KLF13 promotes esophageal cancer progression and regulates triacylglyceride and free fatty acid metabolism through GPIHBP1

Figures

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  Fig. 1. KLF13 is upregulated in EC. A, B KLF13 mRNA levels in EC samples and normal controls from the TCGA database ESCA and GSE45670 datasets. C, D Comparison of KLF13 transcript and protein levels in EC tumors and normal control samples. E Representative KLF13 IHC staining in EC tissues and normal ones. Scale bar, 200 µm. F Survival analysis according to KLF13 expression level in EC using Kaplan-Meier Plotter. G, H KLF13 transcript and protein levels in EC cell lines.

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  Fig. 2. KLF13 silencing suppressed EC cell proliferation. A KLF13 expression in KYSE-450, KYSE-180 and KYSE-150 cells infected with shKLF13#1, shKLF13#2, and shCtrl. B Cell viability of EC cells after KLF13 knockdown. C Cell colony formation assay to determine the effect of KLF13 knockdown on EC cell proliferation. D, E Cell cycle distribution of two EC cells infected with shCtrl, shKLF13#1 and shKLF13#2, assessed by flow cytometry. F, G Apoptosis of EC cells transfected with shKLF13#1, shKLF13#2, and shCtrl, assessed by flow cytometry. H Caspase 3 and cle-caspase 3 protein levels in EC cells infected with shKLF13#1, shKLF13#2, and shCtrl.

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  Fig. 3. Overexpression of KLF13 promotes esophageal cancer proliferation and metastasis. A KLF13 expression in EC cells infected with KLF13 and Ctrl goups. B Cell viability of EC cells following KLF13 overexpression. C Cell colony formation assay to determine the effect of KLF13 overexpression on EC cell proliferation. D, E Transwell and wound-healing assays to examine migration of EC cells transfected with KLF13 and Ctrl. F The protein levels of E-cadherin and N-cadherin in EC cells following KLF13 overexpression.

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  Fig. 4. KLF13 silencing suppressed EC tumor growth. A Comparison of tumor sizes between the shKLF13 and shCtrl groups. B, C Comparison of tumor volumes and weights of mice (n = 5 per group) from the shKLF13 and shCtrl groups. D Ki67 and KLF13 expression in xenograft tumors assessed by IHC analysis. Scale bar = 100 µm.

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  Fig. 5. KLF13 regulates lipid metabolism. A Volcano plot of differential gene expression in EC from WT and KLF13 knockdown cells using RNA-seq. B GSEA plots showing that the fatty-acid metabolism pathway was enriched in the KLF13 knockdown group. C, D Metabolomics analysis showing that lipid metabolism was significantly altered after KLF13 knockdown. E, F TG and FFA secretion in the supernatants of KYSE-450 and KYSE-180 cells after KLF13 knockdown. G, H TG and FFA secretion in the supernatants of KYSE-30 and KYSE-150 cells transfected with Ctrl and KLF13.

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  Fig. 6. KLF13 directly binds to GPIHBP1 and regulates its transcription. A Pie chart showing Chip-seq promoter results, illustrating the KLF13 binding region. B Venn diagram of genes overlapped between the RNA-seq and Chip-seq promoter results. C The motif sequence of KLF13. D ChIP-seq peak in the GPIHBP1 promoter region of the human genome. E Predicted KLF13 binding site in the GPIHBP1 promoter region. F–I GPIHBP1 expression after KLF13 knockdown or KLF13 overexpression in EC cells. J KYSE-450 cells were co-transfected with KLF13 overexpressing plasmids, as well as control and luciferase reporter plasmids containing the GPIHBP1 promoter. K KLF13 ChIP experiments (IgG as an internal control). L Association between KLF13 and GPIHBP1 expression using TCGA samples. M GPIHBP1 immunohistochemistry results in EC and normal tissue samples. Bar = 100 μm. N Immunohistochemistry results for GPIHBP1 in KLF13 high- or low-EC tissue samples. Bar = 100μm.

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  Fig. 7. KLF13 regulates EC cell proliferation, tumor growth, and lipid metabolism through GPIHBP1. A KLF13 and GPIHBP1 protein levels in KYSE450 cells transfected with shCtrl, shKLF13, and shKLF13 +GPIHBP, and KYSE30 cells transfected with Ctrl, KLF13, and KLF13+ shGPIHBP. B Cell viability of KYSE450 and KYSE30 cells. C, D Cell colony numbers of KYSE450 and KYSE30 cells. E–H TG and FFA concentrations in KYSE450 and KYSE30 cells. I Comparison of tumor sizes between the Ctrl, KLF13, and KLF13+shGPIHBP groups. J, K Comparison of tumor volumes and weights in the Ctrl, KLF13 and KLF13 + shGPIHBP groups (n = 5 mice per group). L KLF13, GPIHBP, and Ki67 expression in xenograft tumors evaluated by H&E staining. Scale bar = 100 µm. M, N TG and FFA concentrations in tumors from Ctrl, KLF13, and KLF13+shGPIHBP group mice.