| polycomb group(PCG)proteins, with the counteracting Trithorax group (TrxG) proteins, establish a form of cellular memory by regulating gene expression in a heritable fashion. Dysregulation of the transcriptional memory maintenance system can lead to disastrous consequences, ranging from developmental defects to cancer. Recently, accumulating evidence indicates that enhancer of zeste homologue 2(EZH2), a member of PcG protein family, overexpresses in the aggressive human cancers. Ectopic EZH2 in immortalized human mammary epithelial cell lines promotes anchorage-independent growth and cell invasion, inhibition of EZH2 expression blocked prostate cell growth. In addition, EZH2 expression was an independent predictor of the development of metastases of prostate cancer. Amplification of EZH2 has been shown by fluorescence in situ hybridization (FISH) in multiple types of cancer Therefore, the present research investigates the role of EZH2 in thehepatocellular carcinoma(HCC).Part OneThe expression of EZH2 in the hepatocellular carcinomaMaterials and Methods(DPatients and Specimens28 specimens of fresh HCC tissue, coupled with nontumor tissue from each individual, were obtained from The First Affiliated Hospital of Zhejiang University. All the tissue specimens were immediately frozen in liquid nitrogen and kept at -70°C, which were submitted for routine pathological evaluation and diagnosis confirmation.(2)RT-PCRTotal RNA was extracted using Trizol (Invitrogen) according to the manufacturer' s instructions. cDNA was synthesized with M-MLV (Promega) with lug total RNA. PCR reaction was carried out for EZH2 with 29 cycles, each cycle consisted of preincubation at 94°C for 3 min, denaturation at 94°C for 30s and annealing at 57°C for 30s and extension at 72 °C for 45s. PCR reaction was carried out forP-actin with 25 cycles, each cycle consisted of preincubation at 94°C for 3 min, denaturation at 94°C for 30s and annealing at 56°C for 30s and extension at 72 °C for 45s. The primers were as follows: EZH2 (5' -GACGGCTTCCCAATAACAG-3' and 5' -ATTGAGGCTTCAGCACCAC3-3' ), P-actin (5' -ACTTAGTTGCGTTACACCCTTTC-3' and 5' -CTGTCACCTTCACCGTTCC-3' ). Samples were electrophoresed on an ethidium bromide-stained (0. 1 g/ml) 1. 2%agarose gel. (3)Western blotImmunoblot analysis was performed using standard procedures. Briefly, tissue samples were lysed in buffer (50mM HEPES (pH 7. 0), 250mM NaCl, 0.1% Nonidet P-40, 5mM EDTA, lmM PMSF, lmM DTT) supplemented with protease Inhibitor cocktail (SIGMA) for 30 min on ice to extract total protein. Protein concentration was determined by Bradford assay. Rabbit polyclonal anti-EZH2 (Upstate Biochemistry), rabbit polyclonal anti-ACTIN (Santa Cruz), secondary antibodies HRP-anti-rabbit (Santa Cruz) was used for immunoblotting. The signal was detected by the supersignal-enhanced chemiluminescene system (Pierce).Results(l)Expression of EZH2 in clinical sampleIn transcriptional level, the mean expression of EZH2 in tumor tissue specimens was significantly higher than the mean expression of EZH2 in the corresponding nontumor tissue specimens (1. 17 + 0. 50vs. 0. 50 + 0. 14, p<0. 01). In protein level, the mean expression of EZH2 was also significantly higher than the mean expression of EZH2 in the corresponding nontumor tissue specimens (1. 35 + 0. 65vs. 0. 38 + 0. 20, p<0. 01).(2)EZH2 expression and clinicopathological characteristicsThe expression of EZH2 significantly correlate with the size of cancer diameter (r=0. 802, P<0. 001) The expression of EZH2 in group of tumor diameter >5cm was higher than the group of tumor diameter^5cm (1. 71 + 0. 68 vs. 0. 93 + 0.25, p=0. 001) . However, other pathological variables such as tumor histological differentiation, and metastasis to portal vein, did not significantly correlate with the EZH2 expression in tumor tissue specimens.Conclusion(DEZH2 overexpressed in the hepatocellular carcinoma(2) The expression of EZH2 significantly correlated with the size of cancer diameter(3)The expression of EZH2 in group of tumor diameter>5cm was higher than the group of tumor diameter^5cmPart twoThe effects of EZH2 siRNA on proliferation and distribution of cell cycle of HepG2 cell lineMaterials and Methods(DConstruction of vector producing EZH2 siRNAHairpin siRNA template oligonucleotides were ligated into psilencer?2. 1-U6 neo at BamH I / Hindlll site according to the manufacturer' s instructions. Template oligonucleotides were as follows: EZH2 5' -gatccGAGCnTCAGACGAGCTGATTTCAAGAGAATCAGCTCGTCTCAACCTCt11111ggaaa-3' , 5' -agenttccaacMaaGAGCTTCAGACGACCTGATTCTCTTGAAATCAGCTCGTCTGAACCTCg-3' , targeting the sequences 5' -GAGGTTCAGACGAGCTGAT-3' corresponding to the open reading frame of human EZH2 gene (NM-004456, 256-274);control 5' -gatccGAGGTTCAGACGAGTCGATttcaaBagaATCGACTCGTCTGAACCTCttttttggaaa-3' and 5' -aRcUttccaaaaaat'.AGGTTCAGACGAGTCGATtctcttgaaATCGACTCGTCTGAACCTCg-3> targeting the sequences 5' -GAGGTTCAGACGAGTCGAT-3' , not corresponding to any human genes. The sequences of siRNA template insert were verified by DNA sequencing. EZH2 siRNA plasmid and control were named as pSilence (+) and pSilencer(-), respectively.(2) Cell and TransfectionThe human hepatoma cell line HepG2 maintained in DMEM medium supplemented with 10% foetal calf serum in a humidified incubator at 37°C and 5% C02. Plasmid pSilence (+) and pSilence (-) were transfected into HepG2 cells by Lipofectamine(Invitrogen) as manufacturer's protocol. Stably resistant cells were selected using 800mg/L G418 for first 2 weeks, then 400mg/L G418 expanded cell for further studies. Transfected cells were named as HepG2/Silence (+) and HepG2/Silence(-), respectively. (3)RT-PCRTotal RNA was extracted using Trizol (Invitrogen) according to the manufacturer' s instructions. cDNA was synthesized with M-MLV (Promega) with lug total RNA. PCR reaction was carried out for EZH2 with 29 cycles, each cycle consisted of preincubation at 94°C for 3 min, denaturation at 94°C for 30s and annealing at 57°C for 30s and extension at 72 °C for 45s. PCR reaction was carried out forP-actin 25 with cycles;each cycle consisted of preincubation at 94°C for 3 min, denaturation at 94°C for 30s and annealing at 56°C for 30s and extension at 72 °C for 45s. The primers were as follows: EZH2 (5' -GACGGCTTCCCAATAACAG-3' and 5' -ATTGAGGCTTCAGCACCAC3-3' ), P-actin (5' -ACTTAGTTGCGTTACACCCTTTC-3' and 5' -CTGTCACCTTCACCGTTCC-3' ). Samples were electrophoresed on an ethidium bromide-stained (0. 1 g/ml) 1. 2%agarose gel. (Western blotImmunoblot analysis was performed using standard procedures. Briefly, cells were lysed in buffer (50mM HEPES (pH 7.0), 250mM NaCl, 0. 1% Nonidet P-40, 5mM EDTA, lmM PMSF, lmM DTT) supplemented with protease Inhibitor cocktail (SIGMA) for 30 min on ice to extract total protein. Protein concentrationwas determined by Bradford assay. Rabbit polyclonal anti~EZH2 (Upstate Biochemistry), rabbit polyclonal anti-ACTIN (Santa Cruz), secondary antibodies HRP-anti-rabbit (Santa Cruz) was used for imraunoblotting. The signal was detected by the supersignal-enhanced cherailuminescene system (Pierce). (5)MTT assayHepG2/pSilence(+) , HepG2/pSilence(-) and HepG2 cells (5X103/100ul per well) were plated into a 96-well plate in quadruple. On each day of consecutive 6 days, 20u 1 MTT(5 mg/ml) was added to each well, and the cells were incubated at 37 °C for 4 h, then the OD value was measured at 490nm after the cells were incubated with DMS0(150yl per well) for lOmin.(6) Cell cycle analysisHepG2/pSilence(+), HepG2/pSilence(-) and HepG2 cells were resuspended in PBS buffer with in 5 mg/ml propidium iodide, 0. 1 mg/ml RNAse A. Flow cytometry was performed on flow cytometer. At least 5000 cells were analyzed per sample, three samples per cell line.Results(DpSilence(+) constructionpSilence(+)expressing EZH2 siRNA and control, pSilence(-) were constructed, verified by sequencing;HepG2 cell transfected with pSilence(+) or pSilence(-) with Lipofectamine(Invitrogen). Stably resistant cells were selected using 800mg/L G418 for the first 2 weeks, then 400mg/L G418 expanded cell, named as HepG2/pSilence(+) and HepG2/pSilence(-)(2)pSilence(+) significantly knocked down the EZH2 expressionTo determine the effect of EZH2 siRNA on HepG2 cell proliferation, we examined the proliferation of HepG2/pSilence(+), HepG2/pSilence(-) and HepG2 cellswith MTT assay. We found that EZH2 siRNA markedly inhibited HepG2 proliferation(3) cell cyclecell cycle distribution of HepG2/pSilence(+) cell assessed propidium iodide (PI) staining. Flow cytoraetric analysis showed that HepG2/pSilence(+) cells cycle distribution in the G2/M phase (35. 17%±1.69%), higher to the level of HepG2/pSilence(-)(18. 13%±0. 80%) and HepG2 cells (17. 30% + 0. 17%) , it showed that EZH2 siRNA induced HepG2 cells arrested in the G2/M phasesConclusion(l)The EZH2 siRNA expressing vector was successfully constructed(2)The cell that stable expressed the EZH2 siRNA was successfully constructed(3)EZH2 siRNA significantly knocked down the EZH2 expression(4)EZH2 siRNA markedly inhibited HepG2 proliferation(5)EZH2 siRNA induced HepG2 cells arrested in the G2/M phasesSummary(DEZH2 overexpressed in the hepatocellular carcinoma(2) The expression of EZH2 significantly correlated with the diameter size of cancer(3)The expression of EZH2 in group of tumor diameter>5cm was higher than thegroup of tumor diameter^5cm(4)The EZH2 siRNA expressing vector was successfully constructed(5)The cell that stable expressed the EZH2 siRNA was successfully constructed(6)EZH2 siRNA significantly knocked down the EZH2 expression(7)EZH2 siRNA markedly inhibited HepG2 proliferation(8)EZH2 siRNA induced HepG2 cells arrested in the G2/M phases...
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