MicroRNA-34c targets TGFB-induced factor homeobox 2, represses cell proliferation and induces apoptosis in hepatitis B virus-related hepatocellular carcinoma.

MicroRNAs (miRs) are short, non-coding RNAs with post-transcriptional regulatory functions. Previous studies have demonstrated that miR-34c is involved in diverse biological processes, including carcinogenesis. The aim of the present study was to investigate the role of miR-34c and its target genes in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Expression levels of miR-34c and its predicted target genes were measured. The target genes were validated by a luciferase assay. The effects of miR-34c restoration were evaluated by the detection of HBV antigens, cell proliferation and apoptosis in vitro, in addition to the tumor growth in vivo. The data demonstrated that miR-34c was downregulated in HBV-associated HCC clinical tissues and HCC cell lines compared with their corresponding controls. transforming growth factor-ß-induced factor homeobox 2 (TGIF2), a transcription factor repressing transforming growth factor-ß (TGFß) signaling, was observed to be upregulated and was identified as a target gene of miR-34c. The restoration of miR-34c in HepG2.2.15 cells suppressed TGIF2 expression, HBV replication and viral antigen synthesis; inhibited cell proliferation; and induced apoptosis. miR-34c also inhibited tumor growth in a mouse model. The present study indicates that miR-34c may act as a tumor suppressor by targeting TGIF2 during HBV-associated hepatocellular carcinogenesis. miR-34c and TGIF2 may represent key regulatory factors, diagnostic markers and therapeutic targets for the prevention and treatment of HBV-associated HCC.

miR-122 inhibits viral replication and cell proliferation in hepatitis B virus-related hepatocellular carcinoma and targets NDRG3.

microRNAs (miRNAs) are short, non-coding RNAs with post-transcriptional regulatory functions that participate in diverse biological pathways. miR-122, a liver-specific miRNA, has been found to be down-regulated in hepatocellular carcinoma (HCC) and HCC-derived cell lines. In this study, miR-122 was down-regulated in the hepatitis B virus (HBV)-related HCC cell line HepG2.2.15 compared to HepG2. NDRG3, a member of the N-myc downstream-regulated gene (NDRG) family, was up-regulated in HepG2.2.15 and was identified as a target gene of miR-122. An inverse correlation between the expression of miR-122 and the NDRG3 protein was noted in HBV-related HCC specimens. The transfection of the miR-122 expression vector into the HepG2.2.15 cell line repressed the transcription and expression of NDRG3, which subsequently reversed the malignant phenotype of the cells. The replication of HBV, expression of viral antigens and proliferation of cells were significantly inhibited by restoration of miR-122. The data demonstrate that miR-122 plays an important role in HBV-related hepatocarcinogenesis by targeting NDRG3. Thus, miR-122 and NDRG3 represent key diagnostic markers and potential therapeutic targets for HBV-related HCC.

miR-29c targets TNFAIP3, inhibits cell proliferation and induces apoptosis in hepatitis B virus-related hepatocellular carcinoma.

Recent studies have revealed that microRNA-29c (miR-29c) is involved in a variety of biological processes including carcinogenesis. Here, we report that miR-29c was significantly downregulated in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) cell lines as well as in clinical tissues compared with their corresponding controls. Tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a key regulator in inflammation and immunity, was found to be inversely correlated with miR-29c levels and was identified as a target of miR-29c. Overexpression of miR-29c in HepG2.2.15 cells effectively suppressed TNFAIP3 expression and HBV DNA replication as well as inhibited cell proliferation and induced apoptosis. We conclude that miR-29c may play an important role as a tumor suppressive microRNA in the development and progression of HBV-related HCC by targeting TNFAIP3. Thus miR-29c and TNFAIP3 represent key diagnostic markers and potential therapeutic targets for the prevention and treatment of HBV infection.

Success and complication rates of two precut techniques, transpancreatic sphincterotomy and needle-knife sphincterotomy for bile duct cannulation.

BACKGROUND: The majority of literature on the precut technique is concerned with needle-knife sphincterotomy, whereas the comparison of transpancreatic sphincterotomy and needle-knife sphincterotomy has been rarely reported. Aim The aim of the study was to compare the success and the complication rates of transpancreatic sphincterotomy with needle-knife sphincterotomy. METHODS: During May 2006 and April 2007, 3,178 consecutive endoscopic retrograde cholangiopancreatography (ERCP) procedures were performed in a prospective multicenter study on ERCP-related complications. From the files of these patients, data of cases undergoing precut sphincterotomy, including transpancreatic sphincterotomy and needle-knife sphincterotomy, were retrospectively extracted and analyzed. RESULTS: Overall, 216 patients with precut sphincterotomy were identified; 140 cases received transpancreatic sphincterotomy, and 76 received needle-knife sphincterotomy. There was no significant difference in the initial and eventual success rates between transpancreatic and needle-knife sphincterotomy (82.9% vs. 90.8% and 90.0% vs. 90.8%, respectively). The overall incidences of complications and acute pancreatitis were not significantly different between the two groups (14.3% vs. 18.4% and 11.4% vs. 11.8%, respectively).

PDCD4 inhibits the malignant phenotype of ovarian cancer cells.

Programmed cell death 4 (PDCD4) is a newly identified tumor suppressor that can inhibit activator protein (AP)-1 activation and protein translation. Our previous studies indicate that lost or reduced PDCD4 expression is associated with the progression of ovarian carcinoma. However, direct evidence that PDCD4 inhibits malignant phenotype of human cancer cells is limited. In the present study, we found that PDCD4 expression in ovarian cancer cell lines (SKOV3, 3AO, and CAOV3) inhibited significantly their proliferation and cell cycle progression, and induced apoptosis. More importantly, up-regulation of PDCD4 expression decreased the colony-forming capacity of ovarian cancer cells in vitro and tumorigenic capacity in mice. These results demonstrate that PDCD4 can suppress the malignant phenotype of ovarian cancer cells, and may represent a novel therapeutic target for the treatment of ovarian cancer.

Blockade of Tim-3 pathway ameliorates interferon-gamma production from hepatic CD8+ T cells in a mouse model of hepatitis B virus infection.

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been reported to participate in the pathogenesis of inflammatory diseases. However, whether Tim-3 is involved in hepatitis B virus (HBV) infection remains unknown. Here, we studied the expression and function of Tim-3 in a hydrodynamics-based mouse model of HBV infection. A significant increase of Tim-3 expression on hepatic T lymphocytes, especially on CD8+ T cells, was demonstrated in HBV model mice from day 7 to day 18. After Tim-3 knockdown by specific shRNAs, significantly increased IFN-gamma production from hepatic CD8+ T cells in HBV model mice was observed. Very interestingly, we found Tim-3 expression on CD8+ T cells was higher in HBV model mice with higher serum anti-HBs production. Moreover, Tim-3 knockdown influenced anti-HBs production in vivo. Collectively, our data suggested that Tim-3 might act as a potent regulator of antiviral T-cell responses in HBV infection.

Combined endostatin and TRAIL gene transfer suppresses human hepatocellular carcinoma growth and angiogenesis in nude mice.

Endostatin can inhibit tumor growth by blocking angiogenesis, whereas tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) may function as a soluble cytokine to selectively kill cancer cells without toxicity to most normal cells. To establish the combined anti-tumor therapeutic effect of endostatin and soluble TRAIL (sTRAIL), we performed intra-tumoral human endostatin and sTRAIL gene transfer using plasmid pVAX1 as a vector in a nude mouse model of human liver cancer. For subcutaneously inoculated human BEL7402 cancer, co-expression of both transgenes conferred marked anti-tumor activity with a significant reduction in tumor vessel density and an increase in apoptotic rates, which was accompanied with a strong activation of caspase-3. Importantly, combination therapy employing one-half dose of endostatin and sTRAIL plasmids was more effective than single endostatin or sTRAIL therapy. These results indicate that a pVAX1-mediated combinatorial antiangiogenic and proapoptotic gene therapy approach involving endostatin and sTRAIL can be an effective novel form of treatment for human liver cancer.

Prenatal malnutrition and adult schizophrenia: further evidence from the 1959-1961 Chinese famine.

OBJECTIVE: Evidence from the 1944-1995 Dutch Hunger Winter and the 1959-1961 Chinese famines suggests that those conceived or in early gestation during famines, have a 2-fold increased risk of developing schizophrenia in adult life. We tested the hypothesis in a second Chinese population and also determined whether risk differed between urban and rural areas. METHOD: The risk of schizophrenia was examined in Liuzhou prefecture of Guangxi autonomous region. Rates were compared among those conceived before, during, and after the famine years. Based on the decline in birth rates, we predicted that those born in 1960 and 1961 would have been exposed to the famine during conception or early gestation. All psychiatric case records in Liuzhou psychiatric hospital for the years 1971 through 2001 were examined and clinical/sociodemographic data extracted by psychiatrists blind to exposure status. Data on births and deaths in the famine years were also available, and cumulative mortality was estimated from later demographic surveys. Evidence of famine was verified, and results were adjusted for mortality. Relative risks (RRs) for schizophrenia were calculated for the region as a whole and for urban and rural areas separately. RESULTS: Mortality-adjusted RR for schizophrenia was 1.5 (1960) and 2.05 (1961), respectively. However, the effect was exclusively from the rural areas RR = 1.68 (1960) and RR = 2.25 (1961). CONCLUSIONS: We observe a 2-fold increased risk of schizophrenia among those conceived or in early gestation at the height of famine with risk related to severity of famine conditions.

Risk factors for ERCP-related complications: a prospective multicenter study.

OBJECTIVES: To investigate the potential risk factors for endoscopic retrograde cholangiopancreatography (ERCP) complications and to identify whether the risk factors are different for pancreatitis and asymptomatic hyperamylasemia. METHODS: Consecutive ERCP procedures were studied at 14 centers in China from May 2006 to April 2007. The complications after the patients' first-only procedures were evaluated. Multivariate analysis based on the first-only procedures was used to identify the risk factors. RESULTS: A total of 3,178 procedures were performed on 2,691 patients. Overall, complications developed in 213 (7.92%) patients, pancreatitis in 116 (4.31%), and asymptomatic hyperamylasemia in 396 (14.72%). In the multivariate analysis, female gender (adjusted odds ratios (ORs): 1.52, 95% confidence interval (CI): 1.14-2.02, P=0.004), periampullary diverticulum (OR: 2.02, 95% CI: 1.49-2.73, P<0.001), cannulation time >10 min (OR: 1.51, 95% CI: 1.08-2.10, P=0.016), > or =1 pancreatic deep wire pass (OR: 1.80, 95% CI: 1.33-2.42, P<0.001), and needle-knife precut (OR: 2.70, 95% CI: 1.42-5.14, P=0.002) were risk factors for overall complications. Female gender (OR: 1.84, 95% CI: 1.25-2.70, P=0.002), age < or =60 year (OR: 1.59, 95% CI: 1.06-2.39, P=0.025), cannulation time>10 min (OR: 1.76, 95% CI: 1.13-2.74, P=0.012), > or =1 pancreatic deep wire pass (OR: 2.77, 95% CI: 1.79-4.30, P<0.001), and needle-knife precut (OR: 4.34, 95% CI: 1.92-9.79, P<0.001) were risk factors for pancreatitis. Cannulation time>10 min (OR: 1.96, 95% CI: 1.52-2.54, P<0.001), > or =1 pancreatic deep wire pass (OR: 2.24, 95% CI: 1.74-2.89, P<0.001), needle-knife precut (OR: 2.34, 95% CI: 1.32-4.14, P=0.004), and major papilla pancreatic sphincterotomy (OR: 1.71, 95% CI: 1.23-2.37, P=0.001) were risk factors for asymptomatic hyperamylasemia. CONCLUSIONS: Patient-related factors are as important as procedure-related factors in determining high-risk predictors for post-ERCP overall complications and pancreatitis. However, the risk factors for asymptomatic hyperamylasemia may be mostly procedure related.

pVAX1 plasmid vector-mediated gene transfer of soluble TRAIL suppresses human hepatocellular carcinoma growth in nude mice.

The extracellular domain of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) may function as a soluble cytokine to selectively kill various cancer cells without toxicity to most normal cells. We used a high-biosafety plasmid pVAX1 as a vector and constructed a recombinant plasmid expressing the extracellular domain (95-281 aa) of human TRAIL fused with signal peptides of human IgGgamma, designated as pVAX-sT. Transduction of human BEL7402 liver cancer cells with pVAX-sT led to high levels of sTRAIL protein in the cell culture media and induced apoptosis. The therapeutic potential of pVAX-sT was then evaluated in the BEL7402 transplanted naked mouse model. Subsequent intratumoral administration of naked pVAX-sT resulted in the expression of soluble TRAIL in the sera and the tumor site, as well as effective suppression of tumor growth, with no toxicity to liver. In conclusion, the successful inhibition of liver cancer growth and the absence of detectable toxicity suggest that pVAX-sT could be useful in the gene therapy of liver cancer.

Effect of octreotide administration in the prophylaxis of post-ERCP pancreatitis and hyperamylasemia: A multicenter, placebo-controlled, randomized clinical trial.

OBJECTIVES: Since the introduction of endoscopic retrograde cholangiopancreatology (ERCP) in clinical use, pancreatitis has become a common complication of ERCP. Octreotide is an inhibitor of pancreatic enzyme secretions. Several studies have evaluated the effect of octreotide on the incidence of clinical pancreatitis after ERCP, but with different results. The aim was to determine the efficacy of prophylactic administration of octreotide for the prevention of post-ERCP pancreatitis (PEP) and hyperamylasemia. METHODS: In this study, patients with scheduled ERCP were randomized to receive either octreotide (0.3 mg) via intramuscular injection or a placebo. The study was conducted in 12 digestive endoscopic units in China. Patients were randomized into two groups: an octreotide group (N = 414) and a control group (N = 418). In the octreotide group, octreotide (0.3 mg) was dissolved in 500 mL of 0.9% saline solution and administered by continuous intravenous infusion, beginning 1 h before endoscopic examination and continued for 6 h thereafter; 0.1 mg of octreotide was injected subcutaneously at 6 and 12 h after the intravenous injection was stopped. The control group was given a placebo intravenously. The end point was the development of acute pancreatitis. RESULTS: The overall incidence of acute pancreatitis was 3.85%; this included 2.42% (10/414) in the octreotide group and 5.26% (22/418) in the control group (P = 0.046). The overall incidence of hyperamylasemia was 14.9%; 12.32% (51/414) in the octreotide group and 17.46% (73/418) in the control group (P = 0.041). No side effects were found. CONCLUSION: The results indicate that octreotide can prevent PEP and hyperamylasemia.

Blockade of TRAIL pathway ameliorates HBV-induced hepatocyte apoptosis in an acute hepatitis model.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) may play important roles during hepatitis B virus (HBV) infection. In this study, we used a recombinant human soluble death receptor 5 (sDR5) to explore its effect in a mouse model of HBV-induced acute hepatitis. By measuring blood transaminase activity and hepatocyte apoptosis, we found that sDR5 could alleviate liver damage by blocking TRAIL-induced apoptosis of HBV-transfected hepatocytes. sDR5 injection at 16 mg/kg 24h before HBV transfection was the most effective. Additionally, we showed that sDR5 was equally effective in protecting liver injury as the Stronger Neo-Minophagen C (SNMC), a commonly used drug for patients with liver diseases. Thus, sDR5 represents a potential novel therapeutic drug for patients with fulminant hepatitis.

[Inhibitory effect of endostatin mediated by lipofectin on transplanted ovarian cancer].

OBJECTIVE: To study the inhibitory effect of endostatin mediated by lipofectin on transplanted ovarian cancer in nude mice. METHODS: Constructed recombinant vector pVAX1-sEn expressing human endostatin protein was transfected into ovarian cancer cell line 3AO by lipofectin. mRNA of endostatin was detected by RT-PCR. The expression of endostatin in supernatants was detected by enzyme-linked immunosorbent assay (ELISA). The inhibitory effect of pVAX1-sEn on endothelial cell line ECV-204 was detected by methyl thiazolyl tetrazolium (MTT). By use of lipofectin mediated pVAX1-sEn for intratumor injection, the inhibitory effect on growth of ovarian cancer was observed. RESULTS: The result of RT-PCR showed there was a specific band at 610 bp. The expression quantity of endostatin in transfected cell supernatant was (201 +/- 8) ng/ml by ELISA. MTT showed pVAX1-sEn transfected cell supernatant could effectively inhibit the growth of ECV-204, the highest inhibitory ratio was 42%. The tumor volumes in pVAX1-sEn treatment group was (0.85 +/- 0.18) cm(3), significantly smaller than that in normal saline control group (1.90 +/- 0.28) cm(3) and pVAX1 control group (1.78 +/- 0.32) cm(3) (P < 0.05). HE stain in tumor tissue showed that there were obvious necrosis cells in the pVAX1-sEn treatment group, but there were flourishly growing tumor cells in pVAX1 and normal saline control groups. CONCLUSION: pVAX1-sEn mediated by lipofectin can effectively inhibit the growth of ovarian cancer.

[Application of long distance-PCR to hemophilia A carrier detection and prenatal diagnosis].

OBJECTIVE: To establish an effective laboratory examination system for carrier detection and prenatal diagnosis of haemophilia A (HA). METHODS: Twenty-five carriers of severe HA were directly detected by long-distance PCR (LD-PCR) in search of the factor FVIII (FVIII) gene inversion. Prenatal diagnosis was carried out using pregnant woman's venous blood sample, husband's venous blood sample and fetal navel venous sample at 20-24 weeks of gestation. The plasma coagulation factor VIII activity (FVIII:C) was detected by one-stage method. The concentration of von Willbrand factor (Vwf) was assayed by ELISA. Prenatal diagnosis was finally made by LD-PCR. The results of LD-PCR were proved by DNA sequencing. RESULTS: Eight out of 25 cases were diagnosed as having FVIII geneinversion. Four of these 8 carriers underwent the LD-PCR for prenatal diagnosis, and 2 of them had to terminate pregnancy because their fetuses were diagnosed as having HA. The other two carriers were finally diagnosed to have normal fetuses by combined use of LD-PCR with plasma FVIII:C, vWF in pregnant woman's venous blood, husband's venous blood and fetal navel venous blood, and the one-year follow-up study demonstrated that the babies were normal and living well. CONCLUSION: LD-PCR technique was adopted in this study to detect the factor VIII gene inversion; it could accurately and rapidly diagnose the severe cases of HA and could be used for the HA carriers in need of pregnant diagnosis.

Human endostatin gene transfer, either naked or with liposome, has the same inhibitory effect on growth of mouse liver tumor cells in vivo.

AIM: To explore a safe and efficient strategy of tumor therapy using anti-angiogenetic agents. METHODS: Endostatin gene with a signal sequence of human IgGgamma chain was amplified by PCR and cloned into pVAX1 plasmid which was the only vector authorized by FDA in clinical trial to construct a recombinant plasmid named as pVAX-sEN. The recombinant plasmid was detected with EcoRI/KpnI and DNA sequencing. BALB/c mice bearing hepatocarcinoma cell line H22 were treated with naked pVAX-sEN or liposome-DNA complex in which the dose of DNA and the ratio of DNA and liposome were different from each other. To compare the efficiency of gene transfection, expression of endostatin at the treated tumor site was assayed with ELISA. To investigate the effect of pVAX1-sEN on hepatocellular carcinoma, pVAX-sEN either naked or in liposome-DNA complex was injected into BALB/c mice bearing H22, then the diameter of tumors was measured, microvessel density was detected by immunohistochemistry, endostatin expression in vivo was assayed at different time points. RESULTS: DNA sequencing showed the endostatin gene with the signal peptide was correctly cloned. In situ gene expression assay indicated that both the ratio of DNA and liposome and the dose of DNA could affect the gene transfection efficiency. Interestingly, naked pVAX-sEN had a similar in situ endostatin expression to pVAX-sEN with liposome. Animal experiments showed that pVAX-sEN together with pVAX-sEN-liposome complex could efficiently suppress the growth of mouse hepatoma cells. CONCLUSION: Naked endostatin plasmid intratumoral injection can get a similar gene transfection efficiency to liposome-DNA complex when used in situ.

Establishment of mice model with human viral hepatitis B.

AIM: To establish a mice model of hepatitis B by using HBV-transgenic mice, and to transfer HBV-specific cytotoxic T lymphocytes (CTL) induced from syngeneic BALB/c mice immunized by a eukaryotic expression vector containing HBV complete genome DNA. METHODS: HBV DNA was obtained from digested pBR322-2HBV and ligated with the vector pcDNA3. Recombinant pcDNA3-HBV was identified by restriction endonuclease assay and transfected into human hepatoma cell line HepG2 with lipofectin. ELISA was used to detect the expression of HBsAg in culture supernatant, and RT-PCR to determine the existence of HBV PreS1 mRNA. BALB/c mice were immunized with pcDNA3-HBV or pcDNA3 by intramuscular injection. ELISA was used to detect the expression of HBsAb in serum. MTT assay was used to measure non-specific or specific proliferation ability and specific killing activity of spleen lymphocytes. Lymphocytes from immunized mice were transferred into HBV-transgenic mice (2.5X10(7) per mouse). Forty-eight hours later, the level of serum protein and transaminase was detected with biochemical method, liver and kidney were sectioned and stained by HE to observe the pathological changes. RESULTS: By enzyme digestion with Eco RI, Xho I and Hind III, the recombinant pcDNA3-HBV was verified to contain a single copy of HBV genome, which was inserted in the positive direction. HepG2 cells transfected with the recombinant could stably express PreS1 mRNA and HBsAg. After immunized by pcDNA3-HBV for 4 weeks, HBsAb was detected in the serum of BALB/c mice. The potential of spleen lymphocytes for both non-specific and specific proliferation and the specific killing activity against target cells were enhanced. The transgenic mice in model group had no significant changes in the level of serum protein but had an obvious increase of ALT and AST. The liver had obvious pathological changes, while the kidney had no evident damage. CONCLUSION: A eukaryotic expression vector pcDNA3-HBV containing HBV complete genome is constructed successfully. HepG2 cells transfected with the recombinant can express PreS1 mRNA and HBsAg stably. Specific cellular immune response can be induced in mice immunized by pcDNA3-HBV. A mice model of acute hepatitis with HBV has been established.

Antisense oligonucleotide targeting at the initiator of hTERT arrests growth of hepatoma cells.

AIM: To evaluate the inhibitory effect of antisense phosphorothioate oligonucleotide (asON) complementary to the initiator of human telomerase catalytic subunit (hTERT) on the growth of hepatoma cells. METHODS: The as-hTERT was synthesized by using a DNA synthesizer. HepG2.2.15 cells were treated with as-hTERT at the concentration of 10 micromol/L. After 72 h, these cells were obtained for detecting growth inhibition, telomerase activity using the methods of MTT, TRAP-PCR-ELISA, respectively. BALB/c(nu/nu) mice were injected HepG2.2.15 cells and a human-nude mice model was obtained. There were three groups for anti-tumor activity study. Once tumors were established, these animals in the first group were administered as-hTERT and saline. Apoptosis of tumor cells was detected by FCM. In the 2nd group, the animals were injected HepG2.2.15 cells together with as-hTERT. In the third group, the animals were given as-hTERT 24 hours postinjection of HepG2.2.15 cells. The anti-HBV effects were assayed with ELISA in vitro and in vivo. RESULTS: Growth inhibition was observed in cells treated with as-hTERT in vitro. A significant different in the value of A570-A630 was found between cells treated with as-hTERT and control (P<0.01) by MTT method. The telomerase activity of tumor cells treated with as-hTERT was reduced, the value of A450 nm was 0.42 compared to control (1.49) with TRAP-PCR-ELISA. The peak of apoptosis in tumor cells given as-hTERT was 21.12%, but not seen in saline-treated control. A prolonged period of carcinogenesis was observed in the second and third group animals. There was inhibitory effect on the expression of HBsAg and HBeAg in vivo and in vitro. CONCLUSION: As-hTERT has an anti-tumor activity, which may be useful for gene therapy of tumors.

[Inhibition of HBV DNA replication and expression in 2.2.15 hepatoma cells infected with AFP-mediated HBX antisense RNA].

OBJECTIVE: To study the specific expression of the antisense RNA against hepatitis B virus X (HBX) gene in hepatoblastoma cell line and its anti -HBV activity. METHODS: HBX gene (nt.1370-1827) was amplified by PCR, then cloned into EB virus vector pEBAF which contained human alpha-fetoprotein promoter and enhancer. After transfected into 2.2.15 hepatoma cells and ECV304 human endothelial cells by lipofectin, northern blot, ELISA and real-time qualitative PCR were carried out to assay the expression of HBX mRNA, HBV antigens and HBV DNA level, respectively. RESULTS: The HBX antisense RNA expression vector pEBAF-as-HBX which could be expressed specifically in 2.2.15 hepatoblastoma cells was successfully constructed. Both HBV DNA level and the expressions of hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) in 2.2.15 hepatoblastoma cells were inhibited by pEBAF-as-HBX. Compared with those in sense control (pEBAF-s-HBX), the inhibitory rates of HBsAg, HBeAg, and HBV DNA were 37.9%, 36.8%, and 25%, respectively. CONCLUSIONS: The pEBAF-as-HBX expression vector may lead to targeted-expression of HBX antisense RNA in hepatoma cells and shows great inhibition effect on HBV.

A novel HBV antisense RNA gene delivery system targeting hepatocellular carcinoma.

AIM: To construct a novel HBV antisense RNA delivery system targeting hapatocellular carcinoma and study its inhibitory effect in vitro and in vivo. METHODS: GE7,a 16-peptide specific to EGFR, and HA20,a homologue of N-terminus of haemagglutinin of influenza viral envelope protein, were synthesized and conjugated with polylysin. The above conjugates were organized into the pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system, named AFP-enhancing 4-element complex. Hepatocelluar carcinoma HepG2.2.15 cells was used to assay the in vitro inhibition of the complex on HBV. Expression of HBV antigen was assayed by ELISA. BALB/c nude mice bearing HepG2.2.15 cells were injected with AFP-enhancing 4-element complex. The expression of HBV antisense RNA was examined by RT-PCR and the size of tumor in nude mice were measured. RESULTS: The AFP-enhancing 4-element complex was constructed and DNA was completely trapped at the slot with no DNA migration when the ratio of polypeptide to plasmid was 1:1. The expression of HBsAg and HBeAg of HepG2.2.15 cells was greatly decreased after being transfected by AFP-enhancing 4-element complex. The inhibitory rates were 33.4 % and 58.5 % respectively. RT-PCR showed HBV antisense RNA expressed specifically in liver tumor cells of tumor-bearing nude mice. After 4 injections of AFP-enhancing 4-element complex containing 0.2 micro g DNA, the diameter of the tumor was 0.995 cm+/-0.35, which was significantly smaller than that of the control groups(2.215 cm+/-0.25, P<0.05). CONCLUSION: AFP-enhancing 4-element complex could deliver HBV antisense RNA targeting on hepatocarcinoma and inhibit both HBV and liver tumor cells in vitro and in vivo.

Detection of soluble TRAIL in HBV infected patients and its clinical implications.

AIM: To detect the expression of soluble TRAIL (TNF-related apoptosis inducing ligand, TRAIL) in the peripheral blood of HBV infected patients and try to elucidate whether the expression level of sTRAIL have any correlativity with the clinical staging, the expression level of HBV markers and the degree of liver damage. METHODS: 52 cases of HBV infected patients were investigated, including 8 HBV carriers, 30 chronic hepatitis B, 11 cirrhotics and 3 HBV infection related hepatocellular carcinoma. Expression of soluble TRAIL and markers of the hepatitis B were measured by enzyme-linked immunosorbent assay. RESULTS: The expression level of sTRAIL in the peripheral blood of the HBV infected patients was significantly higher than that of healthy controls (1378.35+/-540.23 pg/ml vs 613.75+/-175.80 pg/ml, P<0.001). In the group of chronic hepatitis, the expression level of sTRAIL was coincident with the status of the disease and was significantly correlated with the level of ALT. In the group of cirrhosis and liver cancer, its expression level was significantly higher than that of the healthy persons and HBV carriers, but lower than that of the hepatitis B patients; meanwhile, the expression of sTRAIL did not have any correlativity with the functional indexes of the liver. CONCLUSION: The soluble TRAIL in the HBV infected people may participate in the liver damage. Our results indicated that the expression level of soluble TRAIL may reflect the ravage of liver caused by host immune reaction to a certain degree.