HCC Cell Migration Research Articles

TEC kinase stabilizes PLK4 to promote liver cancer metastasis.

Aberrated PLK4 expression has been reported in different malignancies and causes centrosome amplification, aneuploidy, and genomic instability. However, the mechanism by which PLK4 is regulated in carcinogenesis remains not fully characterised. Here, we showed that PLK4 was overexpressed in human HCC and overexpression of PLK4 predicted poorer patient prognosis. Unexpectedly, we found that induced expression of PLK4 promotes, but knockdown of PLK4 inhibits, HCC cell migration and invasion. Mechanistically, we found that TEC tyrosine kinase, which also promotes HCC cell migration, stabilizes PLK4 by phosphorylation. TEC directly phosphorylates PLK4 at tyrosine 86 residue, which not only stabilizes the protein but also enhances PLK4-mediated HCC cell invasion. Further investigation by transcriptome sequencing indicated that PLK4 promotes the phosphorylation of focal adhesion kinase to regulate the focal adhesion pathway in HCC cell migration. Taken together, our results demonstrated that PLK4 plays an important role in HCC metastasis and revealed for the first time the mechanism by which PLK4 promotes HCC metastasis via TEC phosphorylation.

Investigating the Association between COMMD3 Expression and the Prognosis of Hepatocellular Carcinoma.

The Copper Metabolism MURR1 Domain (COMMD) family proteins are known to play roles in promoting or inhibiting the proliferation, migration and invasion of tumor cells. However, the role of COMMD3 in hepatocellular carcinoma are still unclear. By investigating the TCGA datasets, we found that the mRNA expression of COMMD3 was significantly upregulated in hepatocellular carcinoma tissue compared with normal liver tissue, which was further supported by Oncomine dataset, Western blot, qRT-PCR, and IHC analysis. Moreover, Kaplan-Meier survival analysis showed that the high expression of COMMD3 was associated with poor overall survival (OS) and disease-free survival (DFS). Consistently, the clinic-pathological analysis found that the overexpression of COMMD3 was correlated with advanced TNM stage, advanced T stage and vascular invasion. By performing multivariate analysis, we found that the expression of COMMD3 was an independent influencing factor on OS and DFS. Furthermore, we knocked down COMMD3 in HCC cells via RNA interference. The results showed that silencing COMMD3 could inhibit the migration, invasion, and angiogenesis of HCC cells. Finally, we established xenograft tumor model in nude mice, and the knockdown of COMMD3 suppressed tumor growth and angiogenesis. In summary, our study showed that the high expression of COMMD3 was correlated with poor prognosis in HCC patients and contributed to migration, invasion and angiogenesis of HCC cells.

MiR-93-5P Represses the Gluconeogenesis of Hepatocellular Carcinoma while Boosting Its Glycolysis and Malignant Progression by Suppressing PCK1.

Herein, we explored effects of miR-93-5p and gluconeogenic rate-limiting enzyme PCK1 on HCC cells. Bioinformatics analysis and cell experiments confirmed that, compared with expression in normal tissue and cells, miR-93-5p in HCC was abnormally upregulated while PCK1 expression was remarkably downregulated. PCK1 overexpression repressed proliferation, migration, and invasion of HCC cells, and blocked cell cycle in G0/G1 phase. During this process, glucose production was boosted while the production of pyruvate, lactic acid, citric acid, and malic acid was reduced, suggesting that the effect was related to inhibition of glycolysis and induction of gluconeogenic pathways. Elevated miR-93-5p level promoted proliferation, migration, and invasion of HCC cells, accelerated development of cell cycle, activated glycolysis, and suppressed gluconeogenesis. In addition, when miR-93-5p and PCK1 were concurrently upregulated, the abovementioned promoting effects were canceled out. These investigations demonstrated that promoting effect of miR-93-5p on HCC cell growth may be carried out by inhibiting the PCK1 expression, suggesting that miR-93-5p and PCK1 could be applied as new biomarkers or novel therapeutic targets for HCC diagnosis.

Mir-454-3p induced WTX deficiency promotes hepatocellular carcinoma progressions through regulating TGF-β signaling pathway.

Background: Wilms tumor gene on X chromosome (WTX) is an X-linked tumor suppressor gene in Wilms tumor; however, however, the molecular mechanism of WTX in the occurrence and development of HCC has not been reported.Methods: The expression of miR-454-3p and WTX wre analyzed in 32 matched human HCC and normal tissue samples. The molecular mechanisms of miR-454-3p/WTX/TGFβ signaling in cell proliferation, migration, invasion and autophagy were investigated in vitro and in vivo.Results: WTX expression was downregulated in HCC tissues; lower WTX levels were associated with poor HCC patient outcomes. WTX loss triggers the activation of TGF-β signaling, which promotes HCC cells proliferation, migration, invasion and autophagy. Further mechanistic study showed that the aberrant upregulation of miR-454-3p was identified as the reason of WTX loss in HCC.Conclusions: WTX is a tumor suppressor gene in HCC, miR-454-3p/WTX/TGFβ signaling will provide a new direction for the diagnosis and treatment of HCC.

Hsa_circ_0007059 sponges miR-421 to repress cell growth and stemness in hepatocellular carcinoma by the PTEN-AKT/mTOR pathway

BackgroundHepatocellular carcinoma (HCC) is a substantial health concern worldwide. Increasing studies have suggested that circle RNAs (circRNAs) function as new regulators in HCC progression. The present work explored the role of hsa_circ_0007059 (circ_0007059) in the developing process of hepatocarcinogenesis. MethodsThe circ_0007059 level in HCC was determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and northern blot. Its biological role in HCC cells was assessed using 3-(4,5-Dimethylthiazol-2-yl)− 2,5-diphenyltetrazolium bromide (MTT), colony formation, flow cytometry, Transwell, sphere formation and western blotting analyses. Bioinformatics analysis, luciferase reporter, and RNA immunoprecipitation (RIP) assays were used to test the regulatory mechanisms of circ_0007059. ResultsOur results revealed that circ_0007059 expression was downregulated in HCC samples and cells. Moreover, circ_0007059 overexpression inhibited HCC cell proliferation, migration, invasion, and stem cell-like property, and strengthened cell apoptosis. In mechanism, circ_0007059 suppressed AKT/mTOR pathway by positively regulating phosphatase and tensin homolog (PTEN) expression. Additionally, circ_0007059 acted as a positive regulator of PTEN through controlling the availability of miR-421. Rescue assays demonstrated that PTEN knockdown or SC79 (AKT agonist) eliminated the effect of circ_0007059 on HCC cell phenotypes. ConclusionCirc_0007059 sponges miR-421 to inhibit oncogenic cellular process in HCC by mediating the PTEN-AKT/mTOR pathway.

Experimental Study of Rhein Inhibits the Invasion and Migration of HepG2 Cells by ERK Signaling Pathway

Objective: To investigate the effectiveness of Rhein on the proliferation, invasion and migration of human hepatoma cell line HepG2 and its possible mechanism.Methods: Human hepatoma cell line HepG2 was treated with different concentrations of Rhein (Rhein treatment group) and culture in culture medium alone (control group).The proliferation activity of the cells was determined by methyl Thiazolyl Tetrazolium (MTT) colorimetry.Transwell assay detected the invasion and migration of cells in each group.Cell scratch test was used to detect the migration ability of cells in each group.Excella-phospho-excellar signal-regulated kinase (P-ERK) activity was determined by ELISA after treatment with 50μ mol/L Rhein at different times.Western blot was used to detect ERK protein expression in HepG2 cells treated with 50 μmol/L Rhein.Results: Compared with the control group, the proliferation activity, invasion and migration ability of HepG2 cells in the Rhein treatment group were all decreased (P< 0.05), and the p-ERK relative activity of HepG2 cells treated with Rhein was decreased (P < 0.05).Conclusion: Rhein inhibits the invasion and migration of HCC cells, possibly by inhibiting the ERK pathway

MiR-221-3p regulates hepatocellular carcinoma cell proliferation, migration and invasion via targeting LIFR

Introduction and ObjectivesHepatocellular carcinoma (HCC) is one of the most common and fatal cancers in the world. This study aims to investigate the mechanism by which miR-221-3p regulates HCC cell proliferation, migration and invasion, so as to provide a new idea for targeted therapy towards HCC. Materials and MethodsExpression quantification data including mature miRNA and mRNA were accessed from TCGA-LIHC dataset, and matched clinical information was obtained as well, which helped identify the miRNA of interest. Thereafter, effect of the miRNA on HCC cell biological functions was assessed with a series of in vitro experiments, such as qRT-PCR, MTT, wound healing assay and Transwell. To gain more insight into the mechanism of the miRNA in HCC, bioinformatics method was conducted to predict downstream target gene. The potential targeting relationship between the miRNA and the predicted mRNA was validated by dual-luciferase reporter assay. Western blot was performed to test protein expression. ResultsMiR-221-3p identified by differential expression analysis was found to be significantly elevated in HCC tissue. Overexpressing miR-221-3p noticeably enhanced HCC cell proliferative, migratory and invasive abilities. Leukemia inhibitory factor receptor (LIFR), confirmed as a downstream target of miR-221-3p in HCC by dual-luciferase reporter assay, was poorly expressed in HCC tissue and cells. Additionally, the expression of LIFR was decreased following the targeted binding between miR-221-3p and LIFR 3’-UTR, while increasing the expression of LIFR attenuated the promoting effect of miR-221-3p on HCC cells. ConclusionMiR-221-3p is an oncogene in HCC cells, and it exerts its role in HCC cell viability and motility via targeting LIFR.

Circular RNA circUBE2J2 acts as the sponge of microRNA-370-5P to suppress hepatocellular carcinoma progression

Accumulating evidences indicate that circular RNAs (circRNAs), a class of non-coding RNAs, play important roles in tumorigenesis. However, the function of circRNAs in hepatocellular carcinoma is largely unknown. CircRNA microarray was performed to identify abnormally expressed circRNAs in HCC tissue samples. We conducted Kaplan–Meier survival analysis to explore the significance of circUBE2J2 in clinical prognosis. Then, we examined the functions of circUBE2J2 in HCC by cell proliferation, migration, and mouse xenograft assay. We identified miR-370-5P as a circUBE2J2-related microRNA by using biotin-labeled circUBE2J2 probe to perform RNA antisense purification (RAP) assay in HCC cells. The dual luciferase reporter assay and RNA pulldown assays were employed to verify the relationships among circUBE2J2, miRNA-370-5P, and KLF7. Microarray analysis and qRT-PCR verified a circRNA termed circUBE2J2 that was downregulated in HCC. Kaplan–Meier survival analysis showed that downregulated circUBE2J2 was correlated with poorer survival. CircUBE2J2 expression in HCC cells was selectively regulated via luciferase reporter assays; circUBE2J2 and KLF7 were observed to directly bind to miR-370-5P. Furthermore, knockdown of circUBE2J2 in HCC could downregulate KLF7, the target of miR-370-5P, thus promoting the proliferation and migration of HCC cells. Then the related experiment suggested that circUBE2J2 could regulate the expression of KLF7 by sponging miR-370-5p. In summary, we infer that circUBE2J2 may act as a competing endogenous RNA (ceRNA) to regulate KLF7 expression through sponging miR-370-5P and play a regulatory functions in HCC. CircUBE2J2 may be a diagnostic biomarker and potential target for HCC therapy.

Delivery of microRNA-21-sponge and pre-microRNA-122 by MS2 virus-like particles to therapeutically target hepatocellular carcinoma cells.

MicroRNAs are related to the development of hepatocellular carcinoma and can serve as potential therapeutic targets. Therapeutic strategies increasing tumor-suppressive microRNAs and reducing oncogenic microRNAs have been developed. Herein, the effects of simultaneously altering two microRNAs using MS2 virus-like particles were studied. The sequences of microRNA-21-sponge and pre-microRNA-122 were connected and cloned into a virus-like particle expression vector. Virus-like particles containing microRNA-21-sponge and pre-microRNA-122 sequences were prepared and crosslinked with a cell-specific peptide targeting hepatocellular carcinoma cells. Delivery effects were studied using RT-qPCR and functional assays to investigate the level of target mRNAs, cell toxicity, and the effects of proliferation, invasion, and migration. Virus-like particles delivered miR-21-sponge into cells, with the Ct value reaching 10 at most. The linked pre-miR-122 was processed into mature miR-122. The mRNA targets of miR-21 were derepressed as predicted and upregulated 1.2-2.8-fold, and the expression of proteins was elevated correspondingly. Proliferation, migration, and invasion of HCC cells were inhibited by miR-21-sponge. Simultaneous delivery of miR-21-sponge and miR-122 further decreased proliferation, migration, and invasion by up to 34%, 63%, and 65%, respectively. And the combination promoted the apoptosis of HCC cells. In conclusion, delivering miR-21-sponge and miR-122 using virus-like particles modified by cell-specific peptides is an effective and convenient strategy to correct microRNA dysregulation in hepatocellular carcinoma cells and is a promising therapeutic strategy for hepatocellular carcinoma.

Clinical Significance of Acylphosphatase 1 Expression in Combined HCC-iCCA, HCC, and iCCA.

Combined hepatocellular and cholangiocarcinoma is a rare primary liver cancer with histological features of both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. Little is known about the prognostic features and molecular mechanism of cHCC-iCCA. Acylphosphatase 1 is a cytosolic enzyme that produces acetic acid from acetyl phosphate and plays an important role in cancer progression. We evaluated the clinical significance of ACYP1 expression in cHCC-iCCA, HCC, and iCCA. ACYP1 immunohistochemistry was performed in 39 cases diagnosed with cHCC-iCCA. The prognosis was evaluated in three different cohorts (cHCC-iCCA, HCC, and iCCA). The relationships between ACYP1 expression and cell viability, migration, invasiveness, and apoptosis were examined using siRNA methods in vitro. In vivo subcutaneous tumor volumes and cell apoptosis were evaluated after downregulation of ACYP1 expression. Almost half of the patients with cHCC-iCCA were diagnosed with high ACYP1 expression. In all three cohorts, the cases with high ACYP1 expression had significantly lower overall survival, and high ACYP1 expression was identified as an independent prognostic factor. Downregulation of ACYP1 reduced the proliferative capacity, migration, and invasiveness of both HCC and iCCA cells. Moreover, knockdown of ACYP1 increased the ratio of apoptotic cells and decreased the expression of anti-apoptosis proteins. In vivo tumor growth was significantly inhibited by the transfection of ACYP1 siRNA, and the number of apoptotic cells increased. High ACYP1 expression could influence the prognosis of cHCC-iCCA, HCC, and iCCA patients. In vitro ACYP1 expression influences the tumor growth and cell viability in both HCC and iCCA by regulating anti-apoptosis proteins.

CHD1L augments autophagy-mediated migration of hepatocellular carcinoma through targeting ZKSCAN3

Autophagy is an important biological process in normal cells. However, how it affects tumor progression still remains poorly understood. Herein, we demonstrated that the oncogenic protein Chromodomain-helicase-DNA-binding-protein 1-like gene (CHD1L) might promote HCC cells migration and metastasis through autophagy. CHD1L could bind to the promotor region of Zinc finger with KRAB and SCAN domain 3 (ZKSCAN3), a pivotal autophagy suppressor, and inhibit its transcription. We established inducible CHD1L conditional knockout cell line (CHD1L-iKO cell) and found that the deletion of CHD1L significantly increased ZKSCAN3 expression both at mRNA and protein level. Deletion of CHD1L impaired the autophagic flux and migration of HCC cells, while specifically inhibiting ZKSCAN3 blocked these effects. Further exploration demonstrated that the enhanced tumor cell migration and metastasis induced by CHD1L was mediated through ZKSCAN3-induced autophagic degradation of Paxillin. In summary, we have characterized a previously unknown function of CHD1L in regulating tumor migration via ZKSCAN3-mediated autophagy in HCC. Further inhibition of CHD1L and its downstream autophagy signaling might shed new light on cancer therapeutics.

Elevated LINC01232 is associated with poor prognosis and HBV infection in hepatocellular carcinoma patients and contributes to tumor progression in vitro

BackgroundHepatocellular carcinoma (HCC) had high prevalence and poor prognosis, and hepatitis B virus (HBV) infection is a major risk factor. The aim of this study is to analyze the role of long intergenic noncoding RNA 01232 (LINC01232) in the prognosis and progression of HCC, and explore the relationship between LINC01232 and HBV infection. MethodsLINC01232 expression and its prognostic value were firstly analyzed using TCGA database. Quantitative real-time PCR was used to evaluate the expression of LINC01232 in HCC patients and cell lines. Kaplan-Meier curves were used to analyze the relationship between LINC01232 expression and HCC overall survival prognosis. Function-loss in vitro experiments were performed to demonstrate the role of LINC01232 in HCC progression. A luciferase reporter assay and Pearson correlation were used to confirm the relationship between LINC01232 and microRNA (miR)-708-5p in HCC. ResultsThe expression of LINC01232 was upregulated in HCC tissues and cell lines, and high LINC01232 was associated with worse overall survival in HCC. LINC01232 reduction inhibited HCC cells proliferation, migration and invasion. LINC01232 expression was significantly correlated with HBV infection and liver cirrhosis, and showed potential to distinguish HBV-infected HCC patients. miR-708-5p, as a HBV-related miRNA, was a potential target of LINC01232, and was negatively correlated with LINC01232 in HCC. ConclusionOur findings found that highly expressed LINC01232 may be a biomarker to indicate survival prognosis in HCC patients, especially in HBV-infected cases. In addition, LINC01232 plays as an oncogene in HCC progression, and its function may exert by sponging miR-708-5p.

RETRACTED ARTICLE: LNCAROD enhances hepatocellular carcinoma malignancy by activating glycolysis through induction of pyruvate kinase isoform PKM2

BackgroundMounting evidence has suggested the essential role of long non-coding RNAs (lncRNAs) in a plethora of malignant tumors, including hepatocellular carcinoma. However, the underlyling mechanisms of lncRNAs remain unidentified in HCC. The present work was aimed to explore the regulatory functions and mechanisms of LncRNA LNCAROD in HCC progression and chemotherapeutic response.MethodsThe expression of LNCAROD in HCC tissues and cell lines were detected by quantitative reverse transcription PCR (qPCR). Cancer cell proliferation, migration, invasion, and chemoresistance were evaluated by cell counting kit 8 (CCK8), colony formation, transwell, and chemosensitivity assays. Methylated RNA immunoprecipitation qRCR (MeRIP-qPCR) was used to determine N6-methyladenosine (m6A) modification level. RNA immunoprecipitation (RIP) and RNA pull down were applied to identify the molecular sponge role of LNCAROD for modulation of miR-145-5p via the competing endogenous RNA (ceRNA) mechanism, as well as the interaction between LNCAROD and serine-and arginine-rich splicing factor 3 (SRSF3). The interaction between insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and LNCAROD was also identified by RIP assay. Gain- or-loss-of-function assays were used to identify the function and underlying mechanisms of LNCAROD in HCC.ResultsWe found that LNCAROD was significantly upregulated and predicted a poorer prognosis in HCC patients. LNCAROD upregulation was maintained by increased m6A methylation-mediated RNA stability. LNCAROD significantly promoted HCC cell proliferation, migration, invasion, and chemoresistance both in vitro and in vivo. Furthermore, mechanistic studies revealed that pyruvate kinase isoform M2 (PKM2)-mediated glycolysis enhancement is critical for the role of LNACROD in HCC. According to bioinformatics prediction and our experimental data, LNCAROD directly binds to SRSF3 to induce PKM switching towards PKM2 and maintains PKM2 levels in HCC by acting as a ceRNA against miR-145-5p. The oncogenic effects of LNCAROD in HCC were more prominent under hypoxia than normoxia due to the upregulation of hypoxia-triggered hypoxia-inducible factor 1α.ConclusionsIn summary, our present study suggests that LNCAROD induces PKM2 upregulation via simultaneously enhancing SRSF3-mediated PKM switching to PKM2 and sponging miR-145-5p to increase PKM2 level, eventually increasing cancer cell aerobic glycolysis to participate in tumor malignancy and chemoresistance, especially under hypoxic microenvironment. This study provides a promising diagnostic marker and therapeutic target for HCC patients.

Knockdown of POLQ interferes the development and progression of hepatocellular carcinoma through regulating cell proliferation, apoptosis and migration

BackgroundDNA Polymerase Theta (POLQ) is a DNA polymerase involved in error-prone translesion DNA synthesis (TLS) and error-prone repair of DNA double-strand breaks (DSBs), whose function in hepatocellular carcinoma has not been investigated.MethodsIn the present study, both the data collected from the Cancer Genome Atlas (TCGA) and our group’s results showed higher POLQ expression in HCC tissues than the para-cancerous tissues, which was associated with higher malignancy and poor prognosis. POLQ knockdown HCC cell model (shPOLQ) was constructed along with the corresponding negative control (shCtrl) through lentivirus infection for loss-of-function study.ResultsWe found that, upon knockdown of POLQ, the proliferation and migration of HCC cells decreased and apoptosis percentage increased. Moreover, the percentage of cells in G2 phase significantly increased in shPOLQ group compared with shCtrl group. Xenografts in mice grafted with shPOLQ cells grew much slower than that transplanted with shCtrl cells, and expressed lower Ki67 level. Furthermore, an apoptosis-related signaling array was used to explore the involvement of downstream signaling pathways, suggesting the enhanced phosphorylation of HSP27 and JNK, and the de-activation of mTOR, PRAS40, ERK1/2 and STAT3 pathways.ConclusionsCollectively, our study revealed that POLQ may participate in the development of HCC, depletion of which may be a promising treatment strategy for HCC.

Circ-AKT3 promotes the proliferation and migration of HCC cells via down-regulating microRNA-335 expression.

Circ-AKT3 promotes the proliferation and migration of HCC cells via down-regulating microRNA-335 expression.

FNDC4 acts as an extracellular factor to promote the invasiveness of hepatocellular carcinoma partly via the PI3K/Akt signalling pathway.

FNDC4 is highly homologous to the exercise‐associated myokine FNDC5/irisin, which is highly expressed and promotes the invasion and metastasis of HCC cells. However, the function of FNDC4 remains unknown. Here, we report that FNDC4, an extracellular factor, plays important roles in the invasion and metastasis of HCC. We found that high FNDC4 expression is associated with poor survival in HCC patients and FNDC4 promotes the migration and invasion of HCC cells. Mechanistically, we found that FNDC4 is related to the PI3K/Akt signalling pathway to a certain extent. Specifically, the extracellular domain of FNDC4 acts as an extracellular factor to promote Akt phosphorylation levels in this pathway. These findings reveal that FNDC4 promotes the invasion and metastasis of HCC partly via the PI3K/Akt signalling pathway.

MARCH6 promotes hepatocellular carcinoma development through up-regulation of ATF2

BackgroundHepatocellular carcinoma (HCC) is a common cause of cancer mortality worldwide. Recent studies have shown that the polytopic enzyme membrane associated ring-CH-type finger 6 (MARCH6) participates in tumorigenesis, but its function in HCC development needs to be investigated. This study aimed to explore the role of MARCH6 in HCC.MethodsExpression of MARCH6 in human HCC samples was checked by immunohistochemical staining assay. Clinical relevance of MARCH6 and activating transcription factor 2 (ATF2) was analyzed from TCGA database. CCK-8, EdU staining, colony formation and transwell were performed to assess cell proliferation, growth and migration. Xenografted tumorigenesis was used to examine in vivo role MARCH6. Immunoblotting was applied to detect protein abundance.ResultsWe found that MARCH6 expression was elevated in human HCC samples. Over-expression of MARCH6 was associated with poor prognosis of HCC patients. Up-expression of MARCH6 promoted cell growth and migration of HCC cells. In contrast, the HCC cell growth and migration were suppressed by MARCH6 knockdown. Furthermore, the DNA synthesis was enhanced by MARCH6. The expression of ATF2 was potentiated by MARCH6 over-expression, while it was suppressed by MARCH6 silencing. TCGA database showed positive correlation between the expression of MARCH6 and ATF2. Importantly, ATF2 expression contributed to the oncogenic function of HCC cells.ConclusionOur findings suggest that MARCH6-mediated ATF2 up-regulation contributes to HCC development. MARCH6 may be a promising target for the diagnosis and treatment of HCC.

Circ-CSPP1 knockdown suppresses hepatocellular carcinoma progression through miR-493-5p releasing-mediated HMGB1 downregulation

BackgroundHepatocellular carcinoma (HCC) accounts for over 80% of primary liver cancers and leads to a high death rate. Research on circular RNAs (circRNAs) suggests that circRNAs are promising biomarkers for cancer treatment. This study aimed to explore the function of a novel circRNA (circ-CSPP1) in HCC. MethodsCirc-CSPP1 was obtained from the microarray data downloaded from the Gene Expression Omnibus (GEO) database. The expression of circ-CSPP1, miR-493-5p and high mobility group box 1 (HMGB1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, colony formation ability, migration and invasion were monitored using cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay and transwell assay, respectively. The protein levels of CyclinD1, Vimentin, matrix metallopeptidase 9 (MMP-9) and HMGB1 were detected by western blot. Xenograft models were established to investigate the function of circ-CSPP1 in vivo. The association between miR-493-5p and circ-CSPP1 or HMGB1 was predicted by the online tool starBase and ensured by dual-luciferase reporter assay. ResultsThe expression of circ-CSPP1 and HMGB1 was elevated, while the expression of miR-493-5p was declined in HCC tissues and cells. Circ-CSPP1 knockdown not only depleted HCC cell proliferation, formation, migration and invasion in vitro but also inhibited tumor growth in vivo. MiR-493-5p was a target of circ-CSPP1, and HMGB1 was a target of miR-493-5p. Rescue experiments presented that miR-493-5p deficiency reversed the effects of circ-CSPP1 knockdown, and HMGB1 overexpression reversed the effects of miR-493-5p restoration. Circ-CSPP1 sponged miR-493-5p to regulate HMGB1 expression. ConclusionKnockdown of circ-CSPP1 suppressed HCC development both in vitro and in vivo by upregulation of miR-493-5p and downregulation of HMGB1, hinting that circ-CSPP1 participated in HCC pathogenesis.

Autophagic Inhibition of Caveolin-1 by Compound Phyllanthus urinaria L. Activates Ubiquitination and Proteasome Degradation of β-catenin to Suppress Metastasis of Hepatitis B-Associated Hepatocellular Carcinoma.

Compound Phyllanthus urinaria L. (CP) is a traditional Chinese medicine (TCM) formula for cancer treatment in the clinic, particularly during progression of hepatitis B-associated hepatocellular carcinoma (HBV-associated HCC). Nevertheless, its anti-metastatic action and mechanisms are not well elucidated. In this study, CP was found to exert remarkable inhibitory effects on the proliferation, migration and invasion of HBV-associated HCC cells. The following network and biological analyses predicted that CP mainly targeted Caveolin-1 (Cav-1) to induce anti-metastatic effects, and Wnt/β-catenin pathway was one of the core mechanisms of CP action against HBV-associated HCC. Further experimental validation implied that Cav-1 overexpression promoted metastasis of HBV-associated HCC by stabilizing β-catenin, while CP administration induced autophagic degradation of Cav-1, activated the Akt/GSK3β-mediated proteasome degradation of β-catenin via ubiquitination activation, and subsequently attenuated the metastasis-promoting effect of Cav-1. In addition, the anti-cancer and anti-metastatic action of CP was further confirmed by in vivo and ex vivo experiments. It was found that CP inhibited the tumor growth and metastasis of HBV-associated HCC in both mice liver cancer xenograft and zebrafish xenotransplantation models. Taken together, our study not only highlights the novel function of CP formula in suppressing metastasis of HBV-associated HCC, but it also addresses the critical role of Cav-1 in mediating Akt/GSK3β/β-catenin axis to control the late-phase of cancer progression.

YTHDF1 promotes hepatocellular carcinoma progression via activating PI3K/AKT/mTOR signaling pathway and inducing epithelial-mesenchymal transition

BackgroundN6-methyladenosine (m6A) modification, as the most abundant RNA modification, widely participates in the physiological process and is involved in multiple disease progression, especially cancer. YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) is a pivotal m6A “reader” protein, which has been reported in multiple cancers. However, the role and molecular mechanism of YTHDF1 in HCC are still not fully elucidated.MethodsBased on various bioinformatics databases, q-RT PCR, western blot, and a tissue microarray containing 90 HCC samples, we examined the expression of YTHDF1 in HCC. Then, we applied the loss-of-function experiments to explore the role of YTHDF1 in HCC by in vitro and in vivo assays. Finally, we performed the gene set enrichment analysis (GSEA) to predict the potential signaling pathway of YTHDF1 involved in HCC and further verified this prediction.ResultsYTHDF1 was overexpressed in HCC and associated with HCC grade. Depletion of YTHDF1 markedly impaired the proliferation, migration, invasion, and cell cycle process of HCC cells. Mechanistically, YTHDF1 promoted the growth of HCC cells via activating the PI3K/AKT/mTOR signaling pathway. Moreover, we also demonstrated that the epithelial-mesenchymal transition (EMT) mediated the promoting effect of YTHDF1 on the migration and invasion of HCC cells.ConclusionsYTHDF1 contributes to the progression of HCC by activating PI3K/AKT/mTOR signaling pathway and inducing EMT.