Fig. 5. IRF5 regulated PLOD2 expression by activating the PLOD2 promoter.

(A) Relative mRNA levels of PLOD2 in HCC and normal tissues were determined based on the TCGA dataset. ***p<0.001. (B) IRF5 mRNA expression was correlated with poor survival in HCC patients based on the TCGA dataset. (C, D) qPCR and western blot assays showing the relative levels of IRF5 and PLOD2 in the siIRF5 Huh7 and MHCC-97H cell lines. *p<0.05, ***p<0.001. (E) Representative IHC staining images showing a positive correlation between IRF5 and PLOD2 levels in human HCC samples (n=200). (F) Correlation of IRF5 and PLOD2 mRNAs in HCC samples from the TCGA database. (G) Schematic view of the luciferase reporter constructs containing various sites of PLOD2. (H) KD of IRF5 reduced PLOD2 promoter activity. HCC cells were reversely transfected with NC or siIRF5 for 24 h, followed by transfection with the full-length PLOD2 promoter (−2,000/+99) for another 48 h, and then were subjected to luciferase activity assay. ***p<0.001. (I) KD of IRF5 decreased the luciferase activity of the site A mutant. HCC cells were reversely transfected with NC or siIRF5 for 24 h, followed by transfection with the site A mutant for another 48 h and then were subjected to luciferase activity assay. ***p<0.001. (J) The sequence of site A and the corresponding sequence of the mutated site are shown. (K) The sequence of site B and the corresponding sequence of the mutated site are shown. (L) KD of IRF5 did not change the luciferase activity of the site B mutant. HCC cells were reversely transfected with NC or siIRF5 for 24 h, followed by transfection with the site B mutant for another 48 h, and then were subjected to luciferase activity assay. HCC, hepatocellular carcinoma; IHC, immunohistochemical; IRF5, interferon regulatory factor 5; KD, knockdown; NC, Negative control; PLOD2, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2; TCGA, The Cancer Genome Atlas.