Fig 2: Pharmacological blocking PRMT5 represses lung cancer cell growth. (A) A549 and ASTC‐a‐1 cells were treated with vehicle or GSK591 and the gene expression was analysed with qRT‐PCR. n = 4. *p < 0.05 versus DMSO. (B) A549 and ASTC‐a‐1 cells were treated with vehicle or GSK591 for indicated concentrations and the protein expression levels of PRMT5, KLF5 and slug were detected by western blotting. (C) A549 and ASTC‐a‐1 cells were treated with vehicle or GSK591 and then the lysate was immunoprecipitated using an anti‐KLF5 antibody. The methylation was evaluated by western blotting using SDMA antibody. (D) A549 and ASTC‐a‐1 cells were treated with vehicle or GSK591 and then treated with CHX for indicated time points. The KLF5 protein expression was evaluated by western blotting. (E) A549 cells were treated with vehicle or GSK591 and then treated with proteasome inhibitor MG132 (10 μM) or lysosome inhibitor BAF‐A1 (100 nM) for 6 h. The KLF5 protein expression levels were detected by western blotting. F. A549 cells were treated with vehicle or GSK591 and the KLF5 expression was measured by immunofluorescence. Bar = 50 μm. n = 3. (G) The expression of KLF5 in nucleus (the intensity of green signal) was quantified. (H) A549 and ASTC‐a‐1 cells were treated with vehicle or GSK591 at the different concentrations and cell proliferation was measured. n = 6. *p < 0.05 versus control.

Fig 3: PRMT5 directly methylates KLF5. (A) The interaction between PRMT5 and KLF5 was captured through co‐immunoprecipitation (Co‐IP) technique. Specifically, the lysate was immunoprecipitated using an anti‐PRMT5 antibody, and then immunoblotted with an anti‐KLF5 antibody in both A549 and ASTC‐a‐1 cells. (B) The Co‐IP was performed using an anti‐KLF5, and then immunoblotted with an anti‐PRMT5 antibody in both A549 and ASTC‐a‐1 cells. (C) The endogenous KLF5 methylation was detected by Co‐IP. The lysate was immunoprecipitated using an anti‐KLF5 antibody, and then immunoblotted with an anti‐SDMA antibody in both A549 and ASTC‐a‐1 cells. (D) The Co‐IP was performed using an anti‐SYM10 antibody, and then immunoblotted with an anti‐KLF5 antibody in both A549 and ASTC‐a‐1 cells. (E) A549 and ASTC‐a‐1 cells were infected with lentivirus containing scramble control or PRMT5‐shRNA1. The lysate was immunoprecipitated using an anti‐KLF5 antibody, and then immunoblotted with an anti‐SDMA antibody. (F) The cell in (E) were transfected with vector or Flag‐PRMT5. The lysate was immunoprecipitated using an anti‐KLF5 antibody, and then immunoblotted with an anti‐SDMA antibody.

Fig 4: PRMT5/KLF5 is overexpressed in lung cancer cells. (A) The relative expression of KLF5 in lung cancer tissues (n = 1017) and normal tissues (n = 627). *p < 0.05 versus normal. (B) KLF5 expression level was negatively correlated with the patient's overall survival with TCGA database analysis. n = 239 for KLF5‐low group and KLF5‐high group. p values were determined by log‐rank test and p = 0.035. (C and D) The correlations between individual gene and pathway score was analysed with Spearman method. The abscissa represents the distribution of the gene expression, and the ordinate represents the distribution of the pathway score. The density curve on the right represents the trend in distribution of pathway immune score; the upper density curve represents the trend in distribution of the gene expression. The value on the top represents the correlation p value, correlation coefficient and correlation calculation method. (E) The correlations between PRMT5 and KLF5 were analysed with Spearman method. (F) The mRNA expression of PRMT5 and KLF5 were measured by qRT‐PCR in different lung cancer cell lines. *p < 0.05 versus IMR90 cells. G and H. The protein expression of PRMT5 and KLF5 were detected by western blotting in different lung cancer cell lines. *p < 0.05 versus IMR90 cells.

Fig 5: Graphic depicts the proposed mechanism by which PRMT5 regulates KLF5 to promote lung cancer cell proliferation and tumourigenesis. In this model, PRMT5 methylates KLF5 at the residue of arginine 41, leading to its stabilisation and activation. This, in turn, enhances the transcription of downstream targets involved in cell proliferation, migration and invasion, ultimately promoting tumour growth and metastasis. The stabilisation of KLF5 and upregulation of KLF5 downstream targets, such as CyclinD1, slug, Nanog and FGF‐BP1 may also contribute to the observed resistance to chemotherapy and radiation therapy in lung cancer cells. Therapeutic targeting of the PRMT5/KLF5 axis may represent a promising strategy for the treatment of lung cancer.