EFFECT OF MATRINE ON EXPRESSION OF HCCR1 AND HCCR2 PROTEINS IN CULTURAL HUMAN HEPATOCELLULAR CARCINOMAS CELLS

Objective： To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas （HCC） cells at the level of gene and protein. Methods： Three methods, representational difference analysis （RDA） of cDNA, microarrays and fluorescence in situ hybridization （FISH） were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results： Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion： Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.

Differential Expression of Genes in HepG2 Cells Caused by UC001kfo RNAi as Shown by RNA-seq

The differential expression of genes in HepG2 cells caused by UC001 kfo RNAi was investigated using RNA-seq. HepG2 cells were infected by Lenti-sh UC001 kfo lentivirus particles. The expression of UC001 kfo m RNA in the HepG2-sh UC001 kfo cell line was detected by real-time PCR. RNA-seq technology was used to identify the difference in the expression of genes regulated by lnc RNA UC001 kfo in the HepG2 cell line. Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different m RNAs. The results showed that m RNAs were differentially expressed between the HepG2-sh UC001 kfo cell line and the HepG2 cell line. The UC001 kfo m RNA was significantly down-regulated in the stable cell line HepG2-sh UC001kfo（P〈0.001）. Additionally, we found 19 signaling pathways or functional classifications encompassing 30 genes that played a role in cancer characteristics, cell adhesion, invasion and migration. The results also showed that the expression of many genes associated with cancer cell invasion and metastasis was decreased with the down-regulation of the lnc RNA UC001 kfo. Lnc RNA UC001 kfo may play a role in regulating cancer cell invasion and metastasis. It was suggested that m RNAs were differentially expressed in the HepG2 cell line after the down-regulation of lnc RNA-UC001 kfo. Some took part in the extracellular matrix, cell adhesion, motility, growth, and localization. The genes encoding of differentially expressed m RNAs may participate in cell invasion and metastasis.

Effects of suppressing glucose transporter-1 by an antisense oligodeoxynucleotide on the growth of human hepatocellular carcinoma cells

BACKGROUND:The glucose transporter-1(Glut-1),a key ratelimiting factor in the transport and metabolism of glucose in cancer cells,is over-expressed in many human cancer cells and this overexpression is correlated with poor biological behavior. The increased levels of Glut-1 expression in hepatocellular carcinoma(HCC)cells functionally affect tumorigenicity.This study was undertaken to investigate effects of suppressing Glut-1 by an antisense oligodeoxynucleotide(AS-ODN)on the growth of human hepatocellular carcinoma(HepG-2)cells. METHODS:We used AS-ODN targeting against the Glut-1 gene in a HepG-2 cell line.There were four experimental groups: empty pcDNA3.1 vector(mock transfection),pcDNA3.1-anti-Glut(+),pcDNA3.1-Glut(+),and non-transfected HepG-2 cells. The Glut-1 mRNA expression was detected by RT-PCR and the Glut-1 protein expression by Western blotting after cell culture, and the glucose uptake was detected after glucose stimulation in each group. RESULTS:Compared with non-transfected HepG-2 or Glut-1 pcDNA3.1,a down-regulation of Glut-1 mRNA in HepG-2 cells transfected with anti-Glut-1 pcDNA3.1 was noted(P<0.05).Glut-1 protein in HepG-2 cells transfected with Glut-1 AS-ODN was decreased compared with non-transfected HepG-2,Glut-1 pcDNA3.1,or empty vectors. Glucose uptake by the HepG-2 cells transfected with AS-ODN was decreased at 1 hour after glucose stimulation.CONCLUSIONS:The application of Glut-1 AS-ODN can down-regulate the expression of Glut-1 at mRNA and protein,and inhibit glucose uptake partially in HepG-2 cells.The Glut-1 gene maybe a potential therapeutic target for HCC.

Possible mechanisms associated with immune escape and apoptosis on anti-hepatocellular carcinoma effect of Mu Ji Fang granules

BACKGROUND Hepatocellular carcinoma(HCC)is one of the most common digestive system cancers with high mortality rates worldwide.The main ingredients in Mu Ji Fang Granules(MJF)are alkaloids,flavonoids,and polysaccharides.MJF has been used in the clinical treatment of hepatitis,cirrhosis and HCC for more than 30 years.Few previous studies have focused on the mechanism of MJF on tumor immunology in the treatment of HCC.AIM To explore the mechanism of action of MJF on tumor immunology in the treatment of HCC.METHODS The absorbable ingredients of MJF were identified using Molecule Network related to High Performance Liquid Chromatography-Electron Spray Ionization-Time of Flight-Mass Spectrometry,and hub potential anti-HCC targets were screened using network pharmacology and pathway enrichment analysis.Forty male mice were randomly divided into the Blank,Model,and MJF groups(1.8,5.4,and 10.8 g/kg/d)following 7 d of oral administration.Average body weight gain,spleen and thymus indices were calculated,tumor tissues were stained with hematoxylin and eosin,and Interferon gamma(IFN-γ),Tumor necrosis factorα(TNF-α),Interleukin-2,aspartate aminotransferase,alanine aminotransferase,alpha-fetoprotein(AFP),Fas,and FasL were measured by Enzyme-linked Immunosorbent Assay.Relevant mRNA expression of Bax and Bcl2 was evaluated by Real Time Quantitative PCR(RTqPCR)and protein expression of Transforming growth factorβ1(TGF-β1)and Mothers against decapentaplegic homolog(SMAD)4 was assessed by Western blotting.The HepG2 cell line was treated with 10 mg/mL,20 mg/mL,30 mg/mL,40 mg/mL of MJF,and another 3 groups were treated with TGF-β1 inhibitor(LY364947)and different doses of MJF.Relevant mRNA expression of TNF-α,IFN-γ,Bax and Bcl2 was evaluated by RT-qPCR and protein expression of TGF-β1,SMAD2,p-SMAD2,SMAD4,and SMAD7 was assessed by Western blotting.RESULTS It was shown that MJF improved body weight gain and tumor inhibition rate in H22 tumorbearing mice,protected immune organs and liver function,reduced the HCC indicator AFP,affected immunity and apoptosis,and up-regulated the TGF-β1/SMAD signaling pathway,by increasing the relative expression of TGF-β1,SMAD2,p-SMAD2 and SMAD4 and decreasing SMAD7,reducing immune factors TNF-αand IFN-γ,decreasing apoptosis cytokines Fas,FasL and Bcl2/Bax,and inhibiting the effect of LY364947 in HepG2 cells.CONCLUSION MJF inhibits HCC by activating the TGF-β1/SMAD signaling pathway,and affecting immune and apoptotic cytokines,which may be due to MJF adjusting immune escape and apoptosis.

Inhibitory effect of metformin on the proliferation of human hepatoma HepG2 cells and its potential mechanism

Objective： This work aimed to study the inhibitory effect and the related mechanism of metformin （MET） on the proliferation of human hepatoma HepG2 cells. Methods： Human hepatoma HepG2 cells were treated with MET （0, 2, 10, and 50 mM）. The inhibitory effect of MET on the proliferation of HepG2 cells was determined by MTT method. The apoptosis of HepG2 cells was detected by flow cytornetry. The expression of cyclin D1 in HepG2 cells was examined by Western blot. ROS-DHE fluorescence probe was used to stain the reactive oxygen species （ROS） generated by HepG2 cells after treat- ment. Results： MET could inhibit the proliferation of HepG2 cells in a dose and time dependent manner. MET promoted the apoptosis of HepG2 cells. In addition, MET suppressed the expression of cell cycle protein cyclin D1 and induced the produc- tion of ROS in HepG2 cells. Conclusion： MET can inhibit the proliferation of human hepatoma HepG2 cells and induce cell apoptosis. Meanwhile, MET has the ability to decrease the expression of cyclin D1 and induce ROS generation, which may be involved in the mechanism of inhibiting hepatoma cells proliferation.

凋亡抑制蛋白Livin在人原发性肝癌细胞系HEPG-2中的表达情况

目的研究凋亡抑制蛋白Livin在人原发性肝癌细胞系HEPG-2中的表达情况。方法以RT-PCR法检测人原发性肝癌细胞系HEPG-2中凋亡抑制蛋白Livin基因mRNA的表达。结果HEPG-2细胞只表达Livin基因的β亚型,即Livinβ。结论人原发性肝癌细胞系HEPG-2细胞阳性表达Livinβ基因。本实验研究结果为原发性肝癌的诊断和治疗提供了新思路、新方法。

Effect of Fanbaicao(Herba Potentillae Discoloris) oil on the expression of p21 and CDK4 in HepG2 cells

OBJECTIVE:To research the anti-cancer mechanism of the Traditional Chinese Medicine Fanbaicao(Herba Potentillae Discoloris) oil in the human hepatoma cell line Hep G2.METHODS:Gas chromatography was used to analyze the components of Fanbaicao(Herba Potentillae Discoloris).We tested the inhibitory effect of Fanbaicao(Herba Potentillae Discoloris) oil on the human hepatoma cell line Hep G2 in vitro using 3-(4,5-Dimet hylt hiazol-2-yl)-2,5-dip henyltetrazoliumbromide assays.Fluorescence activating cell sorter analysis was used to examine the levels of apoptosis,and western blot and immunofluorescence were used to detect the expression of p21,p-p21 and CDK4 proteins.RESULTS:Fanbaicao(Herba Potentillae Discoloris)oil contains 45 ingredients,and L-ascorbic acid 2,6-bispalmitate was the main component and accounted for 44.96% of total drive-off peak area.Other components included(Z)-14-met hyl-8-exadecenal-acetal(8.56%),phytol(7.74%) and lauric acid(6.31%).Fanbaicao(Herba Potentillae Discoloris)oil treatment reduced the proliferation of Hep G2 cells and the half growth inhibition concentration(IC50) was 2.03 mg/m L.Furthermore,we also observed significantly increased Hep G2 cell apoptosis in a dose-dependent manner(P < 0.05).Fanbaicao(Herba Potentillae Discoloris) oil significantly increased the expression of p21 and p-p21 and significantly decreased the expression of CDK4 in Hep G2 cells compared with controls(P < 0.01).CONCLUSION:Our results showed that Fanbaicao(Herba Potentillae Discoloris) oil has anti-cancer activities in Hep G2 cells,which is probably related to the upregulation of p21 and p-p21 and downregulation of CDK4 expression.

pcDNA3.1-Canstatin-3Flag真核表达载体的构建及转染HepG2细胞中的表达

目的:构建pcDNA3.1-Canstatin-3Flag载体并稳定转染肝癌HepG2细胞,检测canstatin在mRNA水平的表达。方法:胎盘中提取总RNA,RT-PCR法获得canstatinDNA,克隆至pcDNA3.1(-)载体中,并测序,重组质粒pcDNA3.1-Canstatin-3Flag转染肝癌HepG2细胞,G418筛选出稳定转染细胞,RT-PCR检测canstatin mRNA表达。结果:1.成功构建出pcDNA3.1-Canstatin-3Flag重组质粒;2.获得稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞;3.发现转染后的肝癌HepG2细胞canstatin在mRNA水平比未转染细胞有明显的增强。结论:获得了稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞,为后期canstatin在肝癌中的研究提供了支持。