Preliminary Study On The Effects And Mechanisms Of Long Non-coding RNAs On Acute Radiation-induced Pulmonary Injury

Objective: In order to screen the differentially expressed long non-coding RNAs(lncRNAs) during early stage of acute radiation-induced pulmonary injury (ARP) and toinvestigate the potential effects of lncRNAs on ARP.Methods:(1) Twenty-four female C57BL/6mice (8-to9-wk old) were dividedrandomly into irradiated group and control group (12mice per group). Using a6-MeVelectron beam accelerator (Siemens KD-2) at a dose rate of200cGy/min and a sourceto skin distance of100cm,12Gy was delivered to the mid-plane. Twenty-four hoursafter irradiation, the right half of mice lungs were fixed in formalin, embeded in paraffin,and then sliced in4m. Immunofluorescence was performed to detect the γ-H2AXfoci. The left lungs were used for RNA extraction, purification, labeling, and microarrayhybridization. The raw hybridization signal was then calculated, selected, andbioinformaticly analyzed to screen differentially expressed lncRNAs. Quantitiverealtime PCR (qRT-PCR) was afterwards performed to confirm related differentiallncRNAs.(2) After consequential bioinformatics analysis and qRT-PCR validation, oneof the verified differentially expressed long intergenic radiation-responsive ncRNAswas selected for further functional study. The sequence specific small interfering RNAs(siRNA) were designed, synthesized and transfected into Beas-2B cells vialipofectamine. Cell survival was detected by cloniogenic assay, and the cell cycledistribution was determined by flow cytometry assay. The γ-H2AX foci formation afterirradiation was visualized via immunofluorescence. Western blot assay was performedto preliminarily study the related proteins.Results:(1) Immunofluorescence analysis found that the γ-H2AX foci in lungtissue of mice after irradiation was significantly increased (P <0.05). There were totally304differentially expressed lncRNAs identified between irradiated and normal micelung tissues, through which62lncRNAs were upregulated and242were downregulated. It was noted that9differential LincRNAs were near coding genes (distance <300kb).As the same time,97upregulated and463downregulated mRNAs were also identifiedin irradiated samples. GO and pathway analysis indicated that the identified mRNAswere mainly related to stress, immune system, p53signaling and metabolism process. Inlung tissues at24h after12Gy,6out of9lincRNAs near the coding genes werevalidated using qRT-PCR in lung tissues. After bioinformatics analysis, one of theupregulation lincRNAs was chosen for further function study.(2) The lncRNAtranscription level in the siRNA transfected Beas-2B cells was significantly decreasedthan that in the NC siRNA transfected control cells. Compared with control cells,siRNA transfection increased cell proliferation and caused an arrest of cell cycle G2phase. The nucleus γ-H2AX foci in siRNA transfected Beas-2B cells were significantlydecreased (P<0.05). The expression of DNA damage related proteins including KU70,KU80, CDK2and MDM2was increased, while a decreased of phosphorylation of p53,RB and RAD50was observed.Conclusions: LncRNAs are differentially expressed in ARP mice lung tissues24hafter exposed to12Gy X-ray. The effects of selected lncRNA on cell proliferation andradiosensitivity in vitro were observed in bronchial epithelial cell line Beas-2B, and itspotential mechanism might primarily be related to cell cycle regulation and DNAdamage repair. Further investigation is needed on the involved molecular mechanisms.