Elevated TRIP13 drives the AKT/mTOR pathway to induce the progression of hepatocellular carcinoma via interacting with ACTN4

Abstract

Background: ATPase associated with a variety of cellular activities (AAA ATPase) family members are closely linked to tumor formation and progression. However, their roles in hepatocellular carcinoma (HCC) largely remain unclear.

Methods: Bioinformatic analyses of public databases were used to excavate the potential AAA ATPases that may contribute to HCC, and thyroid hormone receptor interactor 13 (TRIP13) was selected to following researches because of its most prominently differential expression. Western blot, qRT-PCR and immunohistochemistry were used to detect the expression of TRIP13 in HCC tissues, and then the relationship between TRIP13 expression and clinicopathological parameters were evaluated. Finally, its functions and potential mechanisms were investigated through a series gain- and loss-of-function strategies both in vitro and in vivo.

Results: TRIP13 was significantly overexpressed in HCC tissues and high level of TRIP13 was closely correlated with a worse clinical outcome. Functionally, elevated TRIP13 facilitated cell proliferation, migration, invasion, and promoted cellular epithelial-mesenchymal transition (EMT) in vitro, while promote tumor growth and lung metastasis in vivo. Mechanistically, TRIP13 interacted with ACTN4 and positively regulated its expression, thus activating the AKT/mTOR pathway to drive tumor progression. Moreover, miR-192-5p served as an upstream regulator of TRIP13 by directly binding to TRIP13 mRNA 3' UTR, which may partially explain the high expression of TRIP13 in HCC.

Conclusion: Our findings identified TRIP13 as a promising candidate oncogene in HCC, and TRIP13 induced cell migration, invasion and metastasis of HCC through the AKT/mTOR signaling via interacting with ACTN4.

Keywords: ACTN4; EMT; HCC; TRIP13; miR-192-5p.

Fig. 1

Upregulation of TRIP13 in HCC. a Volcano plot indicated differentially expressed AAA ATPase genes in TCGA LIHC dataset. b Studies from GEO datasets showed higher mRNA expression of TRIP13 in HCC tissues. c-d The mRNA and protein expression of TRIP13 in 15 pairs of HCC (T) and peritumor tissues (P) were analyzed by real-time PCR and western blot. The representative images were shown. e The expression of TRIP13 was detected by IHC in a TMA including 96 HCC cases and typical photos were presented. f IHC results presented the increase of TRIP13 protein in HCC compared with peritumor samples. G Survival analysis of TRIP13 expression in 96 HCC patients. **P < 0.01

Fig. 2

TRIP13 promoted cell proliferation, migration and invasion of HCC cells in vitro. a Western blot analysis of TRIP13 in six different HCC cell lines. b The efficiency of transfection in SMMC7721 and MHCC97H cell lines were confirmed by western blot. c-d Knockdown of TRIP13 in MHCC97H cells inhibited cell proliferation as determined by CCK-8 (c) and colony formation assays (d). e-f Overexpression of TRIP13 in SMMC7721 cells promotes cell proliferation as determined by CCK-8 (e) and colony formation assays (f). g-h Interference with TRIP13 expression in MHCC97H cells inhibited cell migration and invasion as determined by wound healing assay (g) and transwell assay (h). i-j Overexpression of TRIP13 in SMMC7721 cells promotes cell migration and invasion as determined by wound healing assay (i) and transwell assay (j). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

TRIP 13 silencing inhibited tumor growth and lung metastasis in vivo. a MHCC97H cells with different TRIP13 expression were subcutaneously transplanted the xenograft into BALB/c nude mice (n = 6/group) and tumor sizes were measured after 4 weeks. b Tumor weights were measured. c Tumors were formalin-fixed, paraffin-embedded, sliced and stained with TRIP13 and PCNA. Representative images of each group are presented. d-e MHCC97H cells with different TRIP13 expression were injected into BALB/c nude mice (n = 6/group) through the tail vein to establish lung metastasis models. The number of lung metastatic nodules in each group was counted under the microscope. Representative H&E staining images of lungs were shown. **s < 0.01

Fig. 4

TRIP13 induced EMT in HCC cells. a-b The morphology of HCC cells with different TRIP13 expression. c-d Western blot analysis of the EMT markers (E-cadherin, N-cadherin, vimentin) in MHCC97H or SMMC7721 cells with knockdown or overexpressing TRIP13. e IHC staining of EMT markers (E-cadherin, Vimentin, Snail) in serial section of human HCC tissues. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

TRIP13 altered activation of PI3K/AKT/mTOR pathway to induce cell EMT. a Gene set enrichment analysis of TCGA LIHC dataset showed enrichment of PI3K/AKT/mTOR pathway and Wnt/β-catenin pathway gene panels in the TRIP13 high expression group. b Reprehensive genes of PI3K/AKT/mTOR pathway and Wnt/β-catenin pathway were detected in TRIP13 knockdown or overexpression cells. c-d Cells were treated with XAV939 or LY294002 and cell migration and invasion were measured. e Cells were treated with XAV939 or LY294002 and the levels of EMT markers were analyzed. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

TRIP13 activates AKT/mTOR pathway by interacting with ACTN4. a ACTN4 was identified as a binding partner of TRIP13 by combining Co-IP and MS. b The AKT/mTOR pathway was affected by ACTN4 interference. c Co-IP was used to validate the interaction of TRIP13 and ACTN4. d Immunofluorescence used to verify the colocalization of TRIP13 and ACTN4. e-f qRT-PCR and western blot analysis of TRIP13 or ACTN4 mRNA expression in HCC cells transfected with shTRIP13 or siACTN4. g-h Cell migration and invasion were measured in the indicated cells. i EMT markers and reprehensive genes of AKT/mTOR pathway were measured in the indicated cells by western blot. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

miR-192-5p inhibited TRIP13 expression in HCC cells. a-c qRT-PCR and western blot analysis of TRIP13 mRNA in HCC cells transfected with miR-192-5p mimics or miR-192-5p inhibitor. d The putative miR-192-5p binding sequence in the 3′-UTR of TRIP13 (left penal). Luciferase activity of 293 T cells co-transfected with WT or MUT luciferase reporter plasmids and miR-192-5p mimics or negative control (right penal). e-f Cell migration and invasion were measured in HCC cells transfected with miR-192-5p mimics or negative control. g A model depicting the major molecular mechanisms of the miR-192-5p–TRIP13–ACTN4–AKT/mTOR axis in HCC. *P < 0.05, **P < 0.01, ***P < 0.001