Expression In Hepatocellular Carcinoma Cell Lines Research Articles

Ras-Related Protein Rab24 is a Predictor for Hepatocellular Carcinoma Prognosis and Promotes Tumor Growth

Ras-related protein Rab24, a member of the small GTPase family, plays a vital role in regulating intracellular protein trafficking. Recent research has uncovered dysregulation of Rab24 in hepatocellular carcinoma (HCC), but its clinical implications and tumor-related effects require further investigation. We aimed to investigate Rab24’s expression patterns and its role in HCC progression. We analyzed Rab24 expression in HCC and adjacent tissues at the transcriptional, mRNA, and protein levels. The prognostic significance of Rab24 in HCC was assessed through univariate and multivariate analyses, along with Kaplan–Meier survival analysis. Rab24’s impact on cell proliferation was investigated through cellular and xenograft experiments. Our findings revealed elevated Rab24 expression in HCC tissues compared to adjacent liver tissues. High Rab24 expression correlated with larger tumor size and advanced tumor stage. Additionally, HCC patients with high Rab24 expression experienced poorer overall survival, with Rab24 identified as an independent prognostic factor. Manipulating Rab24 expression in HCC cell lines demonstrated its role in promoting tumor proliferation. Silencing Rab24 significantly reduced xenograft growth in vivo. This study highlights the significant association between high Rab24 expression and poorer HCC prognosis, suggesting Rab24’s potential as a novel clinical biomarker and therapeutic target.

DEAD-box helicase 1 inhibited CD8+ T cell antitumor activity by inducing PD-L1 expression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) does not respond well to current treatments, even immune checkpoint inhibitors. PD-L1 (programmed cell death ligand 1 or CD274 molecule)-mediated immune escape of tumor cells may be a key factor affecting the efficacy of immune checkpoint inhibitor (ICI) therapy. However, the regulatory mechanisms of PD-L1 expression and immune escape require further exploration. Here, we observed that DDX1 (DEAD-box helicase 1) was overexpressed in HCC tissues and associated with poor prognosis in patients with HCC. Additionally, DDX1 expression correlated negatively with CD8+ T cell frequency. DDX1 overexpression significantly increased interferon gamma (IFN-γ)-mediated PD-L1 expression in HCC cell lines. DDX1 overexpression decreased IFN-γ and granzyme B production in CD8+ T cells and inhibited CD8+ T cell cytotoxic function invitro and invivo. In conclusion, DDX1 plays an essential role in developing the immune escape microenvironment, rendering it a potential predictor of ICI therapy efficacy in HCC.

Integrating bulk and single-cell RNA sequencing data to establish necroptosis-related lncRNA risk model and analyze the immune microenvironment in hepatocellular carcinoma

BackgroundThe increasing evidence suggests that necroptosis mediates many behaviors of tumors, as well as the regulation of the tumor microenvironment. Long non-coding RNAs (lncRNAs) are involved in a variety of regulatory processes during tumor development and are significantly associated with patient prognosis. It suggests that necroptosis-related lncRNAs (NRlncRNAs) may serve as biomarkers for the prognosis of hepatocellular carcinoma (HCC). MethodslncRNA expression profiles of HCC were obtained from TCGA database. LncRNAs associated with necroptosis were extracted using correlation analysis. Prognostic models were constructed based on least absolute shrinkage and selection operator algorithm (LASSO) and multivariate Cox regression analysis. The differences of tumor microenvironment between high-risk and low-risk groups were further analyzed. Single-cell RNA sequencing data of HCC was performed to assess the enrichment of necroptosis-related genes in immune cell subsets. Finally, real-time RT-PCR was used to detect the prognosis-related lncRNAs expression in different HCC cell lines. ResultsWe constructed a prognostic signature based on 8 NRlncRNAs, which also showed good predictive accuracy. The model showed that the prognosis of patients with high-risk score was significantly worse than that of patients with low-risk score (P < 0.05). Combined with the clinical characteristics and risk score of HCC, Nomogram was drawn for reference in clinical practice. In addition, immune cell infiltration analysis and single cell RNA sequencing analysis showed that a low level of immune infiltration was observed in patients at high risk and that there was a significant correlation between NRlncRNAs and macrophages. The results of RT-qPCR also showed that 8 necroptosis-related lncRNAs were highly expressed in HCC cell lines and human liver cancer tissues. ConclusionThis prognostic signature based on the necroptosis-related lncRNAs may provide meaningful clinical insights for the prognosis and immunotherapy responses in patients with HCC.

SART3, regulated by p53, is a biomarker for diagnosis, prognosis and immune infiltration in hepatocellular carcinoma.

This study aimed to investigate the role of squamous cell carcinoma antigen recognized by T cells 3 (SART3) in hepatocellular carcinoma (HCC). SART3 expression and prognostic value were analyzed in TCGA and GEO datasets. The diagnostic value and prognostic significance of SART3 were determined using immunohistochemistry in the Guangxi cohort. The whole-exome mutation spectrum of SART3 was analyzed in high and low expression groups in both TCGA and Guangxi cohorts. The biological functions of the SART3 gene were validated through in vitro experiments using small interfering RNA technology to downregulate SART3 expression in HCC cell lines. SART3 expression was significantly higher in HCC tissues than in adjacent noncancerous liver tissues in TCGA, GEO and Guangxi cohorts. High expression of SART3 was significantly associated with poor prognosis in HCC patients. In TCGA and Guangxi cohorts, the expression of SART3 in the TP53 mutation group was significantly higher than that in the non-mutation group. Downregulation of SART3 expression significantly inhibited the migration and proliferation of HCC cells. SART3 may be involved mainly in immune infiltration of Th2 cells and macrophages in HCC. Additionally, SART3 can upregulate the expression of immune checkpoints (PD-L1 and TIM-3) and predict potential therapeutic agents for HCC. The findings of this study demonstrate the diagnostic and prognostic value of SART3 in HCC. SART3 may be associated with immune infiltration of Th2 cells and macrophages in HCC, highlighting its potential role in the development and progression of HCC.

SP1/RNASEH2A accelerates the development of hepatocellular carcinoma by regulating EMT

BackgroundThe expression level of Ribonuclease H2, subunit A (RNASEH2A) in hepatocellular carcinoma (HCC) has been reported, but the function of RNASEH2A on HCC cells development and the related molecular mechanisms remain unclear. Herein, we intend to explore the upstream regulator of RNASEH2A and its role in the HCC progression. MethodsGEPIA website was employed to determine the level of RNASEH2A in HCC tissues and get a survival analysis. After reducing RNASEH2A expression by RNA interference, cell counting kit-8, colony formation, Western blot, Transwell and wound healing assays were performed to estimate the malignant properties of HCC cells. The transcriptional factor of RNASEH2A was predicted by UCSC and JASPAR database and confirmed by dual luciferase assay and Ch-IP assay. The expression level of EMT pathway related molecules was determined by western blotting. ResultsAn increased expression of RNASEH2A was presented in HCC and predicted worse prognosis of HCC patients. Functionally, the results demonstrated that depletion of RNASEH2A suppressed HCC cell proliferation, cell cycle, migration and invasion. Moreover, we illustrated that SP1 targeted to the promoter of RNASEH2A and modulated its expression in HCC cell lines. RNASEH2A knockdown counteracted the function of SP1 overexpression in modulating HCC cell growth, cell cycle, and mobility. Then, our data showed that the SP1/RNASEH2A axis affected the malignant behaviors of HCC cells by regulating EMT process. ConclusionsIn summary, these results demonstrated that RNASEH2A promoted HCC cells development through regulating EMT process and was transcriptionally modulated by SP1.

LncRNA TGFB2-OT1 Promotes Progression and Angiogenesis in Hepatocellular Carcinoma by Dephosphorylating β-Catenin.

Hepatocellular carcinoma (HCC) was the sixth most prevalent cancer worldwide. Long non-coding RNA TGFB2-OT1 has been proven to mediate inflammation and autophagy in vascular endothelial cells. However, its function in HCC is still unknown. We analyzed the relationship between TGFB2-OT1 expression and the clinicopathological features of 202 HCC patients. RT-qPCR was used to analyze the TGFB2-OT1 expression in HCC cell lines and tissues. In vitro and in vivo assays were conducted to verify the effect of TGFB2-OT1 on the phenotype of HCC. RNA pull-down assays were applied to reveal the proteins binding to the TGFB2-OT1. Western-blot assays were conducted to analyze the protein expression in HCC cell lines. TGFB2-OT1 was found to be highly expressed in HCC samples and hepatoma cells. TGFB2-OT1 expression was significantly associated with age (P = 0.001), cirrhosis (P = 0.003), tumor size (P < 0.001), tumor encapsulation (P = 0.029), tumor protruding from the liver surface (P = 0.040), and alpha fetoprotein (AFP, P < 0.001) levels. TGFB2-OT1 promoted proliferation, migration, invasion, and angiogenesis in HCC cells, both in vitro and in vivo. TGFB2-OT1 binds to β-catenin and competitively impaired the binding of β-catenin to GSK3β, thus suppressing the phosphorylation of β-catenin at Ser33, Ser37, and Thr41. TGFB2-OT1 is overexpressed in HCC and predicts the poor prognosis of HCC patients. TGFB2-OT1 impedes the phosphorylation of β-catenin and acts as an alternative activator of the Wnt/β-catenin pathway to promote the progression and angiogenesis of HCC.

The role of AURKA/miR-199b-3p in hepatocellular carcinoma cells.

Previous studies proved that AURKA functions as an oncogene in several cancers. This article aimed to probe the miRNA-induced regulatory mechanism of AURKA in hepatocellular carcinoma (HCC). Differentially expressed genes in TCGA-LIHC dataset were screened by bioinformatics methods. Levels of miR-199b-3p and AURKA mRNA were examined by qRT-PCR. Western blot was utilized to evaluate protein levels of AURKA, p-AKT, and AKT. Dual-luciferase assay was introduced to explore their interaction. MTT, colony formation, scratch healing, transwell, and flow cytometry assays were introduced into cell proliferation, migration, invasion, and apoptosis assessment. The impact of miR-199b-3p/AURKA axis on HCC tumor growth was determined in a tumor xenograft model. We found that AURKA was highly expressed in HCC and was coupled to poor prognosis of HCC. As manifested by cellular assays, compared to the normal cells HL-7702, AURKA presented notably high expression in HCC cell lines. Overexpressed AURKA evidently impelled the proliferation, colony formation, migration, and invasion of HCC cells while suppressing apoptosis. The regulatory gene upstream of AURKA was predicted to be miR-199b-3p by bioinformatics method, and there was a markedly negative correlation between the two. Overexpressed miR-199b-3p constrained HCC cell proliferation, migration, and invasion while fostering apoptosis, which could be counteracted by upregulating AURKA. MiR-199b-3p repressed the tumor growth in vivo by targeting AURKA and affected PI3K/AKT signaling pathway. To summarize, this study implied the regulatory mechanism of miR-199b-3p/AURKA axis in HCC, and supplied optional therapeutic targets for HCC patients.

CENPO is Associated with Immune Cell Infiltration and is a Potential Diagnostic and Prognostic Marker for Hepatocellular Carcinoma.

PurposeTo examine the expression, clinical significance, and potential regulatory mechanism of centromere protein O (CENPO) in hepatocellular carcinoma (HCC).MethodsCENPO expression in pan-cancer was studied using the TCGA-GTEx database, in HCC and normal liver tissues using the GEO and TCGA databases, and in clinical HCC samples by RT-qPCR. The diagnostic value of CENPO was assessed using receiver operating characteristic curves. Univariate and multivariate regression analyses of factors associated with HCC prognosis were performed. CENPO function and its mechanism in HCC were explored using GO, KEGG, and GSEA analyses of differentially expressed genes (DEGs). Association of CENPO expression with immune cell infiltration and immune checkpoint-associated molecules was conducted using TCGA data and the TIMER2.0 database. Relationships between CENPO expression and DNA methylation were analyzed using the UALCAN and cBioPortal databases. CENPO expression in HCC cell lines was detected using RT-qPCR.ResultsCENPO is upregulated in most cancers, including HCC and cell lines, and is a potential biomarker for HCC diagnosis (AUC = 0.936, 95% CI: 0.911–0.960). Higher CENPO expression was associated with poorer outcomes in patients with HCC (OS, p = 0.004; DSS, p = 0.002; PFI, p < 0.001), and CENPO was an independent predictor of factors influencing overall survival in HCC. DEGs between samples with high and low CENPO levels were enriched in various biological processes, including activation of the G2M checkpoint and other signaling pathways, while CENPO expression correlated with HCC immune cell infiltration and immune checkpoint-associated molecules, as well as CENPO promoter methylation (p < 0.001).ConclusionIn HCC and cell lines, CENPO is overexpressed, a potential diagnostic marker and an indicator of poor prognosis. CENPO may regulate HCC development by influencing nuclear division and tumor immune infiltration and is regulated by methylation, making it a potential target for HCC immunotherapy.

MiR-3682-3p directly targets FOXO3 and stimulates tumor stemness in hepatocellular carcinoma via a positive feedback loop involving FOXO3/PI3K/AKT/c-Myc.

BACKGROUNDCancer stem cells (CSCs) have been implicated in tumorigenesis and tumor recurrence and metastasis are key therapeutic targets in cancer treatment. MicroRNAs display therapeutic potential by controlling the properties of CSCs; however, whether an association exists between miR-3682-3p and CSCs is unknown. AIMTo investigate the mechanism by which miR-3682-3p promotes stemness maintenance in hepatocellular carcinoma (HCC).METHODSMiR-3682-3p expression in HCC cell lines and 34 pairs of normal and HCC specimens was assayed by quantitative polymerase chain reaction. The functional role of miR-3682-3p was investigated in vitro and in vivo. Dual-luciferase reporter and chromatin immunoprecipitation assays were performed for target asse ssment, and western blotting was utilized to confirm miR-3682-3p/target relationships. RESULTSWe found that miR-3682-3p plays a key role in HCC pathogenesis by promoting HCC cell stemness. The upregulation of miR-3682-3p enhanced CSC spheroid-forming ability, side population cell fractions, and the expression of CSC factors in HCC cells in vitro and the tumorigenicity of transplanted HCC cells in vivo. Furthermore, silencing miR-3682-3p prolonged the survival of HCC-bearing mice. Mechanistically, we found that miR-3682-3p targets FOXO3 and enables FOXO3/β-catenin interaction, which promotes c-Myc expression through PI3K/AKT; c-Myc, in turn, activates miR-3682-3p, forming a positive feedback loop. Intriguingly, miR-3682-3p expression was induced by hepatitis B virus X protein (HBx) and was involved in HBx-induced tumor stemness-related pathogenesis.CONCLUSIONOur findings reveal a novel mechanism by which miR-3682-3p promotes stemness in HCC stem cells. Silencing miR-3682-3p may represent a novel therapeutic strategy for HCC.

RING finger and WD repeat domain 3 regulates proliferation and metastasis through the Wnt/β-catenin signalling pathways in hepatocellular carcinoma

BACKGROUNDHepatocellular carcinoma (HCC) exhibits high invasiveness and mortality rates, and the molecular mechanisms of HCC have gained increasing research interest. The abnormal DNA damage response has long been recognized as one of the important factors for tumor occurrence and development. Recent studies have shown the potential of the protein RING finger and WD repeat domain 3 (RFWD3) that positively regulates p53 stability in response to DNA damage as a therapeutic target in cancers. AIMTo investigate the relationship between HCC and RFWD3 in vitro and in vivo and explored the underlying molecular signalling transduction pathways. METHODS RFWD3 gene expression was analyzed in HCC tissues and adjacent normal tissues. Lentivirus was used to stably knockdown RFWD3 expression in HCC cell lines. After verifying the silencing efficiency, Celigo/cell cycle/apoptosis and MTT assays were used to evaluate cell proliferation and apoptosis. Subsequently, cell migration and invasion were assessed by wound healing and transwell assays. In addition, transduced cells were implanted subcutaneously and injected into the tail vein of nude mice to observe tumor growth and metastasis. Next, we used lentiviral-mediated rescue of RFWD3 shRNA to verify the phenotype. Finally, the microarray, ingenuity pathway analysis, and western blot analysis were used to analyze the regulatory network underlying HCC. RESULTSCompared with adjacent tissues, RFWD3 expression levels were significantly higher in clinical HCC tissues and correlated with tumor size and TNM stage (P < 0.05), which indicated a poor prognosis state. RFWD3 silencing in BEL-7404 and HCC-LM3 cells increased apoptosis, decreased growth, and inhibited the migration in shRNAi cells compared with those in shCtrl cells (P < 0.05). Furthermore, the in vitro results were supported by the findings of the in vivo experiments with the reduction of tumor cell invasion and migration. Moreover, the rescue of RFWD3 shRNAi resulted in the resumption of invasion and metastasis in HCC cell lines. Finally, gene expression profiling and subsequent experimental verification revealed that RFWD3 might influence the proliferation and metastasis of HCC via the Wnt/β-catenin signalling pathway.CONCLUSIONWe provide evidence for the expression and function of RFWD3 in HCC. RFWD3 affects the prognosis, proliferation, invasion, and metastasis of HCC by regulating the Wnt/β-catenin signalling pathway.

Long Non-Coding RNA CASC7 Promotes Proliferation and Inhibits Apoptosis of Hepatocellular Carcinoma Cells via Downregulating miR-340-5p CASC7/miR-340-5p Axis in Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide with a poor prognosis. Amounting studies revealed that long non-coding RNAs (lncRNAs) show important roles in various biological processes. The purpose of this study was to explore the biological function and potential molecular mechanism of CASC7 in HCC. Methods: CASC7 expression in HCC cell lines was detected by qRT-PCR. The expressions of CASC7 and miR-340-5p were changed by transfection of miR-340-5p mimic, the CASC7 overexpression and knockdown plasmids. The interaction between CASC7 and miR-340-5p was assessed by a Dual-Luciferase reporter assay. The biological functions of CASC7 were evaluated by CCK-8, colony formation assay, ROS assay kit, immunofluorescence and flow cytometry (FCM). Results: CASC7 was upregulated in HCC cell lines. CASC7 overexpression significantly promoted cell proliferation, as well as inhibited apoptosis and oxidative stress. In contrast, CASC7 knockdown could reverse these above changes. The result of the Dual-luciferase reporter assay revealed that CASC7 directly targeted miR-340-5p and negatively regulated its expression. In addition, CASC7 promoted proliferation and inhibited apoptosis of HCC cells through activating Nrf2 pathway by downregulating miR-340-5p. Conclusions: In summary, CASC7 promotes HCC tumorigenesis and progression through the Nrf2 pathway by targeting miR-340-5p, which may provide a new target for therapy of HCC.

Tumor-derived PD1 and PD-L1 could promote hepatocellular carcinoma growth through autophagy induction in vitro

BackgroundsAutophagy in tumor was also found to influence immune microenvironment. The relation between autophagy and cancer intrinsic PD1 and PD-L1 expression was not clear. MethodsWith data from TCGA and GTEx databases, mRNA expression levels of autophagy-related genes were compared between tumor samples and normal tissues, which were also correlated with survival status. Expression of autophagy-related genes were also associated with clinical traits in datasets of GSE14520 and ICGC LIRI. Single sample gene set enrichment analysis (ssGSEA) was used to calculate autophagy scores in tumor samples, using signatures from MSigDB database. Lentivirus (PD1 and PD-L1), siRNA (ATG13) and plasmids (LC3A/B) were used to target specific genes in tumor cells; Western blot was used to examine protein expression accordingly. Co-immunoprecipitation was performed to find PD1 or PD-L1 interacting proteins; colony formation and EdU analysis were used to evaluate tumor cell growth abilities. ResultsmRNA levels of autophagy markers were increased in tumor and correlated with worse survival of cancer patients. In hepatocellular carcinoma (HCC), high mRNA expression of autophagy markers was related to poor clinical status; increasing LC3 expression in HCC cell lines could promote tumor growth. Tumor intrinsic PD1 or PD-L1 were related to higher autophagy levels in specific tumor types; over-expression of PD1 or PD-L1 could increase autophagy in tumor cells through ATG13 interaction. ConclusionAutophagy could promote tumor growth in specific cancer types. Tumor intrinsic PD1 or PD-L1 could both increase autophagy through ATG13 interaction.

Smurf2 suppresses the metastasis of hepatocellular carcinoma via ubiquitin degradation of Smad2.

PurposeSmurf2, one of C2-WW-HECT domain E3 ubiquitin ligases, is closely related to the development and progression in different cancer types, including hepatocellular carcinoma (HCC). This study aims to illustrate the expression and molecular mechanism of Smurf2 in regulating the progression of HCC.MethodsThe expression of Smurf2 in human HCC and adjacent non-tumor liver specimens was detected using tissue microarray studies from 220 HCC patients who underwent curative resection. The relationships of Smurf2 and HCC progression and survival were analyzed using the chi-square test, Kaplan–Meier analysis, and Cox proportional hazards model. For Smurf2 was low expression in HCC cell lines, Smurf2 overexpression cell lines were established. The effect of Smurf2 on cell proliferation and migration was detected by Cell Counting Kit-8 and colony formation assay, and the epithelial–mesenchymal transition (EMT) markers and its transcription factors were tested by immunoblotting. The interaction and ubiquitination of Smad2 by Smurf2 were detected by co-immunoprecipitation and immunoprecipitation assay. Finally, the effect of Smurf2 on HCC was verified using the mouse lung metastasis model.ResultsSmurf2 was downregulated in HCC tissues compared to that of corresponding non-tumor liver specimens. The low expression of Smurf2 in HCC was significantly associated with macrovascular or microvascular tumor thrombus and the impairment of overall survival and disease-free survival. In vitro and in vivo analysis showed that Smurf2 overexpression decreased the EMT potential of HCC cells by promoting the ubiquitination of Smad2 via the proteasome-dependent degradation pathway.ConclusionThe expression of Smurf2 was downregulated in HCC specimens and affected the survival of patients. Smurf2 inhibited the EMT of HCC by enhancing Smad2 ubiquitin-dependent proteasome degradation.

AXL Knock-Out in SNU475 Hepatocellular Carcinoma Cells Provides Evidence for Lethal Effect Associated with G2 Arrest and Polyploidization.

AXL, a member of the TAM family, is a promising therapeutic target due to its elevated expression in advanced hepatocellular carcinoma (HCC), particularly in association with acquired drug resistance. Previously, RNA interference was used to study its role in cancer, and several phenotypic changes, including attenuated cell proliferation and decreased migration and invasion, have been reported. The mechanism of action of AXL in HCC is elusive. We first studied the AXL expression in HCC cell lines by real-time PCR and western blot and showed its stringent association with a mesenchymal phenotype. We then explored the role of AXL in mesenchymal SNU475 cells by CRISPR-Cas9 mediated gene knock-out. AXL-depleted HCC cells displayed drastic phenotypic changes, including increased DNA damage response, prolongation of doubling time, G2 arrest, and polyploidization in vitro and loss of tumorigenicity in vivo. Pharmacological inhibition of AXL by R428 recapitulated G2 arrest and polyploidy phenotype. These observations strongly suggest that acute loss of AXL in some mesenchymal HCC cells is lethal and points out that its inhibition may represent a druggable vulnerability in AXL-high HCC patients.

LncSNHG3 promotes hepatocellular carcinoma epithelial mesenchymal transition progression through the miR-152-3p/JAK1 pathway.

The dysregulation of LncRNAs is related to the malignant progression of many cancers. The study aimed to investigate the expression and the biological role of LncSNHG3 in hepatocellular carcinoma (HCC). The TCGA data of the LncSNHG3 in HCC were analyzed. The expression in HCC cell lines was detected by qRT-PCR. Proliferation, migration, and invasion of HepG2 and Huh7 were examined by cell counting kit-8, colony formation, transwell assays, and wound healing assays. At the same time, the interactions among LncSNHG3, miR-152-3p, and JAK1 were confirmed by dual-luciferase reporter assay, RNA immunoprecipitation, subcellular distribution. Xenograft tumor-bearing mice models were used to measure the effect of LncSNHG3 on the growth of HCC in vivo. The apoptosis and epithelial mesenchymal transition (EMT)-associated proteins were checked by WB and IHC. LncSNHG3 was overexpressed in HCC tissues and cell lines. In addition, it is correlated with the tumor stage and survival time of HCC patients. Down-regulated LncSNHG3 could significantly suppress the EMT progression of HCC in vivo and in vitro. LncSNHG3 could promote the JAK1 expression by sponging miR-152-3p. LncSNHG3 acted as an oncogene and promoted the EMT procession in HCC by binding miR-152-3p and promoting JAK1 expression. Predictably, LncSNHG3 was used as a potential marker and will be used as a novel therapy target for HCC in the future.

Long non-coding RNA LINC00630 facilitates hepatocellular carcinoma progression through recruiting transcription factor E2F1 to up-regulate cyclin-dependent kinase 2 expression.

This study is aimed to investigate the role of long non-coding RNA 630 (LINC00630) in hepatocellular carcinoma (HCC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine LINC00630 expression in HCC cell lines and tissues. After LINC00630 was overexpressed or depleted in HCC cell lines, cell counting kit-8 (CCK-8) assay, BrdU assay, and flow cytometry were conducted for detecting HCC cell multiplication, apoptosis, and cell cycle progression. The catRAPID database was adopted to predict the binding relationship between LINC00630 and E2F transcription factor 1 (E2F1), and RNA pull-down and RNA immunoprecipitation (RIP) assays were carried out to verify this binding relationship. The binding of E2F1 to the cyclin-dependent kinase 2 (CDK2) promoter region was verified by dual-luciferase reporter gene assay and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assay. Western blotting was conducted to detect the protein expression of E2F1 and CDK2 in HCC cells. We report that LINC00630 expression was up-regulated in HCC and was significantly correlated with TNM stage and lymph node metastasis. LINC00630 overexpression facilitated HCC cell proliferation and cell cycle progression and inhibited the cell apoptosis, while LINC00630 knockdown had the opposite effects. LINC00630 directly bounds with E2F1. LINC00630 overexpression enhanced the binding of E2F1 to the CDK2 promoter region, thereby promoting CDK2 transcription, whereas knocking down LINC00630 inhibited CDK2 transcription. Collectively, LINC00630 promoted CDK2 transcription by recruiting E2F1 to the promoter region of CDK2, thereby promoting the malignant progression of HCC. Our data suggest that LINC00630 is a promising molecular target for HCC.

Tyrosine Kinase Inhibitors Stimulate HLA Class I Expression by Augmenting the IFNγ/STAT1 Signaling in Hepatocellular Carcinoma Cells.

Combination treatment with tyrosine kinase inhibitors (TKIs) and immunotherapies has shown efficacy in the treatment of multiple cancers, but the immunomodulatory effect of TKIs on the tumor cell phenotype remains unknown in hepatocellular carcinoma (HCC). Given that human lymphocyte antigen class I (HLA-I) is essential for tumor antigen presentation and subsequent antitumor immunity, we examined the effects of regorafenib, as well as other TKIs (sorafenib, lenvatinib and cabozantinib) on HLA-I expression in HCC cell lines. Regorafenib increased cell surface HLA-I and β2-microglobulin protein expression in the presence of interferon γ (IFNγ). The expressions of various genes associated with the HLA-I antigen processing pathway and its transcriptional regulators were also upregulated by regorafenib. Furthermore, we found that regorafenib had an activating effect on signal transducers and activators of transcription 1 (STAT1), and that regorafenib-induced HLA-I expression was dependent on the augmented IFNγ/STAT1 signaling pathway. Trametinib, an inhibitor of the extracellular signal-regulated kinase (ERK) kinase MEK, also activated IFNγ/STAT1 signaling and increased HLA-I expression, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor buparlisib did not. Given that regorafenib directly inhibits Raf/MEK/ERK signaling, the downregulation of the MEK/ERK pathway appears to be one of the mechanisms by which regorafenib promotes STAT1 activation. Sorafenib, lenvatinib, and cabozantinib also showed the same effects as regorafenib, while regorafenib had most potent effects on HLA-I expression, possibly dependent on its stronger inhibitory activity against the MEK/ERK pathway. These results support the clinical combination of TKIs with immunotherapy for the treatment of HCC.

Targeting Hedgehog signalling in CD133-positive hepatocellular carcinoma: improving Lenvatinib therapeutic efficiency.

Lenvatinib has been approved as a first-line treatment for advanced hepatocellular carcinoma (HCC) in recent years. However, Lenvatinib resistance hinders its therapeutic effect, and the underlying mechanism of action of Lenvatinib needs to be better understood. Increasing studies have suggested that cancer stem cells (CSCs) are an important driving force. Hedgehog signalling is important for the maintenance of hepatocellular carcinoma stemness. In the present study, we investigated the therapeutic role of the Hedgehog signalling inhibitor in reversing Lenvatinib resistance in CD133-positive HCC cells. First, we examined the inhibitory impact of Lenvatinib against CD133 expression in HCC cell lines through Western blot. The CCK8 assay showed that GANT61, a Hedgehog signalling inhibitor, has a suppression advantage over other CSCs-related signalling inhibitors regarding cell viability. Moreover, Lenvatinib and GANT61 combined had better inhibitory effects on cell viability and malignant properties, both in vivo and in vitro. In addition, GANT61 reversed the upregulation of CD133 and Hedgehog signalling caused by Lenvatinib in SK-Hep-1 and MHCC97H. Thus, our results suggested that GANT61 reversed Lenvatinib resistance by suppressing Hedgehog signalling in HCC cells, especially in CD133-positive cells and combining Lenvatinib with Hedgehog signalling inhibitors could improve its therapeutic efficacy in HCC patients with high CD133 expression levels.

C2orf40 inhibits hepatocellular carcinoma through interaction with UBR5.

Hepatocellular carcinoma (HCC) urgently needs a marker for early diagnosis and targeted treatment. C2orf40 has been identified as a tumor suppressor gene in many cancers. However, the precise role and regulatory mechanism by C2orf40 contribute to HCC remain elusive and merit exploration. Reverse-transcription PCR, quantitative real-time PCR, and methylation-specific PCR were used to detect expression and methylation of C2orf40 in HCC cell lines or tissues. The effects of C2orf40 in liver cancer cells were examined via colony formation, CCK8, transwell, and flow cytometric assays. The effect of C2orf40 on tumorigenesis in vivo was determined by xenografts and immunohistochemical analysis. Western blot, indirect immunofluorescence, Co-IP, and cycloheximide (CHX) were used to further investigate the potential mechanism of C2orf40. The down-regulation of C2orf40 in hepatocellular cancer tissue samples is often related to the degree of methylation of its promoter CpG. The recovery of C2orf40 expression in HCC cell lines can induce G0/G1 phase arrest and apoptosis and also inhibit cell migration and invasion by reversing the epithelial-mesenchymal transition (EMT) process, both in vivo and in vitro. In addition, C2orf40 can increase the expression of p21 through interaction with UBR5. Low expression levels of C2orf40 are related to the hypermethylation of its promoter. C2orf40 can inhibit HCC through UBR5-dependent or p53-independent mechanisms. C2orf40 may be a diagnostic biomarker and a potential therapeutic target in HCC.

Upregulation of ubiquitin-conjugating enzyme E2T (UBE2T) predicts poor prognosis and promotes hepatocellular carcinoma progression

ABSTRACT Reportedly, ubiquitin-conjugating enzyme E2T (UBE2T) is closely related to the progression of several malignancies. This work is aimed to probe the role of UBE2T in the progression of hepatocellular carcinoma (HCC) patients. The microarray analysis was executed to screen the differentially expressed genes (DEGs) in HCC tissues. The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA2) databases, PCR and immunohistochemistry were utilized to validate the dysregulation of UBE2T in HCC. Kaplan-Meier analysis was employed to determine the relationship between UBE2T expression and the prognosis of HCC patients. PCR was carried out to detect UBE2T protein expression in HCC cell lines. Cell Counting Kit-8 (CCK-8) assay and 5-bromo-2ʹ-deoxyuridine (BrdU) experiments were conducted to examine the proliferation of HCC cells. Scratch healing and Transwell experiments were conducted to examine the migration of HCC cells. Bioinformatics analysis and dual-luciferase reporter gene experiments predicted and validated the targeting relationship with miR-212-5p and UBE2T. We found that UBE2T expression was remarkably up-modulated in HCC tissues and cell lines, and its high expression was linked to a worse prognosis in HCC patients. UBE2T overexpression enhanced HCC cell proliferation and migration. Additionally, UBE2T was verified as a downstream target of miR-212-5p. In conclusion, UBE2T overexpression is markedly linked to unfavorable prognosis in HCC patients. UBE2T, regulated by miR-212-5p, significantly enhances the malignant phenotypes of HCC cells, which can be used as a target for HCC diagnosis and prognosis.