[Screening of mRNA molecules targeted by RAD51AP1 protein in human lung adenocarcinoma A549 cells and its effect on...

Objective: To explore the functional role of RAD51AP1 protein targeting and binding to mRNA in human lung adenocarcinoma A549 cells and its effect on cell proliferation and apoptosis. Methods: In vitro cytological and molecular biological experiments were conducted in the Surgical Research Laboratory of the General Hospital of Ningxia Medical University from January 2024 to September 2025.The expression of RAD51AP1 protein in lung adenocarcinoma tissue was analyzed by Immunohistochemistry(IHC). A549 cell with RAD51AP1 overexpression and knockdown was constructed by Lentiviral transfection, and divided into the RAD51AP1-shRNA group and the RAD51AP1-Over group. Flow cytometry was used to detect cell proliferation and apoptosis. RIP-seq combined with RNA-seq was performed to identify the mRNA molecules targeted by RAD51AP1 protein. The mRNA and protein expression levels of FASN, MUC5B, and RAD51AP1 were detected by RT-qPCR and Western blot, respectively. Actinomycin D assay to detect the RNA stability of FASN, MUC5B, and RAD51AP1 mRNA. Nuclear translocation assay to detect the nuclear import of RAD51AP1 protein. In this study, for normally distributed measurement data, comparisons between two groups were performed using independent samples t-test, while comparisons among multiple groups were conducted using one-way analysis of variance. Count data were presented as frequencies or percentages, and inter-group comparisons were made using the chi-square test. Results: Immunohistochemistry showed that the RAD51AP1 protein is highly expressed in lung adenocarcinoma. Flow cytometry results showed that cell proliferation was significantly increased (27.30±0.72 vs. 47.13±1.04, t=27.13, P<0.000 1)and apoptotic was significantly decreased (9.95±0.87 vs. 3.50±0.41, t=11.62, P<0.000 1)in the RAD51AP1-Over group, while the opposite trends were observed in the RAD51AP1-shRNA group. RIP-seq and RNA-seq results showed that FASN, MUC5B, and RAD51AP1 may be the target mRNA molecules of RAD51AP1 protein. RT-qPCR results showed that compared to the control group, MUC5B mRNA expression was significantly decreased in RAD51AP1-Over group(0.83±0.18 vs. 0.22±0.09, t=5.29, P<0.01), FASN mRNA expression levels was no significant difference in both the RAD51AP1-shRNA and RAD51AP1-Over groups(0.96±0.10 vs. 0.92±0.07, t=0.62;vs. 0.94±0.03, t=0.38;P>0.05). RAD51AP1 mRNA expression showed significant differences consistent with silencing and overexpression(1.02±0.08 vs. 0.15±0.12, t=10.38;vs. 27.51±1.98, t=23.22;P<0.01).Western blot results were consistent with those of RT-qPCR. Actinomycin D assay showed that, compared with the control group, the half-life of MUC5B mRNA was significantly down-regulated in both the RAD51AP1-shRNA and RAD51AP1-Over groups (13.72±0.17 vs. 12.54±0.17, t=8.50;vs. 11.64±0.29, t=10.66;P<0.05). No significant difference was observed in the half-life of FASN mRNA among the groups (8.10±0.19 vs. 7.63±0.23, t=2.75;vs. 7.22±0.60, t=2.43;P>0.05). The half-life of RAD51AP1 mRNA was significantly decreased in the RAD51AP1-shRNA group (10.67±0.52 vs. 5.66±0.12, t=1.93, P<0.05), while no significant difference was found in the RAD51AP1-Over group (10.67±0.52 vs. 9.96±0.37, t=16.21, P>0.05). Nuclear translocation assay showed that the nuclear fluorescence intensity was significantly increased in the RAD51AP1-Over group compared with the Control group (1.00±0.08 vs. 2.24±0.14, t=13.08, P<0.05). Conclusion: Overexpression of RAD51AP1 promoted cell proliferation and inhibited apoptosis, whereas the opposite effects were observed in its knockdown. Furthermore, RAD51AP1 exerts a non-canonical bidirectional regulatory effect on the stability of MUC5B mRNA, whereas no conventional stability regulation was observed for FASN or RAD51AP1's own mRNA, suggesting that it may primarily function through other mechanisms (such as DNA repair or transcriptional regulation) in this cellular system.