Parental HepG2 Research Articles

Exosomal delivery of miR-200b-3p suppresses the growth of hepatocellular carcinoma cells by targeting ERG- and VEGF-mediated angiogenesis

Hepatocellular carcinoma (HCC) remains a lethal malignancy with limited treatment options. Recent discoveries have highlighted the pivotal role of miRNAs in HCC progression. We previously reported that the expression of miR-200b-3p was decreased in HCC cells and exosomal miR‐200b‐3p from hepatocytes inhibited angiogenesis by suppressing the expression of the endothelial transcription factor ERG (erythroblast transformation‐specific (ETS)‐related gene), leading to the hypothesis that the delivery of this miRNA may inhibit angiogenesis and suppress HCC growth in vivo. Here, we tested this hypothesis by using human HCC inoculation models. First, we transfected the human HepG2 HCC cells and established a stable cell line that overexpressed a high level of miR-200b-3p. When miR-200b-3p-overexpressing cells were injected into severe combined immunedeficiency (SCID)-beige mice, tumor growth was significantly reduced compared to tumors of control cells, with a reduction in the expression of ERG and vascular endothelial growth factor (VEGF) and subsequent angiogenesis. Intra-tumoral injection of exosomes containing high levels of miR-200b-3p also reduced the growth of parental HepG2 tumors with reduced ERG and VEGF expression and angiogenesis. These results validate the inhibitory role of miR-200b-3p in tumor angiogenesis, thereby suppressing HCC tumor growth, and provide a novel insight into its potential therapeutic application.

HepG2.2.15-derived exosomes facilitate the activation and fibrosis of hepatic stellate cells.

The role of exosomes derived from HepG2.2.15 cells, which express hepatitis B virus (HBV)-related proteins, in triggering the activation of LX2 liver stellate cells and promoting liver fibrosis and cell proliferation remains elusive. The focus was on comprehending the relationship and influence of differentially expressed microRNAs (DE-miRNAs) within these exosomes. To elucidate the effect of exosomes derived from HepG2.2.15 cells on the activation of hepatic stellate cell (HSC) LX2 and the progression of liver fibrosis. Exosomes from HepG2.2.15 cells, which express HBV-related proteins, were isolated from parental HepG2 and WRL68 cells. Western blotting was used to confirm the presence of the exosomal marker protein CD9. The activation of HSCs was assessed using oil red staining, whereas DiI staining facilitated the observation of exosomal uptake by LX2 cells. Additionally, we evaluated LX2 cell proliferation and fibrosis marker expression using 5-ethynyl-2'-deoxyuracil staining and western blotting, respectively. DE-miRNAs were analyzed using DESeq2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to annotate the target genes of DE-miRNAs. Exosomes from HepG2.2.15 cells were found to induced activation and enhanced proliferation and fibrosis in LX2 cells. A total of 27 miRNAs were differentially expressed in exosomes from HepG2.2.15 cells. GO analysis indicated that these DE-miRNA target genes were associated with cell differentiation, intracellular signal transduction, negative regulation of apoptosis, extracellular exosomes, and RNA binding. KEGG pathway analysis highlighted ubiquitin-mediated proteolysis, the MAPK signaling pathway, viral carcinogenesis, and the toll-like receptor signaling pathway, among others, as enriched in these targets. These findings suggest that exosomes from HepG2.2.15 cells play a substantial role in the activation, proliferation, and fibrosis of LX2 cells and that DE-miRNAs within these exosomes contribute to the underlying mechanisms.

Membrane Association of the Short Transglutaminase Type 2 Splice Variant (TG2-S) Modulates Cisplatin Resistance in a Human Hepatocellular Carcinoma (HepG2) Cell Line.

Hepatocellular carcinoma (HCC) is a heterogeneous malignancy with complex carcinogenesis. Although there has been significant progress in the treatment of HCC over the past decades, drug resistance to chemotherapy remains a major obstacle in its successful management. In this study, we were able to reduce chemoresistance in cisplatin-resistant HepG2 cells by either silencing the expression of transglutaminase type 2 (TG2) using siRNA or by the pre-treatment of cells with the TG2 enzyme inhibitor cystamine. Further analysis revealed that, whereas the full-length TG2 isoform (TG2-L) was almost completely cytoplasmic in its distribution, the majority of the short TG2 isoform (TG2-S) was membrane-associated in both parental and chemoresistant HepG2 cells. Following the induction of cisplatin toxicity in non-chemoresistant parental cells, TG2-S, together with cisplatin, quickly relocated to the cytosolic fraction. Conversely, no cytosolic relocalisation of TG2-S or nuclear accumulation cisplatin was observed, following the identical treatment of chemoresistant cells, where TG2-S remained predominantly membrane-associated. This suggests that the deficient subcellular relocalisation of TG2-S from membranous structures into the cytoplasm may limit the apoptic response to cisplatin toxicity in chemoresistant cells. Structural analysis of TG2 revealed the presence of binding motifs for interaction of TG2-S with the membrane scaffold protein LC3/LC3 homologue that could contribute to a novel mechanism of chemotherapeutic resistance in HepG2 cells.

Extracellular Vesicles altered by a Per- and Polyfluoroalkyl Substance Mixture: In Vitro Dose-Dependent Release, Chemical Content, and MicroRNA Signatures involved in Liver Health.

Per- and polyfluoroalkyl substances (PFAS) have emerged as high priority contaminants due to their ubiquity and pervasiveness in the environment. Numerous PFAS co-occur across sources of drinking water, including areas of North Carolina (NC) with some detected concentrations above the Environmental Protection Agency's health advisory levels. While evidence demonstrates PFAS exposure induces harmful effects in the liver, the involvement of extracellular vesicles (EVs) as potential mediators of these effects has yet to be evaluated. This study set out to evaluate the hypothesis that PFAS mixtures induce dose-dependent release of EVs from liver cells, with exposures causing differential loading of microRNAs (miRNAs) and PFAS chemical signatures. To test this hypothesis, a defined PFAS mixture was prioritized utilizing data collected by the NC PFAS Testing Network. This mixture contained three substances, PFOS, PFOA, and PFHxA, selected based upon co-occurrence patterns and the inclusion of both short-chain (PFHxA) and long-chain (PFOA and PFOS) substances. HepG2 liver cells were exposed to equimolar PFAS, and secreted EVs were isolated from conditioned media and characterized for count and molecular content. Exposures induced a dose-dependent release of EVs carrying miRNAs that were differentially loaded upon exposure. These altered miRNA signatures were predicted to target mRNA pathways involved in hepatic fibrosis and cancer. Chemical concentrations of PFOS, PFOA, and PFHxA were also detected in both parent HepG2 cells and their released EVs, specifically within a 15-fold range after normalizing for protein content. This study therefore established EVs as novel biological responders and measurable endpoints for evaluating PFAS-induced toxicity.

NEAT1_2 and DAZAP1, Paraspeckle Components, Interact with PXR to Negatively Regulate CYP3A4 Induction.

Human pregnane X receptor (PXR) is a major nuclear receptor that upregulates the expression of drug-metabolizing enzymes such as CYP3A4. In our recent study, it was revealed that PXR interacts with DAZ-associated protein 1 (DAZAP1), which is an essential component of the paraspeckle, a membraneless nuclear body, and the interaction was disassociated by rifampicin, a ligand of PXR. The purpose of this study was to clarify the roles of paraspeckles in PXR-mediated transcriptional regulation. Immunoprecipitation assays using PXR-overexpressing HepG2 (ShP51) cells revealed that PXR interacts with not only DAZAP1 but also NEAT1_2, a long noncoding RNA included in the paraspeckle, and that the interaction between PXR and NEAT1_2 was disassociated by rifampicin. These results suggest that PXR is trapped in paraspeckles and that the activation of PXR by its ligands facilitates its disassociation from paraspeckles. Induction of CYP3A4 by rifampicin was significantly enhanced by the knockdown of NEAT1_2 or DAZAP1 in ShP51 cells and their parental HepG2 cells. A luciferase assay using a plasmid containing the PXR response elements of CYP3A4 revealed that the increased CYP3A4 induction by siNEAT1_2 or siDAZAP1 was due to the increased transactivation by PXR. These results suggest that paraspeckles play a role in trapping nuclear PXR in the absence of the ligand to negatively regulate transactivation of its downstream gene. Collectively, this is the first study to demonstrate that the paraspeckle components NEAT1_2 and DAZAP1 negatively regulate CYP3A4 induction by PXR. SIGNIFICANCE STATEMENT: This study revealed that PXR interacts with paraspeckle components NEAT1_2 and DAZAP1 to suppress CYP3A4 induction by PXR, and the interaction is dissociated by PXR ligands. This finding provides a novel concept that paraspeckles formed by liquid-liquid phase separation potentially affect drug metabolism via negative regulation of PXR function.

Drug resistance of hepatoma cells induced by ATP‑binding cassette transporter G2 by reducing intracellular drug concentration.

The side effects and drug resistance during chemotherapy seriously affect the outcome of and may lead to the failure of chemotherapy for patients with hepatoma. The aim of the present study was to investigate the association between the expression of ATP-binding cassette transporter G2 (ABCG2) in hepatoma cells and the drug resistance of hepatoma. An MTT assay was used to determine the half-maximal inhibitory concentration (IC50) of Adriamycin (ADM) in hepatoma HepG2 cells after treatment with ADM for 24 h. An ADM-resistant hepatoma cell subline, HepG2/ADM, was generated from the HepG2 hepatoma cell line through a stepwise selection with ADM doses from 0.01 to 0.1 µg/ml. The HepG2/ABCG2 cell line, an ABCG2-overexpressing hepatoma cell line, was established by transfecting the ABCG2 gene into HepG2 cells. The MTT assay was then used to detect the IC50 of ADM in HepG2/ADM and HepG2/ABCG2 cells after treatment with ADM for 24 h and the resistance index was calculated. The apoptosis, cell cycle and ABCG2 protein expression levels in HepG2/ADM, HepG2/ABCG2 cells, HepG2/PCDNA3.1 and their parental HepG2 cells were detected by flow cytometry. In addition, flow cytometry was used to detect the efflux effect of HepG2/ADM and HepG2/ABCG2 cells after ADM treatment. ABCG2 mRNA expression in cells was detected by reverse transcription-quantitative PCR. After 3 months of ADM treatment, HepG2/ADM cells grew stably in the cell culture medium containing 0.1 µg/ml ADM and the cells were named HepG2/ADM cells. ABCG2 was overexpressed in HepG2/ABCG2 cells. The IC50 of ADM in HepG2, HepG2/PCDNA3.1, HepG2/ADM and HepG2/ABCG2 cells was 0.72±0.03, 0.74±0.01, 11.17±0.59 and 12.75±0.47 µg/ml, respectively. The cell apoptotic rate of HepG2/ADM and HepG2/ABCG2 cells was not significantly different compared with that of HepG2 and HepG2/PCDNA3.1 cells (P>0.05), but the G0/G1 phase population of the cell cycle decreased and the proliferation index increased significantly (P<0.05). The expression levels of ABCG2 gene and protein in HepG2/ADM and HepG2/ABCG2 cells were significantly higher than those in HepG2 and HepG2/PCDNA3.1 cells (P<0.01), but there was no significant difference between HepG2 and HepG2/PCDNA3.1 cells (P>0.05). The ADM efflux effect of HepG2/ADM and HepG2/ABCG2 cells was significantly higher than that of parental HepG2 and HepG2/PCDNA3.1 cells (P<0.05). Therefore, the present study demonstrated that ABCG2 expression is highly increased in drug-resistant hepatoma cells and that high expression of ABCG2 is involved in the drug resistance of hepatoma by reducing the intracellular drug concentration.

Bacterial metabolite butyrate in modulating sorafenib-targeted microRNAs to curtail its resistance in hepatocellular carcinoma.

The host dietary fibre is fermented into short-chain fatty acids (SCFA) by intestinal microbiota as bacterial metabolites like propionate, acetate and butyrate. Among these metabolites, the role of butyrate is well documented to provide energy to intestinal epithelial cells. Also, butyrate has anti-inflammatory and anti-tumour properties and decrease in its level by unbalanced diet can develops cancer. Lately, some research has suggested that sodium butyrate as an inhibitor of histone deacetylase (HDAC) may have anticancer potential for hepatocellular carcinoma (HCC), the most common type of liver cancer. Since, HCC is asymptomatic it is usually diagnosed at its advanced stage. Sorafenib with antiproliferative and antiangiogenic effects is the first line of treatment in advanced HCC. However, prolonged drug treatment to HCC patients develops adaptive resistance towards the sorafenib. Sorafenib resistance can also be enhanced by differentially expressed microRNAs. However, the significance of butyrate in HCC sorafenib resistance and its association with sorafenib-targeted microRNAs is yet to be unfurled. Here, an attempt has been made to explore the role of bacterial metabolite butyrate on sorafenib resistant HCC as well as on sorafenib-targeted microRNAs (miR-7641 and miR-199) to curtail sorafenib resistance in HCC. Initially, in-silico analysis was performed using Human Metabolome Database (HMDB) so to identify specific butyrate producing faecal bacteria. Then, their specific 16s rRNA expression was compared between HCC patients and healthy individuals using qRT-PCR. Additionally, the cell viability (MTT) and apoptosis assays were performed in both parental and sorafenib resistant HepG2 cells to evaluate the role of sodium butyrate in sorafenib resistant HCC. Moreover, the association of sodium butyrate with sorafenib-targeted miR-7641 and miR-199 was also assessed using real time PCR, cell viability, cell apoptosis and transfection assays. In silico analysis demonstrated Roseburia cecical, Roseburia intestinalis, Eubacterium rectal, Faecalibacterium prausnitzii as specific butyrate producing faecal bacteria and their 16s rRNA expression was downregulated in HCC patients. In vitro study revealed the presence of sodium butyrate also decreased the cell viability as well as enhanced cell apoptosis of both parental and resistant HepG2 cells. Interestingly, sodium butyrate also decreased the expression of both sorafenib-targeted miR-7641 and miR-199. Further, combination of both sodium butyrate and antimiR-7641 or antimiR-199 also increased apoptosis and decreased viability of resistant cells. This is first study to unravel the association of butyrate producing bacteria with HCC patients and the significance of bacterial metabolite butyrate as anti-tumour in sorafenib resistant hepatocellular carcinoma. The study also demonstrated the plausible new aspects of bacterial metabolite butyrate association with sorafenib-targeted miRNAs (miR-7641 and miR-199). Hence, the study highlighted the therapeutic potential of bacterial metabolite butyrate that might improve the clinical management of hepatocellular carcinoma.

MiR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line.

Background: Hepatocellular carcinoma (HCC) is the second most common malignancy with increasing cancer deaths worldwide. HCC is mainly diagnosed at its advanced stage, and treatment with FDA-approved sorafenib, the multikinase inhibitor drug, is advised. Acquired resistance against sorafenib develops through several pathways involving hypoxia, autophagy, high glycolysis, or glutaminolysis. Small non-coding RNAs, similar to microRNAs (miRNAs), are also known to affect sorafenib resistance in HCC. However, there is a lack of information regarding the significance of differentially expressed miRNA (if any) on autophagy and glutamine regulation in sorafenib-resistant HCC. Methods: The expression of autophagy and glutaminolysis genes was checked in both parental and sorafenib resistant HepG2 cell lines by real-time PCR. MTT and Annexin/PI assays were also performed in the presence of inhibitors such as chloroquine (autophagy inhibitor) and BPTES (glutaminolysis inhibitor). Next generation sequencing and in silico analysis were performed to select autophagy and glutamine addiction-specific microRNA. Selected miRNA were transfected into both HepG2 cells to examine its effect on autophagy and glutamine addiction in regulating sorafenib-resistant HCC. Results: Our in vitro study depicted a higher expression of genes encoding autophagy and glutaminolysis in sorafenib-resistant HepG2 cells. Moreover, inhibitors for autophagy (chloroquine) and glutaminolysis (BPTES) showed a diminished level of cell viability and augmentation in cell apoptosis of sorafenib-resistant HepG2 cells. NGS and real-time PCR demonstrated the downregulated expression of miR-23b-3p in sorafenib-resistant cells compared to parental cells. In silico analysis showed that miR-23b-3p specifically targeted autophagy through ATG12 and glutaminolysis through GLS1. In transfection assays, mimics of miR-23b-3p demonstrated reduced gene expression for both ATG12 and GLS1, decreased cell viability, and increased cell apoptosis of sorafenib-resistant HepG2 cells, whereas the antimiRs of miR-23b-3p demonstrated contrasting results. Conclusion: Our study highlights the cytoprotective role of autophagy and glutamine addiction modulated by miR-23b-3p (tumor suppressor), suggesting new approaches to curb sorafenib resistance in HCC.

Probing the Mechanism of Action of Cry41Aa on HepG2 through the Establishment of a Resistant Subline.

Cry41Aa, also called parasporin-3, belongs to a group of toxins from the entomopathogenic bacterium Bacillus thuringiensis that show activity against human cancer cells. Cry41Aa exhibits preferential cytocidal activity towards HL-60 (human promyelocytic leukaemia cells) and HepG2 (human liver cancer cells) cell lines after being proteolytically activated. To better understand the mechanism of action of Cry41Aa, we evolved resistance in HepG2 cells through repeated exposure to increasing doses of the toxin. Concentrations of Cry41Aa that killed over 50% of the parental HepG2 cells had no significant effect on the viability of the resistant cells and did not induce either pore formation or p38 phosphorylation (both characteristic features of pore-forming toxins). Preliminary RNA sequencing data identified AQP9 as a potential mediator of resistance, but extensive investigations failed to show a causal link and did not support an enhanced cell repair process as the resistance mechanism.

MiR-381 Reverses Multidrug Resistance by Negative Regulation of the CTNNB1/ABCB1 Pathway in HepG2/Dox Cells, and the Diagnostic and Prognostic Values of CTNNB1/ABCB1 Are Identified in Patients with LIHC.

Multidrug resistance (MDR) is the biggest challenge in cancer therapy. In this study, we explored the molecular mechanism of MDR in human liver cancer and explored the related diagnostic and prognostic values of the targeted genes in patients with hepatocellular carcinoma. We constructed a multidrug-resistant liver cancer cell line, HepG2/Dox, using the parental subline HepG2. The (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay was used to test the viability of the liver cancer cells. Western blotting was performed to test the expression of ABCB1, β-catenin, and β-actin. Luciferase assays were performed to confirm the relationship between miR-381 and its target genes. The diagnostic and prognostic values of target genes were analyzed using publicly available data from The Cancer Genome Atlas. The Mann-Whitney U test and logistic regression were performed to evaluate the association between ABCB1 or CTNNB1 expression and clinical features in patients with liver hepatocellular carcinoma (LIHC). Finally, Kaplan-Meier and Cox regression analyses were performed to test the effect of ABCB1 or CTNNB1 expression on the overall survival of patients with LIHC. ABCB1 expression was upregulated in HepG2/Dox cells. ABCB1 was found to be a direct target of hsa-miR-381 and was negatively regulated by has-miR-381. Moreover, hsa-miR-381 directly targeted the CTNNB1 3' UTR and decreased the luciferase activity of CTNNB1. Transfection with miR-183 partially reversed chemotherapeutic drug resistance by downregulating the expression of ABCB1 and CTNNB1 in HepG2/Dox cells. Spearman's analysis results showed that CTNNB1 and ABCB1 were positively correlated in patients with liver cancer, and increased CTNNB1 and ABCB1 expression occurred in patients with liver cancer. High expression of ABCB1 and CTNNB1 indicated poor prognosis in patients with liver cancer; however, neither ABCB1 nor CTNNB1 expression was an independent diagnostic factor in patients with LIHC. Overexpression of hsa-miR-381 partially reversed the MDR of HepG2 cells by directly targeting and negatively regulating the expression of CTTNB1 and ABCB1. Moreover, high expression of ABCB1 or CTNNB1 indicated poor prognosis in patients with liver cancer.

HBV HBx-Downregulated lncRNA LINC01010 Attenuates Cell Proliferation by Interacting with Vimentin.

Hepatitis B virus (HBV) infection is closely related to hepatocellular carcinoma (HCC) development. To investigate the mechanism of HBV causing HCC, we previously analyzed the transcription of the HBV-transgenic cell line HepG2-4D14 and parental HepG2 cells and identified a subset of long noncoding RNAs (lncRNAs) differentially expressed between them. In this study, we focus on lncRNA LINC01010, as it is significantly downregulated in HepG2-4D14 cells and in liver tissues of HCC patients, and positively correlated with survival. We found that HBV-encoded HBx can reduce the transcription of LINC01010. Functional analysis showed that the overexpression of LINC01010 inhibits proliferation, migration and invasion of HepG2 cells while the knockdown of LINC01010 promotes these processes. By taking the approach of RNA immunoprecipitation (RIP) and mass spectrometry, we identified that LINC01010 can interact with vimentin. Further studies demonstrated that LINC01010 negatively affects the vimentin network extension and causes more rapid subunit exchange and lower stability of vimentin filaments. In addition, LINC01010 can reduce the amount of insoluble vimentin within cells, which suggests that LINC01010 interfers with vimentin polymerization. These data indicate that LINC01010 can inhibit the assembly of vimentin filament. Thus, we revealed that HBV HBx-downregulated LINC01010, which suppresses cell proliferation and migration by negatively regulating the formation of vimentin filament. Taken together, LINC01010 is a potential tumor suppressor that may restrain HBV-related HCC development.

Global analysis of HBV-mediated host proteome and ubiquitylome change in HepG2.2.15 human hepatoblastoma cell line

Hepatitis B virus (HBV) infection remains a major health issue worldwide and the leading cause of cirrhosis and hepatocellular carcinoma (HCC). It has been reported previously that HBV invasion can extensively alter transcriptome, the proteome of exosomes and host cell lipid rafts. The impact of HBV on host proteins through regulating their global post-translational modifications (PTMs), however, is not well studied. Viruses have been reported to exploit cellular processes by enhancing or inhibiting the ubiquitination of specific substrates. Nevertheless, host cell physiology in terms of global proteome and ubiquitylome has not been addressed yet. Here by using HBV-integrated HepG2.2.15 model cell line we first report that HBV significantly modify the host global ubiquitylome. As currently the most widely used HBV cell culture model, HepG2.2.15 can be cultivated for multiple generations for protein labeling, and can replicate HBV, express HBV proteins and secrete complete HBV Dane particles, which makes it a suitable cell line for ubiquitylome analysis to study HBV replication, hepatocyte immune response and HBV-related HCC progression. Our previous experimental results showed that the total ubiquitination level of HepG2.2.15 cell line was significantly higher than that of the corresponding parental HepG2 cell line. By performing a Ubiscan quantification analysis based on stable isotope labeling of amino acids in cell culture (SILAC) of HepG2.2.15 and HepG2 cell lines, we identified a total of 7188 proteins and the protein levels of nearly 19% of them were changed over 2-folds. We further identified 3798 ubiquitinated Lys sites in 1476 host proteins with altered ubiquitination in response to HBV. Our results also showed that the global proteome and ubiquitylome were negatively correlated, indicating that ubiquitination might be involved in the degradation of host proteins upon HBV integration. We first demonstrated the ubiquitination change of VAMP3, VAMP8, DNAJB6, RAB8A, LYN, VDAC2, OTULIN, SLC1A4, SLC1A5, HGS and TOLLIP. In addition, we described 5 novel host factors SLC1A4, SLC1A5, EIF4A1, TOLLIP and BRCC36 that efficiently reduced the amounts of secreted HBsAg and HBeAg. Overall, the HBV-mediated host proteome and ubiquitylome change we reported will provide a valuable resource for further investigation of HBV pathogenesis and host-virus interaction networks.

HBx-upregulated MAFG-AS1 promotes cell proliferation and migration of hepatoma cells by enhancing MAFG expression and stabilizing nonmuscle myosin IIA.

To identify hepatitis B virus (HBV)-related lncRNA(s), we previously examined the transcription profiles of the HBV-transgenic cell line HepG2-4D14 and parental HepG2 cells by RNA deep sequencing and identified 38 upregulated long noncoding RNAs (lncRNAs). In the present study, the lncRNA MAFG-AS1 is investigated in detail because its gene is located adjacent to the MAFG gene, which is an important transcription factor involved in cell proliferation. The level of MAFG-AS1 is significantly higher in HCC tissue than in nontumor tissues. TCGA data show that the expression level of MAFG-AS1 is negatively correlated with survival of HCC patients. GEO cohort data show that compared with healthy tissues, the expression level of MAFG-AS1 is significantly higher in HBV-infected liver tissues. Real-time PCR and luciferase reporter assay data show that HBx can enhance the transcription of MAFG-AS1. Gain-of-function and loss-of-function experiments indicate that MAFG-AS1 promotes proliferation, migration, and invasion of HCC cells. Tumor formation assay results demonstrate that knockdown of MAFG-AS1 significantly inhibits cell proliferation in nude mice. Furthermore, MAFG-AS1 enhances the transcription of adjacent MAFG via E2F1. Additionally, MAFG-AS1 interacts with three subunits (MYH9, MYL12B, and MYL6) of nonmuscle myosin IIA (NM IIA). Knockdown of MAFG-AS1 inhibits ATPase activity of MYH9, interaction of NM IIA subunits, and cell cycle progression. Thus, the lncRNA MAFG-AS1 is upregulated by HBV and promotes proliferation and migration of HCC cells. Our findings suggest that MAFG-AS1 is a potential oncogene that may contribute to HBV-related HCC development.

Valproic acid reverses sorafenib resistance through inhibiting activated Notch/Akt signaling pathway in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common lethal human malignancies worldwide. Sorafenib is the first-line drug approved by the United States Food and Drug Administration for HCC. However, the acquired resistance to sorafenib reduces its beneficial effects and limits clinical use. In this study, we established a sorafenib-resistant HCC cell line HepG2-SR by low-concentration gradient induction. Compared with the parental cell HepG2, the proliferation and anti-apoptosis were increased in drug-resistant cell HepG2-SR. Thorough comparisons of the molecular changes between parental HepG2 and sorafenib-resistant HepG2-SR cells indicated that the Notch signaling pathway and PI3K/Akt signaling pathway were associated with sorafenib resistance mechanisms. Notch1 and Akt were upregulated in sorafenib-resistant cells. However, we surprisingly found that valproic acid (VPA) combined with sorafenib could enhance the sensitivity of drug-resistant cells and reverse the increased levels of Notch1 and Akt in sorafenib-resistant HCC cells. Moreover, Akt inhibitor could suppress Notch1 expression, whereas the level of Akt phosphorylation decreased along with increasing dose of Notch inhibitor. Besides, we found that knockdown of Akt resulted in Notch1 reduction, whereas Notch1 reduction also led to a significant reduction in the phosphorylation of Akt. Collectively, our results indicated that Notch1 and Akt might play vital roles in sorafenib resistance in HCC cells and VPA might overcome the drug resistance to enhance the sensitivity of HCC cells to sorafenib through suppressing Notch/Akt signaling pathway. VPA combined with sorafenib may provide a potential targeting therapeutic regimen for clinically to solve the problem of sorafenib resistance.

The Transfection of HepG2 cells with Truncated β-Catenin Coding Expression Vector

β-catenin is an effector protein in Wnt signaling. β-catenin mutations are reported in the development of many diseases such as autism, colorectal carcinoma, developmental delay, intellectual disability, neurodegeneration, skin, hair and facial anomalies. Exon 3 deletion mediated truncations of the β-catenin associated with these diseases. Therefore understanding the functions of wild type and exon 3 deleted forms of β-catenin may provide an enhancement in the treatment of many diseases. However, to conduct controlled experiments, there could be a demand for the expression vectors that code for wild type and exon 3 deleted forms of β-catenin and originated from the same organism. Since it has long been known that HepG2 cells are heterozygous for β-catenin, in this study, it was found worthy of constructing the expression vectors from the total RNA of HepG2 cells. Then the utility of truncated β-catenin coding pcDNA3.1/CTNNB1 expression vector for upregulation of truncated β-catenin in HepG2 cells was examined. To this end, RNA was isolated from HepG2 cells, cDNA fragments were amplified by polymerase chain reaction (PCR), expression vectors were constructed then sequenced from 5’-prime regions. Following the BLAST analysis, it was concluded that both truncated and wild type β-catenin coding pcDNA3.1/CTNNB1 expression vectors were successfully cloned in E. coli cells. Interestingly, when the parental HepG2 cells were transfected with exon 3 deleted expression vector, β-catenin protein levels were not affected. Moreover, cellular morphology and population doubling time were not significantly altered.

HepG2-1A2 C2 and C7: Lentivirus vector-mediated stable and functional overexpression of cytochrome P450 1A2 in human hepatoblastoma cells

Novel HepG2 cell clones 1A2 C2 and 1A2 C7 were independently generated by lentiviral transduction to functionally overexpress cytochrome P450 1A2 (CYP1A2). We found similar and stable CYP1A2 transcript and protein levels in both cell clones leading to specific enzyme activities of about 370 pmol paracetamol x min-1 x mg-1 protein analyzed by phenacetin conversion. Both clones showed dramatically increased sensitivity to the hepatotoxic compound aflatoxin B1 (EC50 < 100 nM) when compared to parental HepG2 cells (EC50∼5 μM). Thus, newly established cell lines are an appropriate tool to study metabolism and toxicity of substances depending on conversion by CYP1A2.

Claudin-1 silencing increases sensitivity of liver cancer HepG2 cells to 5-fluorouracil by inhibiting autophagy.

Liver cancer is one of the most common cancer types globally. However, the acquisition of drug resistance limits the effectiveness of chemotherapy and commonly results in metastasis. Therefore, an effective therapeutic approach to target chemoresistance-associated cellular molecules is imperative. Claudin-1 (CLDN1) has previously been reported to be associated with the development of drug resistance. The present study investigated the effect of CLDN1 on the sensitivity of 5-fluorouracil (5-FU)-resistant liver cancer cells. Firstly, a 5-FU-resistant HepG2 liver cancer cell line (Hep/5FU) was developed by continuous 5-FU treatment. MTT proliferation, Transwell and Matrigel assays indicated that Hep/5FU cells were significantly resistant to 5-FU, and demonstrated increased migration and invasion abilities, compared with parental HepG2 cells. Furthermore, reverse transcription-quantitative polymerase chain reaction and western blot analysis indicated that mRNA and protein expression levels of CLDN1 were significantly increased in Hep/5FU cells, compared with HepG2 cells. CLDN1 was knocked down by transfection with small interference RNA. MTT and Annexin V-fluorescein isothiocyanate/propidium iodide assays demonstrated that CLDN1 silencing significantly inhibits proliferation and enhances apoptosis induced by 5-FU treatment in Hep/5FU cells, compared with non-silenced Hep/5FU cells. Additionally, CLDN1 silencing attenuated the migration and invasion capabilities of Hep/5FU cells. In addition, it was identified that CLDN1 silencing decreased drug resistance by inhibiting autophagy, which was associated with a decrease in the ratio of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/LC3-I and upregulation of P62. A cell proliferation assay revealed that the addition of autophagy inhibitor 3-methyladenine decreased drug resistance of Hep/5FU cells. By contrast, incubation with the autophagy agonist Rapamycin elevated drug resistance of CLDN1-silenced Hep/5FU cells. In summary, these data indicate that CLDN1 may be a potential target for resensitizing resistant liver cancer HepG2 cells to 5-FU by regulating cell autophagy.

Vimentin as a potential therapeutic target in sorafenib resistant HepG2, a HCC model cell line.

Background/AimsHepatocellular carcinoma (HCC) is the most common liver cancer with high mortality rate in patients suffering from liver diseases. The drug of choice used in advanced-stage of HCC is sorafenib. However, adaptive resistance has been observed in HCC patients undergoing long-term sorafenib treatment, lowering its effectiveness. Hence, it is important to overcome drug resistance to improve overall management of HCC. Here, we have identified a candidate biomarker for sorafenib resistance in a HCC model cell line, HepG2. MethodsInitially, comparative proteomic profiling of parental HepG2 [HepG2 (P)] and sorafenib-resistant HepG2 [HepG2 (R)] cells was performed via MALDI (matrix-assisted laser desorption/ionization) which revealed the deregulation of vimentin in HepG2 (R) cells. Gene and protein level expression of vimentin was also observed through quantitative real-time polymerase chain reaction (qRT PCR) and fluorescence-activated cell sorting (FACS), respectively. Furthermore, withaferin A was used to study regulation of vimentin expression and its significance in sorafenib resistance. ResultsBoth gene and protein level of vimentin expression was found to be downregulated in HepG2 (R) in comparison to HepG2 (P). Interestingly, the study demonstrated that withaferin A further lowered the expression of vimentin in HepG2 (R) cells in a dose-dependent manner. Also, inhibition of vimentin lowered ABCG2 expression and decreased cell viability in parental as well as sorafenib resistant HepG2 cells. ConclusionsHence, our study for the first time highlighted the probable therapeutic potential of vimentin in sorafenib resistant HepG2, a HCC model cell line.

The mitochondrial chaperone Prohibitin 1 negatively regulates interleukin-8 in human liver cancers

Prohibitin 1 (PHB1) is a mitochondrial chaperone whose expression is dysregulated in cancer. In liver cancer, PHB1 acts as a tumor suppressor, but the mechanisms of tumor suppression are incompletely understood. Here we aimed to determine PHB1 target genes to better understand how PHB1 influences liver tumorigenesis. Using RNA-Seq analysis, we found interleukin-8 (IL-8) to be one of the most highly up-regulated genes following PHB1 silencing in HepG2 cells. Induction of IL-8 expression also occurred in multiple liver and nonliver cancer cell lines. We examined samples from 178 patients with hepatocellular carcinoma (HCC) and found that IL-8 mRNA levels were increased, whereas PHB1 mRNA levels were decreased, in the tumors compared with adjacent nontumorous tissues. Notably, HCC patients with high IL-8 expression have significantly reduced survival. An inverse correlation between PHB1 and IL-8 mRNA levels is found in HCCs with reduced PHB1 expression. To understand the molecular basis for these observations, we altered PHB1 levels in liver cancer cells. Overexpression of PHB1 resulted in lowered IL-8 expression and secretion. Silencing PHB1 increased c-Jun N-terminal kinase (JNK) and NF-κB activity, induced nuclear accumulation of c-JUN and p65, and enhanced their binding to the IL-8 promoter containing AP-1 and NF-κB elements. Conditioned medium from PHB1-silenced HepG2 cells increased migration and invasion of parental HepG2 and SK-hep-1 cells, and this was blocked by co-treatment with neutralizing IL-8 antibody. In summary, our findings show that reduced PHB1 expression induces IL-8 transcription by activating NF-κB and AP-1, resulting in enhanced IL-8 expression and release to promote tumorigenesis.

Long noncoding RNA SNHG16 induces sorafenib resistance in hepatocellular carcinoma cells through sponging miR-140-5p.

BackgroundSorafenib is widely used for treatment of hepatocellular carcinoma (HCC), but the acquired resistance remains a major obstacle for its application. Thus it is of critical importance to elucidate the molecular mechanisms underlying sorafenib resistance in HCC. This study aimed to determine the roles of long noncoding RNA SNHG16 in sorafenib-resistant HCC cells.MethodsHCC and matched adjacent normal liver tissue samples were obtained from 103 HCC patients. Sorafenib-resistant HepG2/SOR cell line was established from its parental HepG2 cells by exposure to increasing concentrations of sorafenib. SNHG16 and miR-140-5p expression levels in tissue samples and cells were detected by RT-qPCR analysis. The sensitivity of cells to sorafenib in vitro was evaluated by MTT assay, and the sensitivity of HepG2/SOR cells to sorafenib in vivo was estimated using the nude mouse-based xenograft model. The potential binding relation between SNHG16 and miR-140-5p was validated by dual luciferase reporter assay and biotinylated RNA pull-down assay.ResultsThe results showed that SNHG16 expression was remarkably increased in HCC tissues and cell lines, and its high expression was closely associated with aggressive clinicopathological features and poor prognosis of HCC patients. Further experiments showed that SNHG16 is upregulated in HepG2/SOR cells, whereas knockdown of SNHG16 increases the sensitivity of HepG2/SOR cells to sorafenib in vitro and in vivo. Further mechanistic study identified that SNHG16 functions as an endogenous sponge for miR-140-5p in HepG2 cells, and in HCC tissues, the expression of miR-140-5p is negatively correlated with SNHG16 expression. Moreover, miR-140-5p overexpression also increases the sensitivity of HepG2/SOR cells to sorafenib, and the effects of SNHG16 knockdown on sorafenib resistance could be blocked by miR-140-5p inhibitor.ConclusionCollectively, our findings demonstrated that knockdown of SNHG16 attenuated sorafenib resistance in HCC through sponging miR-140-5p, indicating that SNHG16 might be as a promising therapeutic target to boost the effectiveness of chemotherapy for HCC patients.