LINC00265 targets miR-382-5p to regulate SAT1, VAV3 and angiogenesis in osteosarcoma.

www.aging-us.com AGING 2020, Vol. 12, No. 20 Research Paper LINC00265 targets miR-382-5p to regulate SAT1, VAV3 and angiogenesis in osteosarcoma Ying Xiao1, Chunling Li1, Hongyue Wang2, Yijun Liu3 1 Department of Operating Center, The First Hospital of Jilin University, Changchun 130000, Jilin, China Department of Nephrology, The First Hospital of Jilin University, Changchun 130000, Jilin, China 3 Department of Orthopaedics, The First Hospital of Jilin University, Changchun 130000, Jilin, China 2 Correspondence to: Yijun Liu; email: Keywords: angiogenesis, vasculogenic mimicry, LINC00265, miR-382-5p, spermidine/spermine N1-acetyltransferase-1 Received: April 27, 2020 Accepted: July 6, 2020 Published: August 14, 2020 Copyright: Xiao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT We explored the mechanism by which LINC00265 regulates angiogenesis of osteosarcoma cells via the miR-3825p/spermidine/spermine N1-acetyltransferase-1 (SAT1) and miR-382-5p/vav guanine nucleotide exchange factor 3 (VAV3) axis. Cell scratch assay, Transwell assay and tube formation assay were applied to detect cell migration, invasion and tube formation abilities. The effects of LINC00265 targeting miR-382-5p in osteosarcoma in vivo were studied using a tumour-burden assay. A total of 70 genes potentially involved in osteosarcoma angiogenesis were identified, and a Gene Ontology (GO) analysis found that SAT1 and VAV3 were closely related to angiogenesis. Bioinformatics analysis and clinical experiments confirmed that LINC00265, SAT1 and VAV3 were overexpressed in osteosarcoma and related to a poor prognosis, whereas miR382-5p was downregulated and associated with a poor prognosis. It was confirmed that LINC00265 promoted the proliferation, migration, invasion and angiogenesis of osteosarcoma cells by targeting miR-382-5p to mediate SAT1 and VAV3. Collectively, LINC00265 might promote proliferation, migration, invasion and angiogenesis by targeting miR-382-5p/SAT1 and miR-382-5p/VAV3 in osteosarcoma. INTRODUCTION Osteosarcoma is a malignant tumour originating from mesenchymal cells and has an incidence of approximately 3 % [1]. Osteosarcoma is more likely to occur at the ends of long bones, and the incidence of osteosarcoma in males is higher than that in females [2, 3]. Osteosarcoma progresses rapidly and has high morbidity and mortality [4, 5]. To date, the main treatments for osteosarcoma include surgery, chemotherapy, immunotherapy and gene therapy [6–8]. However, the high metastatic ability and drug resistance of osteosarcoma are the main causes of poor prognosis [9, 10]. MicroRNA (miRNA) can cause message RNA (mRNA) degradation or translational inhibition by targeting the 3' untranslated region (UTR) of mRNA, [11, 12]. Long noncoding RNA (lncRNA) is a type of RNA that is more www.aging-us.com 20212 than 200 nucleotides in length but does not have protein translation capabilities [13–15]. LncRNA has the role of regulating epigenetics by activating or interfering with transcription [16, 17]. LncRNA forms complementary double-strands with the transcripts of exon genes, interfering with mRNA splicing, and then produces different forms of splicing to affect gene expression [18]. LncRNA can also target microRNA (miRNA) and act as a sponge, thereby regulating the process of miRNA targeting mRNA [19–21]. LncRNA can target miRNA by competing endogenous RNA (ceRNA) [22]. MiRNA can cause messenger RNA (mRNA) degradation or translational inhibition by targeting the 3' untranslated region (UTR) of mRNA [11, 12]. The role of lncRNAmiRNA-mRNA in the regulation of cancers is gradually being discovered [23–25]. However, the number of RNAs is large, and bioinformatics analysis helps to screen for meaningful genes [26, 27]. LINC00265 is a AGING newly discovered tumour-associated lncRNA that has a significant promoting effect on colorectal cancer [28], acute myeloid leukaemia [29] and lung adenocarcinoma [30]. However, the role and mechanism of LINC00265 in osteosarcoma is still unclear. The lncRNA-miRNAmRNA network is relatively complex, and the use of bioinformatics can help identify key lncRNA, miRNA, and target genes. The genomic spatial event (GSE) database contains high-throughput gene expression data submitted by research institutions from various countries to the National Center for Biotechnology Information (NCBI), and this database can help to identify key lncRNAs. In this paper, using a bioinformatics analysis, we found that LINC00265, spermidine/spermine N1-acetyltransferase-1 (SAT1) and vav guanine nucleotide exchange factor 3 (VAV3) were overexpressed in osteosarcoma, showed a positive correlation with osteosarcoma, and were associated with a poor prognosis in osteosarcoma patients. Based on this, we revealed a mechanism by which LINC00265 promoted migration, invasion and angiogenesis of osteosarcoma cells via miR-382-5p/SAT1 or/VAV3. These findings may provide new avenues for the discovery of strategies for osteosarcoma treatment. RESULTS Analysis of GEO data GSE119975 contained 3 osteosarcoma samples with vasculogenic mimicry and 3 osteosarcoma samples without vasculogenic mimicry. The samples passed the correction (Figure 1A). There were 529 differentially expressed lncRNAs, of which 274 lncRNAs were downregulated and 255 were upregulated (Figure 1B). There were 91 differentially expressed miRNAs, of which 4 miRNAs were downregulated and 87 were upregulated (Figure 1C). There were 2,214 differentially expressed genes, of which 1378 were downregulated and 836 were upregulated (Figure 1D). Among them, Figure 1. Bioinformatics analysis. (A) Correction of the GSE119975 gene chip. (B–D) Differential expression analysis of the lncRNA, miRNA and mRNA of GSE119975. (E) Correction of the GSE12865 gene chip. (F, G) Differential expression analysis of the miRNA and gene of GSE12865. (H, I) Gene Ontology (GO) analysis of 70 genes overexpressed by GSE119975 and GSE12865. www.aging-us.com 20213 AGING LINC00265 and LINC00342 were significantly overexpressed in the vasculogenic mimicry osteosarcoma cell line. To date, there is little data on LINC00265 and LINC00342, which aroused our interest. GSE12865 contained 2 normal tissue samples and 12 osteosarcoma tissue samples and the samples were corrected (Figure 1E). There were 27 differentially expressed miRNAs, of which 17 were downregulated and 10 were upregulated (Figure 1F). There were 4410 differentially expressed genes, of which 3,088 were downregulated and 1,322 were upregulated (Figure 1G). The upregulated genes in GSE119975 and GSE12865 were analysed to obtain the intersection of 70 genes (Figure 3A). These 70 genes were analysed using Gene Ontology (GO), which showed that they were enriched in 16 pathways. The 70 gene (...truncated)