Gene In HepG2 Cells Research Articles

Direct and macrophage stimulation mediated effects of active, inactive, and cell-free supernatant forms of Akkermansia muciniphila and Faecalibacterium duncaniae on hepcidin gene expression in HepG2 cells.

Hepcidin is a crucial regulator of iron homeostasis with protective effects on liver fibrosis. Additionally, gut microbiota can also affect liver fibrosis and iron metabolism. Although the hepatoprotective potential of Akkermansia muciniphila and Faecalibacterium duncaniae, formerly known as F. prausnitzii, has been reported, however, their effects on hepcidin expression remain unknown. We investigated the direct and macrophage stimulation-mediated effects of active, heat-inactivated, and cell-free supernatant (CFS) forms of A. muciniphila and F. duncaniae on hepcidin expression in HepG2 cells by RT-qPCR analysis. Following stimulation of phorbol-12-myristate-13-acetate (PMA) -differentiated THP-1 cells with A. muciniphila and F. duncaniae, IL-6 concentration was assessed via ELISA. Additionally, the resulting supernatant was treated with HepG2 cells to evaluate the effect of macrophage stimulation on hepcidin gene expression. The expression of genes mediating iron absorption and export was also examined in HepG2 and Caco-2 cells via RT-qPCR. All forms of F. duncaniae increased hepcidin expression while active and heat-inactivated/CFS forms of A. muciniphila upregulated and downregulated its expression, respectively. Active, heat-inactivated, and CFS forms of A. muciniphila and F. duncaniae upregulated hepcidin expression, consistent with the elevation of IL-6 released from THP-1-stimulated cells as a macrophage stimulation effect in HepG2 cells. A. muciniphila and F. duncaniae in active, inactive, and CFS forms altered the expression of hepatocyte and intestinal iron-mediated absorption /exporter genes, namely dcytb and dmt1, and fpn in HepG2 and Caco-2 cells, respectively. In conclusion, A. muciniphila and F. duncaniae affect not only directly but also through macrophage stimulation the expression of hepcidin gene in HepG2 cells. These findings underscore the potential of A. muciniphila and F. duncaniae as a potential therapeutic target for liver fibrosis by modulating hepcidin and intestinal and hepatocyte iron metabolism mediated gene expression.

Knockout of LINC01134 as a potential treatment for Liver Cancer in HepG2 cells

Liver cancer, a major global health issue, continues to contribute significantly to global deaths. Hepatocellular carcinoma (HCC), the most prevalent primary liver cancer, remains a leading cause of cancer-related fatalities. This study explores the potential treatment for HCC by investigating the role of LINC01134, an encoding gene for the LncRNA TLNC1, in mediating the nuclear export of p53. The hypothesis suggests that knocking out the LINC01134 gene in HepG2 cells will decrease p53 nuclear export, leading to increased p53 levels and enhanced cellular responses to DNA damage. The methods include CRISPR CAS-9 for gene knockout, RT-PCR for TLNC1 expression, Immunofluorescence for p53 within the nucleus, and xenograft mouse model for in vivo tumor size reduction. Statistical analysis will be performed using a one-sample T-test. This strategy might effectively improve the cell’s reaction to DNA damage, which could halt or slow the growth of HCC.

Liver regulatory mechanisms of noncoding variants at lipid and metabolic trait loci

Genome-wide association studies (GWASs) have identified hundreds of risk loci for liver disease and lipid-related metabolic traits, although identifying their target genes and molecular mechanisms remains challenging. We predicted target genes at GWAS signals by integrating them with molecular quantitative trait loci for liver gene expression (eQTL) and liver chromatin accessibility QTL (caQTL). We predicted specific regulatory caQTL variants at four GWAS signals located near EFHD1, LITAF, ZNF329, and GPR180. Using transcriptional reporter assays, we determined that caQTL variants rs13395911, rs11644920, rs34003091, and rs9556404 exhibit allelic differences in regulatory activity. We also performed a protein binding assay for rs13395911 and found that FOXA2 differentially interacts with the alleles of rs13395911. For variants rs13395911 and rs11644920 in putative enhancer regulatory elements, we used CRISPRi to demonstrate that repression of the enhancers altered the expression of the predicted target and/or nearby genes. Repression of the element at rs13395911 reduced the expression of EFHD1, and repression of the element at rs11644920 reduced the expression of LITAF, SNN, and TXNDC11. Finally, we showed that EFHD1 is a metabolically active gene in HepG2 cells. Together, these results provide key steps to connect genetic variants with cellular mechanisms and help elucidate the causes of liver disease.

Milk thistle nano-micelle formulation promotes cell cycle arrest and apoptosis in hepatocellular carcinoma cells through modulating miR-155-3p /SOCS2 /PHLDA1 signaling axis

BackgroundHepatocellular Carcinoma (HCC) is a prevalent form of liver cancer that causes significant mortality in numerous individuals worldwide. This study compared the effects of milk thistle (MT) and nano-milk thistle (N-MT) on the expression of the genes that participate in apoptosis and cell cycle pathways in Huh-7 and HepG2 cells.MethodsIC50 values of MT and N-MT were determined using the MTT assay. Huh-7 and HepG2 cell lines (containing mutant and wild-type TP53 gene, respectively) were incubated with MT and N-MT for 24h and 48h and the impact of MT and N-MT on the proliferation of these cell lines was evaluated through a comparative analysis. Cell cycle and apoptosis were assessed by flow cytometry after 24h and 48h treatment in the cell lines mentioned. Real-time PCR was used to analyze miR-155-3p, PHLDA1, SOCS2, TP53, P21, BAX, and BCL-2 expression in the cell lines that were being treated.ResultsN-MT reduces cancer cell growth in a time and concentration-dependent manner, which is more toxic compared to MT. Huh-7 was observed to have IC50 values of 2.35 and 1.7 μg/ml at 24h and 48h, and HepG2 was observed to have IC50 values of 3.4 and 2.6 μg/ml at 24 and 48h, respectively. N-MT arrested Huh-7 and HepG2 cells in the Sub-G1 phase and induced apoptosis. N-MT led to a marked reduction in the expression of miR-155-3p and BCL-2 after 24h and 48h treatments. Conversely, PHLDA1, SOCS2, BAX, and P21 were upregulated in the treated cells compared to untreated cells, which suggests that milk thistle has the potential to regulate these genes. N-MT reduced the expression of TP53 in Huh-7 cells after mentioned time points, while there was a significant increase in the expression of the TP53 gene in HepG2 cells. No gene expression changes were observed in MT-treated cells after 24h and 48h.ConclusionN-MT can regulate cancer cell death by arresting cell cycle and inducing apoptosis. This occurs through the alteration of apoptotic genes expression. A reduction in the expression of miR-155-3p and increase in the expression of SOCS2 and PHLDA1 after N-MT treatment showed the correlation between miR-155-3p and PHLDA1/SOCS2 found in bioinformatics analysis. While N-MT increased TP53 expression in HepG2, reduced it in Huh-7. The findings indicate that N-MT can function intelligently in cancer cells and can be a helpful complement to cancer treatment.

Design, Synthesis, and Antiproliferative Activities of 1,3‐Disubstituted 2‐Thioxoimidazolidin‐4‐one Derivatives against HepG2 and MCF‐7 Cells

AbstractA series of 1,3‐disubstituted‐2‐thioxoimidazolidin‐4‐one and diazine derivatives were synthesized and confirmed through IR, NMR, and mass spectrometry. The in vitro cytotoxic activity of synthesized compounds against HePG2 and MCF‐7 cells were assessed by MTT assay. Furthermore, Aromatase, VEGFR‐2, B‐Raf, cell cycle, Annexin V, caspase‐3 and sulphatase expression by PCR, and β‐catenin expression by western blot were estimated. The results revealed that the synthesized compounds possess cytotoxic activity against HePG2 and MCF‐7 cell lines. Compound 11 (2,2′‐(1E,1′E)‐1,1′‐(hydrazine‐1,2‐diylidene) bis (ethan‐1‐yl‐1‐ylidene) bis (4‐(phenyldiazenyl) phenol) had greater cytotoxic activity than staurosporine (6.24 μg/mL), with IC50 of 2.55 μg/mL against HepG2 cells and (4.21 μg/mL) against MCF‐7 cells. Moreover, the inhibitory activity against aromatase was (0.156 μg/mL), (0.23 μg/mL) against VEGFR2, and (0.25 μg/mL) against B‐Raf. Also, this compound decreased DNA content of HepG2 cells in the S phase and the G2/M phase, and increased the apoptosis in Annexin measurement. By real‐time PCR, This compound up‐regulated the expression of caspase‐3 in HepG2 cells, reduced the expression of sulphatase gene in HepG2 cells, and reduced β‐catenin expression in HepG2 by western blot analysis. Consequently, the synthesized derivatives could be a promising scaffold for the development of potent anti‐cancer agents due to their apoptotic and anti‐inflammatory activities.

Vesicular Integral-Membrane Protein 36 Is Involved in the Selective Secretion of Fucosylated Proteins into Bile Duct-like Structures in HepG2 Cells.

Fucosylated proteins are widely used as biomarkers of cancer and inflammation. Fucosylated alpha-fetoprotein (AFP-L3) is a specific biomarker for hepatocellular carcinoma. We previously showed that increases in serum AFP-L3 levels depend on increased expression of fucosylation-regulatory genes and abnormal transport of fucosylated proteins in cancer cells. In normal hepatocytes, fucosylated proteins are selectively secreted in the bile duct but not blood. In cases of cancer cells without cellular polarity, this selective secretion system is destroyed. Here, we aimed to identify cargo proteins involved in the selective secretion of fucosylated proteins, such as AFP-L3, into bile duct-like structures in HepG2 hepatoma cells, which have cellular polarity like, in part, normal hepatocytes. α1-6 Fucosyltransferase (FUT8) is a key enzyme to synthesize core fucose and produce AFP-L3. Firstly, we knocked out the FUT8 gene in HepG2 cells and investigated the effects on the secretion of AFP-L3. AFP-L3 accumulated in bile duct-like structures in HepG2 cells, and this phenomenon was diminished by FUT8 knockout, suggesting that HepG2 cells have cargo proteins for AFP-L3. To identify cargo proteins involved in the secretion of fucosylated proteins in HepG2 cells, immunoprecipitation and the proteomic Strep-tag system experiments followed by mass spectrometry analyses were performed. As a result of proteomic analysis, seven kinds of lectin-like molecules were identified, and we selected vesicular integral membrane protein gene VIP36 as a candidate of the cargo protein that interacts with the α1-6 fucosylation (core fucose) on N-glycan according to bibliographical consideration. Expectedly, the knockout of the VIP36 gene in HepG2 cells suppressed the secretion of AFP-L3 and other fucosylated proteins, such as fucosylated alpha-1 antitrypsin, into bile duct-like structures. We propose that VIP36 could be a cargo protein involved in the apical secretion of fucosylated proteins in HepG2 cells.

PS-B04-4: A NOVEL HNF4A COREPRESSOR IRF2BP2: ITS ROLE IN THE GLUCONEOGENIC GENE REGULATION VIA BIOCHEMICALLY LABILE INTERACTION

Hepatocyte nuclear factor 4α (HNF4α) has an essential role in controlling the expression of variety of genes involved in key metabolic pathways including gluconeogenesis in the liver. Though mechanistic and physiological significance of proliferator-activated receptor-gamma co-activator-1α (PGC-1α) for HNF4α-mediated transcriptional activation models for gluconeogenic genes is well characterized, transcriptional repression of HNF4α for those genes still remains to be examined. In this study, we applied novel proteomic techniques to evaluate the HNF4α interactome including biochemically labile binding proteins. From the experiment, we identified interferon regulatory factor 2 binding protein 2 (IRF2BP2) as a novel HNF4α corepressor, with which its interaction could not be detected by conventional immunoprecipitation. IRF2BP2 repressed transcriptional activity of HNF4α dependent on its E3 ubiquitin ligase activity. Deficiency of IRF2BP2 gene in HepG2 cells induced gluconeogenic genes comparable to the forskolin-treated wild type HepG2 cells. Together, these results suggest the role of IRF2BP2 as a novel class of nuclear receptor coregulator.

LBODP052 A Novel Hnf4α Corepressor Irf2bp2: Its Role In The Gluconeogenic Gene Regulation Via Biochemically Labile Interaction

Abstract Hepatocyte nuclear factor 4α (HNF4α) has an essential role in controlling the expression of variety of genes involved in key metabolic pathways including gluconeogenesis in the liver. Though mechanistic and physiological significance of proliferator-activated receptor-gamma co-activator-1α (PGC-1α) for HNF4α-mediated transcriptional activation models for gluconeogenic genes is well characterized, transcriptional repression of HNF4α for those genes still remains to be examined. In this study, we applied novel proteomic techniques to evaluate the HNF4α interactome including biochemically labile binding proteins. From the experiment, we identified interferon regulatory factor 2 binding protein 2 (IRF2BP2) as a novel HNF4α corepressor, with which its interaction could not be detected by conventional immunoprecipitation. IRF2BP2 repressed transcriptional activity of HNF4α dependent on its E3 ubiquitin ligase activity. Deficiency of IRF2BP2 gene in HepG2 cells induced gluconeogenic genes comparable to the forskolin-treated wild type HepG2 cells. Together, these results suggest the role of IRF2BP2 as a novel class of nuclear receptor coregulator. Presentation: No date and time listed

IRF2BP2 is a novel HNF4α co-repressor: Its role in gluconeogenic gene regulation via biochemically labile interaction

Hepatocyte nuclear factor 4α (HNF4α) has essential roles in controlling the expression of a variety of genes involved in key metabolic pathways, including gluconeogenesis in the liver. The mechanistic and physiological significance of peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) for HNF4α-mediated transcriptional activation models for gluconeogenic genes is well characterized. However, the transcriptional repression of HNF4α for those genes remains to be examined. In this study, we applied novel proteomic techniques to evaluate the interactions of HNF4α, including those with biochemically labile binding proteins. Based upon our experiments, we identified interferon regulatory factor 2 binding protein 2 (IRF2BP2) as a novel HNF4α co-repressor. This interaction could not be detected by conventional immunoprecipitation. IRF2BP2 repressed the transcriptional activity of HNF4α dependent on its E3 ubiquitin ligase activity. Deficiency of the IRF2BP2 gene in HepG2 cells induced gluconeogenic genes comparable to that of forskolin-treated wild-type HepG2 cells. Together, these results suggest that IRF2BP2 represents a novel class of nuclear receptor co-regulator.

Aldo-keto reductase family 1 B10 participates in the regulation of hepatoma cell cycle through p27/p-Rb signaling pathway

Objective: Aldo-keto reductase family 1 member B10 (AKR1B10) pathogenesis, early diagnosis and prognosis are closely related with hepatoma. Therefore, this study explores the effect and mechanism of AKR1B10 on cell cycle in hepatoma cells. Methods: HepG2 cells were infected with lentivirus LV-AKR1B10-shRNA or treated with epalrestat, an AKR1B10 inhibitor. The expression level of AKR1B10 was detected by Western blot assay and real-time fluorescence quantitative PCR (RT-qPCR). Decreased AKR1B10 activity was detected by reduced coenzyme II (NADPH) absorbance at 340 nm. The low expression of AKR1B10 and the effect of different concentrations of epalrestat on cell proliferation and cell cycle were detected by CCK-8 method and flow cytometry. The protein expression levels of p-rb, cyclin D1, E1, p27 in HepG2 cells were detected by Western blot. The mean of the two samples was tested using independent sample t-test. Results: AKR1B10 expression level in hepatoma cells was significantly increased compared to normal liver cells, and the relative expression level of AKR1B10 protein in HepG2 cells was 6.71 ± 1.11 (P = 0.012). Epalrestat was significantly inhibited with the enzymatic activity of AKR1B10 in a dose-dependent manner. AKR1B10 gene in HepG2 cells was effectively silenced. HepG2 cells treated with different concentrations of epalrestat (AKR1B10 inhibitor) for 24, 48 and 72 h had inhibited cell proliferation, promoted G0/G1 cell cycle arrest, reduced the expression of p-Rb, cyclin D1, and cyclin E1 and increased the expression of cyclin dependent kinase inhibitor p27 expression. Conclusion: AKR1B10 inhibitory expression and activity can promote G0/G1 cell cycle arrest in HepG2 cells through the p27 / p-Rb pathway.

RORα phosphorylation by casein kinase 1α as glucose signal to regulate estrogen sulfation in human liver cells.

Estrogen sulfotransferase (SULT1E1) metabolically inactivates estrogen and SULT1E1 expression is tightly regulated by multiple nuclear receptors. Human fetal, but not adult, livers express appreciable amounts of SULT1E1 protein, which is mimicked in human hepatoma-derived HepG2 cells cultured in high glucose (450 mg/dl) medium. Here, we have investigated this glucose signal that leads to phosphorylation of nuclear receptor RORα (NR1F1) at Ser100 and the transcription mechanism by which phosphorylated RORα transduces this signal to nuclear receptor HNF4α, activating the SULT1E1 promoter. The promoter is repressed by non-phosphorylated RORα which binds a distal enhancer (−943/−922 bp) and interacts with and represses HNF4α-mediated transcription. In response to high glucose, RORα becomes phosphorylated at Ser100 and reverses its repression of HNF4α promoter activation. Moreover, the casein kinase CK1α, which is identified in an enhancer-bound nuclear protein complex, phosphorylates Ser100 in in vitro kinase assays. During these dynamic processes, both RORα and HNF4α remain on the enhancer. Thus, RORα utilizes phosphorylation to integrate HNF4α and transduces the glucose signal to regulate the SULT1E1 gene in HepG2 cells and this phosphorylation-mediated mechanism may also regulate SULT1E1 expressions in the human liver.

Mining a human transcriptome database for chemical modulators of NRF2.

Nuclear factor erythroid-2 related factor 2 (NRF2) encoded by the NFE2L2 gene is a transcription factor critical for protecting cells from chemically-induced oxidative stress. We developed computational procedures to identify chemical modulators of NRF2 in a large database of human microarray data. A gene expression biomarker was built from statistically-filtered gene lists derived from microarray experiments in primary human hepatocytes and cancer cell lines exposed to NRF2-activating chemicals (oltipraz, sulforaphane, CDDO-Im) or in which the NRF2 suppressor Keap1 was knocked down by siRNA. Directionally consistent biomarker genes were further filtered for those dependent on NRF2 using a microarray dataset from cells after NFE2L2 siRNA knockdown. The resulting 143-gene biomarker was evaluated as a predictive tool using the correlation-based Running Fisher algorithm. Using 59 gene expression comparisons from chemically-treated cells with known NRF2 activating potential, the biomarker gave a balanced accuracy of 93%. The biomarker was comprised of many well-known NRF2 target genes (AKR1B10, AKR1C1, NQO1, TXNRD1, SRXN1, GCLC, GCLM), 69% of which were found to be bound directly by NRF2 using ChIP-Seq. NRF2 activity was assessed across ~9840 microarray comparisons from ~1460 studies examining the effects of ~2260 chemicals in human cell lines. A total of 260 and 43 chemicals were found to activate or suppress NRF2, respectively, most of which have not been previously reported to modulate NRF2 activity. Using a NRF2-responsive reporter gene in HepG2 cells, we confirmed the activity of a set of chemicals predicted using the biomarker. The biomarker will be useful for future gene expression screening studies of environmentally-relevant chemicals.

Aberrant NSUN2-mediated m5C modification of H19 lncRNA is associated with poor differentiation of hepatocellular carcinoma

RNA methylation is an important epigenetic modification. Recent studies on RNA methylation mainly focus on the m6A modification of mRNA, but very little is known about the m5C modification. NSUN2 is an RNA methyltransferase responsible for the m5C modification of multiple RNAs. In this study, we knocked down the NSUN2 gene in HepG2 cells by CRISPR/Cas9 technology and performed high-throughput RNA-BisSeq. An important tumor-related lncRNA H19 was identified to be targeted by NSUN2. Studies have shown that the expression of H19 lncRNA is abnormally elevated and has a carcinogenic effect in many types of tumors. Our results demonstrated that m5C modification of H19 lncRNA can increase its stability. Interestingly, m5C-modified H19 lncRNA can be specifically bound by G3BP1, a well-known oncoprotein which further leads to MYC accumulation. This may be a novel mechanism by which lncRNA H19 exerts its oncogenic effect. Besides, both the m5C methylation level and the expression level of H19 lncRNA in hepatocellular carcinoma tissues were significantly higher than those in adjacent non-cancer tissues, which were closely associated with poor differentiation of hepatocellular carcinoma (HCC). In conclusion, we found that H19 RNA is a specific target for the NSUN2 modifier. The m5C-modified H19 lncRNA may promote the occurrence and development of tumors by recruiting the G3BP1 oncoprotein. Our findings may provide a potential target and biomarker for the diagnosis and treatment of HCC.

Sorafenib- Taurine Combination Model for Hepatocellular Carcinoma Cells: Immunological Aspects.

Sorafenib (Sor) is a multi-kinase inhibitor. It is recommended for the treatment of advanced hepatocellular carcinoma (HCC). However, Sor has severe and marked side effects. On the other hand, taurine (Tau) has been shown to enhance the therapeutic effects of cancer chemotherapy and also to enhance the function of leukocytes. Here, we aimed to investigate the enhancing efficacy of Sor as well as minimizing its marked side effects by using Tau in combination in an immunological aspect. We evaluated the influence of Sor and Tau combination on the expression pattern of FOXP3 gene in HepG2 cells compared to peripheral blood mononuclear leukocytes (PBMCs) as control normal cells. Also, the levels of TGF-β and IL-10 released in culture media of both cells were determined. Our results revealed that, Tau reduced cytotoxicity of Sor on PBMC indicated by lactic dehyrogenase (LDH) release assay. In addition, Sor-Tau combination led to FOXP3 down-regulation in hepatic cancer cells (HepG2). The results showed also that, TGF-β levels decreased significantly in their culture media. In contrary, the cytokine increased in PBMCs culture media. Moreover, IL-10 was significantly elevated in the culture media of both cells. This study could open new avenues for the improvement of therapeutic efficacy of Sorafenib treated HCC patients by using Tau in combination.

Increased Expression of Serine Hydroxymethyltransferase 2 (SHMT2) is a Negative Prognostic Marker in Patients with Hepatocellular Carcinoma and is Associated with Proliferation of HepG2 Cells.

BackgroundSerine hydroxymethyltransferase 2 (SHMT2) is a key enzyme in one-carbon cell metabolism, including in liver cancer. However, the associations between SHMT2 expression at the gene and protein level and prognosis in patients with hepatocellular carcinoma (HCC) remains unknown. This study aimed to investigate the expression levels of SHMT2 in tumor tissue samples from patients with HCC and clinical outcome and the effects of silencing the expression of the SHMT2 gene in HepG2 cells.Material/MethodsExpression levels of SHMT2 were evaluated in 144 cases of HCC using immunohistochemistry and correlated with clinicopathological factors using the chi-squared (χ2) test. The prognostic significance of SHMT2 expression was analyzed by univariate analysis and multivariate analysis. Twenty pairs of HCC tissue and adjacent normal liver tissue were compared for SHMT2 expression levels using quantitative reverse transcription polymerase chain reaction (qRT-PCR). HepG2 cells underwent SHMT2 gene silencing and MTT and transwell assays investigated cell proliferation and migration. Western blot was used to detect the expression of markers of epithelial-mesenchymal transition (EMT).ResultsExpression levels of SHMT2 in HCC tissues were significantly correlated with tumor grade and hepatitis B virus (HBV) infection, and increased expression was an independent negative prognostic factor in patients with HCC (P=0.003). Increased expression of the SHMT2 gene promoted the proliferation and migration of the HepG2 HCC cell line.ConclusionsIncreased expression of SHMT2 was a negative prognostic biomarker in patients with HCC. Expression of the SHMT2 gene promoted the proliferation and migration of HepG2 HCC cells.

1874-P: A Novel Role for SerpinB1 in the Regulation of Insulin Signaling in Hepatocytes

Compensatory expansion of β cell mass has been pursued as a potential therapeutic strategy to cure diabetes. We previously identified SerpinB1 as a hepatocyte-derived circulating factor that contributes to adaptive beta cell expansion in the liver-specific insulin receptor knockout (LIRKO) mouse. To understand how SerpinB1 is regulated and secreted, we began by first investigating the mechanism(s) and signaling pathways that modulate the gene and protein expression of SerpinB1 in hepatocytes. Hepatic SerpinB1 expression was elevated in insulin resistant mouse models such as db/db and ob/ob, and circulating interleukin-6 (IL-6) levels were higher in these mice. Indeed, IL-6 induced SerpinB1 expression in HepG2 cells and human primary hepatocytes via the JAK/STAT3 pathway, indicating an important role for this cytokine in inducing the expression of the protease inhibitor in insulin resistant states. Conversely, silencing of SERPINB1 gene in hepatocytes decreased INSR gene and protein expression with a consequent alteration of expression of several downstream signaling genes related to gluconeogenesis and cell proliferation in hepatocytes. Ribosomal protein S3 (RPS3), identified as a binding partner of SerpinB1 in HepG2 cells, exhibited reduced expression in db/db and ob/ob mice liver along with reduced insulin receptor (IR) expression. Silencing of RPS3 gene in HepG2 cells, phenocopied the effects of SerpinB1 gene silencing, and decreased IR protein expression with consequent alterations in downstream signaling proteins. Additional studies are required to examine the precise regulation of IR expression by SerpinB1 and how the latter is secreted from insulin resistant hepatocytes. These results indicate that SerpinB1 expression is upregulated in insulin resistance by IL-6 and the SerpinB1/RPS3 complex plays a role in regulating insulin signaling in hepatocytes. Disclosure K. Orime: None. D.F. De Jesus: None. U. Jhala: None. R. Kulkarni: None.

Construction of Traf3 knockout liver cancer cell line using CRISPR/Cas9 system.

The gene editing technology in CRISPR/Cas9 system was used to construct the Traf3 knockout HepG2 cell line to explore the role of Traf3 in the development of liver cancer. Five sgRNA sites were designed for the exons of Traf3. The recombinant plasmid of Lentiviral vector2-Traf3-sgRNA was constructed and transformed into Stbl3 competent cells. The recombinants were screened and sequenced, and the effectiveness of the designed gRNA was verified by sequencing. The constructed vector was transfected into HepG2 cells by lentiviral, and the monoclonal antibody was selected to detect the knockout effect of Traf3 gene in HepG2 cells by Western blot. PCR amplification and gene sequencing were performed to obtain the cell line, which the Traf3 gene was knocked out. MTT and Transwell assays were used to detect the effect of Traf3-knockout on HepG2 cell proliferation and cell invasion, respectively. The Lentiviral vector2-sgRNA expression vector was successfully constructed. PCR amplification electrophoresis and gene sequencing showed that the Trep3-knockdown HepG2 cells were successfully constructed. Compared with the wild HepG2 cells group, the proliferation and invasion ability of HepG2 cells were enhanced in the Traf3 knockout group. Knockout Traf3 gene by CRISPR/Cas9 system enhanced the proliferation, migration, and invasion of HepG2 cells, and provided an effective tool for studying the function and mechanism of Traf3.

Expression profiles of long noncoding RNAs associated with the NSUN2 gene in HepG2 cells.

NOP2/Sun domain family member 2 (NSUN2) is upregulated in numerous types of tumors and may be implicated in multiple biological processes, including cell proliferation, migration and human tumorigenesis. However, little is known about how NSUN2 serves a role in these processes. In the present study, expression profiles of long noncoding RNAs (lncRNAs) and mRNAs were developed in NSUN2-deficient HepG2 cells by RNA-sequencing analysis. A total of 757 lncRNAs were differentially expressed, 392 of which were upregulated, and 365 were downregulated compared with wild-type HepG2 cells. Moreover, 212 lncRNAs were co-expressed with 368 target mRNAs. It was also observed that 253 pairs of lncRNAs and mRNAs exhibited negative correlations and that 290 pairs had positive correlations. Bioinformatics analysis indicated that these lncRNAs regulated by NSUN2 were associated with ‘signal transduction’, ‘extracellular exosome’ and ‘calcium ion binding’, and were enriched in ‘pathways in cancer’, ‘PI3K-Akt signaling pathway’ and ‘ECM-receptor interaction pathway’. These results illustrate the landscape and co-expression network of lncRNAs regulated by NSUN2 and provide invaluable information for studying the molecular function of NSUN2.

Three New Polyynes from Codonopsis pilosula and Their Activities on Lipid Metabolism.

Three new polyynes, named choushenpilosulynes A–C (1–3), were isolated from an 85% aqueous EtOH extract of the roots of Codonopsis pilosula cultivated in Xundian County of Yunnan province, China. Their structures, including the absolute configuration of the glucose residue in 1 and 2, were determined by spectroscopic analysis and gas chromatography (GC). In addition, biological evaluation shows that all the compounds can inhibit the expression of the squalene monooxygenase (SQLE) gene in HepG2 cells, suggesting that these compounds may be involved in lipid metabolism.

Persian shallot, Allium hirtifolium Boiss, induced apoptosis in human hepatocellular carcinoma cells.

This study investigated the potential of Persian shallot extract as an anticancer agent in HepG2 tumor cell line, an in vitro human hepatoma cancer model system. The inhibitory effect of Persian shallot on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of Persian shallot, the activity of Persian shallot in inducing apoptosis was investigated through the detection of annexin V signal by flow cytometry and expression of some apoptosis related genes such p21, p53, puma, caspase-8 family-Bcl-2 proteins like bid, bim, bcl-2 and bax were measured by real-time PCR in HepG2 cells. Persian shallot extract inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 149μg/ml. The results of real-time PCR revealed a significant up-regulation of bid, bim, caspase-8, puma, p53, p21 and bax genes and a significant downregulation of bcl-2 gene in HepG2 cells treated with Persian shallot extract significantly. Therefore, this is the first report on an increased expression of bid, bim, caspase-8, puma, p53, p21 and bax genes and down regulation of bcl-2 gene indicating that the Persian shallot extract possibly induced the process of cell death through the intrinsic and extrinsic apoptosis pathways and triggers the programmed cell death in HepG2 tumor cell lines by modulating the expression of pro-/anti-apoptotic genes. Furthermore, we showed that Persian shallot extract increased annexin V signal and expression, resulting in apoptotic cell death of HepG2 cells after 24h treatment. Therefore, according to the results of this study, the Persian shallot extract could be considered as a potential candidate for production of drug for the prevention or treatment of human hepatoma.