG1 gene in gastric adenocarcinoma tissues was significantly higher than normal tissues of paired or unpaired samples.(2)The high expression of TUG1 was significantly correlated with age(P = 0.044),antireflux therapy(P = 0.001),and disease-specific survival(P = 0.038).(3)High TUG1 expression was correlated with adverse overall survival,progression survival and first progression-factor factor(FP).(4)GO and KEGG enrichment analysis indicated that TUG1 was involved in regulating cell cycle and DNA replication.(5)GSEA analysis showed that TUG1 was involved in the positive regulation of proliferation,substrate dependent cell migration and the regulation of the execution phase of apoptosis.(6)RT-qPCR results showed that the expression of TUG1 in gastric cancer resistant strains was higher than that in gastric cancer cells(7)CCK-8,wound-healing and migration experiments showed that TUG1 could affect the cell drug resistance by participating in the regulation of proliferation and migration of gastric cancer cells.Conclusions:(1)Bioinformatics analysis showed that TUG1 can be used as a biomarker for diagnosis in gastric cancer,and participate in the regulation of proliferation,migration and DNA replication related pathways(2)Experimental assays results showed that TUG1 promoted proliferation and migration,which induced drug resistance of gastric cancer.