E74-like ETS transcription factor 1 promotes the progression of pancreatic cancer by regulating doublecortin-like kinase 1/Janus kinase/signal transducer and activator of transcription pathway

Abstract

This study was targeted at investigating the biological functions of E74-like ETS transcription factor 1 (ELF1) in pancreatic cancer (PC) and its underlying mechanism. ELF1 expression in PC tissues was detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry. Cell counting kit-8 (CCK-8) method, EdU method and flow cytometry were used to detect the cell proliferation and apoptosis of PC cell lines after transfection. A subcutaneous tumorigenesis model was constructed to validate the oncogenic role of ELF1 in vivo. PROMO database was used to predict the binding site of ELF1 on the promoter region of doublecortin-like kinase 1 (DCLK1). Dual-luciferase reporter gene assay, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assay and quantitative real-time PCR were performed to detect the binding of ELF1 to the promoter region of DCLK1. The effect of ELF1 on DCLK1 expression was detected by Western blot assay. It was found that ELF1 expression in PC tissues and cells was up-regulated. ELF1 overexpression promoted the proliferation and inhibited the apoptosis of PC cells, while knocking down ELF1 had the opposite effects. ELF1 could bind to the promoter region of DCLK1 and ELF1 overexpression promoted the expression of DCLK1. Bioinformatics analysis suggested that Janus kinase (JAK) - signal transducer and activator of transcription (STAT) signaling pathway was associated to DCLK1 expression, and overexpression of ELF1 promoted the expression of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). In conclusion, ELF1 promoted the malignant progression of PC via regulating DCLK1/ JAK/STAT signaling pathway.

Keywords: DCLK1; ELF1; apoptosis; pancreatic cancer; proliferation.

Figure 1

ELF1 is up-regulated in PC, and it is a potential prognostic factor. A. ELF1 expression characteristics in PC tissues and non-cancerous tissues were analyzed using GEPIA database. B. Representative IHC staining of ELF1 in PC tissues and non-cancerous tissues were provided (scale bars =50 μm and 20 μm, respectively). C. The expression of ELF1 mRNA in PC tissues (n=44) and normal tissues (n=44) was determined by qRT-PCR. D. The expression of ELF1 mRNA in PC cell lines (PANC-1, BxPc-3, Capan-2) and hTERT-HPNE cells was determined by qRT-PCR. E. The association between the prognosis of PC patients and the expression of ELF1 was analyzed using StarBase database. *P < 0.05 and ***P < 0.001

Figure 2

Effects of ELF1 on the proliferation and apoptosis of PC cells. A. The transfection efficiency of ELF1 overexpression plasmid and si-ELF1 was detected by Western blot. B. The effects of ELF1 overexpression or knockdown on the viability of Capan-2 and PANC-1 cells were detected by CCK-8 assay. C. The effects of ELF1 overexpression or knockdown on the proliferation of Capan-2 and PANC-1 cells were detected by EdU assay (scale bars =50 μm). D. The effects of ELF1 overexpression or knockdown on the apoptosis of Capan-2 and PANC-1 cells were detected by flow cytometry. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

ELF1 transcriptionally promotes DCLK1 expression. A. PROMO database was used to predict the binding sites between ELF1 and DCLK1 promoter region. B. The correlation between ELF1 expression and DCLK1 expression in PC samples was analyzed by the StarBase database. C. Dual-luciferase reporter gene assay was performed to detect the effects of ELF1 overexpression and knockdown on the luciferase activities of DCLK1-WT and DCLK1-MUT. D. ChIP-qPCR assay was applied to detect the binding of ELF1 to the DCLK1 promoter region. E. Western blot was used to detect the effect of ELF1 overexpression or knock-down on the expression of DCLK1 in Capan-2 and PANC-1 cells. **P < 0.01 and ***P < 0.001.

Figure 4

ELF1 interferes with cell proliferation and apoptosis by targeting DCLK1. The ELF1 overexpression plasmid and si-DCLK1 were co-transfected into Capan-2 cells, and the si-ELF1 and DCLC1 overexpression plasmids were co-transfected into PANC-1 cells. A. DCLK1 mRNA expression was probed by qRT-PCR after transfection. B, C. The viability of Capan-2 and PANC-1 cells was detected by CCK-8 and EdU assays. D. The apoptosis of Capan-2 and PANC-1 cells was detected by flow cytometry. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5

ELF1 modulates JAK-STAT signaling pathway via DCLK1. A. Gene set enrichment analysis was adopted to analyze the signaling pathway related to DCLK1. B. The ELF1 overexpression plasmid and si-DCLK1 were co-transfected into Capan-2 cells, and the si-ELF1 and DCLK1 overexpression plasmids were co-transfected into PANC-1 cells. JAK2 expression and STAT3 expression were analyzed by Western blot assay. C. After ELF1 overexpression plasmids were transfected into Capan-2 cells, the Capan-2 cells with ELF1 overexpression and the control Capan-2 cells were subcutaneously transplanted into nude mice, and the tumor size of the two groups (3 mice per group) was compared. D. The expression levels of ELF1, DCLK1, STAT3 and JAK2 in the tumor tissues of the two groups of mice were detected by Western blot and compared.