Fig 2: Screening of key HRGs to construct a PCA prognostic model. (A) Heatmap and (B) volcano map of 18 differentially expressed HRGs. (C) Five HRGs closely related to OS in PCA by univariate Cox regression. (D) Spearman correlation analysis of 5 HRGs in the TCGA database. (E) Selection of the optimal genes used to construct the final prediction model by LASSO regression analysis. Ten-fold cross-validation for tuning parameter selection. (F) LASSO coefficient profiles of the key genes. Each colored line represents the variation trajectory of each gene coefficient; (G-J) comparison of the expression levels of BHLHE40, ENO1, SDC4, and TGM2 between PCA tissue and normal pancreatic tissue in the training set. (K-N) KM survival curves for of BHLHE40, ENO1, SDC4, and TGM2 in the training set. ***, P<0.001. HRGs, hypoxia-related genes; PCA, pancreatic cancer; FC, fold-change; FDR, false discovery rate; OS, overall survival; TCGA, The Cancer Genome Atlas; LASSO, least absolute shrinkage and selection operator; KM, Kaplan–Meier.

Fig 3: Identifying the downstream genes of BHLHE40. (A) Volcano map of 77 DEGs between the control group and the BHLHE40-knockdown group in GSE107300; (B-E) Spearman correlation analyses between the expression of BHLHE40 and 4 downstream genes in TCGA cohort, including CYP24A1, GCNT4, TLR3, and TMEM139; (F-I) KM survival curves of CYP24A1, GCNT4, TLR3, and TMEM139 based on the TCGA cohort; (J-L) GSEA of CYP24A1, GCNT4, and TLR3, based on the median expression of each gene. DEGs, differentially expressed genes; FC, fold-change; FDR, false discovery rate; TCGA, The Cancer Genome Atlas; KM, Kaplan–Meier; GSEA, gene set enrichment analysis.

Fig 4: TLR3 served as a downstream gene of BHLHE40 and promoted migration and invasion of PCA cells. (A) The JASPAR database predicts binding sites between BHLHE40 and the promoters of GCNT4 and TLR3; (B) knockdown of BHLHE40 repressed the expression of TLR3 in PANC-1 cells; (C) by using ChIP assay, BHLHE40 was immunoprecipitated from PANC-1 cells, and the binding of BHLHE40 to the promoter of TLR3 was detected using qRT-PCR with the indicated primers; (D) knockdown of TLR3 by using siTLR3 repressed the expression of TLR3 in PANC-1 cells; (E) cell migration was assessed using a wound healing assay upon treatment with siTLR3 (×40); (F) the invasion ability of PANC-1 cells transfected with siTLR3 was evaluated by using transwell assay (×200), in which cells were stained with 0.1% crystal violet; (G) the invasion ability of PANC-1 cells co-transfected with siBHLHE40 and siTLR3 was determined by transwell assay (×200), in which cells were stained with 0.1% crystal violet. All data are presented as the means ± SD of 3 independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001. PCA, pancreatic cancer; TSS, transcription start site; ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time polymerase chain reaction; siRNAs, small interfering RNAs; SD, standard deviation; siTLR3, siRNAs targeting TLR3.

Fig 5: BHLHE40 triggered metastasis of pancreatic cancer cells. (A) The expression levels of BHLHE40, ENO1, SDC4, and TGM2 under normoxic or hypoxic conditions were evaluated in 9 PCA cell lines; (B) the protein expression level of BHLHE40 in PCA (https://www.proteinatlas.org/ENSG00000134107-BHLHE40/pathology/pancreatic+cancer#img) and normal pancreatic tissue (https://www.proteinatlas.org/ENSG00000134107-BHLHE40/tissue/pancreas#img); (C-F) the expression of BHLHE40 of PCA patients with different grades, stages, T stages, and M stages in the TCGA cohort; (G) the expression levels of BHLHE40 in MIA PaCa-2, SW1990, and PANC-1 cell lines were determined by qRT-PCR; (H) knockdown of BHLHE40 by using siRNAs targeting BHLHE40 (siBHLHE40) repressed the expression of BHLHE40 in PANC-1 cells; (I) Transwell assays were performed to assess the invasion ability of PANC-1 cells transfected with siRNAs targeting BHLHE40 (siBHLHE40) (×200), in which cells were stained with 0.1% crystal violet; (J) cell migration was assessed using a wound healing assay (×40). All data are presented as the mean ± SD of 3 independent experiments. *P<0.05; **, P<0.01; ***, P<0.001. PCA, pancreatic cancer; TCGA, The Cancer Genome Atlas; siRNA, small interfering RNA; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation.