E2F1 promotes cell migration in hepatocellular carcinoma via FNDC3B

Abstract

FNDC3B (fibronectin type III domain containing 3B) is highly expressed in hepatocellular carcinoma (HCC) and other cancer types, and fusion genes involving FNDC3B have been identified in HCC and leukemia. Growing evidence suggests the significance of FNDC3B in tumorigenesis, particularly in cell migration and tumor metastasis. However, its regulatory mechanisms remain elusive. In this study, we employed bioinformatic, gene regulation, and protein-DNA interaction screening to investigate the transcription factors (TFs) involved in regulating FNDC3B. Initially, 338 candidate TFs were selected based on previous chromatin immunoprecipitation (ChIP)-seq experiments available in public domain databases. Through TF knockdown screening and ChIP coupled with Droplet Digital PCR assays, we identified that E2F1 (E2F transcription factor 1) is crucial for the activation of FNDC3B. Overexpression or knockdown of E2F1 significantly impacts the expression of FNDC3B. In conclusion, our study elucidated the mechanistic link between FNDC3B and E2F1. These findings contribute to a better understanding of FNDC3B in tumorigenesis and provide insights into potential therapeutic targets for cancer treatment.

Keywords: ChIP; E2F1; FNDC3B; ddPCR; hepatocellular carcinoma; transcription factor.

Fig. 1

Flowchart of the integrated strategy used to reveal TFs for FNDC3B. There are three major steps for our TFs screening: bioinformation, regulation, and DNA‐binding. By summarizing the ChIP‐seq data from the public domain databases, candidate TFs were selected for further verification. TF knockdowns and qPCR were applied to validate the regulatory relationship between TFs and FNDC3B. ChIP–ddPCR was applied to verify that TFs could bind to the promoter of FNDC3B.

Fig. 2

Regulation Screening by TFs Knockdown coupled with QPCR. (A) For each TF, two shRNAs were designed to transiently knock down its expression. qPCR was used to calculate the expression of FNDC3B, and the results are presented as relative fold changes. All the experiments were repeated three times independently and presented as mean ± SD. (B) The shRNA of E2F1, MAX, and MAFF were delivered by the lentivirus system in Huh7, SKhep1, and HrpG2 cells. The expression levels of FNDC3B were calculated using the $2^{-\mathrm{\Delta \Delta }{C}_{\mathrm{q}}}$ method, and the results are presented as relative fold changes. All the experiments were repeated three times independently and presented as mean ± SD.

Fig. 3

E2F1 predicted binding sites on the promoter of FNDC3B. (A) E2F1‐binding elements, E1 and E2, were predicted by GTRD (http://gtrd.biouml.org/). (B) JASPAR (http://jaspar.genereg.net/) E2F1 motif was used to determine the binding site in E1 and E2.

Fig. 4

DNA‐binding screening. (A) PCR primer pairs were designed to amplify E1, E2, and a negative control fragment. The ddPCR was carried out with DNA fragments immunoprecipitated by anti‐E2F1 or IgG. Each PCR was repeated three times, and the error bars represent SD. *** represents an extremely significant difference (Student's t‐test, P < 0.001). (B) Dual‐luciferase reporter assay was used to detect E2F1 binding sites on the FNDC3B promoter region.

Fig. 5

E2F1 regulated the expression and function of FNDC3B. (A) Violin plot showing the mRNA expression of E2F1 in HCC and normal tissues from the TCGA database. The dashed line and dotted lines within the violin plot represent the median and interquartile range, respectively. (B) E2F1 mRNA expression was upregulated in HCC tissues (**** for P < 0.0001) compared with 50 paired non‐cancerous adjacent tissues using Wilcoxon designed‐rank tests. (C) Western blot of FNDC3B in the E2F1 overexpressed and knockdown cells. Experiments were repeated three times. (D) Huh7 cells were treated for 24 h with the indicated concentration of HLM006474. Experiments were repeated three times. (E) Transwell migration assay performed using cells transfected with shE2F1. Migration ability was determined by calculating migration cells after 24 h of incubation. All the experiments were repeated three times independently and presented as mean ± SD. ** represent an extremely significant difference (Student's t‐test, P < 0.01). Scale bar: 100 μm.