IGF2BP2 Promotes Liver Cancer Growth Through an m6A-FEN1-Dependent Mechanism

Abstract

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. N6-methyladenosine (m6A) plays an important role in posttranscriptional gene regulation. METTL3 and IGF2BP2 are key genes in the m6A signal pathway and have recently been shown to play important roles in cancer development and progression. In our work, higher METTL3 and IGF2BP2 expression were found in HCC tissues and were associated with a poor prognosis. In addition, IGF2BP2 overexpression promoted HCC proliferation in vitro and in vivo. Mechanistically, IGF2BP2 directly recognized and bound to the m6A site on FEN1 mRNA and enhanced FEN1 mRNA stability. Overall, our study revealed that METTL3 and IGF2BP2, acting as an oncogene, maintained FEN1 expression through an m6A-IGF2BP2-dependent mechanism in HCC cells, and indicated a potential biomarker panel for prognostic prediction in liver cancer.

Keywords: FEN1; IGF2BP2; METTL3; N6-methyladenosine; liver cancer.

Figure 1

Elevated IGF2BP2 expression correlates with poor prognosis of patients with HCC. (A) METTL3 (left) and IGF2BP2 (right) mRNA levels in the TCGA databases. (B) Kaplan-Meier survival curves of OS based on METTL3 and IGF2BP2 expression using the online bioinformatics tool GEPIA. (C) Representative IHC images of IGF2BP2 protein expression in HCC tissues (T) and adjacent normal tissue (N) of two patients (P1–P2). (D) IGF2BP2 protein levels were measured in HCC tissues and paired normal tissues by western blotting. (E) An m6A RNA methylation assay revealed the m6A content in HCC tissues and adjacent normal tissues. (****P < 0.0001, Student’s t-test).

Figure 2

DNA Copy Number Aberrations Promote IGF2BP2 Overexpression in HCC. (A) IGF2BP2 copy numbers in the TCGA databases. (B) Correlation between the DNA copy number and mRNA expression of IGF2BP2 in TCGA. (C, D) The comparison of the IGF2BP2 DNA copy number between HCC tissues (T) and adjacent normal tissues (N) in 20 patients and the correlation between the DNA copy number and mRNA expression of IGF2BP2 in 20 HCC tissue. (****P < 0.0001, ***P < 0.001, Student’s t-test).

Figure 3

IGF2BP2 Promotes HCC Cells Tumorigenicity in vitro. (A) The expression of IGF2BP2 mRNA in HCC cells was determined using qRT-PCR (The experiment was repeated three times). (B, C) Expression level of IGF2BP2 knockdown efficiency in HepG2 and Hun7 cell lines was detected by western blot and qRT-PCR. (D) The influences of IGF2BP2 knockdown on cell proliferation were confirmed using the CCK-8 assay (**P < 0.01, Student’s t-test). (E, F) The influences of IGF2BP2 knockdown on cell proliferation were confirmed by EdU assay (**P < 0.01, Student’s t-test). (G) The representative picture of colony formation assay, and the quantification of colonies per well (***P < 0.001, Student’s t-test).

Figure 4

IGF2BP2 Promotes HCC Cells Tumorigenicity in vivo. (A, B) Knockdown of IGF2BP2 effectively inhibited HepG2 cells subcutaneous tumor growth in nude mice (***P < 0.001, Student’s t-test). (C) IHC analysis of IGF2BP2 in tumor tissue samples. (D) qRT-PCR analysis of KI-67 in tumor samples. (*P <0.05, Student’s t-test).

Figure 5

FEN1 is regulated by METTL3-mediated m6A modification and recognized by IGF2BP2 via an m6Adependent manner. (A) Volcano plots displaying enrichment of dysregulated target genes in IGF2BP-knockdown (shIGF2BP) versus control (shNS) HepG2 cells. The numbers of significantly downregulated or upregulated genes (|log2 FC| > 1, P < 0.01, two-tailed Student’s t-test) in the RIP-seq target group. (B) RNA-seq, MeRIP-seq and RIP-seq identified differentially expressed genes in HepG2 cells when compared with their corresponding controls. (C) Relative changes in FEN1 and E2F1 mRNA levels upon IGF2BP2 silencing in other HCC cell lines (***P < 0.001, Student’s t-test). (D) The protein levels of FEN1 in other HCC cell lines were measured by western blotting. (E) RIP–qPCR showing the binding of IGF2BP2 to FEN1 (***P < 0.001, Student’s t-test). (F) IGF2BP2 expression was positively correlated with FEN1 expression in HCC in TCGA database.

Figure 6

FEN1 plays an oncogenic role in ovarian cancer cells in HCC. (A) Expression level of FEN1 knockdown efficiency in HepG2 and Hun7 cell lines was detected by western blot and qRT-PCR (***P < 0.001, **P < 0.01, Student’s t-test). (B) The influences of FEN1 knockdown on cell proliferation were confirmed using the CCK-8 assay. (C) The influences of FEN1 knockdown on the quantification of colonies per well. (D) FEN1 mRNA levels in the oncomine databases. (E) Kaplan-Meier survival curves of OS based on FEN1 expression using the online bioinformatics tool GEPIA.