Epithelial-mesenchymal Transition Of Hepatocellular Carcinoma Cells Research Articles

TGF-β Promotes Hepatocellular Carcinoma Metastasis Through Through m6A Modification of PCDHGA9.

Objective: Hepatocellular carcinoma (HCC) is a highly lethal cancer with significant mortality, primarily attributed to metastasis. Although Protocadherin Gamma Subfamily A, 9 (PCDHGA9) has been identified as a tumor suppressor gene in cancer metastasis, its role in HCC remains ambiguous. This study aims to clarify the role of PCDHGA9 in HCC by examining its expression, clinical significance, and molecular activities. Methods: Tissue microarray immunofluorescence analysis evaluated the expression of PCDHGA9 and its clinical relevance. In vitro experiments involved manipulating PCDHGA9 levels in SK-HEP-1 cells to assess migration through wound-healing and transwell assays. In vivo, shPCDHGA9 cell injections were utilized to observe effects on tumor growth and metastasis. Protein analysis and Western Blot validated epithelial-mesenchymal transition (EMT)-related proteins. Subsequent to TGF-β treatment, cell proliferation and apoptosis were quantified using Cell counting kit-8 and flow cytometry, respectively, followed by investigation of TGF-β effects on PCDHGA9 N6-methyladenosine (m6A) modification via Methylated RNA immunoprecipitation, RT-qPCR, and Western blot analysis. Results: Downregulation of PCDHGA9 expression in HCC tissues is correlated with poor prognosis. In vitro experiments demonstrated that modulating PCDHGA9 expression influenced HCC cell migration. In vivo, PCDHGA9 knockdown is correlated with increased metastasis. Furthermore, TGF-β stimulation promoted cell proliferation and inhibited apoptosis. Mechanistically, TGF-β-mediated m6A modification led to PCDHGA9 decay, promoting EMT in HCC cells. Conclusion: PCDHGA9 serves as a potential tumor suppressor in HCC by inhibiting EMT. During this process, TGF-β is observed to exert regulatory control over m6A modifications of PCDHGA9.

Long non-coding RNAFOXD1-AS1 modulated CTCs epithelial-mesenchymal transition and immune escape in hepatocellular carcinoma in vitro by sponging miR-615-3p.

Hepatocellular carcinoma (HCC) is widely recognized as a globally prevalent malignancy. Immunotherapy is a promising therapy for HCC patients. Increasing evidence suggests that lncRNAs are involved in HCC progression and immunotherapy. The study reveals the mechanistic role of long non-coding RNA (lncRNA) FOXD1-AS1 in regulating migration, invasion, circulating tumor cells (CTCs), epithelial-mesenchymal transition (EMT), and immune escape in HCC in vitro. This study employed real-time PCR (RT-qPCR) to measure FOXD1-AS1, miR-615-3p, and programmed death-ligand 1 (PD-L1). The interactions of FOXD1-AS1, miR-615-3p, and PD-L1 were validated via dual-luciferase reporter gene and ribonucleoprotein immunoprecipitation (RIP) assay. In vivo experimentation involves BALB/c mice and BALB/c nude mice to investigate the impact of HCC metastasis. The upregulation of lncRNA FOXD1-AS1 in malignant tissues significantly correlates with poor prognosis. The investigation was implemented on the impact of lncRNA FOXD1-AS1 on the migratory, invasive, and EMT of HCC cells. It has been observed that the lncRNA FOXD1-AS1 significantly influences the generation and metastasis of MCTC in vivo analysis. In mechanistic analysis, lncRNA FOXD1-AS1 enhanced immune escape in HCC via upregulation of PD-L1, which acted as a ceRNA by sequestering miR-615-3p. Additionally, lncRNA FOXD1-AS1 was found to modulate the EMT of CTCs through the activation of the PI3K/AKT pathway. This study presents compelling evidence supporting the role of lncRNA FOXD1-AS1 as a miRNA sponge that sequesters miR-655-3p and protects PD-L1 from suppression.

Plumbagin Regulates Snail to Inhibit Hepatocellular Carcinoma Epithelial-Mesenchymal Transition in vivo and in vitro.

Plumbagin (PL) has been shown to effe ctively inhibit autophagy, suppressing invasion and migration of hepatocellular carcinoma (HCC) cells. However, the specific mechanism remains unclear. This study aimed to investigate the effect of PL on tumor growth factor (TGF)-β-induced epithelial-mesenchymal transition (EMT) in HCC. Huh-7 cells were cultured, and in vivo models of EMT and HCC-associated lung metastasis were developed through tail vein and in situ injections of tumor cells. In vivo imaging and hematoxylin and eosin staining were used to evaluate HCC modeling and lung metastasis. After PL intervention, the expression levels of Snail, vimentin, E-cadherin, and N-cadherin in the liver were evaluated through immunohistochemistry and Western blot. An in vitro TGF-β-induced cell EMT model was used to detect Snail, vimentin, E-cadherin, and N-cadherin mRNA levels through a polymerase chain reaction. Their protein levels were detected by immunofluorescence staining and Western blot. In vivo experiments demonstrated that PL significantly reduced the expression of Snail, vimentin, and N-cadherin, while increasing the expression of E-cadherin at the protein levels, effectively inhibiting HCC and lung metastasis. In vitro experiments confirmed that PL up-regulated epithelial cell markers, down-regulated mesenchymal cell markers, and inhibited EMT levels in HCC cells. PL inhibits Snail expression, up-regulates E-cadherin expression, and down-regulates N-cadherin and vimentin expression, preventing EMT in HCC cells and reducing lung metastasis.

METTL3-mediated m6A modification of lncRNA TSPAN12 promotes metastasis of hepatocellular carcinoma through SENP1-depentent deSUMOylation of EIF3I.

In a previous study, we discovered that the level of lnc-TSPAN12 was significantly elevated in hepatocellular carcinoma (HCC) and correlated with a low survival rate. However, the function and mechanism of lnc-TSPAN12 in modulating epithelial-mesenchymal transition (EMT) and metastasis in HCC remains poorly understood. This study demonstrates that lnc-TSPAN12 positively influences migration, invasion, and EMT of HCC cells in vitro and promotes hepatic metastasis in vivo. The modification of N6-methyladenosine, driven by METTL3, is essential for the stability of lnc-TSPAN12, which may partially contribute to the upregulation of lnc-TSPAN12. Mechanistically, lnc-TSPAN12 exhibits direct interactions with EIF3I and SENP1, acting as a scaffold to enhance the SENP1-EIF3I interaction. As a result, the SUMOylation of EIF3I is inhibited, preventing its ubiquitin-mediated degradation. Ultimately, this activates the Wnt/β-catenin signaling pathway, stimulating EMT and metastasis in HCC. Our findings shed light on the regulatory mechanism of lnc-TSPAN12 in HCC metastasis and identify the lnc-TSPAN12-EIF3I/SENP1 axis as a novel therapeutic target for HCC.

Stromal interaction molecule 1/microtubule-associated protein 1A/1B-light chain 3B complex induces metastasis of hepatocellular carcinoma by promoting autophagy.

Metastasis is the leading cause of death in hepatocellular carcinoma (HCC) patients, and autophagy plays a crucial role in this process by orchestrating epithelial-mesenchymal transition (EMT). Stromal interaction molecule 1 (STIM1), a central regulator of store-operated calcium entry (SOCE) in nonexcitable cells, is involved in the development and spread of HCC. However, the impact of STIM1 on autophagy regulation during HCC metastasis remains unclear. Here, we demonstrate that STIM1 is temporally regulated during autophagy-induced EMT in HCC cells, and knocking out (KO) STIM1 significantly reduces both autophagy and EMT. Interestingly, STIM1 enhances autophagy through both SOCE-dependent and independent pathways. Mechanistically, STIM1 directly interacts with microtubule-associated protein 1A/1B-light chain 3B (LC3B) to form a complex via the sterile-α motif (SAM) domain, which promotes autophagosome formation. Furthermore, deletion of the SAM domain of STIM1 abolishes its binding with LC3B, leading to a decrease in autophagy and EMT in HCC cells. These findings unveil a novel mechanism by which the STIM1/LC3B complex mediates autophagy and EMT in HCC cells, highlighting a potential target for preventing HCC metastasis.

Plectin Promotes the Migration of Hepatocellular Carcinoma Cells Through Inducing F-actin Polymerization

To explore the relationship between the expression of plectin and the migration of hepatocellular carcinoma (HCC) cells and to elucidate the molecular mechanisms by which plectin expression affects the migration of HCC cells. First of all, Western blot was performed to determine the expression of plectin in normal hepatocytes and HCC cells. Secondly, a plectin-downregulated HCC cell strain was established and the control group (shNC group) and shPLEC group were set up. Each group was divided into a vehicle control group (shNC+DMSO group or shPLEC+DMSO group) and a F-actin cytoskeleton polymerization inducer Jasplakinolide group (shNC+Jasp group or shPLEC+Jasp group). Western blot was performed to determine the expression of plectin and epithelial-mesenchymal transition (EMT)-related proteins, including N-cadherin, vimentin, and E-cadherin. HCC cell migration was evaluated by Transwell assay. KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to analyze the signaling pathways related to plectin gene. The polymerization of F-actin was analyzed by immunofluorescence assay. Compared with the normal hepatocytes, HCC cells showed high expression of plectin. Compared with those in the shNC group, the expression of plectin in the shPLEC group was decreased (P<0.05), the migration ability of HCC cells was weakened (P<0.05), and the EMT process was inhibited (with the expression of N-cadherin and vimentin being decreased and the expression of E-cadherin being increased) (P<0.05). KEGG analysis showed that the regulation of cytoskeletal F-actin was most closely associated with plectin and cytoskeletal F-actin depolymerized in the shPLEC group. After treatment with Jasplakinolide, an inducer of F-actin cytoskeleton polymerization, the migration ability of HCC cells in the shPLEC+Jasp group was enhanced compared with that of shPLEC+DMSO group (P<0.05) and the EMT process was restored (with the expression of N-cadherin and vimentin being increased and the expression of E-cadherin being decreased) (P<0.05). In addition, the polymerization of cytoskeletal F-actin in HCC cells was also restored. Plectin is highly expressed in HCC cells. Plectin promotes the migration and the EMT of HCC cells through inducing F-actin polymerization.

The role and mechanism of HIF-1α-mediated glypican-3 secretion in hypoxia-induced tumor progression in hepatocellular carcinoma

ObjectiveTo explore the expression and secretion mechanism of glypican-3 (GPC3) in hepatocellular carcinoma (HCC) cells under hypoxic conditions, and its role in tumor progression. MethodsHuh7 cells with and without the knockdown of hypoxia-inducible factor 1-alpha (HIF-1α) were cultured under 1% O2 for varying durations to induce hypoxia. The expression levels of GPC3, HSP70, CD63, STX11 and SYT7 in the cytoplasm and exosomes of Huh7 cells were evaluated by western blotting and immunofluorescence. GPC3 protein expression was further measured in cells treated with GW4869 under hypoxic conditions. Huh7 cells and human umbilical vein endothelial cells (HUVECs) were cultured with the exosomes extracted from the control and GPC3-knockdown cells, the cell proliferation, migration, epithelial-mesenchymal transition (EMT), invasion, and in vitro angiogenesis were analyzed. Tumor xenografts were established to assess the role of GPC3-deficient exosomes in tumor growth. ResultsHypoxic culture conditions downregulated GPC3, STX11 and SYT7 protein levels in the Huh7 cells and upregulated GPC3 mRNA, and also increased GPC3 protein expression in the exosomes. HIF-1α knockdown, as well as treatment with GW4869, upregulated GPC3 protein in the Huh7 cells grown under 1% O2, but downregulated exosomal GPC3. Furthermore, exosomes derived from the GPC3-knockdown cells inhibited the proliferation and migration of Huh7 cells, decreased the expression of N-cadherin, vimentin, β-catenin, c-Myc and cyclin D1, and increased that of E-cadherin. Likewise, the GPC3-deficient exosomes also suppressed the invasion and tube formation ability of the HUVECs compared to that of control cells. Consistent with the in vitro results, the GPC3-deficient exosomes also repressed tumor growth in vivo. ConclusionHypoxia promoted secretion of exosomal GPC3 through the activation of HIF-1α. GPC3-deficient exosomes inhibited the proliferation, migration and EMT of HCC cells via the Wnt/β-catenin signaling pathway, and suppressed the angiogenic potential of HUVECs. This provided a novel understanding of the role of exosomal GPC3 in HCC progression.

ATRA sensitized the response of hepatocellular carcinoma to Sorafenib by downregulation of p21-activated kinase 1

BackgroundSorafenib resistance greatly reduces the efficacy of treatments in advanced hepatocellular carcinoma (HCC) patients, but the underlying mechanisms are not thoroughly understood. All-trans retinoic acid (ATRA), an anti-leukaemia agent, has attracted considerable attention due to its role in sensitizing cells to other anticancer treatments. We aimed to investigate the combined effect of ATRA and Sorafenib on HCC and the underlying mechanisms.MethodsCCK-8, cell sphere formation, trans-well migration, and wound-healing assays were used to analyse the biological behaviours of HCC cells in vitro. Western blotting and qRT-PCR analysis were conducted to measure the expression of p21 activated kinase 1 (PAK1) and phospho-p21 activated kinase 1 (pPAK1). Xenograft models were established to confirm the synergistic effects of ATRA and Sorafenib in vivo. TUNEL assays and immunohistochemistry were utilized to determine apoptosis, proliferation, PAK1 and pPAK1 levels in tumour tissues.ResultsWe observed that PAK1 was overexpressed in HCC, and its expression was negatively correlated with the survival of patients. PAK1 promoted the proliferation, self-renewal and epithelial-mesenchymal transition of HCC cells. Correlation analysis indicated that the IC50 of Sorafenib was positively correlated with the level of pPAK1 in HCC cell lines. ATRA inhibited the progression of HCC and sensitized HCC response to Sorafenib by downregulation of PAK1, as shown by the calculated coefficient of drug interaction and the data obtained from xenograft models.ConclusionsOur findings indicated that instead of treatment with Sorafenib alone, the combination of ATRA and Sorafenib provides a more effective treatment for HCC patients.2_mPhFeBDQdvWNSurq11yxVideo

E2F1-mediated KDM4A-AS1 up-regulation promotes EMT of hepatocellular carcinoma cells by recruiting ILF3 to stabilize AURKA mRNA

Hepatocellular carcinoma (HCC) is a gastrointestinal tumor with high clinical incidence. Long non-coding RNAs (lncRNAs) play vital roles in modulating the growth and epithelial-mesenchymal transition (EMT) of HCC. However, the underlying mechanism of lncRNA KDM4A antisense RNA 1 (KDM4A-AS1) in HCC remains elusive. In our study, the role of KDM4A-AS1 in HCC was systematically investigated. The levels of KDM4A-AS1, interleukin enhancer-binding factor 3 (ILF3), Aurora kinase A (AURKA), and E2F transcription factor 1 (E2F1) were determined by RT-qPCR or western blot. ChIP and dual luciferase reporter experiments were performed to detect the binding relationship between E2F1 and KDM4A-AS1 promoter sequence. RIP and RNA-pull down confirmed the interaction of ILF3 with KDM4A-AS1/AURKA. Cellular functions were analyzed by MTT, flow cytometry, wound healing and transwell assays. IHC was performed to detect Ki67 in vivo. We found that KDM4A-AS1 was increased in HCC tissues and cells. Elevated KDM4A-AS1 level was correlated to poor prognosis of HCC. Knockdown of KDM4A-AS1 inhibited the proliferation, migration, invasion and EMT of HCC cells. ILF3 bound to KDM4A-AS1 and AURKA. KDM4A-AS1 maintained the stability of AURKA mRNA by recruiting ILF3. E2F1 transcriptionally activated KDM4A-AS1. Overexpressed KDM4A-AS1 reversed the contribution of E2F1 depletion to AURKA expression and EMT in HCC cells. KDM4A-AS1 promoted tumor formation in vivo through the PI3K/AKT pathway. These results revealed that E2F1 transcriptionally activated KDM4A-AS1 to regulate HCC progression via the PI3K/AKT pathway. E2F1 and KDM4A-AS1 may serve as good prognostic targets for HCC treatment.

Microtubule-associated protein 4 promotes epithelial mesenchymal transition in hepatocellular cancer cells via regulating GSK3β/β-catenin pathway

Metastasis is a major obstacle in the treatment of hepatocellular carcinoma (HCC). Microtubule-associated protein 4 (MAP4) plays an important role as a coordinator between microtubules and microfilaments. However, the role of MAP4 in HCC migration and epithelial mesenchymal transition (EMT) is unclear. We compared the protein and mRNA levels of MAP4 in human HCC and adjacent normal tissues using western blotting, immunohistochemistry and RT-qPCR. The migration and invasion abilities and the levels of EMT markers (E-Cadherin, N-Cadherin, Vimentin, and Snail) were compared between MAP4-knockdown and MAP4-overexpressed HCC cells. Finally, we examined whether β-catenin and glycogen synthase kinase 3β (GSK3β) are involved in the stimulatory effects of MAP4 on HCC migration, invasion and EMT. The results revealed that MAP4 levels were higher in the HCC tissues than in the normal hepatic tissues. More importantly, MAP4 knockdown suppressed migration and invasion abilities and EMT processes in HCC cells, which were confirmed by the stimulatory effects of MAP4 overexpression on EMT processes in HCC cells. Further evidence demonstrated that the up-regulation of β-catenin activity induced by the interaction between MAP4 and GSK3β possibly accounted for the pro-migration and pro-EMT effects of MAP4 on HCC cells. Taken together, these results suggest that MAP4 promotes migration, invasion, and EMT in HCC cells by regulating the GSK3β/β-catenin pathway.

Bigelovin inhibits hepatocellular carcinoma cell growth and metastasis by regulating the MAPT-mediated Fas/FasL pathway.

Bigelovin (BigV), as traditional Chinese medicine, has been shown to inhibit the malignant progression of hepatocellular carcinoma (HCC). This study aimed to investigate whether BigV affects the development of HCC by targeting the MAPT and Fas/FasL pathway. Human HCC cell lines HepG2 and SMMC-7721 were used for this study. Cells were treated with BigV, sh-MAPT, and MAPT. The viability, migration, and apoptosis of HCC cells were detected by CCK-8, Transwell, and flow cytometry assays, respectively. Immunofluorescence and immunoprecipitation were used to verify the relationship between MAPT and Fas. Subcutaneous xenograft tumor and tail vein-injected lung metastases mouse models were constructed for histological observation. Hematoxylin-eosin staining was used to assess lung metastases in HCC. Western blotting was used to measure the expression of migration, apoptosis, and epithelial-mesenchymal transition (EMT) marker proteins, as well as Fas/FasL pathway-related proteins. BigV treatment inhibited the proliferation, migration, and EMT of HCC cells, whereas enhanced cell apoptosis. Moreover, BigV downregulated MAPT expression. The negative effects of sh-MAPT on HCC cell proliferation, migration, and EMT were enhanced by BigV treatment. Conversely, BigV addition attenuated the positive effects of MAPT overexpression on the malignant progression of HCC. In vivo experiments showed that BigV and/or sh-MAPT reduced tumor growth and lung metastasis while promoting tumor cell apoptosis. Furthermore, MAPT could act with Fas and inhibit its expression. sh-MAPT upregulated the expression of Fas/FasL pathway-associated proteins, which were enhanced by BigV administration. BigV suppressed the malignant progression of HCC via activating the MAPT-mediated Fas/FasL pathway.

Circular RNA circ_0003028 regulates cell development through modulating miR-498/ornithine decarboxylase 1 axis in hepatocellular carcinoma.

Circular RNA has been revealed to participate in multiple biological functions and contribute to various diseases' progression. This study aims to clarify the role of circ_0003028 and its potential molecular mechanism in hepatocellular carcinoma (HCC). The levels of circ_0003028, miR-498, and ornithine decarboxylase 1 (ODC1) mRNA were examined by quantitative real-time PCR. The cell proliferation ability was detected via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, colony formation, and 5-ethynyl-2'-deoxyuridine assays. The apoptotic rate was evaluated through flow cytometry. The migration and invasion capacity was tested by using wound healing assay and transwell assay. The protein levels of E-cadherin, N-cadherin, and vimentin were measured by western blot assay. The ceRNA regulatory mechanism of circ_0003028 was observed via dual-luciferase reporter and RNA pull-down assays. The mice xenograft models were constructed to confirm the oncogenicity of circ_0003028 in HCC in vivo . Circ_0003028 and ODC1 were upregulated, whereas miR-498 was downregulated in HCC tissues and cells. Circ_0003028 knockdown inhibited cell proliferation and metastasis, and promoted apoptosis. MiR-498 was a direct target of circ_0003028, and inhibition of miR-498 reversed the inhibitory effect of circ_0003028 silencing on HCC progression. Moreover, ODC1 was a direct target of miR-498 and ODC1 overexpression abated the anticancer roles of miR-498 in HCC. Additionally, circ_0003028 regulated ODC1 expression by sponging miR-498. Finally, we found that circ_0003028 could induce epithelial-mesenchymal transition of HCC cells by exosome pathway. In brief, the results demonstrated that circ_0003028 exerted tumourigenicity roles via miR-498/ODC1 signaling axis, providing a promising biomarker and therapeutic target for HCC.

MiR-23b-3p suppresses epithelial-mesenchymal transition, migration, and invasion of hepatocellular carcinoma cells by targeting c-MET

BackgroundAberrant expression of c-MET is known to be associated with tumor recurrence and metastasis by promoting cell proliferation, epithelial-mesenchymal transition (EMT), and migration in hepatocellular carcinoma (HCC). Recently, miR-23b-3p has been identified as a tumor suppressor, but detailed role of miR-23b-3p in HCC is still unclear. Our study aimed to investigate how miR-23b-3p is associated with the malignant potential of HCC cells. MethodsHCC tissues and their adjacent non-tumor tissues were acquired from 30 patients with HCC. Expression of EMT- or stemness-related genes were examined in the two HCC cell lines. Migration of HCC cells was analyzed using transwell and wound healing assays. Resultsc-MET was overexpressed in HCC tissues compared to the adjacent non-tumor tissues. c-MET knockdown inhibited EMT and reduced migration and invasion of HCC cells. Furthermore, c-MET was a target of miR-23b-3p, and miR-23b-3p expression was decreased in HCC tissues compared to non-tumor tissues. Treatment of miR-23b-3p inhibitor in HCC cells promoted EMT, cell migration, and invasion. In contrast, miR-23b-3p overexpression suppressed EMT, cell migration, and invasion, concomitantly reducing c-MET expression. Transfection of miR-23b-3p inhibitor with concomitant c-MET knockdown mitigated the effects of miR-23b-3p inhibitor on EMT in HCC cells. In addition, transforming growth factor beta1 (TGF-β1) stimulation after miR-23b-3p overexpression induced neither the mesenchymal phenotype nor migratory property of HCC cells. ConclusionIn this study, we confirmed that miR-23b-3p downregulation significantly increased EMT, migration, and invasion of HCC cells. In addition, c-MET was confirmed to be a target of miR-23b-3p in HCC cells and regulated the functional effects of miR-23b-3p. These results suggest that miR-23b-3p can be used as a prognostic biomarker and candidate target for HCC treatment.

LOXL3-promoted hepatocellular carcinoma progression via promotion of Snail1/USP4-mediated epithelial-mesenchymal transition.

Lysyl-oxidase-like 3 (LOXL3) was reported to be essential in epithelial-mesenchymal transition (EMT) of cancers. However, the role of LOXL3 in hepatocellular carcinoma (HCC) remained unclear. In this study, we explored clinical significance, biological functions, and regulatory mechanisms of LOXL3 in HCC. Our study found that LOXL3 expression was markedly associated with the tumor size and clinical stage of HCC, and it was highly expressed in tumor tissues of metastatic HCC patients. High expression of LOXL3 predicted a poor prognosis of HCC. TGF-β1 treatment elevated LOXL3 protein expression and cell invasion, and reduced cell apoptosis in HCC cell lines (SMMC-7721 and Huh-7), while downregulation of LOXL3 reversed the promotive effects of TGF-β1 treatment on LOXL3 protein expression and cell invasion, and the inhibitory effect on cell apoptosis. Mechanistically, LOXL3 interacted with snail family transcriptional repressor 1 (Snail1) through STRING database and RIP assay, and Snail1 bound to ubiquitin-specific peptidase 4 (USP4) promoter by JASPAR database, luciferase reporter gene and Co-IP assays. Overexpression of USP4 reversed the inhibitory effect of LOXL3 silence on EMT in HCC cells through deubiquitinating and stabilizing the expression of Snail1. Moreover, LOXL3-promoted HCC EMT through Wnt/β-catenin/Snail1 signaling pathway. In vivo study revealed that silence of LOXL3-inhibited HCC tumor growth. In conclusion, LOXL3 silence inhibited HCC invasion and EMT through Snail1/USP4-mediated circulation loop and Wnt/β-catenin signaling pathway.

The long non-coding RNA LIMT inhibits metastasis of hepatocellular carcinoma and is suppressed by EGF signaling

BackgroundThe long non-coding RNA LIMT (lncRNA inhibiting metastasis) acts as a tumor suppressor factor in some cancers. However, the biological role of LIMT in hepatocellular carcinoma (HCC) has not been explored.Methods and ResultsQuantitative real-time PCR was performed to evaluate the expression of LIMT in HCC tissue. The effects of LIMT on tumor growth and metastasis were assessed by in vitro experiments, including colony formation and transwell assays, and in vivo in nude mouse models. Western blot analysis was used to evaluate the expression levels of proteins associated with epithelial-mesenchymal transition (EMT). LIMT expression was significantly lower in HCC than in normal liver tissue. Functionally, overexpression of LIMT repressed the proliferation, invasion, and EMT of HCC cells, while LIMT knockdown increased proliferation, invasion, and EMT of HCC cells in vitro. Furthermore, LIMT overexpression suppressed HCC growth and metastasis while silencing of LIMT had an opposite effect in vivo. Finally, LIMT overexpression reversed EGF-induced EMT.ConclusionsOur results suggest that LIMT could play an anti-cancer effect in HCC and might be a potential novel therapeutic target in HCC.

Tollip promotes hepatocellular carcinoma progression via PI3K/AKT pathway.

The activation of signaling pathways induced by Toll-like receptor (TLR) has been demonstrated to play essential roles in multiple liver diseases. Toll-interacting protein (Tollip) acts as an endogenous negative modulator of TLR signaling and is implicated in various cardio-metabolic diseases. However, the effect of Tollip in hepatocellular carcinoma (HCC) remains elusive. In the current study, enhanced Tollip expression was observed in HCC cells and tissues examined by RT-PCR, western blot, and immunohistochemistry staining. Moreover, the co-immunofluorescence staining demonstrated that increased Tollip expression was primarily located in hepatocytes. Functionally, Tollip overexpression significantly increased proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of HCC cells, which ultimately accelerated tumorigenesis. Mechanistically, Tollip overexpression dramatically promoted the activation of PI3K/AKT signaling pathway in HCC cells which was attenuated by Tollip silencing. Importantly, the inhibition of PI3K/AKT axis can abolish the promoted effects of Tollip on proliferation and EMT of HCC cells. Our current study demonstrated that Tollip played an important role in the regulation of HCC development by engaging PI3K/AKT signaling pathway. These evidences suggested that the blockade of Tollip-PI3K/AKT axis was an ideal therapeutic treatment for management of HCC.

Methylation‑associated inactivation of JPH3 and its effect on prognosis and cell biological function in HCC.

In recent years, researchers have found that epigenetics plays an important role in the occurrence and development of hepatocellular carcinoma (HCC). DNA methylation is involved in the proliferation and metastasis of HCC. However, the junctophilin 3 (JPH3) level and the potential regulatory mechanism of its DNA methylation in HCC remain uncertain. In the present study, 73 HCC samples were enrolled to analyze the expression of JPH3. Reverse-transcription quantitative PCR, western blotting and immunohistochemistry were used to detect the expression of JPH3 in HCC. Kaplan-Meier method and Cox regression analysis were applied to evaluate the prognostic impact of JPH3 on HCC patients. DNA methylation-specific PCR and bisulfite Sanger sequencing were used to detect the degree of DNA methylation of JPH3 in HCC. The demethylation drug 5-Aza-2′-deoxycytidine (5-Aza) was used to reduce the DNA methylation of JPH3. The role of JPH3 in the malignant biological behavior of HCC by promoting epithelial-mesenchymal transition (EMT) was confirmed by functional cell experiments. The results showed that JPH3 exhibited low levels in HCC tissues and cell lines. HCC patients with low expression of JPH3 had poor survival outcomes. JPH3 had higher DNA methylation levels in HCC tissues and cell lines. When the demethylation drug 5-Aza was used to reduce the degree of methylation of JPH3, its protein expression level was significantly increased and it significantly inhibited the malignant biological behavior of HCC cells. Additionally, effective increase in the expression of JPH3 through gene regulation technology also inhibited the proliferation, invasion and migration of HCC cells. After altering the DNA methylation level of JPH3, the EMT of HCC cells was also affected. Therefore, our study demonstrated the inactivation of JPH3 by promoter methylation and its function as a tumor suppressor in HCC. JPH3 may serve as a biomarker for early diagnosis and as a potential therapeutic target for HCC.

LncRNA LINC00924 upregulates NDRG2 to inhibit epithelial-mesenchymal transition via sponging miR-6755-5p in hepatitis B virus-related hepatocellular carcinoma.

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is a life-threatening cancer. Long noncoding RNAs participate in HBV-related HCC progression. Based on the bioinformatics analysis, LINC00924 downregulation is positively related to unfavorable outcomes in patients with HBV-related HCC. Herein, we detected the biological functions and regulatory system of LINC00924 in HCC. The LINC00924 downregulation in HBV-related HCC tissues and cells was revealed by reverse transcription-quantitative polymerase chain reaction. Functionally, as Transwell assays and western blotting indicated, LINC00924 elevation inhibited HCC cell invasion and epithelial-mesenchymal transition (EMT). The binding site between LINC00924 and miR-6755-5p was determined by luciferase reporter assays. miR-6755-5p was confirmed to target NDRG2. miR-6755-5p upregulation decreased NDRG2 messenger RNA (mRNA) and protein levels. The mRNA and protein levels of NDRG2 were downregulated in tissues and cells. NDRG2 knockdown attenuated the inhibition induced by LINC00924 overexpression on invasion and EMT of HCC cells. In summary, LINC00924 increases NDRG2 expression to inhibit EMT by targeting miR-6755-5p in HBV-related HCC.

18[Formula: see text]-Glycyrrhetinic Acid Inhibits TGF-[Formula: see text]-Induced Epithelial-to-Mesenchymal Transition and Metastasis of Hepatocellular Carcinoma by Targeting STAT3.

18[Formula: see text]-glycyrrhetinic acid (GA) is the active ingredient of the traditional Chinese medicinal herb Glycyrrhizae radix et rhizoma. We previously demonstrated that GA inhibited tumor growth in hepatocellular carcinoma (HCC). However, the effect of GA on transforming growth factor-[Formula: see text] (TGF-[Formula: see text]-induced epithelial-mesenchymal transition (EMT) and metastasis were still unclear. In this study, in vitro transwell assays and immunofluorescence (IF) demonstrated that GA inhibited TGF-[Formula: see text]-induced migration, invasion and EMT of HCC cells. However, it had little effect on the inhibition of proliferation by TGF-[Formula: see text]. Moreover, we confirmed that GA suppressed the metastasis of HCC cells in vivousing an ectopic lung metastasis model. Furthermore, we found that GA inhibited TGF-[Formula: see text]-induced EMT mainly by reducing the phosphorylation of signal transducer and activator of transcription 3 (STAT3), which played an essential role in TGF-[Formula: see text]-induced EMT and cell mobility. Mechanistically, GA inhibited the phosphorylation of STAT3 by increasing the expression of Src homology 2 domain-containing protein tyrosine phosphatases 1 and 2 (SHP1 and SHP2). Therefore, we concluded that GA inhibited TGF-[Formula: see text]-induced EMT and metastasis via the SHP1&SHP2/STAT3/Snail pathway. Our data provide an attractive therapeutic target for future multimodal management of HCC.

MiR-369-5p inhibits the proliferation and migration of hepatocellular carcinoma cells by down-regulating HOXA13 expression

MicroRNA (miRNA) is vital to the progression of hepatocellular carcinoma (HCC). Thereinto, miR-369-5p could yield assorted effects on various cancers, but there are few reports concerning the effect of miR-369-5p on HCC. Thus this study aimed to investigate the effect and mechanism of miR-369-5p in HCC. The data of miR-369-5p and HOXA13 expressions in liver hepatocellular carcinoma (LIHC) were analyzed by starBase, and then the miR-369-5p expression in HCC tissues and cells was detected by quantitative real-time PCR. Subsequently, miR-369-5p mimic was transfected into HCC cells and then its effects on cell activities were evaluated by cell counting kit-8, colony formation, wound healing, transwell assays, respectively. Expressions of epithelial-mesenchymal transition (EMT)-related genes were determined by western blot. The targeting relationship between miR-369-5p and HOXA13 was predicted by Targetscan and verified by dual-luciferase reporter assay. Pearson correlation test was used to analyze the correlation between HOXA13 and miR-369-5p. The above assays were experimented again to investigate the effects of HOXA13 on biological activity and EMT of HCC cells. MiR-369-5p expression was down-regulated and HOXA13 expression was up-regulated in LIHC. MiR-369-5p targeted HOXA13 and the expression of miR-369-5p was negatively correlated with the HOXA13 expression. MiR-369-5p inhibited the viability, proliferation, migration and invasion of HCC cells, increased E-cadherin level and decreased N-cadherin and Vimentin expressions. Concurrently, HOXA13 overexpression could counteract the effects of miR-369-5p on biological activity and EMT-related biomarkers of HCC cells. To conclude, miR-369-5p inhibits the viability, proliferation, migration and invasion of HCC cells by repressing the expression of HOXA13.