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Role of tight junction-associated MARVEL protein marvelD3 in migration and epithelial-mesenchymal transition of hepatocellular carcinoma.

ABSTRACT: MarvelD3, a recently identified tight junction membrane protein, could be associated with hepatocellular carcinoma (HCC). We aimed to investigate the role of marvelD3 in Epithelial-Mesenchymal Transition (EMT) and migration of HCC and explore the underlying molecular mechanisms. First, we assessed marvlD3 expression in HCC and normal liver tissues and found loss of marvelD3 expression was significantly correlated with the occurrence and TNM stage of HCC. Second, we detected that marvelD3 was downregulated in HCC cells with transforming growth factor β1 and snail/slug-induced EMT. Finally, we analyzed expression of marvelD3 protein was significantly associated with EMT and the NF-κB signaling pathway. Our study demonstrated that MarvelD3 inhibited EMT and migration of HCC cells along with inhibiting NF-κB signaling pathway.Abbreviations: HCC, Hepatocellular carcinoma; TJ, Tight junction; MARVEL, MAL and related proteins for vesicle trafficking and membrane link; EMT, Epithelial-mesenchymal transition; NF-κB, Nuclear factor kappa B; TAMPs, Tight junction-associated marvel proteins; TGF-β1, Transforming growth factor-β1; MMP9, matrix metallopeptidase 9; RT-PCR, Real-time PCR; IHC, Immunohistochemistry; IF, Immunofluorescence.

SUBMITTER: Li Y

PROVIDER: S-EPMC8331009 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeThe aim of this work was to investigate the clinicopathological significance of fibrinogen gamma chain (FGG) and its biological roles during hepatocellular carcinoma (HCC) development and progression.MethodsThe expression of FGG was examined by Western blot and reverse transcription quantitative PCR in two different sample sets, including 24 or 35 pairs of HCC tumor tissues and their corresponding adjacent non-tumorous tissues. Afterward, association analysis between the expression of FGG and clinicopathological characteristics was systematically analyzed in 79 HCC patients. Subsequently, the mobility and invasiveness of SK-HEP-1 cells with FGG overexpression or knockdown were evaluated by transwell assay and wound healing assay. Additionally, the expressions of epithelial to mesenchymal transition (EMT)-associated markers were also detected in FGG overexpressed or silenced SK-HEP-1 cells.ResultsThe expression of FGG was significantly increased in primary HCC tissues comparing with its corresponding adjacent non-tumorous tissues. Clinical pathological analysis demonstrated that upregulation of intracellular FGG was significantly associated with increased vascular invasion, more satellite nodules, and more advanced TNM stage, and HCC patients with stronger expression of FGG had a higher recurrence rate and correspondingly a shorter overall survival time. Meanwhile, the high expression of FGG was also proved to be an independent risk factor for disease-free survival after surgical resection. In vitro phenotype studies showed that overexpression of FGG could promote the migration and invasion in SK-HEP-1 cells; conversely, these phenotypes could be significantly inhibited by knocking down the expression of FGG. Mechanism studies indicated that FGG could promote the migration and invasion through EMT signaling pathway by regulating the expressions of Slug and ZEB1.ConclusionFGG played important roles in enhancing cancer cell motility and invasiveness through EMT signaling, and might serve as a potential prognostic biomarker for HCC patients.

Project description:ObjectiveInhibition of tumor metastasis is a useful strategy to improve the efficacy of cancer therapy. Ventilagolin, a natural 1, 4-naphthoquinone derivative extracted from Ventilago leiocarpa Benth, has shown promising antitumor effects in previous studies. However, the effects and underlying mechanisms of Ventilagolin against migration, invasion and epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) remain unclear. The present study has examined these effects and determined whether the proto-oncogene Pim-1 is involved.MethodsThe effects of Ventilagolin on migration, invasion, Pim-1 and EMT-related proteins (eg, E-cadherin, N-cadherin, Vimentin) expression were assessed by scratch wound healing, Transwell, qRT-PCR and Western blot assays, respectively. Pim-1 stably overexpressed HepG2 and SMMC-7721 cells were generated to explore whether Ventilagolin inhibited migration, invasion and EMT of HCC cells via regulating Pim-1. Subcutaneous xenograft tumor model in nude mice was established. Histopathological changes of tumor tissues were examined by H&E staining and expressions of Pim-1 and EMT-related proteins were detected by immunohistochemistry.ResultsVentilagolin significantly (P < 0.01) reduced the expression of Pim-1 levels in HepG2 and SMMC-7721 cells. Compared with the control group, the migration and invasion abilities of Pim-1-overexpressing HepG2 and SMMC-7721 cells were significantly (P < 0.05, P < 0.01) enhanced, the expression of E-cadherin was decreased (P < 0.01), and the levels of N-cadherin and Vimentin were upregulated (P < 0.05, P < 0.01). Ventilagolin treatment effectively reversed these effects of Pim-1 overexpression. In vivo experiments showed that Ventilagolin could effectively suppress HCC tumor growth, downregulate Pim-1, N-cadherin and Vimentin expression, and upregulate E-cadherin expression.ConclusionVentilagolin suppresses HCC cell proliferation, migration and invasion and reverses EMT process by downregulating Pim-1, suggesting Ventilagolin is a potential therapeutic agent for treatment of HCC.

Project description:Rationale: The role of SLUG in epithelial-mesenchymal transition during tumor progression has been thoroughly studied, but its precise regulation remains poorly explored. Methods: The affinity purification, mass spectrometry and CO-IP were performed to identify the interaction between SLUG and ubiquitin-specific protease 5 (USP5). Cycloheximide chase assays and deubiquitination assays confirmed that the effect of USP5 on the deubiquitin of SLUG. The dual-luciferase reporter and chromatin immunoprecipitation assays were employed to observe the direct transcriptional regulation of E-cadherin by SLUG effected by USP5. EMT related markers was detected by western blotting and immunofluorescence. Molecular docking, SPR sensor (biacore) and co-location were detected to prove Formononetin targets USP5. Bioinformatics analysis was used to study the relation of USP5 and SLUG to malignancy degree of HCC. Cell migration, invasion in HCC cells and xenografts model in nude mouse were conducted to detect the promotion of USP5 and the inhibition of Formononetin on EMT. Results: USP5 interacts with and stabilizes SLUG to regulate its abundance through USP5 deubiquitination activities in epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC). USP5 is highly expressed and positively correlated with SLUG expression in HCC with high malignancy. Knockdown of USP5 inhibits SLUG deubiquitination and inhibits HCC cells proliferation, metastasis, and invasion, while overexpression of USP5 promotes SLUG stability and EMT in vitro and in vivo. Through virtual screening, we found that Formononetin exhibits excellent binding to USP5. Moreover, Formononetin inhibits deubiquitinating activities of USP5 to SLUG and consequently impedes the EMT and malignant progression of HCC. Conclusion: Our findings reveal that USP5 serve as a potential target for tumor intervention and provide a preliminary antitumor therapy for inhibit EMT by targeting USP5 or its interaction with SLUG in HCC.

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Role of tight junction-associated MARVEL protein marvelD3 in migration and epithelial-mesenchymal transition of hepatocellular carcinoma.

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