Effect of ARID2 knockout on proliferation gene expression of hepatocarcinoma cells Hep3B / 解放军医学杂志

Objective To construct ARID2 knockout human liver cancer cell line Hep3B by CRISPR/Cas9 system, explore the effect of ARID2 knockout on the proliferation of Hep3B, and the differences in gene expression between wild type and ARID2 knockout Hep3B cell lines. Methods The plasmid lentiCRISPRv2 was constructed with ARID2 knockout plasmid, and then transfected Hep3B cells; The positive cells were selected by puromycin, sorted by flow cytometry and cultured to obtain the monoclonal cell lines; ARID2 knockout Hep3B cell lines were identified by Western blotting and Sanger sequencing; The effect of ARID2 knockout on the proliferation of Hep3B cell line was detected by CCK-8 method; RNA-seq was used to analyze the differentially expressed genes between wild type Hep3B cells and ARID2 knockout Hep3B cells, and the results of RNA-seq were validated by Real-time quantitative PCR (RT-qPCR). The possible biology functions of ARID2 were explored through Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG Pathway), GO Biological Processes and Gene set enrichment analysis (GSEA). Results Two ARID2 knockout Hep3B cell lines were successfully constructed. Compared with the wild type cell line, the proliferation of ARID2 knockout cell lines was significantly accelerated (P<0.05). A total of 85 differentially expressed genes were identified by RNA-seq analysis between ARID2 knockout cell lines and wild type cell line, of which 17 genes were up-regulated and 68 down-regulated. The mRNA expression of 10 differentially expressed genes were validated by RT-qPCR, the verification results were consistent with that by RNA-seq. Results of KEGG Pathway, GO Biological Processes and GSEA indicated that ARID2 genes might be involved in such biological processes as protein processing and transport, chemokine signaling pathway, Wnt signaling pathway, complement and coagulation cascade reaction, epithelial-mesenchymal transition (EMT), glycolysis, TGF-β signaling pathway, and TNF-α/NF-KB signaling pathway, et al. Conclusion ARID2 knockout can promote proliferation of Hep3B cell line; and ARID2 may play an important role by variety of biological processes in tumor proliferation, invasion, metastasis and tumor microenvironment.

Effects of RNA interference of targeting Cyclin D1 gene on hepatoma cell proliferation and Mdm2 gene expression / 重庆医学

Objective To investigate the effects of down-regulating Cyclin D1 expression on Mdm2 gene expression and proliferation of human hepatoma cells.Methods Cyclin D1-siRNA was transfected into the hu man hepatoma cell line Hep3B s with liposome.The experiment was divided into the blank control group,negative control siRNA (NC-siRNA) group and Cyclin D1-siRNA group.RT-PCR and Western blot were used to detect the expressions of Cyclin D1,Mdm2,Mdm4,P53 and P21.The cell cycle was measured by flow cytometer;the cellular activity was tested with MTT;the cell apoptosis was examined by TUNEL.Results Compared with the blank control group and NC-siRNA group,the expressions of P53 and P21 in the Cyclin D1-siRNA group were increased (P＜0.05),while the expressions of Cyclin D1,Mdm2 and Mdm4 were decreased (P＜0.01);there were no significant differences in the G1,S and G2 phases among 3 groups(P＞0.05);the cell vitality in the in the Cyclin D1-siRNA group was significantly weakened compared with the other two groups(P＜0.01),while the cell apoptosis was significantly enhanced(P＜0.01).Conclusion The down-regulation of Cyclin D1 gene expression could inhibit the expressions of Mdm2 and Mdm4,and up-regulates the expressions of P53 and P21.Down-regulating Cyclin D1 expression can inhibit the proliferation of liver cancer cells and promotes their apoptosis.

Ubiquitinated proteomics research of Hep3B / 生物工程学报

Ubiquitination is one of the most major post-translational modifications playing important role in regulation of intra-cellular proteins' stability, degradation, localization and biological activity. However, these proteins are difficult to be detected due to their low abundance, short half-life. In this study, ubiquitin-binding domains (UBDs) were constructed to purify the ubiquitinated proteins from Hep3B cells. Ubiquitinated proteins and sites were detected by LC-MS/MS. A total of 1 900 potential ubiquitinated proteins were identified. Among them, 158 ubiquitinated sites were identified, belonging to 102 proteins. Bioinformatics analysis revealed that the enriched pathways of ubiquitinated proteins were closely related to tumor occurrence and development. The dysfunction of ubiquitin-proteasome has a high correlation with cell signaling and extracellular matrix changing in tumor cells.

Anticancer effects of crude extract from Melia toosendan Sieb. et Zucc on hepatocellular carcinoma in vitro and in vivo / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc and its possible molecular mechanisms in vitro and in vivo.</p><p><b>METHODS</b>Transonic alcohol-chloroform extraction method was used to extract toosendanin from the bark of Melia toosendan Sieb. et Zucc, and the content of toosendanin in the crude extract was measured by high performance liquid chromatography (HPLC). Anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc were investigated in in vivo and in vitro studies. In the in vitro experiment, human hepatocellular carcinoma cell lines SMMC-7721 and Hep3B were co-incubated with toosendanin crude extract of different concentrations, respectively. In the in vivo experiment, BALB/c mice were subcutaneously inoculated with mouse hepatocellular carcinoma H22 cells and treated with crude extract.</p><p><b>RESULTS</b>HPLC revealed the content of toosendanin was about 15%. Crude extract from Melia toosendan Sieb. et Zucc inhibited cancer cells growth in a dose- and time-dependent manner. The 50% inhibitory concentration (IC50, 72 h) was 0.6 mg/L for SMMC-7721 cells and 0.8 mg/L for Hep3B cells. Both high-dose [0.69 mg/(kg d)] and low-dose [0.138 mg/(kg d)] crude extract could markedly suppress cancer growth, and the inhibition rate was greater than 50%. Hematoxylin and eosin staining showed necrotic area in cancers and transmission electron microscopy displayed necrotic and apoptotic cancer cells with apoptotic bodies. Immunohistochemistry showed that the expression of Bax and Fas increased and the expression of Bcl-2 reduced.</p><p><b>CONCLUSIONS</b>Toosendanin extract has potent anti-cancer effects via suppressing proliferation and inducing apoptosis of cancer cells in vivo and in vitro. The mechanism of apoptosis involves in mitochondrial pathway and death receptor pathway.</p>

Human coagulation factor VIII domain-specific recombinant polypeptide expression

BACKGROUND: Hemophilia A is caused by heterogeneous mutations in F8. Coagulation factor VIII (FVIII), the product of F8, is composed of multiple domains designated A1-A2-B-A3-C1-C2. FVIII is known to interact with diverse proteins, and this characteristic may be important for hemostasis. However, little is known about domain-specific functions or their specific binding partners. METHODS: To determine F8 domain-specific functions during blood coagulation, the FVIII domains A1, A2, A3, and C were cloned from Hep3B hepatocytes. Domain-specific recombinant polypeptides were glutathione S-transferase (GST)- or polyhistidine (His)-tagged, over-expressed in bacteria, and purified by specific affinity chromatography. RESULTS: Recombinant polypeptides of predicted sizes were obtained. The GST-tagged A2 polypeptide interacted with coagulation factor IX, which is known to bind the A2 domain of activated FVIII. CONCLUSION: Recombinant, domain-specific polypeptides are useful tools to study the domain-specific functions of FVIII during the coagulation process, and they may be used for production of domain-specific antibodies.

miR-224 affects Hep3B cell proliferation and apoptosis / 中国癌症杂志

Background and purpose:MiR-224 is overexpressed in hepatocellular carcinoma, and participate in invasion and metastasis of cancer. The aim of this study was to investigate the effects of miR-224 antisense oligonucleotide (ASO) on the proliferation and apoptosis of Hep3B cells.Methods:After transfection with miR-224 ASO, and detecting the miR-224 mRNA expression of Hep3B cells by real-time quantitative PCR; the miR-224 expression in Hep3B cells was measured and cell proliferation was analyzed by MTT assay and the colony formation experimentin vitro andin vivo. The cell apoptosis was analyzed by flow cytometry.Results:Compared with the control group, miR-224 ASO significantly reduced the miR-224 mRNA expression in the Hep3B cell(P<0.05), MTT assay results showed that Hep3B cells survived rate decreased greatly after transfection with miR-224 ASO. Clone formation assay revealed that the colony formation rate in miR-224 ASO group was significantly lower than that in the control group.In vivo study further confirmed that miR-224 ASO could inhibit the proliferation of Hep3B cells,and miR-224 ASO group grew substantially slow compared with the negative control. Flow cytometry indicated that miR-224 ASO group promoted apoptosis significantly.Conclusion:miR-224 was overexpressed in Hep3B cells. Reducing the expression of miR-224 can effectively inhibit the growth of Hep3B cells and promote apoptosis. miR-224 may become a new target for the regulation of gene expression in hepatocellular carcinoma.

Effects of Sufentanil in inhibiting Hep3B cell viability and promoting cell apoptosis in liver cancer / 西安交通大学学报(医学版)

Objective To investigate the effects and mechanism of Sufentanil on Hep3B cell viability in liver cancer.Methods Sufentanil of different concentrations (0.01,0.1,1,5,10,1 5 μmol/L)was used to treat Hep3B cells.The changes of cell cycle,apoptosis and protein expression were detected to explore the potential mechanisms by MTT method,flow cytometry and Western blot,respectively.Results Reduced viability of Hep3B cells,arrested cell cycle in the G0/G1 phase,decreased expression of survivin protein,and increased expression of caspase-3 protein were observed with the increase of Sufentanil concentration. Conclusion Sufentanil inhibited the viability of Hep3B cells through affecting cell cycle,promoting cell apoptosis and changing protein expressions of survivin and caspase-3.

Cell-type specific regulation of thrombospondin-1 expression and its promoter activity by regulatory agents

Thrombospondin-1 (TSP-1), a multifunctional protein that is able to function as a negative regulator of solid tumor progression and angiogenesis, is normally present at a very low level but rapidly elevated in pathological tissues. To understand the cellular regulation of TSP-1 expression, the mode of it's expression in Hep3B, SK-HEP-1, and porcine aortic endothelial (PAE) cells was examined in the presence of all-trans retinoic acid (ATRA), interleukin-6 (IL-6), interferon-gamma (IFN-gamma), or phorbol 12-myristate 13-acetate (PMA). ATRA or IL-6 induced a dose-dependent increase of TSP-1 protein and mRNA levels in PAE cells, while they negatively regulated TSP-1 expression in the Hep3B and SK-HEP-1 cells. In contrast, PMA showed just the opposite effects on the TSP-1 expression in the same cells. IFN-gamma had little effect on TSP-1 level in Hep3B and PAE cells. The TSP-1 expression in SK-HEP-1 cells by these agents showed a close resemblance to that of liver cells rather than that of the endothelial cell line. Possible TSP-1 promoter-mediated responses by ATRA, IL-6, IFN-gamma, or PMA in Hep3B and PAE cells examined with luciferase activity of TSP-LUC reporter plasmid showed that levels of TSP-1 promoter activity were lower than that of the expressed TSP-1 protein and mRNA levels. Transfection of c-Jun and/or RARalpha expression vectors into Hep3B and PAE cells resulted in the enhanced TSP-1 promoter activity as well as the increments of of its protein and mRNA level. These results suggest that regulatory agents-induced TSP-1 expression may be attributed to mRNA stability and/or translational activation in concert with transcriptional activation and TSP-1 expression may be independently controlled via each signal pathway stimulated by PMA or ATRA.

Studies on the Mechanism of Hypoxic Increase of VEGF Expression in the Hep3B Human Hepatoma Cells / 대한암학회지

PURPOSE: Hepatocellular carcinoma (HCC), a typical hypervasculized tumor is very sensitive to hypoxia and vascular endothelial growth factor (VEGF) has previously been identified to be up-regulated in response to hypoxia in several cell types. However, the molecular mechanisms by which hypoxia is sensed by the cells remain enigmatic. To investigate whether calcium and AP-1 are involved in hypoxia-sensing mechanism, we performed following experiments. MATERIALS AND METHODS: Hep3B cells were grown in hypoxic condition. To assess cell viability, MTT assay was performed. To investigate the effect of calcium and AP-1, northern blot analysis was performed after treatment with BAPTA/AM. RESULTS: The expression of VEGF was significantly up-regulated by hypoxia in Hep3B, hepatocellular carcinoma cell line. The increased expression of VEGF induced by hypoxia was blocked by the addition of BAPTA/AM, a cytosolic calcium chelator to the media. In addition, we found that the expression of c-jun protooncogene was also up-regulated by hypoxia. Hypoxic increase of c-jun expression was also normalized by the treatment with BAPTA/AM. CONCLUSION: These results suggest that the increased expression of VEGF by hypoxia is mediated through the calcium and c-jun signalling pathway in the Hep3B human hepatoma cell lines.