Expression In HepG2 Research Articles

Ascorbic acid enhances low-density lipoprotein receptor expression by suppressing proprotein convertase subtilisin/kexin 9 expression

Ascorbic acid, a water-soluble antioxidant, regulates various biological processes and is thought to influence cholesterol. However, little is known about the mechanisms underpinning ascorbic acid-mediated cholesterol metabolism. Here, we determined if ascorbic acid can regulate expression of proprotein convertase subtilisin/kexin 9 (PCSK9), which binds low-density lipoprotein receptor (LDLR) leading to its intracellular degradation, to influence low-density lipoprotein (LDL) metabolism. At cellular levels, ascorbic acid inhibited PCSK9 expression in HepG2 and Huh7 cell lines. Consequently, LDLR expression and cellular LDL uptake were enhanced. Similar effects of ascorbic acid on PCSK9 and LDLR expression were observed in mouse primary hepatocytes. Mechanistically, ascorbic acid suppressed PCSK9 expression in a forkhead box O3-dependent manner. In addition, ascorbic acid increased LDLR transcription by regulating sterol regulatory element-binding protein 2. In vivo, administration of ascorbic acid reduced serum PCSK9 levels and enhanced liver LDLR expression in C57BL/6J mice. Reciprocally, lack of ascorbic acid supplementation in L-gulono-γ-lactone oxidase deficient (Gulo-/-) mice increased circulating PCSK9 and LDL levels, and decreased liver LDLR expression, whereas ascorbic acid supplementation decreased PCSK9 and increased LDLR expression, ameliorating LDL levels in Gulo-/- mice fed a high fat diet. Moreover, ascorbic acid levels were negatively correlated to PCSK9, total and LDL levels in human serum samples. Taken together, these findings suggest that ascorbic acid reduces PCSK9 expression, leading to increased LDLR expression and cellular LDL uptake. Thus, supplementation of ascorbic acid may ameliorate lipid profiles in ascorbic acid-deficient species.

RETRACTED: Growth Arrest-Specific Transcript 5 (GAS5) Exerts Important Roles on the Treatment of BM45 Cells of Liver Cirrhosis

Bone marrow (BM)-derived CD45 (BM45) cells were demonstrated to exhibit an improved antifibrotic effect on the treatment of CCL4-induced liver fibrosis by significantly increasing the level of matrix metalloproteinase 9 (MMP-9). In this study, we aimed to validate the therapeutic effect of BM45 on the treatment of liver cirrhosis and to further investigate the molecular mechanism underlying the effect of growth arrest-specific transcript 5 (GAS5) on BM45. Accordingly, GAS5 significantly suppressed miR-222 and miR-21 expression but enhanced p27 and MMP-9 expression in HepG2 and LX2 cells. Additionally, GAS5 obstructed transforming growth factor (TGF)-β-induced dysregulation of miR-222, p27, and α-smooth muscle actin (α-SMA) in mice. GAS5 showed a considerable potential to enhance the capability of BM45 in restoring the normal expression of CCL4, miR-222, miR-21, MMP-9, p27, and α-SMA that was dysregulated by alanine aminotransferase (ALT), albumin, and fibrosis. In summary, our study validated the regulatory relationship between miR-21 and MMP-9, as well as between miR-222 and p27. The overexpression of GAS5 upregulated the expression of MMP-9 and p27 via respectively reducing the miR-222 and miR-21 expression, resulting in higher BM45-induced activation of hepatic stellate cells (HSCs). Accordingly, same results were obtained in an animal model, indicating that GAS5 may exert a positive effect on the treatment of BM45 of liver cirrhosis.

GB-2 inhibits ACE2 and TMPRSS2 expression: In vivo and in vitro studies

After the first case of Coronavirus disease 2019 (COVID-19) was reported in Wuhan, COVID-19 has rapidly spread to almost all parts of world. Angiotensin converting enzyme 2 (ACE2) receptor can bind to spike protein of SARS-CoV-2. Then, the spike protein of SARS-CoV-2 can be cleaved and activated by transmembrane protease, serine 2 (TMPRSS2) of the host cells for SARS-CoV-2 infection. Therefore, ACE2 and TMPRSS2 are potential antiviral targets for treatment of prevention of SARS-CoV-2 infection. In this study, we discovered that 10−250 μg/mL of GB-2, from Tian Shang Sheng Mu of Chiayi Puzi Peitian Temple, can inhibit ACE2 mRNA expression and ACE2 and TMPRSS2 protein expression in HepG2 and 293 T cells without cytotoxicity. GB-2 treatment could decrease ACE2 and TMPRSS2 expression level of lung tissue and kidney tissue without adverse effects, including nephrotoxicity and hepatotoxicity, in animal model. In the compositions of GB-2, we discovered that 50 μg/mL of theaflavin could inhibit protein expression of ACE2 and TMPRSS2. Theaflavin could inhibit the mRNA expression of ACE2. In conclusion, our results suggest that GB-2 and theaflavin could act as potential compounds for ACE2 and TMPRSS2 inhibitors in the further clinical study.

Involvement of APOBEC3B in mutation induction by irradiation.

To better understand the cancer risk posed by radiation and the development of radiation therapy resistant cancer cells, we investigated the involvement of the cancer risk factor, APOBEC3B, in the generation of radiation-induced mutations. Expression of APOBEC3B in response to irradiation was determined in three human cancer cell lines by real-time quantitative PCR. Using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutation assay, mutations in the HPRT gene caused by irradiation were compared between APOBEC3B-deficient human hepatocellular carcinoma (HepG2) cells [APOBEC3B knocked out (KO) using CRISPR-Cas9 genome editing] and the parent cell line. Then, HPRT-mutated cells were individually cultured to perform PCR and DNA sequencing of HPRT exons. X-Irradiation induced APOBEC3B expression in HepG2, human cervical cancer epithelial carcinoma (HeLa) and human oral squamous cell carcinoma (SAS) cells. Forced expression of APOBEC3B increased spontaneous mutations. By contrast, APOBEC3B KO not only decreased the spontaneous mutation rate, but also strongly suppressed the increase in mutation frequency after irradiation in the parent cell line. Although forced expression of APOBEC3B in the nucleus caused DNA damage, higher levels of APOBEC3B tended to reduce APOBEC3B-induced γ-H2AX foci formation (a measure of DNA damage repair). Further, the number of γ-H2AX foci in cells stably expressing APOBEC3B was not much higher than that in controls before and after irradiation, suggesting that a DNA repair pathway may be activated. This study demonstrates that irradiation induces sustained expression of APOBEC3B in HepG2, HeLa and SAS cells, and that APOBEC3B enhances radiation-induced partial deletions.

α‑enolase is highly expressed in liver cancer and promotes cancer cell invasion and metastasis

The expression levels of α-enolase, also known as enolase 1 (ENO1), in liver cancer tissues and the autoantibody levels of ENO1 in the sera of patients with liver cancer were detected to investigate the function of ENO1 in the invasion and metastasis of liver cancer, as well as its clinical diagnostic value. Small interfering RNA (siRNA) was used to disrupt ENO1 gene expression in HepG2 and Huh7 liver cancer cells. The proliferation ability of liver cancer cells was assessed using Cell Counting Kit-8 (CCK-8); the migration ability of liver cancer cells was assessed using scratch tests; and the migration and invasion abilities of liver cancer cells were assessed using Transwell assays. ENO1 expression in liver cancer tissues (43.8%) was significantly higher than that in benign liver lesions (15.2%) (P=0.005). The serum anti-ENO1 antibody levels in the liver cancer group were significantly higher than those in the control and benign liver lesion groups (P<0.001). After ENO1 gene interference, the proliferation, migration and invasion abilities of HepG2 and Huh7 liver cancer cells exhibited different degrees of suppression. The results revealed that ENO1 promotes liver cancer invasion and metastasis; ENO1 plays an important role in liver cancer and can be used as a potential liver cancer-associated marker.

Chlorogenic Acid Decreases Malignant Characteristics of Hepatocellular Carcinoma Cells by Inhibiting DNMT1 Expression.

BackgroundHepatocellular carcinoma (HCC) is the most common malignant tumor of the adult liver, exhibiting rapid progression and poor prognosis. Chlorogenic acid (CGA), a polyphenol, has several biological activities, including the suppression of liver cancer cell invasion and metastasis. Increased levels or alterations in the function of DNMT1 are associated with the inactivation of tumor suppressor genes. However, the CGA-affected DNMT1 expression mediated mechanism is still unclear.MethodsThe human hepatocellular carcinoma (HCC) HepG2 cells were treated with a positive control drug (5-AZA) or varying doses of CGA. DNA methyltransferase 1 (DNMT1) protein levels and other relevant proteins were evaluated using Western blotting and immunocytochemistry. Cell-cycle analysis was performed by flow cytometry-based PI staining, and cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The transwell invasion and wound healing assays were used to evaluate cell migration and invasion. In vivo proliferation of the HCC cells was detected. We investigated the expression of DNMT1, p53, p21, p-ERK, MMP-2, and MMP-9 in tumors using immunohistochemical analysis.ResultsOur results showed that CGA inhibited the proliferation, colony formation, invasion, and metastasis of HepG2 cells both in vitro and in vivo by down-regulating DNMT1 protein expression, which enhanced p53 and p21 activity, and resulting in a significant reduction in cell proliferation and metastasis. Moreover, CGA inactivated ERK1/2 and reduced MMP-2 and MMP-9 expression in HepG2 cells.ConclusionsCGA can suppress liver cancer cell proliferation, invasion, and metastasis through several pathways. CGA could serve as a candidate chemopreventive agent for HCC.

Estrogen-induced epigenetic silencing of FTH1 and TFRC genes reduces liver cancer cell growth and survival

ABSTRACT Estrogen (E2) regulates hundreds of genes involved in cell metabolism and disrupts iron homoeostasis in various cell types. Herein, we addressed whether E2-induced epigenetic modifications are involved in modulating the expression of iron-regulatory genes. Epigenetic status of FTH1 and TFRC genes was assessed in E2-treated cancer cells. E2-induced DNA methylation was associated with decreased FTH1 and TFRC expression in Hep-G2 and Huh7 cells, but not in AGS or MCF7 cells. Demethylation with 5-Aza-2-deoxycytidine upregulated the expression of both these genes in Hep-G2 cells. The expression of DNMT3B, PRMT5, and H4R3me2s increased in E2-treated cells. Chromatin immunoprecipitation showed that E2 treatment recruited PRMT5 and H4R3me2s on FTH1 but not on TFRC. Knockdown of PRMT5, DNMT3B, and Estrogen-receptor alpha rescued FTH1 from E2-induced silencing. However, knockdown of DNMT3B alone blocked the inhibitory effects of E2 on TFRC. Analysis of human liver tissues in publicly available datasets showed that FTH1 and TFRC are highly expressed in primary liver tumours, but a lower expression is associated with better survival. Interestingly, we showed that the silencing of FTH1 and/or TFRC inhibited carcinogenesis in Hep-G2 cells. For the first time, our findings uncovered the novel signalling pathway involved in the protective effects of E2 against liver cancer.

MicroRNA-148a regulates low-density lipoprotein metabolism by repressing the (pro)renin receptor.

High plasma LDL cholesterol (LDL-c) concentration is a major risk factor for atherosclerosis. Hepatic LDL receptor (LDLR) regulates LDL metabolism, and thereby plasma LDL-c concentration. Recently, we have identified the (pro)renin receptor [(P)RR] as a novel regulator of LDL metabolism, which regulates LDLR degradation and hence its protein abundance and activity. In silico analysis suggests that the (P)RR is a target of miR-148a. In this study we determined whether miR-148a could regulate LDL metabolism by regulating (P)RR expression in HepG2 and Huh7 cells. We found that miR-148a suppressed (P)RR expression by binding to the 3’-untranslated regions (3’-UTR) of the (P)RR mRNA. Mutating the binding sites for miR-148a in the 3’-UTR of (P)RR mRNA completely abolished the inhibitory effects of miR-148a on (P)RR expression. In line with our recent findings, reduced (P)RR expression resulted in decreased cellular LDL uptake, likely as a consequence of decreased LDLR protein abundance. Overexpressing the (P)RR prevented miR-148a-induced reduction in LDLR abundance and cellular LDL uptake. Our study supports a new concept that miR-148a is a regulator of (P)RR expression. By reducing (P)RR abundance, miR-148a decreases LDLR protein abundance and consequently cellular LDL uptake.

Intestinal Inflammation Alters the Expression of Hepatic Bile Acid Receptors Causing Liver Impairment.

The gut-liver axis has been recently investigated in depth in relation to intestinal and hepatic diseases. Key actors are bile acid (BA) receptors, as farnesoid-X-receptor (FXR), pregnane-X-receptor (PXR), and G-protein-coupled-receptor (GPCR; TGR5), that control a broad range of metabolic processes as well as inflammation and fibrosis. The present study aims to investigate the impact of intestinal inflammation on liver health with a focus on FXR, PXR, and TGR5 expression. The strategy to improve liver health by reducing gut inflammation is also considered. Modulation of BA receptors in the inflamed colonic tissues of inflammatory bowel disease (IBD) pediatric patients is analyzed. A dextran sodium sulphate (DSS) colitis animal model was built. Co-cultures with Caco2 and HepG2 cell lines were set up. Modulation of BA receptors in biopsies of IBD pediatric patients was assessed by real-time PCR and immunohistochemistry. Histology showed inflammatory cell infiltration in the liver of DSS mice, where FXR and PXR were significantly decreased and oxidative stress was increased. Exposure of Caco2 to inflammatory stimuli resulted in the reduction of BA receptor expression in HepG2. Caco2 treatment with dipotassium glycyrrhizate (DPG) reduced these effects on liver cells. Inflamed colon of patients showed altered FXR, PXR, and TGR5 expression. This study strongly suggests that gut inflammation affects hepatic cells by altering BA receptor levels as well as increasing the production of pro-inflammatory cytokines and oxidative stress. Hence, reducing gut inflammation is needed not only to improve the intestinal disease but also to protect the liver.

Bidirectional interaction of lncRNA AFAP1-AS1 and CRKL accelerates the proliferative and metastatic abilities of hepatocarcinoma cells

Actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1), a long non-coding RNA transcribed from the antisense strand of protein coding gene AFAP1, has attracted attention in cancer research. Despite, its biological function and regulatory mechanism in hepatocellular carcinoma still unknown. The present study revealed AFAP1-AS1 mediated hepatocarcinoma progression through targeting CRKL. The bidirectional interaction of AFAP1-AS1 and oncogenic protein CRKL, and the deregulation of AFAP1-AS1 effects on Ras, MEK and c-Jun activities were investigated in depth. AFAP1-AS1 was upregulated in surgical tumorous tissues from hepatocarcinoma patients compared with the paired paracancerous non-tumor liver tissues, and in hepatocarcinoma Huh7, HCCLM3 and HepG2 cell lines compared with LO2, a normal liver cell line. AFAP1-AS1 knockdown noticeably suppressed the proliferative, migratory and invasive properties, and the epithelial-mesenchymal transition (EMT) process of HepG2 and HCCLM3 through upregulating E-cadherin and downregulating N-cadherin and vimentin. CRKL knockdown reduced AFAP1-AS1 expression levels in HepG2 and HCCLM3 cells. AFAP1-AS1 suppression impaired CRKL expression in HepG2 and HCCLM3. AFAP1-AS1 level change was positively correlated with the expression level changes of Ras, MEK and c-Jun in mediating the invasiveness of hepatocarcinoma cells. Current work demonstrated AFAP1-AS1 to be an applicable progression indicator of hepatocarcinoma. AFAP1-AS1 probably promotes the proliferation, EMT progression and metastasis of hepatocarcinoma cells via CRKL mediated Ras/MEK/c-Jun and cadherin/vimentin signaling pathways. AFAP1-AS1-CRKL bidirectional feedback signaling is worthy of further study on the monitoring, diagnosis and treatment of cancers.

Transcriptional regulation of microRNA-126a by farnesoid X receptor in vitro and in vivo.

Recent research has indicated the microRNA-126a (miR-126a) is an endothelial cell-specific and highly conserved endogenous small non-coding RNA molecule. It contributes to the vascular integrity and angiogenesis, but the molecular regulation mechanism of miR-126a remains unknown. Herein, quantitative real-time polymerase chain reaction (qRT-PCR) results showed that Farnesoid X Receptor (FXR) activation promoted miR-126a expression in HepG2, LO2, and Hep1-6 cells. Furthermore, FXR was found to transcriptionally regulate the miR-126a by binding to its DR8 site. The binding site of FXR was confirmed on intron 6 or 7 of miR-126a host gene epidermal growth factor-like domain 7 (EGFL7) by luciferase reporter assays, electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays. All these data collectively suggest that FXR regulates transcripts of miR-126a by binding to DR8 in miR-126a gene promoter. This study may provide a molecular therapeutic target for angiogenic disorders, aging, and liver failure.

Minicircle DNA vector expressing interferon-lambda-3 inhibits hepatitis B virus replication and expression in hepatocyte-derived cell line

BackgroundInterferon-alpha (IFNα) is a first-line treatment option for chronic hepatitis B virus (HBV) infection, but the severe systemic side-effects limited its clinical application. Interferon-lambda (IFNλ) with comparable antiviral activity and less toxic side-effects is thought to be a good alternative interferon to IFNα. Additionally, the gene vector mediated sustainably expression of therapeutic product in the target cells/tissue may overcome the shortcomings resulted from the short half-life of IFNs.ResultsWe constructed a liver-specific IFNλ3-expressing minicircle (MC) vector under the control of a hepatocyte-specific ApoE promoter (MC.IFNλ3) and investigated its anti-HBV activity in a HBV-expressing hepatocyte-derived cell model (HepG2.2.15). As expected, the MC.IFNλ3 vector capable of expressing IFNλ3 in the recipient hepatocytes has demonstrated robust anti-HBV activity, in terms of suppressing viral antigen expression and viral DNA replication, via activation the interferon-stimulated gene (ISG) expression in HepG2.2.15 cells.ConclusionsGiven the MC vector can be easily delivered into liver, the liver-targeted IFN gene-transfer (MC.IFNλ3), instead of systemic administrating IFN repeatedly, provides a promising concept for the treatment of chronic HBV infection.

MiR-664b-5p Inhibits Hepatocellular Cancer Cell Proliferation Through Targeting Oncogene AKT2.

Background: miR-664b-5p accelerates the development of certain cancers, but the role of miR-664b-5p in hepatocellular carcinoma (HCC) has been less reported. Therefore, the authors aimed to study the role of miR-664b-5p in HCC progression. Materials and Methods: miR-664b-5p expression in liver cancer and adjacent tissues, and in HepG2 and SUN-475 cells, was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Relationship between miR-664b-5p and AKT2 was predicted by TargetScan and confirmed by dual-luciferase reporter assay, and gene or protein expressions were determined by performing qRT-PCR and Western blotting. The viability and apoptosis, and the migration and invasion of HepG2 and SUN-475 cells were determined by CCK-8 assay and flow cytometry, and transwell assay, respectively. Results: Downregulated miR-664b-5p was observed in hepatocellular cancer tissues. Functional analyses revealed that miR-664b-5p mimic suppressed viability, migration, and invasion, but promoted apoptosis in HepG2 and SUN-475 cells. AKT2 was a target of miR-664b-5p, whose mimics inhibited the expression of AKT2. However, upregulated AKT2 promoted viability, migration, and invasion, but inhibited apoptosis in HepG2 and SUN-475 cells, and such effects were reversed by miR-664b-5p mimics. Conclusions: miR-664b-5p acts as a cancer suppressor through negatively regulating AKT2 expression in HepG2 and SUN-475 cells, suggesting that miR-664b-5p could be a protective target for HCC patients.

Factors affecting expression and transcription of uncoupling protein 2 gene.

Previous studies suggest a negative relationship between hepatic oxidative stress and productivity in beef cattle. Uncoupling protein 2 (UCP2) is involved in the disappearance of reactive oxygen species, suggesting the defensive role of UCP2 against oxidative stress. The present study examined the relationship between oxidative stress and expression levels of UCP2/Ucp2 in cultured human and mouse liver-derived cells. We also explored factors regulating bovine Ucp2 transcription. As oxidative stress inducers, hydrogen peroxide, ethanol, and cumene hydroperoxide (CmHP) were used. Expression levels of hemoxygenase 1 (HMOX1), a representative gene induced by oxidative stress, were not affected by any oxidative stress inducers in HepG2 human liver-derived cells. The levels of UCP2 mRNA were also unaffected by the oxidative stress inducers. Treatment with CmHP increased expression of Hmox1 in Hepa1-6 mouse liver-derived cells, but Ucp2 expression was not changed. Stimulus screening for regulator of transcription (SSRT) revealed that expression of p50 or p65, transcription factors conferring response to oxidative stress, did not stimulate bovine Ucp2 transcrition in HepG2 cells. SSRT also showed 11 molecules that induced Ucp2 transcription more than 4-fold; among them, endoplasmic reticulum (ER) stress-related transcription factors such as XBP1, c-JUN, JUNB, and C/EBPβ were identified. However, treatment with ER stress inducers did not increase Ucp2 expression in HepG2 and Hepa1-6 cells. The present results suggest that 1) neither oxidative stress nor ER stress induces Ucp2 expression in liver-derived cells, and 2) Ucp2 transcription is stimulated by several transcription factors.

MiR-206 inhibits cell proliferation, invasion, and migration by down-regulating PTP1B in hepatocellular carcinoma.

Protein tyrosine phosphatase 1B (PTP1B) has been reported as an oncogene in hepatocellular carcinoma (HCC). However, how PTP1B is regulated in HCC remains unclear. MicroRNAs (miRNAs) are a class of small non-coding RNAs involved many biological processes including tumorigenesis. In this study, we investigated whether miRNA participated in the regulation of PTP1B in HCC. We found that miR-206, which was down-regulated during tumorigenesis, inhibited HCC cell proliferation and invasion. Overexpression of miR-206 inhibited proliferation, invasion, and migration of HCC cell lines HepG2 and Huh7. Mechanistically, we demonstrated that miR-206 directly targeted PTP1B by binding to the 3′-UTR of PTP1B mRNA as demonstrated by the luciferase reporter assay. Overexpression miR-206 inhibited PTP1B expression while miR-206 inhibition enhanced PTP1B expression in HepG2 and Huh7 cells. Functionally, the regulatory effect on cell proliferation/migration/invasion of miR-206 was reversed by PTP1B overexpression. Furthermore, tumor inoculation nude mice model was used to explore the function of miR-206 in vivo. Our results showed that overexpression of miR-206 drastically inhibited tumor development. In summary, our data suggest that miR-206 inhibits HCC development by targeting PTP1B.

ALTERING THE EXPRESSION OF P-GLYCOPROTEIN RESPONSIBLE FOR CHEMOTHERAPEUTIC DRUG RESISTANCE IN HEPG2 CELL LINE POST-TREATMENT WITH UREA AND β-MERCAPTOETHANOL

Objective: The objective of this study is to alter the expression of p-glycoprotein (p-gp) pump proteins in HepG2 cells after treating with urea and β-mercaptoethanol (BME) (lead compounds). The most common cause for resistance to a broad range of anticancer drugs is influenced by overexpression of p-gp pumps that detect and eject anticancer drugs from the cancer cell. Altering the expression of these proteins will reduce the efflux action and enhance the drug retention eventually killing the cancer cell.&#x0D; Materials and Methods: 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyl tetrazolium bromide (MTT) assay was carried out to measure the cell viability (HepG2 cells) post-treatment with the lead compounds followed by flow cytometric analysis for protein expression studies.&#x0D; Results: MTT assay confirms that the viability of HepG2 cells reduces as the concentrations of the lead compounds are increased. Flow cytometric analysis confirms reduced p-gp expression in HepG2 cells post-treatment with urea and BME. Compare to BME, urea turns out to be a potential compound in altering the expression of p-gp.&#x0D; Conclusion: The present cell line study confirms that urea and BME are potential compounds which are able to reduce the p-gp expression inHepG2 cells.

Expression and function of long non-coding RNA epigenetically-induced 1 in hepatocellular carcinoma

Objective To observe the expression of long non-coding RNA (lncRNA EPIC1) in hepatocellular carcinoma (HCC) and its effects on the proliferation, invasion and apoptosis of hepatoma cell lines. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was adopted to detect the expression levels of EPIC1 in 7 cases of HCC tissues and their paracancerous tissues, 5 liver cancer cell lines (HepG2, SK-HEP-1, HuH-7-80, SMMC-7721, SNU-387) and normal human liver cell line (HL-7702). Hep G2 and HuH-7-80 cells were transfected with recombinant lentiviral particles carrying the EPIC1 genes [LV-small interfering RNA (siRNA)] as the experimental group, and Hep G2 and HuH-7-80 cells transfected with negative control lentiviral particles (LV-NC) were used as the control group. The proliferative capacity of HepG2 and HuH-7-80 cells was measured by cell counting kit-8 (CCK-8) proliferation assay after transfection. The invasion capacity of HepG2 and HuH-7-80 cells was examined by Transwell invasion assay. The apoptosis rate of HepG2 and HuH-7-80 cells was detected by flow cytometry. Results The expression level of EPIC1 in HCC tissues (8.77±0.36) was significantly higher than that in their adjacent tissues (2.39±0.45), and the difference was statistically significant (P<0.01). The expression levels of EPIC1 in HepG2, SK-HEP-1, HuH-7-80, SMMC-7721 and SNU-387 cells (7.24±0.81, 3.11±0.35, 5.17±1.12, 4.38±0.48 and 2.69±0.65) was significantly [about (5.61±0.12) and (4.01±0.16) times] higher than that of HL-7702 cells (1.29±0.13), among which HepG2 and HuH-7-80 cells had the highest expression level, and the difference was statistically significant (P<0.01). The proliferative capacity of HepG2 cells in the EPIC1 group with low expression at 24, 48, 72 h (LV-siRNA1: 0.43±0.02, 0.72±0.02, 1.31±0.01, P<0.05; LV-siRNA2: 0.42±0.02, 0.76±0.04, 1.03±0.02, P<0.01) was significantly lower than HepG2 cells (0.59±0.02, 0.98±0.02, 1.63±0.02) in the control group. The proliferative capacity of HuH-7-80 cells with low expression in the EPIC1 group at 24, 48, 72 h (LV-siRNA1: 0.59±0.01, 0.73±0.02 and 1.31±0.05, P<0.05; LV-siRNA2: 0.43±0.04, 0.74±0.04 and 1.26±0.04, P<0.01) was significantly lower than that of the HuH-7-80 cells (0.64±0.02, 0.82±0.02 and 1.78±0.02) in the control group. The number of cells across the basement membrane in the EPIC1 group with low expression in HepG2 and HuH-7-80 cells (43.35±6.16, 55.40±8.18) was significantly less than that in the control group (80.67±5.14 and 74.73±6.13) (P<0.05). The apoptosis rate of HepG2 and HuH-7-80 cells with low expression in EPIC1 group was significantly higher than that in the control group [(2.60±0.96)% and (3.60±0.33)%], and the difference was statistically significant (P<0.01). Conclusion lncRNA EPIC1 is highly expressed in HCC tissues and cells. EPIC1 with low expression can reduce the capacity of proliferation and invasion of hepatoma cells and promote the apoptosis of hepatoma cells. Key words: Carcinoma, hepatocellular; Long non-coding RNA EPIC1; Proliferation; Invasion; Apoptosis

Non-Thermal Plasma, as a New Physicochemical Source, to Induce Redox Imbalance and Subsequent Cell Death in Liver Cancer Cell Lines.

Alteration of cancer cell redox status has been recognized as a promising therapeutic implication. In recent years, the emerged field of non-thermal plasma (NTP) has shown considerable promise in various biomedical applications, including cancer therapy. However, understanding the molecular mechanisms procuring cellular responses remains incomplete. Thus, the aim of this study was a rigorous biochemical analysis of interactions between NTP and liver cancer cells. The concept was validated using three different cell lines. We provide several distinct lines of evidence to support our findings; we use various methods (epifluorescent and confocal microscopy, clonogenic and cytotoxicity assays, Western blotting, pharmacological inhibition studies, etc.). We assessed the influence of NTP on three human liver cancer cell lines (Huh7, Alexander and HepG2). NTP treatment resulted in higher anti-proliferative effect against Alexander and Huh7 relative to HepG2. Our data clearly showed that the NTP-mediated alternation of mitochondrial membrane potential and dynamics led to ROS-mediated apoptosis in Huh7 and Alexander cells. Interestingly, plasma treatment resulted in p53 down-regulation in Huh7 cells. High levels of Bcl-2 protein expression in HepG2 resulted in their resistance in response to oxidative stress- mediated by plasma. We show thoroughly time- and dose-dependent kinetics of ROS accumulation in HCC cells. Furthermore, we show nuclear compartmentalization of the superoxide anion triggered by NTP. NTP induced apoptotic death in Huh7 liver cancer cells via simultaneous downregulation of mutated p53, pSTAT1 and STAT1. Contrary, hydrogen peroxide treatment results in autophagic cell death. We disclosed detailed mechanisms of NTP-mediated alteration of redox signalling in liver cancer cells.

The Critical Role of X Chromosome-Linked Inhibitor of Apoptosis (XIAP) in Differential Synergism Induced by Pentachlorophenol and Copper-1,10-Phenanthroline Complex in Normal and Cancer Liver Cells.

Chemical pollutants often co-occur and can interact to cause unexpected combined toxic effects. Both pentachlorophenol (PCP) and copper-1,10-phenanthroline [Cu(OP)2], used as wood preservatives, coexist in fluids and tissues of ordinary population. Our previous studies demonstrate that a combination of subtoxic PCP and Cu(OP)2 causes synergistic toxicity on Escherichia coli and hepatocarcinoma cells. However, it is not clear whether this effect also occurs in normal hepatocytes; and if so, what are the differences as compared with the hepatocarcinoma cells. We demonstrate that the combination of low-toxic PCP and Cu(OP)2 (0-1.6 µM; PCP/Cu(OP)2 molar ratio: 2:1) induces a concentration-dependent intracellular copper accumulation, apoptosis, caspase-3/9 activation, depolarization of mitochondrial membrane potential, and oxidative stress (reactive oxygen species increasing and glutathione/oxidized glutathione ratio decreasing) in both normal hepatocytes HL-7702 and hepatocarcinoma HepG2 cells. However, HepG2 cells are more susceptible to the above molecular events as compared with HL-7702 cells. Further data reveal that PCP/Cu(OP)2 markedly decreases X chromosome-linked inhibitor of apoptosis (XIAP), p-ERK-1/2, and p-JNK protein expression in HepG2, but not HL-7702. Overexpression of XIAP gene in HepG2 significantly blocks PCP/Cu(OP)2-induced cytotoxicity, caspase activity, apoptosis, ROS accumulation, and antioxidant genes expression. These results suggest that the combination of low-toxic PCP and Cu(OP)2 preferentially induce synergistic cytotoxicity in human hepatocarcinoma cells by XIAP-ROS-apoptosis pathway, compared with the normal hepatocytes. The present data not only confirm the synergistic toxicity of PCP/Cu(OP)2 combination in normal liver cells, but also suggest a possible opportunity in developing new therapeutic approaches for liver cancer by sensitizing cancer cells to chemotherapy.

Development of Novel DNA-Encoded PCSK9 Monoclonal Antibodies as Lipid-Lowering Therapeutics.

Elevated low-density lipoprotein cholesterol (LDL-C) is one of the major contributors to cardiovascular heart disease (CHD), the leading cause of death worldwide. Due to severe side effects of statins, alternative treatment strategies are required for statin-intolerant patients. Monoclonal antibodies (mAbs) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) have shown great efficacy in LDL-C reduction. Limitations for this approach include the need for multiple injections as well as increased costs associated with patient management. Here, we engineered a DNA-encoded mAb (DMAb) targeting PCSK9 (daPCSK9), as an alternative approach to protein-based lipid-lowering therapeutics, and we characterized its expression and activity. A single intramuscular administration of mouse daPCSK9 generated expression in vivo for over 42 days that corresponded with a substantial decrease of 28.6% in non-high-density lipoprotein cholesterol (non-HDL-C) and 10.3% in total cholesterol by day 7 in wild-type mice. Repeated administrations of the DMAb plasmid led to increasing expression, with DMAb levels of 7.5 μg/mL at day 62. daPCSK9 therapeutics may provide a novel, simple, less frequent, cost-effective approach to reducing LDL-C, either as a stand-alone therapy or in combination with other LDL-lowering therapeutics for synergistic effect.