TFAIP6 facilitates hepatocellular carcinoma cell glycolysis through upregulating c-myc/PKM2 axis

Abstract

Background: Hepatocellular carcinoma (HCC) is the most prevalent liver cancer. Despite of the improvement of therapies, the durable response rate and survival benefit are still limited for HCC patients. It's urgent to clarify the molecular mechanisms and find therapeutic strategies to improve the clinical outcome. TNFα-stimulated gene-6 (TNFAIP6) plays a critical role in the prognosis of various tumors, but its roles in HCC are still unclear.

Methods: Quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC) analysis were employed to evaluate the clinical relevance of TNFAIP6 expressions in HCC patients. Cell counting kit-8 (CCK-8), Edu assay, and transwell assay were performed to evaluate the malignancy of HCC cells. Glucose uptake, lactate production, ATP production, extracellular acidification rate (ECAR) by Seahorse XF analyzer were employed to evaluate the role of TNFAIP6 in the regulation of aerobic glycolysis. The expressions of key proteins involved in glycolysis were examined by Western blot. Co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) were used for protein-protein interactions or protein-RNA interactions respectively. Knockdown and overexpression of TNFAIP6 in HCC cells were employed for analyzing the functions of TNFAIP6 in HCC.

Results: TNFAIP6 was significantly upregulated in HCC and predicted a poor clinical prognosis. Knockdown of TNFAIP6 inhibited in vitro cell proliferation, invasion, migration, as well as glycolysis in HCC cells. Mechanistically, we clarified that TNFAIP6 interacted with heterogeneous nuclear ribonucleoprotein C (HNRNPC), stabilized c-Myc mRNA and upregulated pyruvate kinase M2 (PKM2) to promote glycolysis.

Conclusions: Our study reveals a molecular mechanism by which TNFAIP6 promotes aerobic glycolysis, which is beneficial for malignance of HCC and provides a potential clinical therapy for disease management.

Keywords: Glycolysis; Hepatocellular carcinoma; PKM2; TNFAIP6; c-Myc.

Fig. 1

Ectopically overexpressed TNFAIP6 indicates the poor prognosis of HCC patients. (A) The expression of TNFAIP6 was examined by qRT-PCR, compared between 60 pairs of HCC and matched noncancerous liver tissues. (B,C,D) TNFAIP6 expression was evaluated in patients with different stages of HCC (B), lymph node metastasis (C), and distant metastasis (D) respectively. (E) The positive expression of TNFAIP6 was examined by immunohistochemistry in HCC tissues and normal tissues. (F) The overall survival of patients was analyzed by Kaplan-Meier analyses. (G) The expression of TNFAIP6 was examined by qRT-PCR in normal liver cell line and HCC cell lines. n = 60, *, P < 0.05; **, P < 0.01; ***, p < 0.001.

Fig. 2

TNFAIP6 confers to the proliferation, migration and invasion of HCC cells. Huh7 and Hep3B cells were transfected with vector, TNFAIP6, sh-NC or sh-TNFAIF6 respectively. (A) Analysis of mRNA level of TNFAIP6 using qRT-PCR. (B) Analysis of protein level of TNFAIP6 upon overexpression or knockdown using Western blot. Cell proliferation was detected by CCK-8 analysis (C) and Edu assays (D). (E) Trans-well analysis of migration and invasion. (F) The expression of EMT related proteins was analyzed by Western blot. n = 3, mean ± SEM *, P < 0.05; **, P < 0.01; ***, p < 0.001.

Fig. 2

TNFAIP6 confers to the proliferation, migration and invasion of HCC cells. Huh7 and Hep3B cells were transfected with vector, TNFAIP6, sh-NC or sh-TNFAIF6 respectively. (A) Analysis of mRNA level of TNFAIP6 using qRT-PCR. (B) Analysis of protein level of TNFAIP6 upon overexpression or knockdown using Western blot. Cell proliferation was detected by CCK-8 analysis (C) and Edu assays (D). (E) Trans-well analysis of migration and invasion. (F) The expression of EMT related proteins was analyzed by Western blot. n = 3, mean ± SEM *, P < 0.05; **, P < 0.01; ***, p < 0.001.

Fig. 3

TNFAIP6 promotes HCC cells glycolysis. Huh7 and Hep3B cells were transfected with vector, TNFAIP6, sh-NC or sh-TNFAIF6 respectively. (A,B,C) Glucose uptake levels (A), Lactic acid (B) and ATP production (C) was measured by commercial kits. (D) The ECAR was measured by seahorse assay. (E) The expression of glycolysis related proteins was analyzed by Western blot n = 3, mean ± SEM, *, P < 0.05; **, P < 0.01; ***, p < 0.001.

Fig. 4

TNFAIP6 stabilizes c-Myc mRNA by interacting with HNRNPC. (A) c-Myc mRNA expression level was detected by qRT-PCR. (B) c-Myc protein expression level was detected by Western blot. (C) The STRING online software predicted the interactions among TNFAIP6, HNRNPC and c-Myc. (D) The potential interaction between HNRNPC, TNFAIP6 and c-Myc was assessed by Co-IP assay. (E) The interaction between HNRNPC and c-Myc after TNFAIP6 overexpression or knockdown was assessed by Co-IP assay. (F) c-Myc mRNA expression level was examined by qRT-PCR. (G) The mRNA stability of c-Myc was examined by qRT-PCR after treating with actinomycin D for indicated time periods. n = 3, mean ± SEM, *, P < 0.05; **, P < 0.01; ***, p < 0.001.

Fig. 5

c-Myc transcription activates PKM2 to induce HCC cells glycolysis. (A) JASPAR software predicted the binding site of c-Myc to the PKM2 promoter region. (B) The binding between c-Myc and PKM2 promoter region was validated by ChIP assay. (C) Luciferase reporter assay to measure activity of 293T cells expressing wild type PKM2 or PKM2 mutant. (D and E) mRNA and protein expression levels of c-Myc and PKM2 in c-Myc overexpressing or silencing cells. (F,G,H) Glucose uptake levels (F), Lactic acid (G) and ATP production (H) were measured in Huh7 and Hep3B cells overexpressing or knocked-down c-Myc by commercial kits. (I) The expression of Glycolysis related proteins was analyzed by Western blot. n = 3, mean ± SEM, *, P < 0.05; **, P < 0.01; ***, p < 0.001.

Fig. 6

The graphical abstract of TNFAIP6 in HCC. TNFAIP6 binds to HNRNPC, stabilizes c-Myc mRNA and upregulates c-Myc/PKM2 axis to promote glycolysis in hepatocellular carcinoma.