Detection of eukaryotic translation initiation factor 4E and its clinical significance in hepatocellular carcinoma.

AIM: To study the expression of eukaryotic translation initiation factor 4E (eIF4E), which is closely correlated with malignant tumors, and its relationship to prognosis in hepatocellular carcinoma. METHODS: Western blotting was performed to quantify the elF4E protein expression in the normal human liver cell line L02 and the hepatoma cell lines Hep3B, HepG2, and Huh7. Forty-six hepatocellular carcinoma samples with complete clinical data were obtained from Changzheng Hospital during the period of December 2008 to July 2009. The expression of eIF4E in the tumor samples and their adjacent tissues were detected by immunohistochemistry. The relationship between the test results and hepatocellular carcinoma (HCC) prognosis was statistically analysed by using a COX proportional hazard model. RESULTS: Western blotting analysis showed that there were distinct eIF4E protein bands in all three of the hepatoma cell lines. In particular, the HepG2 cell line had the highest level of eIF4E protein expression. The L02 cell group had a low eIF4E expression. Immunohistochemical assay showed that there were 32 cases in which the tumour tissue expression was higher than their adjacent tissues, accounting for 69.57%. There were also 14 cases in which the tumour tissue expression was lower or no significant difference was found, accounting for 30.43%. COX proportional hazards model analysis showed that HCC prognosis was related to the depth of invasion, the overexpression of eIF4E and p53, possibly as independent HCC prognostic predictors. CONCLUSION: In summary, eIF4E expression is associated with liver cancer, and patients with high eIF4E expression levels have a higher risk of recurrence.

Cellular prion protein promotes glucose uptake through the Fyn-HIF-2α-Glut1 pathway to support colorectal cancer cell survival.

Cellular prion protein (PrPc) is a glycosylphosphatidylinositol-anchored membrane protein that has various physical functions, including protection against apoptotic and oxidative stress, cellular uptake of copper ions, transmembrane signaling, and adhesion to the extracellular matrix. In this study, we show that PrPc is highly expressed in colorectal adenocarcinomas. Transcriptome profiling of PrPc-depleted DLD-1 cells revealed downregulation of glucose transporter 1 (Glut1). PrPc is shown to be involved in regulating Glut1 expression through the Fyn-HIF-2α pathway. As Glut1 is the natural transporter of glucose and is required for the high glycolytic rate seen in colorectal tumors, silencing of PrPc reduced the proliferation and survival rate of colorectal cancer cells in vitro. In vivo, knockdown of PrPc by hydrodynamic injection with a cocktail of PrPc-shRNA-encoding plasmids also inhibited tumorigenicity in a xenograft model in nude mice. In summary, our data characterize a novel molecular mechanism that links PrPc expression to the regulation of glycolysis. Targeting PrPc will therefore be a promising strategy to overcome the growth and survival advantage in colorectal tumors.

[Liposome-mediated glial growth factor 2 gene therapy in brain injury: an experimental study with rats].

OBJECTIVE: To explore the protective effect of glial growth factor-2 (GGF2) on brain injury. METHODS: Thirty-four SD rats underwent lateral fluid percussion to establish brain injury models and then were randomly divided into 4 groups: treatment group (n = 10, the plasmid pEGFP-N1-GGF2 mixed with liposome was injected into the brain tissue directly), vector control group (n = 10, the vector pEGFP-N1 mixed with liposome was injected into the brain tissue directly), liposome control group (n = 10, liposome was injected), and sham operation group (n = 4). Three assessment tasks were performed for neurobehavioral evaluation: Clivas Test, Beam Balance Test and Beam Walking Test. 10 days after brain injury, the rats were sacrificed and their brains were embedded in paraffin for HE staining, Nissle staining and immunohistochemical examination of MBP, NSE, and GFAP. RESULTS: The Clivas test score of the treatment group was 66.25 +/- 3.54, significantly higher than those of the vector control group and. liposome control group (58.31 +/- 3.72 and 57.21 +/- 3.93 respectively, both P < 0.05). The beam test score of the treatment group was 2.59 +/- 0.21, significantly lower than those the vector control group and liposome control group (3.41 +/- 0.25 and 3.24 +/- 0.22 respectively, both P < 0.05). The walking test score of the treatment group was 20.15 +/- 2.59, significantly lower than those of control group and liposome control group (27.00 +/- 3.47 and 27.80 +/- 3.00 respectively, both P < 0.05). The improvement in beam walking test was the greatest. The neuron number in the external granular layer and external pyramidal layer in cortex of the treatment group was 98 +/- 10, significantly more than those of the vector control group and liposome group (75 +/- 7 and 67 +/- 8, both P < 0.05). The neuron number in the internal pyramidal layer in cortex of the treatment group was 37 +/- 4, significantly more than those of the vector control group and liposome group (19 +/- 3 and 23 +/- 4 respectively, both P < 0.05). The neuron number in the CA1 region in hippocampus of the treatment group was 102 +/- 11, significantly more than those of the vector control group and liposome group (67 +/- 8 and 58 +/- 9 respectively, both P < 0.01). Higher level of immunoreactivity with MBP was also detected in the cortex in the rats of the treatment group. CONCLUSION: Cationic liposome-mediated GGF2 gene therapy effectively promotes the recovery of brain injury.

[Estrogenic activities of parabens].

OBJECTIVE: Studies indicated that parabens, used as anti-microbial agents in food, cosmetics, and pharmaceuticals, produced a positive uterotrophic response in vivo. They also damaged the late stages of spermatogenesis, altered proportion of pups born alive, and body weight of offspring. They reduced the number of sperm in the epididymis, and the sperm motile activity in male offspring. Parabens could compete with [3H] 17beta-estradiol for binding to the estrogen receptor. The proliferation of two estrogen-dependent cell lines MCF-7 and ZR-75-1 could be increased by parabens. They also increased expression of both transfected and endogenous estrogen-regulated genes in MCF-7 cells. The studies demonstrated parabens were weakly estrogenic.

Replication and gene expression of mutant hepatitis B virus in a transgenic mouse containing the complete viral genome with mutant s gene.

AIM: To establish the transgenic mouse line harbouring complete hepatitis B virus (HBV) genome with mutant s gene (adr subtype). METHODS: Transgenic mice were generated by microinjecting HBV genome into fertilized eggs. Integration, expression, replication of HBV gene and histological changes in transgenic mice were estimated by genomic DNA PCR, serum DNA PCR, Southern blot, ELISA, HE staining, immunohistochemistry and transmission electron microscopy. Transgenic mice with HBsAg positive in serum were bred and analyzed. RESULTS: A total of 288 eggs survived from microinjections were transplanted into the oviducts of 13 pseudopregnant mice and 49 pups were produced. Twenty-six mice were identified to have the integrated HBV gene. Serum HBsAg and HBeAg were detected in 2 of 43 mice. HBsAg and HBcAg in cytoplasm or nuclei of hepatocytes were detected in 10 mice. Founders with HBsAg in serum were named lineages G145R-15 and G145R-18. Of the 16 F1 offsprings generated by G145R-15 founder, 12 were positive for HBV genome with PCR, 10 were positive for HBsAg and HBcAg with immunohistochemistry and 7 were positive for HBsAg and HBeAg with ELISA. Only 1 of 8 F1 offsprings generated by G145R-18 founder was survived and it was detected positive for HBV genome, HBsAg, HBcAg and HBeAg. Both of the two lineages had some pathological characteristics of mild chronic hepatitis B in the liver, such as swelling of hepatocytes and focal hepatocellular necrosis and parenchymal lymphomononuclear cell infiltrate. CONCLUSION: Transgenic mice harbouring HBV with mutant s gene can be generated. The HBV genes are integrated in the transgenic mice genome and can be expressed, replicated, packaged and excreted. HBV DNA can be stably transmitted in the transgenic mice.

Antigenic and immunogenic changes due to mutation of s gene of HBV.

AIM: To investigate the change of immunological characteristics of HBsAg caused by the mutation at codon 145 of HBsAg using DNA-based immunization. METHODS: Plasmids expressing mutant and wild type envelope antigens were transfected into human hepatocellular carcinoma cells via electrotransformation. The antigenicity of HBsAg was studied with EIA and immunocytochemical staining. Then plasmids were used to immunize 5 C57BL/6 mice. Sera of mice were detected for anti-HBs and anti-preS2 with ELISA. RESULTS: The mutant HBsAg could be detected by native antibody in EIA and immunocytochemical study. But the A((450 nm)) value of the mutant HBsAg in the supernatant was apparently lower than that of the wild-type. Both mutant and native HBsAg expression plasmid could stimulate a strong humoral immune response to HBsAg and preS2 antigen in mice. Protective antibodies against HBsAg elicited by the native HBsAg occurred earlier than that elicited by the mutant HBsAg about one to two weeks. The occurrence of protective antibodies against preS2 antigen was one to two weeks earlier than that of anti-HBs. CONCLUSION: The amino acid substitution causes changes of the antigenicity and immunogenicity of HBsAg, but mutant HBsAg can still induce a protective humoral immune response in mice.

[Induction of specific humoral immune response in mice by recombinant mutant HBV s gene vaccine].

AIM: To construct the recombinant eukaryotic expression vector pCMV-S2. S + 145R(PR) containing mutant HBV s gene and detect the specific humoral immune response to the PR in mice. METHODS: The PR was constructed by positional cloning of restriction endonuclease, and then it was transfected into human hepatocellular carcinoma cell line Hep G2 through electrotransformation. The antigenicity of PR was examined by EIA, ELISA and immunocytochemical staining. The PR and empty vector pcDNA3.0 were then used respectively to immunize intramuscularly 5 C57BL/6 mice, dosage being 100 pg purified plasmid each mouse. The titers of serum anti-HBs and anti-HBs2 antibodies were detected by ELISA. RESULTS: In vitro experiment showed that mutant HBsAg could bind to anti-HBs antibody. The PR could induce anti-HBs and anti-HBs2 antibody production in immunized mice. But the appearance time of serum anti-HBs2 antibody one or two weeks earlier than that of serum anti-HBs antibody. CONCLUSION: The expression product of PR had a good antigenicity, which can induce specific hu-moral immune response in C57BL/6 mice.