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.