Abstract
BackgroundAccumulating evidences indicate that non-coding RNAs (ncRNAs) including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) acting as crucial regulators in osteosarcoma (OS). Previously, we reported that Rho associated coiled-coil containing protein kinase 1 (ROCK1), a metastatic-related gene was negatively regulated by microRNA-335-5p (miR-335-5p) and work as an oncogene in osteosarcoma. Whether any long non-coding RNAs participate in the upstream of miR-335-5p/ROCK1 axial remains unclear.MethodsExpression of differentiation antagonizing non-protein coding RNA (DANCR) and miR-335-5p/miR-1972 in osteosarcoma tissues were determined by a qRT-PCR assay and an ISH assay. Osteosarcoma cells’ proliferation and migration/invasion ability changes were measured by a CCK-8/EDU assay and a transwell assay respectively. ROCK1 expression changes were checked by a qRT-PCR assay and a western blot assay. Targeted binding effects between miR-335-5p/miR-1972 and ROCK1 or DANCR were verified by a dual luciferase reporter assay and a RIP assay. In vivo experiments including a nude formation assay as well as a CT scan were applied to detect tumor growth and metastasis changes in animal level.ResultsIn the present study, an elevated DNACR was found in osteosarcoma tissue specimens and in osteosarcoma cell lines, and the elevated DNACR was closely correlated with poor prognosis in clinical patients. Functional experiments illustrated that a depression of DANCR suppressed ROCK1-mediated proliferation and metastasis in osteosarcoma cells. The results of western blot assays and qRT-PCR assays revealed that DANCR regulated ROCK1 via crosstalk with miR-335-5p and miR-1972. Further cellular behavioral experiments demonstrated that DNACR promoted ROCK1-meidated proliferation and metastasis through decoying both miR-335-5p and miR-1972. Finally, the outcomes of in vivo animal models showed that DANCR promoted tumor growth and lung metastasis of osteosarcoma.ConclusionsLncRNA DANCR work as an oncogene and promoted ROCK1-mediated proliferation and metastasis through acting as a competing endogenous RNA (ceRNA) in osteosarcoma.
Highlights
Accumulating evidences indicate that non-coding RNAs including long non-coding RNAs and microRNAs acting as crucial regulators in osteosarcoma (OS)
We focused on the competing endogenous RNA (ceRNA) network which is comprising of differentiation antagonizing non-protein coding RNA (DANCR), miR-335-5p/ miR-1972 and Rho associated coiled-coil containing protein kinase 1 (ROCK1), as well as explored the potential effect of DANCR to ROCK1 - mediated proliferation and migration/invasion in osteosarcoma
The expression level of DANCR in 4 osteosarcoma cell lines MG-63, U2OS, MNNG/HOS, 143B and a normal human osteoblastic cell line hFOB 1.19 were measured by quantitative real-time PCR (qRT-PCR)
Summary
Accumulating evidences indicate that non-coding RNAs (ncRNAs) including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) acting as crucial regulators in osteosarcoma (OS). We reported that Rho associated coiled-coil containing protein kinase 1 (ROCK1), a metastatic-related gene was negatively regulated by microRNA-335-5p (miR-335-5p) and work as an oncogene in osteosarcoma. Long non-coding RNAs (lncRNAs), which are a group of transcribed RNA molecules and with length of more than 200 nucleotides, are involved in diverse biological processes in tumor initiation, growth and metastasis through epigenetic, transcriptional and post transcriptional mechanisms [4,5,6]. LncRNAs present multiple functions including posttranscriptional regulation, chromatin modification in various malignant tumors such as lung adenocarcinoma, esophageal squamous cell carcinoma, renal cell carcinoma, hepatocellular carcinoma and osteosarcoma [7,8,9,10,11]. CeRNA theory hypothesizes that RNA transcriptions includes lncRNAs communication through a new “language” mediated by microRNA response elements (MREs)
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