Evaluation of the effect of Helicobacter pylori -derived OMVs and released exosomes from stomach cells treated with OMVs on the expression of genes related to the TGF-β/SMAD signaling pathway in hepatocellular carcinoma

Etomidate elicits anti-tumor capacity by disrupting the JAK2/STAT3 signaling pathway in hepatocellular carcinoma

Currently, chemokines and their receptors, CXCL12-CXCR4 and CCL21-CCR7 axes, are deemed vital factors in the modulation of angiogenesis and are crucial for the growth and development of liver cancer. Tumor-derived DNA can be recognized by immune cells to induce an autoimmune response. In this study, we demonstrated the mechanism of tumor-derived DNA on the CXCL12-CXCR4 and CCL21-CCR7 axes of hepatocellular carcinoma (HCC) cells and the regulatory effect of sinomenine hydrochloride. Tumor-derived DNA was separated from HCCLM cell lines. Tumor-derived DNA was transfected into SK-Hep1 cells by Lipofectamine 2000. We found that sinomenine hydrochloride reduced the expression of CXCR4, CXCR12, CCR7, and CCL21 in HCC cells, suppressed the growth and invasion of HCC cells, and increased apoptosis. In contrast to the controls, the protein expressions of CXCR4, CXCL12, CCR7, CCL21, P-ERK1/2, MMP-9, and MMP-2 in SK-Hep1 cells were significantly increased after transfection of tumor-derived DNA, while the increase was reversed by sinobine hydrochloride. Acid sinomenine interferes with tumor-derived DNA and affects ERK/MMP signaling via the CXCL12/CXCR4 axis in HCC cells. CXCR4 siRNA and CCR7 siRNA attenuated tumor-derived DNA activation of ERK1/2/MMP2/9 signaling pathways in HCC cells. CXCR4-oe and CCR7-OE enhance the stimulation of erK1/2/MMP2/9 signaling pathway by tumor-derived DNA in HCC cells. Tumor-derived DNA reduced apoptosis and increased invasion of SK-Hep1 cells by CXCL12-CXCR4 axis and CCL21-CCR7 axis, and sinobine hydrochloride reversed this regulation. These results strongly suggest that tumor-derived DNA can increase the growth and invasion of oncocytes via the upregulation of the expression of CXCL12-CXCR4 and CCL21-CCR7 axis and through ERK1/2/MMP2/9 signaling pathway in HCC cells, and sinobine hydrochloride can inhibit this signaling pathway, thus inhibiting HCC cells. These results provide new potential therapeutic targets for blocking the progression of HCC induced by CXCL12-CXCR4 axis and CCL21-CCR7.

The purpose of this study was to investigate the role of microRNA-148a (miR-148a) in hepatocellular carcinoma (HCC) metastasis and explore its potential mechanism in HCC cells. Expression levels of miR-148a were measured using qRT-PCR in 120 HCC tissue samples and two HCC cell lines. Migration and invasion assays were used to determine the role of miR-148a in HCC cells. Flow cytometry was used to access the effect of miR-148a on cell cycle of HCC cells. Western blot was performed to analyze the effect of miR-148a on epithelial-to-mesenchymal transition (EMT) and PI3K/AKT signaling pathways in HCC cells. Luciferase reporter assay was conducted to explore the downstream targets and biological function of miR-148a in HCC cells. The results showed that level of miR-148a was significantly downregulated in both HCC tissue and plasma samples in HCC patients. A higher level of miR-148a was positively correlated with better survival time and prognosis of HCC patients. Transfection of miR-148a inhibited the proliferation, migration and invasion of HCC cell lines. Transfection of miR-148a arrested HCC cells at S phase and promoted apoptosis of HCC cells. Death receptor-5 (DR-5) was identified as a direct target of miR-148a in HCC cell lines. Western blot and qRT-PCR analyses showed that miR-148a upregulated EMT and downregulated PI3K/AKT signaling pathways in HCC cell lines. In conclusion, data in the current study indicate that miR-148a inhibits HCC cells growth via downregulation of EMT and PI3K/AKT signaling pathways by targeting death receptor. These data suggest that miR-148a may serve as a therapeutic target for HCC cancer therapy in the future.

Hepatocellular carcinoma (HCC) is one of the most prevalent human malignancies worldwide and has high morbidity and mortality. Elucidating the molecular mechanisms underlying HCC recurrence and metastasis is critical to identify new therapeutic targets. This study aimed to determine the roles of aminopeptidase N (APN, also known as CD13) in HCC proliferation and metastasis and its underlying mechanisms. We detected APN expression in clinical samples and HCC cell lines using immunohistochemistry, flow cytometry, real-time PCR, and enzyme activity assays. The effects of APN on HCC metastasis and proliferation were verified in both in vitro and in vivo models. RNA-seq, phosphoproteomic, western blot, point mutation, co-immunoprecipitation, and proximity ligation assays were performed to reveal the potential mechanisms. We found that APN was frequently upregulated in HCC tumor tissues and high-metastatic cell lines. Knockout of APN inhibited HCC cell metastasis and proliferation in vitro and in vivo. Functional studies suggested that a loss of APN impedes the ERK signaling pathway in HCC cells. Mechanistically, we found that APN might mediate the phosphorylation at serine 31 of BCKDK (BCKDKS31), promote BCKDK interacting with ERK1/2 and phosphorylating it, thereby activating the ERK signaling pathway in HCC cells. Collectively, our findings indicate that APN mediates the phosphorylation of BCKDKS31 and activates its downstream pathway to promote HCC proliferation and metastasis. Therefore, the APN/BCKDK/ERK axis may serve as a new therapeutic target for HCC therapy, and these findings may be helpful to identify new biomarkers in HCC progression.

Inhibition of B7-H4 promotes hepatocellular carcinoma cell apoptosis and autophagy through the PI3K signaling pathway

BackgroundCartilage oligomeric matrix protein (COMP) is known to promote fibrosis in skin, lung and liver. Emerging evidence shows that COMP plays critical roles in tumor development, including breast cancer, colon cancer and hepatocellular carcinoma (HCC). Nevertheless, the role of COMP in HCC proliferation and metastasis and its underlying mechanisms remain fully unclear.MethodsSerum COMP was determined by ELISA. Cell Counting Kit-8 and plate colony formation were performed to evaluate cell proliferation. Wound healing and transwell assays were used to determine migration and invasion of HCC cells. Western blotting and immunofluorescence were carried out for detection of epithelial-to-mesenchymal transition (EMT) markers and MMPs in HCC cells. The in vivo role of COMP was evaluated using mouse models. We also measured effects of hepatic stellate cells (HSCs)-conditioned medium (CM) on HCC progression using transwell coculture system.ResultsHere, we found that serum COMP levels in HCC patients were significantly higher than those in healthy controls. Accordingly, high serum COMP levels in HCC patients significantly correlated with malignant clinical characteristics and poor clinical outcomes. Next, we investigated that recombinant human COMP protein (rCOMP) treatment resulted in increased abilities of proliferation, invasion and migration of HCC cells. Furthermore, rCOMP treatment enhanced proliferative and metastatic colonization of HCC cells in vivo. Mechanistically, CD36 receptor played an essential role in COMP-mediated HCC cell proliferation and metastasis. Functionally, COMP/CD36 signaling caused phosphorylation of ERK and AKT, resulting in the upregulation of tumor-progressive genes such as EMT markers, MMP-2/9, Slug and Twist in HCC cells. Interestingly, we revealed that COMP was secreted by HSCs. CM of LX2 cells with COMP knockdown showed weaker effects on the activation of MEK/ERK and PI3K/AKT signaling pathways in HCC cells compared to control CM.ConclusionsOur findings indicated that HSCs-derived COMP collaborated with CD36 and subsequently played an essential role in MEK/ERK and PI3K/AKT-mediated HCC progression. COMP might act as a promising target for the diagnosis and treatment of aggressive HCC.

Sevoflurane Inhibits Metastasis in Hepatocellular Carcinoma by Inhibiting MiR-665-Induced Activation of the ERK/MMP Pathway

Current evidence finds that circulating exosomal lncRNA focally amplified lncRNA on chromosome 1 (FAL1) promotes the progression of hepatocellular carcinoma (HCC). However, the underlying mechanism of serum extracellular vesicular FAL1 in HCC progression remains elusive. Here, we extracted extracellular vesicles (EVs) from serum samples of HCC patients and healthy volunteers, and found that FAL1 was highly enriched in the serum EVs of HCC patients. Then, macrophages were treated with EVs alone or together with small interfering RNA against FAL1 (si-FAL1). The data indicated that FAL1-enriched EVs induced macrophage M2 polarization, while silencing FAL1 in macrophages antagonized the role of EVs. Moreover, HepG2 cells were co-cultured with the conditioned macrophages, and co-culturing with EVs-incubated macrophages promoted HepG2 cell proliferation, invasion, cell cycle progression, and colony formation, and inhibited cell apoptosis and sorafenib sensitivity, while interfering FAL1 in macrophages reversed these effects. Consistently, ectopic expression of FAL1 in macrophages also induced macrophage M2 polarization, and co-culture of FAL1-overexpressing macrophages with HepG2 cells facilitated the malignant progression of HepG2 cells. Furthermore, co-culturing HepG2 cells with EVs-incubated macrophages activated the Wnt/β-catenin signaling pathway, and treatment with a Wnt/β-catenin pathway inhibitor IWP-2 partially neutralized the effect of EVs-incubated macrophages on HepG2 cell malignant behaviors. Additionally, FAL1 enriched EVs-incubated macrophages markedly increased mouse xenograft tumor growth. In conclusion, extracellular vesicular lncRNA FAL1 promotes macrophage M2 polarization and further activates the Wnt/β-catenin signaling pathway in HCC cells, thus promoting HCC progression.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers in the world. MicroRNAs play a pivotal role in the progression of various cancers. To date, very little attention has been paid to miR-4458. Therefore, the aim of our study was to explore the function and underlying molecular mechanism of miR-4458 in HCC. We found that the expression of miR-4458 was reduced in HCC tissues and cell lines. Forced overexpression of miR-4458 inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of HCC cells, while downregulation of miR-4458 promoted the aggressive phenotype. Furthermore, transforming growth factor beta receptor 1 (TGFBR1), the modulator of the TGF-β signaling pathway, was verified to be a novel target gene of miR-4458 by dual-luciferase reporter gene assay. Upregulated miR-4458 dramatically abolished TGFBR1 and p-Smad2/3 expression, thus blocking the TGF-β signaling pathway. Moreover, restoration of TGFBR1 partially rescued the miR-4458-mediated suppressive effect on the migration, invasion, and EMT and reactivated the TGF-β signaling pathway in HCC cells. In summary, our findings first demonstrated a mechanism of miR-4458 in HCC cell migration, invasion, and EMT by regulating the TGF-β signaling pathway via directly targeting TGFBR1.

BackgroundOur previous studies have reported the down-regulation of EGFL8 correlates to the development and prognosis of colorectal and gastric cancer. The present study is carried out to explore the expression pattern and role of EGFL8 in hepatocellular carcinoma (HCC).Methods and materialsEGFL8 expression in 102 cases of HCC tissues matched with adjacent non-tumorous liver tissues, a normal liver cell line and three liver cancer cell lines with different metastatic capacity was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. Moreover, the clinicopathological features and prognosis of HCC patients were correlated with expression of EGFL8. Subsequently, the gain-and loss-of-function experiments were carried out to investigate the biological function of EGFL8 in HCC. We also used N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)- phenylglycine t-butyl ester (DAPT), an inhibitor for Notch signaling pathway, in these experiments to verify the involvement of Notch signaling pathway in the effects of EGFL8. Additionally, a mouse model was established to investigate the effect of EGFL8 on metastasis of HCC cells. The expression of Notch signaling pathway in HCC cells and xenograft mouse tumors were detected by Western blot and immunohistochemistory.ResultsThe expression of EGFL8 was significantly decreased in HCC tissues and cell lines and EGFL8 down-regulation correlated to multiple nodules, vein invasion, high TNM stage and poor prognosis of HCC. Interestingly, the expression levels of EGFL8 in three liver cancer cell lines were negatively associated with their metastatic capacity. In vitro and in vivo experiments indicated that EGFL8 obviously suppressed metastasis and invasion of HCC cells but slightly promoted apoptosis. Meanwhile, the expression of Notch signaling pathway was obviously suppressed in EGFL8 overexpressed HCCLM3 cells and xenograft mouse tumors generated from these cells but markedly elevated in EGFL8 depleted Hep3B cells. Furthermore, the up-regulated expression of Notch signaling pathway and effects induced by EGFL8 knockdown in Hep3B cells could be counteracted by DAPT treatment.ConclusionThe down-regulation of EGFL8 was correlated to progression and poor prognosis of HCC and regulates HCC cell migration, invasion and apoptosis through activating the Notch signaling pathway, suggesting EGFL8 as a novel therapeutic target and a potential prognostic marker for HCC.

FDX1 downregulation activates mitophagy and the PI3K/AKT signaling pathway to promote hepatocellular carcinoma progression by inducing ROS production

Long noncoding RNAs (lncRNAs) have been associated with hepatocellular carcinoma (HCC), but the underlying molecular mechanisms of their specific association with hepatocarcinogenesis have not been fully explored.Methods: miR503HG was identified by microarray and validated by real-time PCR. Survival analysis was evaluated using the Kaplan-Meier method and assessed using the log-rank test. In vitro and in vivo assays were preformed to explore the biological effects of miR503HG in HCC cells. The interaction of miR503HG with HNRNPA2B1 was identified by RNA pull-down and RNA immunoprecipitation. Expression of HNRNPA2B1 was examined by western blotting, immunofluorescence and immunohistochemical analyses, while HNRNPA2B1 ubiquitination was detected by immunoprecipitation.Results: We have identified 713 differentially expressed lncRNAs in 12 pairs of HCC tissues compared with corresponding noncancerous liver tissues. One of these lncRNAs, miR503HG, the host gene of miR503, is markedly decreased in HCC. Expression level of miR503HG is significantly associated with the time to recurrence and overall survival and is an independent risk factor for recurrence and survival. Enhanced expression of miR503HG could noticeably inhibit HCC invasion and metastasis in vitro and in vivo. Further investigation suggested that miR503HG could specifically interact with the heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1). miR503HG promoted HNRNPA2B1 degradation via the ubiquitin-proteasome pathway, which reduced the stability of p52 and p65 mRNA, and simultaneously suppressed the NF-κB signaling pathway in HCC cells. In addition, miR503HG can function synergistically with miR503 to inhibit HCC migration.Conclusion: Our findings support a role for miR503HG in tumor recurrence risk and survival prediction in HCC patients. We demonstrate a novel mechanism by which miR503HG inhibits the NF-κB signaling pathway and exerts its metastatic tumor suppression function through modulating the ubiquitination status of HNRNPA2B1.

Simultaneous activation of apoptosis and cGAS-STING pathway with a mitochondria-targeted dehydrocholic acid compound for enhanced hepatocellular carcinoma chemoimmunotherapy.

High‐mobility group protein A2 (HMGA2) is highly expressed in hepatocellular carcinoma (HCC) cells and contributes to tumor metastasis and poor patient survival. However, the molecular mechanism through which HMGA2 is transcriptionally regulated in HCC cells remains largely unclear. Here, we showed that the expression HMGA2 was upregulated in HCC, and that elevated HMGA2 could promote tumor metastasis. Incubation of HCC cells with epidermal growth factor (EGF) could promote the expression of HMGA2 mRNA and protein. Mechanistic studies suggested that EGF can phosphorylate p300 at Ser1834 residue through the PI3K/Akt signaling pathway in HCC cells. Knockdown of p300 can reverse EGF‐induced HMGA2 expression and histone H3‐K9 acetylation, whereas a phosphorylation‐mimic p300 S1834D mutant can stimulate HMGA2 expression as well as H3‐K9 acetylation in HCC cells. Furthermore, we identified that p300‐mediated H3‐K9 acetylation participates in EGF‐induced HMGA2 expression in HCC. In addition, the levels of H3‐K9 acetylation positively correlated with the expression levels of HMGA2 in a chemically induced HCC model in rats and human HCC specimens.

The ubiquitin-like protein FAT10 in hepatocellular carcinoma cells limits the efficacy of anti-VEGF therapy