Mcl-1 as a potential therapeutic target for human hepatocelluar carcinoma / 华中科技大学学报（医学）（英德文版）

Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality in part due to its high resistance to chemotherapeutic drugs. The anti-apoptotic Mcl-1 expression has been reported as a resistance factor in various types of tumors. Here, we investigated the expression of Mcl-1 in hepatoma cells and HCC tissues and its relationship with p53, and analyzed the possibility of the gene as a molecular target for HCC therapy. HCC specimens of 30 patients were examined by immunohistochemistry for Mcl-1 and p53 expression. Mcl-1 expression in hepatoma cell lines was measured by RT-PCR and Western blotting. The suppression of Mcl-1 by RNA interference or specific phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, was evaluated as monotherapy, and it was combined with mitomycin C (MMC) in treating hepatoma cell line HepG2. Cell viability and apoptosis were assessed by MTT and FACS analysis. Finally, changes of Mcl-1 or p53 expression in various hepatoma cell lines were examined after transfection with Mcl-1 siRNA, the Mcl-1 expression plasmid, or the wide-type p53 expression plasmid, respectively. Mcl-1 protein was remarkably enhanced in HCC tissues as compared with adjacent non-tumor liver tissues. In addition, Mcl-1 was prominently expressed in HepG2 and Hep3B cells, weakly in SMMC7721 cells, and not in L02 cells. P53 protein was also overexpressed in HCC tissues and there was a significant correlation between the expression of p53 and Mcl-1. Silencing Mcl-1 by RNAi or LY294002 downregulated Mcl-1 expression and led to decreased cell viability and increased apoptosis. Combination of MMC and Mcl-1 RNAi or LY294002 exhibited a significant chemosensitizing effect. The expression of p53 was not influenced by Mcl-1 siRNA in HepG2 cells or transfection with the Mcl-1 expression plasmid in L02 cells. Furthermore, the expression of Mcl-1 in Hep3B cells was also not significantly changed after transfection with the wild-type p53 expression plasmid. It is concluded that Mcl-1 is overexpressed in HCC tissues. The mechanisms by which silencing Mcl-1 sensitizes hepatoma cells towards chemotherapy may be not attributed to the upregulated expression of p53 but the dysfunction of p53 through Mcl-1/p53 interaction. Mcl-1 may be a potential target of gene therapy for HCC.

The effect of hepatitis B virus X protein on the expression of CtIP in HepG2 Cells / 中华肝脏病杂志

To investigate the effect of hepatitis B virus X protein(HBx) on CtBP-interacting protein(CtIP) which is an important repair factor of DNA double strand break damage in HepG2 cells induced by bleomycin. A HBx stably expressing HepG2 cell line and a control HepG2 cell line with empty vector transfected were established. After the double strand break (DSB) damage occurred, the mRNA and protein levels of CtIP were detected by Real-time PCR and Western blot assay respectively, cell cycle profiles and apoptotic cell death were determined by a flow cytometry, and the position of CtIP in cells was observed by confocal laser scanning microscopy. It showed that HepG2 cells transfected with hepatitis B virus X gene could stably express HBx protein. After being induced by bleomycin, the percentage of apoptotic cell was 16.90%+/-0.89% in HBx stably expressing HepG2 cell line and 15.30%+/-0.86% in control cell line, respectively (q = 2.074, P is more than to 0.05). While the percentage of death cell was 8.71%+/-0.74% in HBx stably expressing HepG2 cell line and 4.90%+/-0.46% in control cell line, respectively (q = 7.126, P is less than to 0.01). The two cell lines manifested the increase of G2/M arrest and significant difference existed between the two cell lines. HBx down regulated the expression levels of CtIP and its mRNA. The CtIP level was 0.66+/-0.04 in HepG2-HBx cell and 0.73+/-0.05 in HepG2-vec cell, respectively (t = 2.314, P is less than to 0.05). The relative mRNA level was 1.00+/-0.06 in HepG2-HBx cell and 1.23+/-0.08 in HepG2-vec cell, respectively (t = 2. 732, P is less than to 0.05). We also found that CtIP was concentrated in the cell nucleus. The research suggests that HBx may affect DNA-repair pathways by disrupting the expression of CtIP.

HBx gene down-regulates miR-192 expression and inhibits apoptosis of human hepatoma cell line HepG2 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the mechanism by which HBV X gene(HBx) inhibits apoptosis of human hepatoma cell line HepG2 in terms of miRNA.</p><p><b>METHODS</b>Three cell lines were prepared: HepG2 cells stably transfected with HBx (HepG2/HBx), HepG2 cells stably transfected with pcDNA3.1 (HepG2/pcDNA3.1) and HepG2 cells. Flow cytometry was adopted to measure the apoptosis of these three cells and Taqman fluorescence quantitative PCR was used to examine miR-192 expression. After HepG2 cells was transfected with miR-192, the apoptosis was analyzed by flow cytometry and the expressions of p53 and PUMA at mRNA and protein levels were evaluated by SYBR Green quantitative PCR and Western blot, respectively.</p><p><b>RESULTS</b>Compared with HepG2/pcDNA3.1 cells (11.46% ± 0.69%) and HepG2 cells (12.5% ± 0.66%), the apoptosis rate of HepG2/HBx cells (2.37% ± 0.35%) was significantly reduced (F = 171.722, P < 0.01). The level of miR-192 was 49.1% ± 5.9% in HepG2 cells, which was dramatically down-regulated (F = 14.319, P = 0.019) as compared to the other two groups (HepG2/pcDNA3.1: 98.0% ± 8.9%; HepG2: 100%). Compared with HepG2 cells transfected with miR-NC (10.74% ± 1.15%), transfection of miR-192 into HepG2 cells led to increased apoptosis (15.74% ± 1.17%) (F = 18.415, P = 0.013) and higher p53 and PUMA expressions at mRNA (p53: 1.68 ± 0.12 vs 0.90 ± 0.09, F = 43.115, P = 0.003, PUMA: 1.66 ± 0.10 vs 0.98 ± 0.06, F = 22.541, P = 0.009) and protein (p53: 3.07 vs 1, PUMA: 2.13 vs 1) levels.</p><p><b>CONCLUSION</b>HBx could inhibit apoptosis of HepG2 cells through down-regulation of miR-192 which induces apoptosis of HepG2 cells.</p>

MiR-122 regulates the expression of PEG10 in hepatoma cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Our previous work indicated that overexpression of imprinting gene PEG10 is associated with malignant phenotype of hepatocellular carcinoma. The aim of this study is to explore whether disregulation of PEG10 leads to dysregulation of microRNAs.</p><p><b>METHODS</b>In silico analysis using TargetScan indicated that miR-122 could regulate the expression of PEG10. The expression of miR-122 in three hepatoma cell lines, Huh7, Hep3B and HepG2 and in primary human normal liver cell were compared using real time RT-PCR. After pre-miR-122 was transfected into HepG2 cell, the levels of PEG10 mRNA and protein were measured.</p><p><b>RESULTS</b>In silico analysis revealed that miR-122 could regulate the expression of PEG10. Real time RT-PCR indicated that miR-122 was not expressed in Hep3B and HepG2 cells, and only weakly expressed in Huh7 cells, but highly expressed in primary human normal liver cells. The expression of miR-122 was negatively correlated with the expression of PEG10. After pre-miR122 was transfected into HepG2, the mRNA level of PEG10 was not increased, whereas the protein level of PEG10 was increased.</p><p><b>CONCLUSION</b>miR-122 may be involved in regulation of PEG10 expression.</p>

The imprinting status of genetic imprinted gene PEG10 in human hepatocellular carcinomas / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the imprinting status of genetic imprinted gene PEG10 (perternally expressed gene 10) in human hepatocellular carcinoma (HCC) tissues and liver cancer cell lines.</p><p><b>METHODS</b>Genomic DNA was extracted from 20 HCC tissues and its adjacent tissues, 15 normal liver tissues, 5 liver cancer cell lines (PLC/PRF/5, smmc-7721, HepG2, Hep3B, SK-HEP-1) and 2 normal human liver cell lines (changliver, HL7702). The DNA fragments containing single nucleotide polymorphism (SNP) site of PEG10 were amplified by polymerase chain reaction (PCR) and the genotype of samples was detected by DNA sequencing. Total RNA was extracted from heterozygous samples, the imprinting status and expression level of PEG10 were evaluated by quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) and DNA sequencing.</p><p><b>RESULTS</b>It was found that 16/40 of HCC and its adjacent tissues were heterozygous, 3/15 of normal liver tissue were heterozygous. A site of heterozygous mutation was found in HepG2 cells by DNA sequencing. The other liver tissues and cell lines were all homozygous. PEG10 was biallelically expressed and showed loss of imprinting (LOI) in 82.4% (14/17) liver cancer samples (16 HCC tissues and HepG2), however it was a monoallelic expression and showed genomic imprinting in17.6% (3/17) liver cancer samples. In HCC, the expression levels of PEG10 were increased apparently, but it was negative expressed in cancer adjacent tissues and normal liver tissues. Expression levels of PEG10 were not significantly different between imprinted HCC tissues and HCC tissues with LOI (t = 1.311, P value is more than 0.05).</p><p><b>CONCLUSION</b>PEG10 imprinting is lost in a majority of HCC and no correlation exists between the imprinting status of PEG10 and its expressions in HCC tissues.</p>

Establishment of PEG10 transgenic mouse and effects of PEG10 on growth, metastasis of transplanted tumor in mice / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish PEG10 transgenic mice model and study the effect of PEG10 transgene on tumor growth and metastasis in mice.</p><p><b>METHODS</b>The linearized expression element of pALB-PEG10, which contained mouse albumin promoter, structural gene of PEG10, and polyaenylation signal sequence, was microinjected into 3741 KM mouse fertilized ova. The manipulated embryos were then transplanted into the oviducts of 94 pseudopregnant recipient mice. All the newborn mice were screened by PCR to detect genomic DNA in tail tissue, then PEG10 mRNA and protein expression were detected by RT-PCR and western blot, respectively in the positive mice. Hepatoma cell H22 was subcutaneously inoculated into the right armpit of wild type mice and No.17, No.33 transgenic mice. Tumor size was measured every week. Mice were sacrificed on day 12 and then the tumors were exercised and weighted. Tumors and livers were fixed in formaldehyde and sectioned. The sections were stained with hematoxylin/eosin and examined under microscope. The expression of PEG10 protein was detected with immunohistochemistry method.</p><p><b>RESULTS</b>Among the 43 off-springs, 3 were positive for tail tissue PEG10 gene examination, PEG10 was successfully expressed in the liver of the randomly selected transgenic mouse. H22 tumor grew faster in all the transgenic mice than in wild type mice. The average size and weight of tumors between the transgenic mice and wild type mice were significantly different (P < 0.05). Most tumors in the transgenic mice invaded the surrounding tissues and showed liver metastasis, PEG10 protein was expressed in liver. In contrast, nearly all the tumors in wild type mice were capsulized and PEG10 was not expressed in liver.</p><p><b>CONCLUSION</b>Our results showed that the PEG10 gene could be expressed in the liver of the transgenic mice. PEG10 promotes growth, invasion, and metastasis of transplanted H22 tumors in mice.</p>

Insertion of TC motif into hepatitis B virus core protein c/e1 site does not affect the expression of S and e antigen / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether insertion of TC motif into hepatitis B virus (HBV) core protein c/e1 site affects the expression of S and e antigen.</p><p><b>METHODS</b>Different oligonucleotides encoding TC motif were inserted into the c/e1 site of the core gene of a 1.3 copy wild-type HBV genome vector. HepG2 cells were divided into several groups of cells to transiently transfect with the wild-type and mutant HBV vectors, respectively. In each group, the expression level of core protein inside cells was detected by western blotting, and the levels of S and e antigen in culture medium were analyzed by ELISA assay.</p><p><b>RESULTS</b>Western blotting showed that these TC-tagged core proteins were expressed at similar level of wild-type one. ELISA assay indicated that the level of S and e antigen in culture medium of different groups were not significantly different.</p><p><b>CONCLUSION</b>Insertion of TC motif into HBV core protein c/e1 site does not interference with the expression of viral protein encoded by HBV genome.</p>

Establishment of an I-SceI system and its application to introduce DNA double-strand break into human hepatoma cell line HepG2 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct a system of I-SceI and induce a site-specific DNA double-strand break (DSB) in the genome of HepG2 for using this system in future exploration of the potential mechanisms of HBV integration by DSB repair.</p><p><b>METHODS</b>The eukaryotic expression plasmid pEGFP2 was constructed and transfected into human hepatoma cell line HepG2. The positive neomycin-resistant transfected cell clones were generated by G418 selection. Then the positive cells containing an 18-bp I-SceI endonuclease site were transfected transiently with pCMV(3NLS) I-SceI, an I-SceI expression plasmid. At 24 h post-transfection with pCMV (3NLS) I-SceI, gamma-H2AX, as an early cellular marker of DSB, was detected using immunocytochemistry and Western blot analysis.</p><p><b>RESULTS</b>Restriction analysis and DNA sequencing verified that the plasmid pEGFP2 was successfully constructed. gamma-H2AX increased significantly in cells transfected with the I-SceI system.</p><p><b>CONCLUSIONS</b>Genomic DSB can be induced into HepG2 by introducing an I-SceI system. The cell model could provide us with a practical tool for further study to see if DSB is a potential target for HBV integration.</p>

Construction of recombinant plasmid human imprinted gene PEG10 and the primary functional identification in transfected cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct a plasmid expressing human imprinted gene PEG10 and to study the effect of overexpression of PEG10 in a stable transfected human normal liver cell line L02 and in non-liver derived cell line 293.</p><p><b>METHODS</b>Full length cDNA of PEG10 open reading frame 1 was amplified and subcloned into a mammalian expression vector pcDNA3.1hisC. Recombinant plasmid was stably transfected into L02 cells and control cells via Lipofectamine 2000. The expression and the function of PEG10 in L02 cells and control group cells were examined using RT-PCR, Western blot, MTT and TUNEL.</p><p><b>RESULTS</b>Recombinant plasmid was successfully constructed and confirmed through DNA sequencing and restriction digesting. PEG10 gene accelerated the growth of L02 cells and inhibited their apoptosis but it had no conspicuous effect on the non-liver derived cells.</p><p><b>CONCLUSION</b>The constructed expressing vector pcDNA3.1hisC-PEG10 provides a useful tool for further study on the effects and mechanisms of PEG10. Over-expression of PEG10 may promote L02 cells' proliferation and inhibit their apoptosis, but not in the non-liver derived cell line 293.</p>

The simultaneous knock-down of Ku70 and Ku80 by a tandem Ku-shRNA-encoding plasmid expression system / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To extend the use of vector-derived siRNA by generating multiple shRNAs in the same plasmid.</p><p><b>METHODS</b>Construct a vector that expresses shRNAs targeting on Ku70 and Ku80 in tandem. The gene silencing efficiency of each shRNA was verified previously. After identification by restriction digestion and DNA sequencing, the reconstructed plasmid, named psiRNAKus, was transfected into the human hepatoma cell line HepG2. The tandem-shRNA-induced silencing of targeted genes was determined by RT-PCR at RNA level and Western blot at protein level.</p><p><b>RESULTS</b>The shRNAs encoded by psiRNAKus down-regulated both the expression of Ku70 and Ku80.</p><p><b>CONCLUSION</b>The vector-derived siRNA delivery system that allows multiple shRNA species to be expressed from the same vector may be of value in experimental and therapeutic applications.</p>

The deletion of La protein binding site in HBV RNA leads to instability of S gene mRNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of deletion of the La protein binding site of HBV RNA, caused by its mutation, on the HBV S-mRNA stability of S gene, to study the role of the site in hepatitis B virus life cycle, and to try to find a new anti-HBV target in the future.</p><p><b>METHODS</b>A HBV vector with mutation related to the La protein binding site was constructed using molecular cloning and PCR based site directed mutagenesis, and the vector was named pHBV-mLa. The HBV RNA secondary structure of the site was calculated using a computer. Normal HBV vectors and mutant vectors were respectively transfected into HepG2 cells by Lipofectamine 2000. HBV S-mRNA levels in the two groups were analyzed using semi-quantitative RT-PCR, and HBsAg secretion into the culture media was tested using ELISA.</p><p><b>RESULTS</b>A HBV vector with mutation related to the La protein binding site was successfully constructed, and it was identified and confirmed using restriction analysis and sequencing. The HBV RNA secondary structure of the mutant vector was completely different to the stem-loop structure of the normal HBV vector. Semi-quantitative RT-PCR and ELISA analyses showed that the level of HBV S-mRNA in the mutant vector group was significantly lower than that in the normal HBV vector group (t'=12.703, P<0.05), and the expression efficacy of HBsAg was reduced in the mutant vector group (t= 44.648, P<0.01).</p><p><b>CONCLUSIONS</b>The change of La protein binding site in the HBV RNA caused by the mutation in HBV DNA disorganizes the stem-loop structure in the HBV RNA site. With the structural change, the La protein cannot bind the site and stabilize the HBV RNA (HBV S-mRNA), as the cleavage site in the upstream of the stem-loop structure is exposed to endoribonuclease. This results in HBV S-mRNA decay and affects the expression of the S gene. This study shows that only the sequence of this site in the HBV DNA is reserved, then the stem-loop structure in the La protein binding site will remain intact, and the disorganization of the stem-loop structure affects the stability of the transcripted HBV RNA. The La protein binding site in HBV RNA and the special secondary structure of the site are crucial to the life cycle of the hepatitis B virus.</p>

Hammerhead ribozyme-mediated cleavage of transforming growth factor beta1 RNA in a cell-free system and in hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To identify the activity of hammerhead ribozyme against transforming growth factor beta1 (TGFbeta1) in a cell-free system and in activated hepatic stellate cells (HSCs).</p><p><b>METHODS</b>The ribozyme against TGFb1 was designed with computer software. The transcripts of ribozyme, disabled ribozyme and target RNAs were prepared using the RiboMAX large scale RNA production system. The in vitro cleavage reactions were performed through incubation of 32P-labeled target RNAs with ribozyme or disabled ribozyme in different conditions. The eukaryotic expression vector encoding ribozyme and disabled ribozyme were constructed, and then transfected into HSC-T6 cells which exhibited characteristics of activated HSCs. The intracellular activity of the ribozyme was determined by detecting the ribozyme, disabled ribozyme and the TGFbeta1 expression.</p><p><b>RESULTS</b>The ribozyme cleaved target RNAs into anticipated products effectively. As expected, the disabled ribozyme possessed no cleavage activity in vitro. Further study demonstrated that the ribozyme expressed efficiently and inhibited TGFbeta1 expression in activated HSCs, while the disabled ribozyme displayed only a slight effect on TGFbeta1 expression.</p><p><b>CONCLUSION</b>The ribozyme with perfect cleavage activity in the cell-free system used inhibited TGFbeta1 expression effectively in activated HSCs. This ribozyme can provide a potential therapeutic approach for liver fibrosis.</p>

Construction and expression of EGFP controlled HBV promoters in eukaryotic cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct four expression vectors carrying enhanced green fluorescent protein (EGFP) gene under the control of different HBV promoters, and to detect and analyze their expressions in hepatoma cell lines.</p><p><b>METHODS</b>Four HBV promoters were amplified using PCR, and they were inserted into the T-vector and identified using restriction enzymes and sequencing, then cloned into the expression vector pEGFP-1. The four recombinant plasmids were transfected into human hepatoma cell line HepG2 by lipofectamine2000, and the positive cell clones were detected using fluorescence microscopy.</p><p><b>RESULTS</b>All target fragments were separately obtained and successfully cloned into the expression vector. The expressions of EGFP under the control of the four promoters were detected. The expressions of EGFP controlled by different promoters had some differences.</p><p><b>CONCLUSIONS</b>Reporter gene EGFP under the control of four HBV promoters can be specifically expressed in hepatoma cell line HepG2, and different promoters give different results; this may provide another option in gene therapy of liver diseases.</p>

Relationship between death receptor 5 and apoptosis in hepatocellular carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the antitumor efficacy of death receptor 5, its ligand (TRAIL) and DR5mAb in human hepatocellular carcinoma.</p><p><b>METHODS</b>Expression of DR5 in the HCC cell lines HepG2, SMMC 7721 and normal human liver cell line LO2 was measured at mRNA and protein level by semi-quantitative RT-PCR and Western blot, respectively. MTT method was used to measure the cell viability and flow cytometry assay was used to detect apoptosis so as to observe the inhibitory effect of TRAIL and DR5mAb on HCC cells.</p><p><b>RESULTS</b>Death receptor 5 was highly expressed in the HCC cell lines, but rarely expressed in normal human liver cell line (P < 0.01). With the increase of TRAIL concentration, the cell viability of HCC cells decreased gradually. However, when the concentration of TRAIL was above 1000 ng/ml, HCC cells were resistant to TRAIL, but still sensitive to DR5mAb. After incubation with DR5mAb (1000 ng/ml) for 24 h, the rate of apoptosis in HCC cells reached to 52.45% +/- 0.57%, which was higher than that incubated with TRAIL under the same condition (14.74% +/- 0.48%) (P < 0.05). The cell viability of normal human liver cell line treated with TRAIL tended to decline with the increase of the concentration, which was significantly different from that of matched control group. But DR5mAb had little effect on normal human liver cell line.</p><p><b>CONCLUSION</b>Death receptor 5 as a target plays an important role in the course of HCC apoptosis induction. Agonistic monoclonal antibody specific for human DR5 can selectively and effectively kill hepatocellular carcinoma cells in vitro, while is not harmful to normal human hepatocytes. It reveals that DR5mAb might provide a new direction in hepatocellular carcinoma treatment research.</p>

A study of the association of iNOS and eNOS gene polymorphism with portal hypertension in liver cirrhosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study whether polymorphism of iNOS and eNOS genes is associated with portal hypertension in liver cirrhosis.</p><p><b>METHODS</b>A case control study of 106 patients with liver cirrhosis due to HBV was performed in comparison with 108 controls using PCR-RFLP to detect iNOS promoter -969G --> C and eNOS exon7 894G --> T polymorphism.</p><p><b>RESULTS</b>The frequency of the C allele and GC genotype in the iNOS promoter -969G --> C was significantly higher in the portal hypertension group than in the control group. (P < 0.05). The frequency of the T allele and GT genotype in eNOS exon7 894G --> T was dramatically higher in the portal hypertension group than in the control group (P < 0.05). Multivariate logistic regression analysis revealed that iNOS polymorphism in iNOS promoter -969G --> C and eNOS exon7 894G --> T was an independent new risk factor for portal hypertension. We discovered that iNOS promoter -969G --> C led to an increase in its functional activity.</p><p><b>CONCLUSIONS</b>The polymorphism of iNOS promoter -969G --> C and eNOS exon7 894G --> T is associated with portal hypertension of liver cirrhosis, which is a new independent risk factor found related to the occurrence of portal hypertension. The polymorphism of iNOS promoter -969G --> C results in functional activity increase of the iNOS promoter.</p>

Inhibition of mutant-type p53 by a chimeric U6 maxizyme in hepatocellular carcinoma cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the inhibition of maxizyme (Mz) directed against the mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG --> AGT) both in cell-free system and in MHCC97 cell lines.</p><p><b>METHODS</b>Maxizyme and control mutant maxizyme (G5 --> A5) were designed by computer and cloned into the eukaryotic expression vector pBSKneoU6 (pU6Mz, pU6asMz). Mz was driven by T7 RNA polymerase promoter in vitro. In the cell lines, U6 promoter was driven by RNA PolIII. The mutant type p53 gene fragment was cloned into the pGEM-T vector under the T7 promoter control. The 32P-labeled mtp53 transcript was the target RNA. Cold maxizyme transcripts were incubated with 32P-labeled target RNA in vitro. pU6Mz was introduced into MHCC97 cells by Lipofectamine2000 and mtp53 expression was analyzed by RT-PCR and Western blot.</p><p><b>RESULTS</b>In vitro cleavage showed that pU6Mz was very active with cleavage efficiency of 42% while pU6asMz was not. The wild type p53 was not cleaved. Partial down-regulation of mtp53 mRNA and mtp53 protein were observed in MHCC97 cells transfected with pU6Mz but not those with pU6asMz. The proliferation of MHCC cells was inhibited by MTT analysis.</p><p><b>CONCLUSION</b>Our findings suggest that the chimeric U6 maxizyme against the mtp53 is a new promising gene therapeutic agent in treating hepatocellular carcinoma.</p>

The relationship between the plasma homocysteine level and the polymorphism of MTHFR gene C667T in liver cirrhosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the relationship between the plasma homocysteine (HCY) level and the polymorphism of N(5), N(10)-methylenetetrahydrofolate reductase (MTHFR) gene C667T in liver cirrhosis.</p><p><b>METHODS</b>112 normal subjects and 87 liver cirrhosis patients were recruited in the study. Their plasma HCY levels were measured using high performance liquid chromatography with fluorescence detection and polymorphisms of their MTHFR gene were analyzed using PCR-RFLP.</p><p><b>RESULTS</b>The mean level of plasma HCY was significantly higher in patients with liver cirrhosis (21.71+/-4.86) micromol/L than that in healthy individuals (8.34+/-3.59) micromol/L. There were three kinds of MTHFR genotypes: +/+ (TT, homozygous mutation), +/- (CT, heterozygous mutation) and -/- (CC, wild type). The frequencies of the three genotypes were as follows: +/+, 29.9%; +/-, 52.9%; -/-, 17.2% in cirrhosis patients and +/+, 19.6%; +/-, 33.9%; -/-, 46.4% in normal subjects. The frequency of homozygous or heterozygous mutation was significantly higher in cirrhosis patients than that in the normal control. Moreover, plasma homocysteine level was markedly higher in patients with MTHFR genetic mutation than those without mutation.</p><p><b>CONCLUSIONS</b>Hyperhomocysteinemia may be an independent risk factor for liver cirrhosis. MTHFR is the main enzyme related to homocysteine metabolism. The genetic mutation of MTHFR C667T is possibly an important mechanism of hyperhomocysteinemia in liver cirrhosis. The level of plasma homocysteine may be an early indicator for liver cirrhosis.</p>

Synthesis of a novel L-nucleoside, beta-L-D4A and its inhibition on the replication of hepatitis B virus in vitro / 药学学报

<p><b>AIM</b>Nucleoside analogues have become the most promising candidates of anti-HBV drugs. In this study, beta-L-D4A was synthesized and explored its inhibitiory action against hepatitis B virus (HBV) in 2. 2. 15 cells derived from HepG2 cells transfected with HBV genome.</p><p><b>METHODS</b>beta-L-D4A was stereo-controlled synthesized from D-glutamic acid, and the structure was identified by IR, 1H NMR and MS. 2. 2. 15 Cells were placed at a density of 5 x 10(4) per well in 12-well tissue culture plates, and treated with various concentrations of beta-L-D4A for 6 days. At the end, medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with Hind III. Both of the above DNA were subjected to Southern blot, hybridized with a 32P-labeled HBV probe and autoradiographed. The intensity of the autoradiographic bands was quantitated by densitometric scans of computer and EC50 was calculated. 2. 2. 15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. IC50 was calculated.</p><p><b>RESULTS</b>The synthesized compound structure conformed with beta-L-D4A; Autoradiographic bands showed similar for supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. EC50 0.2 micromol x L(-1). The experiment of cytotoxicity gained IC50 200 micromol x L(-10.</p><p><b>CONCLUSION</b>beta-L-D4A has been synthesized successfully. beta-L-D4A possessed potent inhibitory effect on replication of HBV in vitro with low cytotoxicity, TI value was 1 000. It is expected to be developed clinically into a new anti-HBV drug.</p>

Clinical study of lamivudine and interferon combinate administration to inhibit hepatitis B virus replication / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore a new strategy for effective and economical anti-virus therapy for HBV infection, we conducted a sequence administration of lamivudine and interferon alpha 1b to evaluate its effects on HBV replication and rebound as well as YMDD mutation induced by lamivudine.</p><p><b>METHODS</b>150 HBV patients having at least 6 months history of infection were assigned randomly into 5 groups. Each group of these patients was either treated with lamivudine, interferon alpha 1b, lamivudine combined with interferon, sequence administration of lamivudine and interferon (sequence group) or no anti-virus therapy (control group) for 12 months. The serum samples were collected at 0, 3, 6, 9, 12 and 18th months and were assayed for ALT, AST, HBeAg, HBV DNA (quantitive PCR) as well as YMDD mutation types by microarray.</p><p><b>RESULTS</b>The anti-virus replication effects were shown as early as the 3rd month in the sequence group but not in the IFN and control groups. The significant and persistent inhibition effect of it on HBV replication and improvement of liver function was shown. It was more effective than lamivudine or IFN treatments at the end of the drug administration and 6 months later after the drug was withdrawn. We also found that this sequence administration pattern can significantly shorten the period of treatment of lamivudine as well as reduce the rate of YMDD mutation and rebound of HBV replication after lamivudine withdrawal. It is also more economical than a combined therapy of lamivudine with IFN.</p><p><b>CONCLUSION</b>This sequence administration of lamivudine and IFN pattern can significantly improve the anti-virus effect on HBV replication, shorten the period of treatment with lamivudine, reduce the mutation rate of YMDD and prevent the rebound of HBV after drug withdrawal.</p>

Hammerhead ribozyme against human telomerase catalytic subunit (hTERT) induced apoptosis of liver cancer cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct vector pEGFP-C1-hTERT-ribozyme (pGTRz-U6) and its mutant (pGTmRz-U6) against hTERT containing U6 promoter, then transfect them into human liver cancer cell line SMMC7721 to observe the action of the human telomerase catalytic subunit (hTERT) hammerhead ribozyme on proliferation and apoptosis of human liver cancer cell SMMC7721.</p><p><b>METHODS</b>Eukaryotic expressing vector pGTRz-U6 and mutant pGTmRz-U6 were constructed and transfected into SMMC7721 using Lipofectamine2000 Reagent, with pEGFP-C1 as the control group. After strict screening by G418, positive clones were cultured; the amount of expression of ribozyme and hTERT was detected by RT-PCR; cell proliferation by MTT; telomerase activity by TRAP and silver staining assay; cell apoptosis by FCM.</p><p><b>RESULTS</b>We found that the two ribozymes were expressed persistently in SMMC7721; different expression levels (P < 0.01) of hTERT among SMMC7721-Rz, SMMC7721-mRz and SMMC7721-pEGFP-C1 was exhibited by the analysis of variance with SPSS software. The difference between SMMC7721-Rz and the others is significant in t-test (P < 0.01), while there was no difference between SMMC7721-mRz and SMMC7721-pEGFP-C1 (P > 0.05). With the advance of cell division, telomerase activities of the cells treated by SMMC7721-Rz and SMMC7721-mRz decreased gradually, and the percentage of apoptosis of the cells transfected with Rz and mRz increased gradually. The apoptosis percentage of 7PDS SMMC7721-Rz was 29.86%, while those of SMMC7721-mRz and SMMC7721-pEGFP-C1 were 9.87% and 3.36%, respectively.</p><p><b>CONCLUSION</b>The apoptosis level of SMMC7721 induced by hTERT ribozyme increases as cells divide, and this ribozyme maybe a potential approach for liver cancer gene therapy.</p>