Abstract
Long non-coding RNAs (lncRNAs) have been regarded as the primary genetic regulators of several important biological processes. However, the biological functions of lncRNAs in radiation-induced lung damage remain largely unknown. The present study aimed to investigate the potential effects of lncRNAs on radiation-induced lung injury (RILI). Female C57BL/6 mice were exposed to 12 Gy single doses of total body irradiation (TBI). LncRNA microarray screening was conducted at 24 h post-irradiation (IR) to investigate the differentially-expressed lncRNAs during RILI. Following the subsequent bioinformatics analysis and reverse transcription-polymerase chain reaction (RT-PCR) validation, one of the verified differentially-expressed long intergenic radiation-responsive ncRNAs (LIRRs), LIRR1, was selected for further functional study. The normal human bronchial epithelial BEAS-2B cell line was used as the cell model. The recombinant eukaryotic expression vector for the lncRNA was designed, constructed and transfected using lipofectamine. RT-PCR, clonogenic and flow cytometry assays, immunofluorescence detection and western blot analysis were performed to reveal the role of the lncRNA in the radiosensitivity regulation of the RILI target cells. In lung tissues 24 h after 12 Gy TBI, six of the identified differentially-expressed LIRRs near the coding genes were validated using quantitative (q)PCR. The upregulation of two LIRRs was observed and confirmed using qPCR. LIRR1 was chosen for further functional study. Following the stable transfection of LIRR1, identified through G418 screening, increased radiosensitivity, evident cell cycle G1 phase arrest and increased γ-H2AX foci formation were observed in the bronchial epithelial BEAS-2B cell line subsequent to IR. LIRR1 overexpression also led to decreased expression of the KU70, KU80 and RAD50 DNA repair proteins, marked activation of p53, decreased mouse double minute 2 homolog (MDM2) expression, and substantially induced p21 and suppressed cyclin-dependent kinase 2 in BEAS-2B following IR. Subsequent to the use of Pifithrin-α, a specific inhibitor of p53 activation, increased MDM2 expression was observed in the LIRR1-overexpressing cells, suggesting that LIRR1 could mediate the DNA damage response (DDR) signaling in a p53-dependent manner. The present study provides a novel mechanism for RILI, using the concept of lncRNAs.
Highlights
Radiation‐induced lung injury (RILI) is a common dose‐limiting complication of thoracic radiotherapy that usually consists of radiation pneumonitis and radiation fibrosis [1,2,3,4]
LIRR1 was chosen for further functional study
The differentially‐expressed long intergenic non‐coding RNA (lincRNA) was identified in C57BL/6 mouse lung tissue 24 h after total body irradiation (TBI) using microarray screening and reverse transcription‐polymerase chain reaction (RT‐PCR) validation
Summary
Radiation‐induced lung injury (RILI) is a common dose‐limiting complication of thoracic radiotherapy that usually consists of radiation pneumonitis and radiation fibrosis [1,2,3,4]. In contrast to small non‐coding RNAs, including microRNA and transcription initiation RNAs, lncRNAs are usually >200 nucleotides in length and lack an open reading frame [6,7,8]. In the last decade, increasing evidence has established that, apart from microRNAs, lncRNAs may be primary genetic regulators of several important biological processes, including metabolism, development and carcinogenesis [7,9]. LncRNAs have previously been reported to conduct a broad spectrum of molecular and cellular roles by implementing different modes of action, including chromatin modification and epigenetic regulation, subcellular and structural organization of transcripts, and regulation of the expression of neighboring genes either in cis or trans form, a phenomenon known as transvection, their physiological functions remain poorly understood [6,7]. In terms of the ionizing IR‐induced DDR, the deregulation and biological functions of IR‐responsive lncRNAs remain largely unknown
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