长链非编码RNA PANDA通过促进p53-MDM2反馈环促使肺癌细胞放射治疗抵抗及其机制研究

Previous studies have confirmed that radiation resistance is a key factor in the recurrence and metastasis of lung cancer, but the specific mechanism is not clear. Radiation will activate ATM, ATR, which will promote tumor suppressor gene P53 phosphorylation, thus promotes P53 transferred to nucleus to promote tumor cell apoptosis and proliferation inhibition. However, expression of P53 was fine-tuned by the P53-MDM2 feedback loop, but how P53 is identified and combined with MDM2 is not clear. Our previous study found that long non-coding RNA Panda expression in lung cancer after radiotherapy would be increased, and the expression of A549 radiation resistance cells was significantly higher than control cells, suggesting that it may play a key role in radiotherapy resistance of lung cancer. P53 can directly promote PANDA transcription, while interference with PANDA expression will reverse the P53 protein level but there was no effect on P53 transcription level, this suggests that a P53-PANDA negative feedback loop may exist. Thus, we make the assumption: radiation stimulation will promote P53 phosphorylation, and then P53 transffered into nuclear and up-regulated PANDA, MDM2 transcription, and PANDA will recruitment MDM2 and P53 to bind together to promote P53 degradation by ubiquitination, thereby inhibites the pro-apoptotic gene transcription, resulting in lung cancer resistance to radiation.

放疗抵抗是肺癌复发转移的关键因素，然而具体机制尚不清楚。放疗激活ATM、ATR后，将促使抑癌基因p53磷酸化入核，导致肿瘤细胞发生凋亡及增殖抑制。p53的表达受到P53-MDM2反馈环的精细调节，但P53是如何识别并与MDM2结合的，目前尚不明确。我们前期研究发现，长链非编码RNA PANDA在A549放疗抵抗细胞株及放疗后肺癌组织中的表达均显著上调，提示其可能在肺癌放疗抵抗中扮演关键角色。p53直接促使PANDA转录及表达。PANDA 表达后将反向调节P53蛋白水平，然而对p53转录水平并无影响，提示可能存在p53-PANDA负反馈环。RIP实验证实PANDA与p53和MDM2均有直接结合，由此，我们提出假设：放疗刺激将促使p53磷酸化入核，上调PANDA、MDM2转录，PANDA表达上调后将募集MDM2与p53结合，促使p53泛素化降解，进而抑制促凋亡基因转录，导致肺癌放疗抵抗的发生。

既往研究证实放射治疗后发生的抵抗是肺癌复发转移的关键因素，然而其具体机制尚不清楚。我们的研究发现，长链非编码RNA “熊猫”（PANDA）在肺癌放疗后表达上调，且在A549放疗抵抗细胞株表达显著高于对照细胞株。过表达PANDA可降低放疗对肺癌细胞的促凋亡作用。敲降PANDA表达将反向调节P53的蛋白水平，然而对P53转录水平并无影响。通过RIP实验、RNA pull down 实验、Co-IP实验，我们证实PANDA可通过P53-MDM2反馈环，促使P53降解，从而促使放疗抵抗。RNA-Seq及 P53 ChIP-Seq提示，PANDA可通过P53调控其靶基因，包括凋亡基因的转录。最后，我们通过在体实验证实，PADNA可作为肺癌放疗抵抗的防治靶点。