Abstract
Osteosarcoma (OS) is a common malignant bone tumor that commonly occurs in children and adolescents. Long noncoding RNAs (lncRNAs) are recognized as a novel class of regulators of gene expression associated with tumorigenesis. However, the effect and mechanism of lncRNAs in OS tumorigenesis and drug resistance have not been characterized. The purpose of the study is to screen potential biomarker and therapeutic target against OS. We compared the lncRNA expression profiles between OS cell lines with different drug resistance levels using RNA-seq analysis and found that lncRNA DICER1-AS1 was significantly differentially expressed in multi-drugresistant OS cells SJSA-1 versus multi-drugsensitive OS cells G-292. Bisulfite Sequencing PCR (BSP) assay was performed to analyze the differential methylation status of the promoter region of DICER1-AS1 in four OS cells. Subsequently, in vitro gain- and loss-of-function experiments demonstrated the roles of DICER1-AS1 and miR-34a-5p in the multi-drugresistance of OS cells. The main findings is that DICER1-AS1 directly binds to miR-34a-5p, and their expression has a negative correlation with each other. The hypermethylation of the promoter region of DICER1-AS1 silenced its expression in the drugresistant cells SJSA-1 and MNNG/HOS. Moreover, we found that growth arrest and DNA damage-inducible alpha (GADD45A) participates in the DICER1-AS1/miR-34a-5p-regulated drug resistance of OS cells, probably via the cell cycle/pRb-E2F pathway. Our results revealed DICER1-AS1/miR-34a-5p-regulated drug resistance of OS cells, a new lncRNA-regulated network in OS tumorigenesis, suggested that DICER1-AS1 can be considered as a potential biomarker and therapeutic target against OS cells.
Highlights
Noncoding RNAs, especially microRNAs and long noncoding RNAs, are reported to participate in the proliferation, apoptosis, metastasis, invasion and drug resistance of tumors [1, 2]
Among the putative long noncoding RNA (lncRNA), we predicted the potential miR-34a-5p targets using the following online databases: targetScan, miRanda and picTar, which might be negatively correlated with the expression of miR-34a-5p (Supplementary Figure 6), twelve lncRNAs were the proposed as the targets of miR-34a-5p, as demonstrated in the hierarchical clustering profiling (Supplementary Figure 5B)
We further verified the expression of these 12 lncRNAs and confirmed 7 of them, including LRP4-AS1, DICER1-AS1, TERC, ANK3, LINC00693, TTN-AS1 and CRHR1-IT1
Summary
Noncoding RNAs, especially microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), are reported to participate in the proliferation, apoptosis, metastasis, invasion and drug resistance of tumors [1, 2]. LncRNAs play roles in regulating gene expression at different levels, including chromatin modification, transcriptional and posttranscriptional processing [9, 10]. An increasing number of studies have demonstrated the functional roles of lncRNAs in OS tumorigenesis. LncRNA PVT1 is upregulated in OS cells and contributes to cell metastasis via the miR-497/HK2 pathway [18]. These reports suggest the important roles of lncRNAs in OS tumorigenesis
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