KLF16 promotes pancreatic adenocarcinoma cell proliferation and migration by positively regulating SMAD6

Figure 1 Krüppel-like factor 16 expression in pancreatic adenocarcinoma. A: Expression of Krüppel-like factor (KLF)16 in various The Cancer Genome Atlas (TCGA) cancers; B: Expression of KLF16 in pancreatic adenocarcinoma (PAAD) from the Gene Expression Profiling Interactive Analysis (GEPIA) database. ^aP < 0.05.

Figure 2 Krüppel-like factor 16 regulates pancreatic adenocarcinoma cell proliferation. A, B: The efficiency of Krüppel-like factor (KLF)16 knockdown in pancreatic adenocarcinoma (PAAD) cells by (A) qPCR and (B) western blotting. GAPDH used as the internal control; C, D: Cell proliferation of PAAD cells after KLF16 knockdown. (E, F) Colony numbers of PAAD cells after KLF16 knockdown; G, H: The efficiency of KLF16 overexpression in the cells by (G) qPCR and (H) western blotting. GAPDH used as the internal control; I, J: Cell proliferation of PAAD cells after KLF16 overexpression by the CCK-8 assay; K, L: Colony numbers of PAAD cells after KLF16 overexpression by colony formation assay. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 3 Krüppel-like factor 16 regulates the cell cycle progression and apoptosis in pancreatic adenocarcinoma cells. A, B: Cell cycle progression of pancreatic adenocarcinoma (PAAD) cells with (A) Krüppel-like factor (KLF)16 knockdown or (B) KLF16 overexpression; C, D: Cell apoptosis of PAAD cells with (C) KLF16 knockdown or (D) KLF16 overexpression. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 4 Krüppel-like factor 16 regulates pancreatic adenocarcinoma cell migration. A, B: Cell migration of pancreatic adenocarcinoma (PAAD) cells with (A) knockdown of Krüppel-like factor (KLF16) or (B) overexpression of KLF16 by transwell assay. Bar, 100 μm. ^bP < 0.01, ^cP < 0.001.

Figure 5 SMAD6 is a downstream target of Krüppel-like factor 16 in pancreatic adenocarcinoma. A: Volcano analysis of differentially expressed genes with Krüppel-like factor (KLF)16 knockdown in MIA PaCa-2 cells by RNA-seq; B: The top 10 pathways or biological processes of upregulated or downregulated genes; C and D: SMAD6 mRNA and protein levels in pancreatic adenocarcinoma (PAAD) cells with KLF16 knockdown or overexpression. GAPDH used as the internal control; E: The expression of SMAD6 in PAAD from The Cancer Genome Atlas; F: Spearman relationship between KLF16 and SMAD6 in PAAD. ^bP < 0.01; ^cP < 0.001.

Figure 6 SMAD6 regulates pancreatic adenocarcinoma cell malignancy. A: The efficiency of SMAD6 knockdown or SMAD6 overexpression in pancreatic adenocarcinoma (PAAD) cells by qPCR; B: Cell proliferation of PAAD cells with knockdown or overexpression of SMAD6; C, D: The migration capacity of PAAD cells with (C) SMAD6 knockdown or (D) SMAD6 overexpression by transwell assay. Bar, 100 μm. ^bP < 0.01; ^cP < 0.001.

Figure 7 SMAD6 knockdown rescues the effects of Krüppel-like factor 16 overexpression on pancreatic adenocarcinoma. (A) Protein levels of Krüppel-like factor (KLF)16 and SMAD6 in control (Ctrl), KLF16, and KLF16+shSAMD6 cells by western blotting in MIA PaCa-2 cells. GAPDH used as the internal control; B: Cell viability of Ctrl, KLF16, and KLF16+shSAMD6 cells by CCK-8 assay in MIA PaCa-2 cells; C: Protein levels of KLF16 and SMAD6 in siCtrl, siKLF16, and siKLF16+SAMD6 cells by western blotting in AsPC-1 cells. GAPDH used as the internal control; D: Cell viability of siCtrl, siKLF16, and siKLF16+SAMD6 cells by the CCK-8 assay in AsPC-1 cells; E: Tumor size from three groups of nude mice that were injection with MIA PaCa-2 cells transfected with Ctrl, KLF16, and KLF16+shSAMD6, separately; F: Tumor growth curve in three groups; G: Tumor weight among the three groups. NS: Not significant. ^bP < 0.01; ^cP < 0.001.