Study on mechanism of Hippo/YAP pathway inhibition by salvianolic acid B in HuH-7 cells / 中国药理学通报

Aim To investigate whether salvianolic acid B ( Sal B) has inhibitory effect on hepatoma HuH- 7 cells and explore whether it works via Hippo/YAP signaling pathway. Methods HuH-7 cells were induced by TGF-β1 (9 pmol · L

miR-3612 regulates the malignant biological behaviors of hepatocellular carcinoma cells via targeting SEMA4C / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨miR-3612靶向信号素（SEMA）4C对肝细胞癌细胞增殖与侵袭能力的影响。方法: 收集2020年5月至2021年9月间在皖南医学院第一附属医院弋矶山医院手术切除的肝细胞肝癌的40对癌组织和相应癌旁组织，常规培养肝细胞癌Hep3B和Huh7细胞，将其分为对照组、miR-3612 mimics-NC组、miR-3612 mimics组、miR-3612 inhibitor-NC组、miR-3612 inhibitor组、si-NC组、si-SEMA4C组、mimics-NC+pcDNA-NC组、miR-3612 mimics+pcDNA-NC组和miR-3612 mimics+pcDNA-SEMA4C组，用转染试剂将相应的核酸和质粒转染各组细胞。qPCR法检测miR-3612和SEMA4C mRNA在肝细胞癌组织和Hep3B和Huh7细胞中的表达，双荧光素酶报告基因实验和免疫共沉淀（RIP）实验验证miR-3612与SEMA4C的结合及调控关系，qPCR法和WB法检测转染后各组Hep3B和Huh7细胞中miR-3612、SEMA4C mRNA和蛋白的表达，CCK-8法、细胞划痕实验和Transwell小室实验分别检测各组Hep3B和Huh7细胞的增殖、迁移和侵袭能力。结果: miR-3612在肝细胞癌组织和Hep3B和Huh7细胞中呈低表达（P<0.001），而SEMA4C则呈高表达（P<0.001），过表达miR-3612可抑制Hep3B和Huh7细胞的增殖、迁移、侵袭和vimentin、SEMA4C蛋白的表达，促进E-cadherin蛋白的表达（P<0.05或P<0.01或P<0.001），敲低miR-3612则促进Hep3B和Huh7细胞的增殖、迁移、侵袭和SEMA4C蛋白的表达（P<0.05或P<0.01或P<0.001）。双荧光素酶报告基因实验和RIP实验证实miR-3612与SEMA4C可直接结合（P<0.001），miR-3612与SEMA4C的表达呈负相关也间接证明了这一点（P<0.001）。敲减SEMA4C能明显抑制Hep3B、Huh7细胞的增殖、侵袭和迁移能力（P<0.05或P<0.01或P<0.001），过表达SEMA4C可逆转过表达miR-3612对Hep3B和Huh7细胞增殖、迁移、侵袭和EMT的抑制作用（P<0.05或P<0.01或P<0.001）。结论: miR-3612通过调控SEMA4C表达影响Hep3B和Huh7细胞的恶性生物学行为，miR-3612有望成为临床肝细胞癌治疗的潜在靶点。

Neobavaisoflavone induces pyroptosis in hepatocellular carcinoma Huh-7 cells through caspase-3/GSDME signal pathway / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨新补骨脂异黄酮（NBIF）对肝细胞癌（HCC）Huh-7细胞焦亡的影响及其分子机制。方法：体外培养Huh-7细胞，用CCK-8法检测不同浓度的NBIF处理48 h时对细胞存活率的影响，光学显微镜下观察NBIF处理后Huh-7细胞的形态变化，乳酸脱氢酶（LDH）释放实验检测细胞的LDH释放量，WB实验检测细胞中GSDME、caspase-3的蛋白水平变化。采用siRNA干扰Huh-7细胞中caspase-3、GSDME表达后，CCK-8法检测NBIF处理对细胞存活率的影响，WB实验检测GSDME蛋白表达水平，观察NBIF处理对细胞形态的影响，并检测细胞LDH释放量。结果：60 μmol/L以上的NBIF均能显著抑制Huh-7细胞的增殖（均P<0.01），光学显微镜下观察到NBIF处理后的细胞出现肿胀、吐泡现象，且LDH释放增加（P<0.01）；WB实验结果表明，NBIF能够激活caspase-3蛋白并切割GSDME蛋白，增加GSDME-N的表达（均P<0.01）。干扰caspase-3、GSDME表达后，NBIF对细胞的抑制作用减弱（均P<0.01），GSDME-N蛋白表达受到抑制（P<0.01），显微镜下细胞肿胀、吐泡现象几乎消失，LDH释放明显减少（P<0.05）。结论：NBIF能够通过caspase-3/GSDME途径诱导Huh-7细胞发生焦亡，从而抑制HCC细胞的增殖，为HCC的治疗提供一种新思路。

Anti-hepatocellular Carcinoma Activity and Related Mechanism of Apigenin-7-O-Glucoside / 肿瘤防治研究

Objective To investigate the inhibitory effect of apigenin-7-o-glucoside (AGL) on the viability of Huh7 cells and tumor growth in Huh7-xenograft tumor nude mice. Methods CCK-8 was used to detect the proliferation inhibitory effect and the half inhibitory concentration of AGL on Huh7 cells. The mitochondrial membrane potential measurement was used to analyze the early apoptosis of Huh7 cells after AGL treatment. Flow cytometry was used to analyze the effect of AGL on Huh7 cell apoptosis, and Western blot was used to explore the expression level of the proteins associated with apoptosis and inflammation, as well as the possible related mechanism. In Huh7-xenograft tumor nude mice, vernier caliper was used to measure tumor volume to analyze the effect of AGL on tumor growth rate. HE staining was used to observe the pathological state of mouse organs, and the inflammation-related factors in serum were detected with ELISA. Results After Huh7 cells were treated with AGL, the mitochondrial membrane potential reduced, the content of ROS increased and the apoptosis rate was increased to 25.23% by 50 μmol/L AGL treatment; while the expression levels of Bax, Bad, Cleaved Caspase-3 and Cleaved Caspase-9 increased, and the expression levels of Bcl2 and Bcl-xL decreased, the phosphorylation level of NF-κB, IKKα/β and IκBα decreased; the tumor growth rate decreased, the serum IL-6 and TNF-α levels significantly decreased, while the IL-2 and IL-10 levels increased. Conclusion AGL could promote the apoptosis of Huh7 cells and relieve the tumor development in Huh7-xenograft tumor nude mice, which may be related to the NF-κB pathway.

Study on synergistic anti-tumor effect of Shuangdan Capsules combined with 5-FU on hepatocellular carcinoma cells Huh-7 and xenograft mice / 中国中药杂志

This paper discussed the synergistic anti-tumor effect of Shuangdan Capsules combined with 5-fluorouracil(5-FU) on human liver cancer cell line Huh-7 and tumor bearing mice. The effects of Shuangdan Capsules combined with 5-FU on the activity and vascular endothelial growth factor(VEGF) receptor protein expression of Huh-7 cells were investigated, and the effects of drug combination on tube formation of HUVEC cell were also verified. In addition, the mice model of Huh-7 was established to observe the anti-tumor effect of drug combination and the distribution of tumor blood flow in tumor bearing mice by using molecular imaging. HPLC analysis showed that Shuangdan Capsules mainly consisted of danshensusodium, protocatechuic aldehyde, paeoniflorin, rosmarinic acid, alkannic acid, salvianolic acid B, and paeonol. In MTT experiment, the inhibition rate of Shuangdan Capsules(20 mg·L~(-1)) and 5-FU(1 μmol·L~(-1)) on Huh-7 cells was 60%, and the CI value was 0.59, suggesting that these two drugs had synergistic anti-hepatoma cells effect. The expression of VEGF receptor in Huh-7 cells was inhibited by the combination of these two drugs. In addition, the process of HUVEC was slow, and the number, length and area of the lumen branches decreased significantly. In vivo, Shuangdan Capsules combined with 5-FU inhibited the growth and prolongation of survival of Huh-7 cells in subcutaneous transplanted tumor nude mice; serum expression of CD31 and VEGF in nude mice were decreased, while caspase-3 was increased. Meanwhile, the drug combination significantly inhibited the expressions of MMP2 and VEGF in tumor tissues. Ultrasound showed that Shuangdan Capsules combined with 5-FU also inhibited tumor angiogenesis and reduced blood flow of tumor tissue. The results showed that Shuangdan Capsules combined with 5-FU may inhibit tumor angiogenesis by inhibiting VEGF and MMP2 expressions, thereby blocking tumor growth.

Effect of Compound Phyllanthus urinaria Ⅱ(CPU Ⅱ) on Proliferation, Apoptosis and Autophagy of Hepatocellular Carcinoma Huh7 Cells / 中国实验方剂学杂志

Objective:To observe the effect and investigate the mechanism of compound Phyllanthus urinaria Ⅱ(CPU Ⅱ)on proliferation, apoptosis and autophagy of human hepatoma cell line Huh7. Method:Huh7 cells were cultured in vitro and divided into blank control group, high-dose CPU Ⅱ group (40 g·L-1),low-dose CPU Ⅱ group (20 g·L-1), and 5-FU group (0.04 g·L-1). Methye thiazolye telrazlium(MTT) assay was used to detect the inhibitory effect of CPU Ⅱ on proliferation of human hepatoma Huh7 cells. The apoptosis rate was observed by Annexin V-FITC/PI flow cytometry; the changes of autophagosomes in each group were observed by monodansylcadaverin (MDC) staining; Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression of phosphatidylinositol 3-kinase (PI3K), Serine-threonine protein kinase 2(Akt2), B-cell lympoma-2(Bcl-2) and Microtubule-associated protein 1 light chian 3(LC3Ⅱ) and Western blot was used to detect the protein expression of Akt2 and LC3Ⅱ. Result:CPU Ⅱ(40, 20 g·L-1) significantly inhibited the hepatoma cell line Huh7 and induced apoptosis, with an apoptosis rate of 51.72% and 19.74% respectively, significantly higher than that of control group (PPPPConclusion:CPUⅡ had obvious inhibitory effect on the proliferation of hepatoma cell line Huh7, and the mechanism may be related to inhibiting the activation of PI3K/Akt signaling pathway to induce apoptosis and autophagy.

Inhibitory effect of asiatic acid on human liver carcinoma cells via cell cycle blockage and apoptosis induction / 中国肿瘤生物治疗杂志

@# Objective: To investigate the inhibitory effect of asiatic acid (AA) on malignant biological behaviors of human liver cancer cells and to explore the mechanism. Methods: Human liver cancer cell line (Huh7) was used as research subject, and treated with different concentrations of AA (0, 5, 10, 25, 50, 100 μmol/L) in vitro. The effect of AA on cell proliferation was determined by CCK-8 and EdU assay; the apoptosis and cell cycle distribution were detected by flow cytometry, while the expressions of apoptosis-related proteins (AKT, P-ERK 1/2 , p38, cleaved-caspase3, cleaved-caspase9, BAX, Bcl-2, AKT, ERK, p38, pro-caspase 3 and pro-caspase 9) were examined by WB. Results: AA could inhibit the proliferation of Huh7 cells in a dose- and time-dependent manner (all P<0.05). After being incubated with 10 μmol/L AA for 24 h, the proliferation of Huh7 cells was significantly inhibited (P<0.05), the apoptosis rate was significantly increased (P<0.05), and cell cycle was arrested in G1 phase (P<0.05).AAinduced p-p38 expression, but inhibited the expression of p-AKT and p-ERK in a dose-dependent manner (all P<0.05). In addition, as the concentration of AA increased, the levels of cleaved-caspase 3, cleaved-caspase 9 and BAX increased, while the level of Bcl-2 decreased (all P<0.05). Conclusion:AAinhibits the proliferation of human liver cancer cells and promotes its apoptosis, which is associated with the MAPK and PI3K/AKT pathways.

miR-144-3p targets FZD4 to inhibit malignant biological behaviors of liver cancer Huh-7 cells via blocking Wnt/β-catenin pathway / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effect of miR-144-3p modulating proliferation, migration and apoptosis of liver cancer Huh-7 cells through blocking frizzled class receptor 4 (FZD4)/Wnt/β-catenin pathway and the possible mechanism. Methods: A total of 18 pairs of cancer tissues and corresponding para-cancerous tissues from liver cancer patients, who underwent surgery in Workers' Hospital of Liuzhou City from March 2012 to July 2017, were collected for this study; in addition, hepatic cancer cell lines (Huh-7, SMMC7721 and MHCC97) and human normal liver epithelial cell line THLE-3 were also collected. The expression of miR-144-3p in liver cancer tissues and cell lines was detected by qPCR. MiR-144-3p mimics/inhibitor and FZD4 siRNA were transfected into liver cancer Huh-7 cells; the proliferation, migration and apoptosis of Huh-7 cells were evaluated by CCK-8 assay, Transwell assay, wound healing assay and Annexin V-FITC/PI double staining flow cytometry assay, respectively. The interaction between miR-144-3p and FZD4 was verified by dual-luciferase reporter gene assay. Results: The expression of miR-144-3p was down-regulated in liver cancer tissues and cell lines (P<0.05 or P<0.01). Over-expression of miR-144-3p significantly inhibited cell proliferation viability, migration but induced apoptosis of Huh-7 cells (all P<0.01). Moreover, dual-luciferase reporter gene assay showed that miR-144-3p directly interacted with FZD4 and suppressed its expression. Furthermore, in vitro experiments verified that miR-144-3p targeted FZD4 to suppress the proliferation, migration and promote apoptosis of Huh-7 cells via blocking Wnt/β-catenin pathway (all P<0.01). Conclusion: miR-144-3p inhibits malignant biological behaviors of liver cancer Huh-7 cells via blocking Wnt/FZD4/β-catenin signaling pathway, which may provide potential molecular targets for early diagnosis or treatment of liver cancer.

Effect and mechanism of dauricine on proliferation and apoptosis of hepatoma Huh7 cells / 中草药

Objective To study the effect of dauricine on the proliferation and apoptosis of hepatoma Huh7 cells, and explore its anti-tumor mechanism and its relationship with Hedgehog signaling pathway. Methods The effects of different concentrations of dauricine (2, 4, 8 μg/mL) on the proliferation of Huh7 cells were detected by MTT assay. Apoptosis of Huh7 cells was analyzed by flow cytometry. Real-time PCR and Western blotting were used to detect the levels of Hedgehog signaling pathway-related genes and proteins. Results With the increase of the concentration of dauricine and the duration of action, the inhibition rate of Huh7 cell proliferation was increased. Among them, 8 μg/mL dauricine had the highest inhibition rate (48.8%) at 48 h. Dauricine induced the apoptosis in Huh7 cells. With the increase of the concentration of dauricine, the apoptotic rate of cells was increased significantly (P < 0.05, 0.01). The mRNA and protein expression levels of PTCH1, GLi1, SMO and SHH genes in Hedgehog signaling pathway were significantly decreased, while the level of cleaved Caspase-3 protein was significantly increased, accompany with the decreased expression of Bcl-2 in dauricine concentration-dependent pattern (P < 0.05, 0.01) in dauricine group compared with the control group. Conclusion Dauricine could significantly inhibit the proliferation and promote apoptosis of Huh7 cells, which may play a role by blocking Hedgehog signaling pathway.

Inhibitory Effects and Mechanism of Anemoside B 4 on Hepatocellular Carcinoma Huh- 7 Cells and Tumor-bearing Nude Mice / 中国药房

OBJECTIVE： To study the anti-tumor effect of anemoside B4 （AB4） on hepatocellular carcinoma Huh-7 and tumor-bearing nude mice and its mechanism. METHODS： Huh-7 cells were divided into AB4 treatment group， negative control group （constant volume of culture medium） and positive control group （5-fluorouracil 50 μmol/L）. The inhibitory rate of Huh-7 cell proliferation was tested by MTT after treated with 0， 3， 6， 12， 25， 50， 100， 200， 400， 800， 1 600 μmol/L AB4 for 12， 24， 36， 48 h； IC50 were calculated. The number of clone cells was evaluated by clone formation tests after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h. The apoptosis rate of Huh-7 cells was tested by Annexin Ⅴ-FITC/PI double staining after treated with 50 μmol/L AB4 for 24 h. The expression of apoptotic proteins such as Bcl-2， Bax， Caspase-3， Cleaved Caspase-3 and Cleaved PARP were tested by Western blot after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h. Tumor-bearing nude mice were randomly divided into negative control group （normal saline）， positive control group （5-fluorouracil 50 mg/kg）， AB4 lwo-dose， medium-dose and high-dose groups （25， 50， 100 mg/kg）， with 3 mice in each group. They were given relevant medicine intraperitoneally， once a day， for consecutive 19 d. The growth of tumor was observed， and anti-tumor rate was also calculated. HE staining was used to observe the morphology of tumor cells. RESULTS： The inhibition rate of AB4 on Huh-7 cell proliferation increased with the increase of concentration of AB4， but it did not increase significantly after reaching 50 μmol/L； it increased with the increase of time， but it did not increase significantly after 24 h. The IC50 of AB4 was （56.76±1.756） μmol/L. Compared with negative control group， the number of clone cells was decreased significantly after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h， while the protein expression of Bcl-2 was decreased significantly （P＜0.05）； the apoptotic rate， the protein expression of Bax， Caspase-3， Cleaved Caspase-3 and Cleaved PARP were increased significantly （P＜0.05 or P＜0.01）， there was no statistical significance， compared with positive control group. Compared with negative control group， the volume of tumor was decreased significantly in AB4 low-dose， medium-dose and high-dose groups， positive control group （P＜0.05）； the outline of tumor cells become more and more blurred； there were nuclear pyknosis， unclear nucleoplasm and nuclear fragmentation； its anti-tumor rates were 25.93％， 39.15％， 46.26％， 42.83％， respectively. CONCLUSIONS： AB4 inhibits Huh-7 cells and tumor-bearing mice， the mechanism of which may be associated with up-regulating the proportion of Bax/Bcl-2， activating Caspase-3， cracking PARP and inducing apoptosis.

Study on Improvement Effect of Methylated Urolithin A on Oleic Acid-induced Lipid Accumulation in Huh- 7 Cells and Its Me- chanism / 中国药房

OBJECTIVE： To study the improvement effect and mechanism of methylated urolithin A on oleic acid-induced lipid accumulation in human liver cancer Huh-7 cells. METHODS： Oleic acid was adopted to induce lipid accumulation model cells. Huh-7 cells were divided into control group （culture medium）， model group （1 mmol/L oleic acid）， low-dose group （1 mmol/L oleic acid+10 μmol/L methylated urolithin A） and high-dose group （1 mmol/L oleic acid+20 μmol/L methylated urolithin A）. Oil red O staining was used to observe lipid accumulation in cells. Triglyceride（TG） enzyme assay was applied to determine the TG content in cells. PCR was employed to detect the mRNA expression of FASN， SREBP-1， PPAR-α and PPAR-γ in cells. Western blotting was used to determine the protein expression of FASN in cells. RESULTS： After induced by oleic acid， a large amount of lipid droplet accumulated around the cells； the intracellular lipid and TG content， mRNA expression levels of FASN， SREBP-1 and PPAR-γ， protein expression levels of FASN were increased significantly， while mRNA expression level of PPAR-α was decreased significantly （P＜0.01）. After intervened with methylated urolithin A， lipid droplet around the cells decreased significantly； the contents of lipid and TG in cells were decreased significantly， while the mRNA expression levels of FASN， SREBP-1 and PPARγ and protein expression level of FASN were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS： Methylated urolithin A can improve oleic acid-induced lipid accumulation in Huh-7 cells， the mechanism of which may be associated with inhibiting fat synthesis， promoting lipid metabolism and down-regulating the expression of metabolism-related factors as FASN， SREBP-1 and PPAR-γ.

Analysis of Mechanism of Artesunate in Treatment of Liver Cancer Based on Metabolomics / 中国实验方剂学杂志

Objective: To study on the antitumor mechanism of artesunate in the treatment of liver cancer based on gas chromatography-mass spectrometry (GC-MS). Method: CellTiter-Glo® Luminescent Cell Viability Assay was used to detect activity of artesunate with different concentrations (0, 12.5, 25, 50, 100 μmol·L-1) on human liver cancer Huh7, SMMC-7721 cells for 24, 48, 72 h. GC-MS was employed to analyze the changes of metabolites of artesunate in two kinds of hepatoma cells (Huh7, SMMC-7721) for 24 h. The data was preprocessed by Postrun Analysis 4.41 workstation. Partial least squares-discriminant analysis (PLS-DA) was used to analyze two sets of differential metabolites and to analyze metabolic pathways of differential metabolites based on MetaboAnalyst 3.0 software. Result: Compared with the normal group, after two kinds of liver cancer cells was treated by artesunate, a total of 39 identical metabolites in the cells have undergone significant changes, which were mainly related to five metabolic pathways,including biosynthesis of aminoacyl-transfer RNA (tRNA), metabolism of alanine, aspartic acid and glutamic acid, metabolism of glycine, serine and threonine, metabolism of arginine and proline, metabolism of glutathione. Conclusion: Artesunate (12.5-100 μmol·L-1) can inhibit the growth of liver cancer cells (Huh7, SMMC-7721), it mainly involves five metabolic pathways, which may be the pathway of artesunate against liver cancer.

Effect of cinobufagin combined with Sorafenib on proliferation and apoptosis of hepatocellular carcinoma Huh7 cells via AURKA/Ras/Raf/ERK signaling pathway / 中草药

Objective To investigate the effect and mechanism of cinobufagin combined with Sorafenib on the proliferation and apoptosis of hepatocellular carcinoma Huh7 cells. Methods The proliferation of Huh7 cells was measured using MTT assay; The apoptosis morphological changes of Huh7 cells were detected using Hoechst33342/PI fluorescence staining; The cells cycle was detected by flow cytometry; The expression of Ki67 protein was detected by immunocytochemistry; The expressions of Bax, Bcl-2, Caspase-8, AURKA, Ras, Raf, ERK, and p-ERK proteins were measured using Western blotting. Results Cinobufagin, Sorafenib, and combination therapy inhibited the proliferation of Huh7 cells, and the inhibitory effect of the combination group was more obvious with synergistic effect. Fluorescence staining showed morphological changes of apoptosis. Sorafenib induced the cell cycle S phase arrest, cinobufagin and combination therapy induced the cell cycle G2/M phase arrest, combination group had more obvious cell cycle arrest in G2/M phase than single drug groups. Both cinobufagin and Sorafenib attenuated the expression of Ki67, and the effect of combination group was more significant. Cinobufagin, Sorafenib, and combination therapy up-regulated the expression of Bax and Caspase-8 proteins; down-regulated the expression of Bcl-2 protein; up-regulated the ratio of Bax/Bcl-2; had no obvious effect on the expression of ERK protein; significantly down-regulated the expression of AURKA, Ras, Raf, and p-ERK proteins; And the effect of combination group was more significant (P < 0.05). Conclusion Cinobufagin combined with Sorafenib could inhibit the proliferation and induce the apoptosis of hepatocellular carcinoma Huh7 cells through AURKA/Ras/Raf/ERK signaling pathway.

In vitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype

Objective To explore inhibitory effects of genome-specific, chemically synthesized siRNAs (small interference RNA) against NS3 gene of hepatitis C virus (HCV) 1a genotype in stable Huh-7 (human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. Methods Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin (G418) selection. The cell clones resistant to 1 000 μg antibiotic concentration (G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. Results RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested (50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24 (80%, P = 0.013) and 48 h (75%, P = 0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70% (P < 0.05) viral RNA reduction as compared to NS3-is33, which showed a 64% (P < 0.05) decrease in viral copy number. siRNA synergism (NS3-is33 + NS3-is44) decreased viral load by 84% (P < 0.05) as compared to individual inhibition by each siRNA (i.e., 64%–70% (P < 0.05)) in serum-inoculated cells. Synthetic siRNAs mixture (NS5B-is88 + NS3-is33) targeting different region of HCV genome (NS5B and NS3) also decreased HCV viral load by 85% (P < 0.05) as compared to siRNA inhibitory effects alone (70% and 64% respectively, P < 0.05). Conclusions siRNAs directed against NS3 gene significantly decreased mRNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.

In vitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype

OBJECTIVE@#To explore inhibitory effects of genome-specific, chemically synthesized siRNAs (small interference RNA) against NS3 gene of hepatitis C virus (HCV) 1a genotype in stable Huh-7 (human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells.@*METHODS@#Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin (G418) selection. The cell clones resistant to 1000 μg antibiotic concentration (G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR.@*RESULTS@#RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested (50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24 (80%, P = 0.013) and 48 h (75%, P = 0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70% (P < 0.05) viral RNA reduction as compared to NS3-is33, which showed a 64% (P < 0.05) decrease in viral copy number. siRNA synergism (NS3-is33 + NS3-is44) decreased viral load by 84% (P < 0.05) as compared to individual inhibition by each siRNA (i.e., 64%-70% (P < 0.05)) in serum-inoculated cells. Synthetic siRNAs mixture (NS5B-is88 + NS3-is33) targeting different region of HCV genome (NS5B and NS3) also decreased HCV viral load by 85% (P < 0.05) as compared to siRNA inhibitory effects alone (70% and 64% respectively, P < 0.05).@*CONCLUSIONS@#siRNAs directed against NS3 gene significantly decreased mRNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.

Cissampelos sympodialis has anti-viral effect inhibiting dengue non-structural viral protein-1 and pro-inflammatory mediators

ABSTRACT Dengue is the most important viral infection transmitted among humans by arthropod-borne. There are currently no vaccines or specific therapeutical treatment. Therefore, immunomodulatory compounds from plants have been widely examined for their antiviral effects. Cissampelos sympodialis Eichler, Menispermaceae, has scientifically proven to present immunomodulatory activities. Here we assessed the antiviral activity of leaf hydroalcoholic extract, warifteine or methylwarifteine from C. sympodialis in an in vitro dengue virus infection model. The results demonstrated that leaf hydroalcoholic extract or warifteine/methylwarifteine treatment did not reduce dengue virus-Ag+ hepatocyte (Huh-7 cell) rates in present experimental conditions. However, we assessed the potential antiviral effect of leaf hydroalcoholic extract or warifteine/methylwarifteine on dengue virus-infection by the production of inflammatory molecules, TNF-α, MIF, IL-8 and PGE2. Dengue virus infection enhanced TNF-α, MIF, IL-8 and PGE2 production in infected Huh-7 cells and leaf hydroalcoholic extract but not warifteine/methylwarifteine treatments, significantly reduced these molecules in infected cells. In dengue virus-infected Huh-7 cells, non-structural protein-1 is produced and leaf hydroalcoholic extract significantly inhibited it independently of alkaloids. Our findings imply that leaf hydroalcoholic extract may attenuate dengue virus infection in Huh-7 cells by inhibiting the enhanced of pro-inflammatory mediators and non-structural protein-1 production induce by dengue virus independently of warifteine/methywarifteine its major compound.

Mebiolgel, a thermoreversible polymer as a scaffold for three dimensional culture of Huh7 cell line with improved hepatocyte differentiation marker expression and HCV replication.

Purpose: A novel three dimensional (3D) culture system purely synthesised from co‑polymer which is free from biological contamination for Huh7 cell cultivation and hepatitis C virus (HCV) replication has been attempted. Materials and Methods: Mebiolgel, a thermo‑reversible gelation polymer was used as a 3D scaffold for culturing Huh7, a liver carcinoma cell line used in our study. The 3D culture of the cells were infected with cell culture derived HCV. Result: The scaffold supported the cell growth as 3D spheroids for up to 63 days. Moreover mebiolgel was found to be improving the hepatocyte differentiation of Huh7 cells at the transcript level. Three dimensional culture was susceptible for HCV infection, and this was confirmed by detecting the HCV replication intermediate viral core antigen. Conclusion: Mebiolgel based culture system was proven to be suited for 3D culture of Huh7 cells by improvising liver specific genotypic expression and was susceptible for HCV replication. Since mebiolgel based Huh 7 express better hepatocyte differentiation markers genotypically, this can be implemented as an alternate for primary hepatocytes in studies such as viral isolation from patient serum.

Effect of GPR49 gene on proliferation and invasive ability of hepatoma cells / 器官移植

Objective To discuss the effect of G-protein-coupled receptor 49 (GPR49)gene on proliferation and invasive ability of hepatoma cell line Huh7 and its molecular biological mechanism.Methods According to the different transfected small interfering RNA(si-RNA),Huh7 cells were divided into the GPR49-siRNA(si-GPR49)group and the NC-siRNA (si-NC)group.Untransfected Huh7 cells were set as the control group. Messenger RNA (mRNA )and protein expression of GPR49, cyclin D1 and matrix metalloproteinase 9 (MMP9)in the cells of the three groups were respectively detected by reverse transcription-polymerase chain reaction (RT-PCR)and Western blot method.The proliferation and invasive ability of the cells of each group were respectively detected by MTT method and Transwell method.Results The relative expression of GPR49 mRNA of the si-GPR49 group was (23.8 ±3.1)% of the control group (P <0.05). Compared with the control group,the protein expression of GPR49,cyclin D1 and MMP9 of the si-GPR49 group decreased significantly (all in P <0.05).The proliferation experiment results by MTT indicated that the optical density(OD)of the cells of the si-GPR49 group at 72 h was (0.53 ±0.12),which was significantly lower than that of the control group (1.35 ±0.28).The difference had statistical significance (P <0.05). The average invaded cell counts of the si-GPR49 group were (13.6 ±2.5),which was significantly lower than (65.3 ±6.1 )of the control group.The difference had statistical significance (P <0.05 ).Conclusions GPR49-siRNA may inhibit the gene expression of GPR49 in Huh7 cells.Its mechanism may be that the proliferation of Huh7 cells is inhibited by reducing the level of cyclin D1;the migration and invasive ability of Huh7 cells is inhibited by affecting the expression level of MMP9.

The Applications of Hepatitis C Virus (HCV) Replication System in Developing Anti-HCV Reagents

Hepatitis C virus (HCV) is known to be a major cause of chronic hepatitis, liver cirrhosis and hepatocarcinoma. Therapeutic reagents are improving, but are still limited, and the protective vaccine against HCV is not available yet. However, the research of HCV life cycle and pathogenesis has been difficult due to obstacles, which are the lack of effective cell culture systems and small-animal models. Recently, breathtaking progress in terms of HCV replication system has been made using various forms of HCV clones and human hepatocarcinoma 7 cell lines (huh 7). The establishment of complete cell-culture system for HCV replication gave researchers opportunities to study the entire viral life cycle including entry, assembly, release of viral particles and the interaction with host cells. In fact, these efforts now appear to move into the identification and the development of innovative anti-HCV reagents. In this review, we go over the biological characters of HCV, a variety of in vitro cell culture, in vivo animal models of HCV infection, HCV immune-pathogenesis and the application of HCVcc system in terms of developing anti-HCV reagents.

Inhibitory Effect of siRNA Expression Vector Inhibiting IGF2 Gene on the Proliferation of Hepatoma Cell Line Huh-7 / 中国药房

OBJECTIVE:To investigate the inhibitory effect of siRNA expression vector inhibiting human insulin-like growth factor 2(IGF2)gene on the proliferation of hepatoma cell line Huh-7. METHODS:siRNA expression vector pGL3-hAFP-hTERT-siRNA3(“siRNA3”)which inhibited IGF2 gene by dual promoter regulation of recombinant human alpha-foetoprotein(hAFP)and human telomerase reverse transcriptase(hTERT)was transfected into the Huh-7 cell and normal hepatocyte L-02,and then a nega-tive control group(vector pGL3-hAFP-hTERT)and a blank control group were set up. IGF2 mRNA expression was detected by re-al-time fluorescent quantitative polymerase chain reaction 48 h after transfection into the cells in all groups;the activity of the cells by the microplate reader 0,24,48 and 72 h thereafter;and the cell cycle and apoptosis by the flow cytometer 48 h thereafter,and the changes in the protein levels of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in the cell were detected by Western blot. RESULTS:Compared with the negative control group and blank control group,IGF2 mRNA expression in the Huh-7 cell transfected with siRNA3 was obviously weaker;at 48 and 72 h after transfection,the activity of Huh-7 cell signigicantly reduced, Huh-7 cells at G1 phase obviously increased and those at S phase markedly decreased;the occurrence of early,late and total apopto-sis in Huh-7 cells apparently increased,and the protein expression of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in cells significantly weakened,with statistically significance(P0.05). CONCLUSIONS:siRNA which inhibited IGF2 gene by dual promoter regulation of recombinant hAFP and hTERT can specially inhibit IGF2 gene expression and the prolifer-ation of Huh-7 cells,which may be involved with down-regulated protein expression of cell proliferation-associated gene PCNA, cell cycle control-associated genes Cyclin E2,Cyclin D2 and Cdc2 and apoptosis regulation-associated gene Bcl-2 as a result of down-regulated IGF2 mRNA expression and protein expres-sion.