长链非编码RNA-X53654在放射性肺损伤中的功能研究

Radiation-induced lung injury is a common dose-limiting complication of thoracic radiotherapy, but until now its underlying molecular mechanisms and signaling pathways are insufficiently understood. Long non-coding RNAs (lncRNAs) are a novel class of messenger RNA (mRNA)-like transcripts . LncRNAs are usually more than 200 nucleotides in length and lacking an open reading frame. They have been regarded as primary genetic regulators of several important biological processes in the last decades. However, regarding ionizing irradiation induced lung injury, the deregulation and biological function of lncRNAs remains largely unknown. In our preliminary study, lncRNA-X53654 was identified as negatively correlated with the initiation and progression of radiation-induced lung injury via high throughput lncRNA microarray screening, bioinformatics analysis, and real-time PCR confirmation. Increased radiosensitivity, obvious cell cycle G2 / M phase arrest and subG1 apoptotic peak were also been observed in bronchial epithelial cell line Beas-2B with knockdown X53654 gene. Based on these data, we proposed a hypothesis that X53654 might play a key role in the development of radiation-induced lung injury. In the present study, C57BL/6 mice and pulmonary cell lines are used to construct radiation induced lung injury models. The precise biological functions, definite downstream effectors and regulatory pathway(s) of X53654 gene in radiation-induced lung injury are to be identified from different level, using methods such as recombinant eukaryotic plasmid transfection, RNA interference, Northern blot , real-time PCR, and Western blot etc.. It will provide novel mechanisms of radiation-induced lung injury in regards to the concept of LncRNAs.

放射性肺损伤是胸部肿瘤放疗最主要的并发症和剂量限制性毒性反应，其发生机制至今尚未完全明确。长度大于200nt的长链非编码RNA（LncRNA）参与调控机体多种重要生命过程，但其与电离辐射所致机体损伤的关系鲜见报道。前期研究筛选鉴定的LncRNA -X53654与放射性肺损伤发生、发展负相关；抑制该基因可增加支气管上皮细胞Beas-2B的放射敏感性，引起细胞周期G2/M期阻滞，诱导细胞凋亡；提示X53654可能在放射性肺损伤发生、发展中起关键调控作用。本研究拟采用动物和细胞模型，通过重组真核质粒转染、RNA干扰、Northern blot、real-time PCR、Western blot等手段，从动物、细胞、分子水平深入研究X53654在放射性肺损伤中的功能，初步确定上下游作用分子及调控通路。预期本研究将从LncRNA这个新的角度揭示放射性肺损伤的相关机制，为其临床防治提供新的研究思路。

本课题组前期通过基因芯片筛选和荧光定量PCR法，从小鼠放射性肺损伤模型的肺组织中获得了一批可能与放射性肺损伤发生发展有关的基因长链非编码RNA (Long non-coding RNA, lncRNA)，X53654就是是其中一个，功能尚无报道。国内外未见该分子在放射损伤尤其是放射性肺损伤发生和发展中作用的报道。. 本研究采用支气管上皮细胞为研究模型，通过gain-of-function和loss-of-function的手段，在体外进一步研究X53654的生物学功能。结果发现：敲减X53654后，受照后肺上皮细胞克隆形成能力增加，细胞发生明显的G2期阻滞，细胞核中由电离辐射引起的γ-H2AX焦点的数目显著下降，细胞中周期相关RAD50、磷酸化P53和RB蛋白表达水平明显减少，而KU70、KU80、MDM2、CDK2的表达水平明显增加；而X53654高表达的肺上皮细胞受照射后存活分数明显下降，细胞G2期阻滞比对照组明显改善，细胞核中由电离辐射引起的γ-H2AX焦点的数目比对照组显著增加，细胞中KU70、KU80、MDM2蛋白表达水平明显减少，而磷酸化p53和p21的表达水平明显增加。此外，对高表达X53654的支气管上皮细胞进行lncRNAs高通量芯片筛选联合生物信息学分析，结合后续相关功能研究，初步发现X53654的功能可能与其对细胞凋亡、细胞周期、细胞增殖以及上皮间质细胞转化（epithelial mesenchymal transition, EMT）相关信号通路等重要信号分子的转录调控或转录后修饰有关。本研究所获得的相关结果，从lncRNA这个新的角度探讨了放射性肺损伤发送发展过程中可能存在的lncRNA-mRNA/或lncRNA-蛋白质等新型调控网络的作用及机制，为放射性肺损伤的发生分子机制提供了新的基础研究数据。