A preliminary investigation of the RBP function of RAD51AP1 protein in human lung adenocarcinoma A549 cells and its effect on cell proliferation and apoptosis

OBJECTIVE To explore the functional role of RAD51AP1 on the proliferation and apoptosis of human lung adenocarcinoma A549 cells, and to verify whether RAD51AP1 possesses RNA-binding protein (RBP) function.METHODS Immunohistochemistry was used to detect the expression of RAD51AP1 protein in lung adenocarcinoma and squamous carcinoma. Lentiviral vectors were used to construct A549 cell models with RAD51AP1 overexpression and silencing, Western blot to verify the expression efficiency of RAD51AP1, flow cytometry for cell proliferation and apoptosis detection, RIP-seq and RNA-seq combined to detect RAD51AP1 target mRNA, RT-qPCR, Western blot to detect the expression level of target mRNA molecules, actinomycin D assay to detect the RNA stability of target mRNA molecules, and nuclear translocation assay to detect the entry of RAD51AP1 into the nucleus.RESULTS Immunohistochemistry results showed that the RAD51AP1 protein is highly expressed in lung adenocarcinoma. Western blot results showed successful construction of RAD51AP1 overexpression and silencing of A549 cell line; flow results showed that overexpression and silencing of RAD51AP1 did not have any significant changes on the proliferative and apoptotic ability of A549 cells (p>0.05), but both led to a significant increase in necrotic cells ( p<0.05); RIP-seq and RNA-seq results showed that the target mRNAs might be FASN, MUC5B, RAD51AP1, RT-qPCR results showed that the expression of FASN and MUC5B was significantly reduced (p<0.05), Western blot results showed no change in FASN protein level (p>0.05), and MUC5B protein level was significantly reduced (p<0.05), and RAD51AP1 maintained the difference between the transfected groups both in mRNA level and protein level (p<0.05); Actinomycin D assay showed that the stability of MUC5B, FASN, and RAD51AP1 mRNAs was decreased with the increase of time; nuclear translocation assay showed that the RAD51AP1 protein into the nucleus phenomenon increased.CONCLUSION RAD51AP1 protein cannot maintain the mRNA stability and expression of the target molecules MUC5B, FASN, and RAD51AP1. Therefore, our findings do not support an RNA-binding protein (RBP) function between RAD51AP1 and these target molecules.

【研究目的】探讨RAD51互作蛋白1（RAD51AP1）对人肺腺癌A549细胞增殖与凋亡的功能作用，并验证RAD51AP1是否具备RNA结合蛋白（RNA-binding protein, RBP）功能。
【研究方法】采用免疫组织化学法检测RAD51AP1蛋白在肺腺癌及肺鳞状细胞癌组织中的表达水平；通过慢病毒载体构建RAD51AP1过表达与沉默的A549细胞模型，采用蛋白质印迹法（Western blot）验证RAD51AP1的表达效率；利用流式细胞术检测细胞增殖与凋亡情况；联合RNA免疫沉淀测序（RIP-seq）与RNA测序（RNA-seq）筛选RAD51AP1的靶mRNA；采用实时定量聚合酶链反应（RT-qPCR）、蛋白质印迹法检测靶mRNA分子的表达量；采用放线菌素D实验检测靶mRNA分子的RNA稳定性；通过核移位实验探究RAD51AP1的入核过程。
【研究结果】免疫组织化学结果显示，RAD51AP1蛋白在肺腺癌组织中呈高表达。蛋白质印迹法结果证实，RAD51AP1过表达及沉默的A549细胞系构建成功。流式细胞术结果表明，RAD51AP1过表达或沉默对A549细胞的增殖与凋亡能力无显著影响（p>0.05），但均可显著升高坏死细胞比例（p<0.05）。RNA免疫沉淀测序与RNA-seq结果显示，候选靶mRNA包括FASN、MUC5B、RAD51AP1。实时定量聚合酶链反应结果显示，FASN与MUC5B的表达量显著降低（p<0.05）；蛋白质印迹法结果显示，FASN蛋白水平无显著变化（p>0.05），而MUC5B蛋白水平显著下调（p<0.05）；RAD51AP1在转染组间的mRNA及蛋白水平差异均具有统计学意义（p<0.05）。放线菌素D实验结果显示，随着培养时间延长，MUC5B、FASN及RAD51AP1 mRNA的稳定性均有所下降。核移位实验结果显示，RAD51AP1的入核现象显著增强。
【结论】RAD51AP1蛋白无法维持靶分子MUC5B、FASN及RAD51AP1的mRNA稳定性与表达水平。因此，本研究结果不支持RAD51AP1与上述靶分子之间存在RNA结合蛋白功能。