Gene transfer and expression of recombined human proinsulin in hepatoma cells of rats

AIM:To explore the recombined human proinsulin gene containing glucose reaction element (GLRE) expression in transfected CBRH7919 cells. METHODS:The packaged retrovirus encoding genetically modified human proinsulin PLXSN-(GLRE)3-BP-1MpINS3 and PLXSN-(GLRE)3-BP-1MpINS2 were transfected into CBRH7919 cells. Insulin values in cells after transient and steady expression screened by G418 at different glucose levels were detected. Chromosome DNA was isolated from transfected and untransfected cells and polymerase chain reaction (PCR) was performed. PCR products were analyzed by electrophoresis. RESULTS:38 h after transfection, at the glucose levels of 0-25 mmol/L, the levels of insulin produced by cells including PLXSN-(GLRE)3-BP-1MpINS3 were (3.57±0.21)U/L, (5.30±0.20)U/L, (16.27±0.87)U/L, (23.23±1.12)U/L, respectively (P<0.05). One month after transfection, under above glucose levels, insulin values were (3.57±0.21)U/L, (5.30±0.20)U/L, (16.27±0.87)U/L, (23.23±1.12)U/L (P<0.05). CBRH7919 cells including PLXSN-(GLRE)3-BP-1MpINS2 secreted detectable insulin value at the level of 25 mmol/L, they were (2.10±0.23)U/L and (2.05±0.17)U/L, respectively. PCR products of transfected cells showed target band, but control cells did not. CONCLUSIONS:Recombined proinsulin gene was transfected successfully in CBRH7919 cells. The cells combined human proinsulin gene has the ability of producing insulin with increase in glucose concentration in vitro.     

Effect of NP9 on gene expression profile of NPC cell line CNE1

AIM: To identify the gene expression profiles of CNE1 cells stably transfected with NP9 expressing plasmid, and to explore the potential molecular function of NP9 gene.METHODS: CNE1 cells stably expressing NP9 protein and CNE1 cells transfected with empty vector were used as test and control. Differentially expressed genes were screened with high- throughout human genome array. Differential expression of 6 genes was analyzed with quantitative RT-PCR. RESULTS: Of all the 14 500 human genes in array, 266 genes were revealed differential expression between test and control , of which 82 genes (RA>1)were up-regulated in test and 184 genes (RA<1) were down-regulated. 34 genes and 75 genes were found distinctively up-regulation (RA>1.5) and down-regulation (RA<1.5),respectively.CONCLUSION: NP9 expression in CNE1 cells leads to changes of some genes involved in regulation of cell cycle, cell proliferation and differentiation, cell signal transduction, cell adhesion. Some new clues may be provided for further studying the potential function and molecular mechanism of NP9 gene.     

Proliferation-inhibitory and apoptosis-inducing effects of cinnamic acid combined with cisplatin on human hepatocellular carcinoma cell line MHCC97

AIM：To investigate the effects of cinnamic acid (CA) combined with cisplatin on the proliferation and apoptosis of human hepatocellular carcinoma cell line MHCC97. METHODS：Human hepatocellular carcinoma cell line MHCC97 was culured and divided into CA group, cisplatin group, CA+cisplatin group and control group. MTT assay, inverted microscopy, annexin V-FITC/PI staining and flow cytometry were applied to identify the viability, morphology and apoptosis of the cells. The apoptosis-related signaling protein caspase-3 was detected by Western blotting. RESULTS：CA and cisplatin either alone or in combination significantly inhibited the proliferation and induced obvious apoptosis of MHCC97 cells, while CA alone or combined with cisplatin had no significant inhibitory effect on normal human liver L-02 cells. The rates of mid-and late apoptosis or necrosis were higher in cisplatin group than that in CA group or combination group, but the early apoptotic rate was just the opposite. Pro-apoptotic activity in combination group was much stronger than that in CA group or cisplatin group at lower concentration, and combination group promoted apoptosis and decreased the cytotoxic side effects of cisplatin. CA and cisplatin either alone or in combination also up-regulated the cleaved caspase-3 expression in a time-dependent manner, and the effects in CA group and combination group were higher than that in cisplatin group. CONCLUSION：CA and cisplatin either alone or in combination inhibit the growth of MHCC97 cells by inducing apoptosis, and the activation of caspase-3 may play important roles in these processes.     

Analysis of gene expression profile of multidrug resistant MCF/DOX cell line after benflumetol derivative LY980503 treatment

AIM:To investigate the effect of LY980503(a benflumetol derivative)on multidrug resistance of tumor cell line using DNA microarray.METHODS:Total RNA was extracted from multidrug resistant MCF/DOX cell line. cDNA microarray containing 320 cDNAs was used to detect the gene expression profile.RESULTS:9 down-regulated genes and 1 up-regulated gene were identified after multidrug resistant MCF/DOX cells were treated with LY980503.CONCLUSION:LY980503 can effectively reverse the resistance of MCF/DOX to DOX in vitro by adjusting the expression of multi-genes.     

Effects of sulphated heparin on proliferation and apoptosis of human hepatocellular carcinoma cell line (HepG2)

AIM:To explore the effect and the mechanism of sulphated heparin on the proliferation and the apoptosis of human hepatocellular carcinoma cells.METHODS:The human hepatocellular carcinoma cell line (HepG-2) was used to identify the expression ofrasgene protein and to study the effect of sulphated heparin on proliferation and the apoptosisin vitro.RESULTS:The sulphated heparin downregulated the ras protein expression and inhibited the cell growth in HepG2 cells. In the presence of sulphated heparin, the apoptosis rate of HepG2 increased.CONCLUSION:The data suggest that the effects of sulphated heparin on the proliferation and the apoptosis of the human hepatocellular carcinoma cell are correlated with the signaling transduction mediated byrasgene protein.     

Expression of human leukocyte antigen E in hepatocellular carcinoma cell lines

AIM: To investigate the human leukcyte antigen E (HLA-E) expression in hepatocellular carcinoma cell lines. METHODS: The techniques of real-time PCR and Western blotting were used to study the HLA-E expression in the 5 cell lines of hepatocellular carcinoma and a fetal liver cell line at mRNA and protein levels. RESULTS: The results of real-time PCR showed that no statistical difference of HLA-E mRNA level between fetal liver cell L02 and other 4 cell lines of hepatocellular carcinoma (HepG2, Bel7402, PLC and MHCC97) was observed, and almost absence of HLA-E mRNA expression in Hep3B2.1-7 cells was detected. However, the results of Western blotting showed that there was a significant statistical difference of HLA-E protein levels between L02 cells and the 5 cell lines of hepatocellular carcinoma (HepG2, Bel7402, PLC, MHCC97 and Hep3B2.1-7), and no HLA-E protein in Hep3B2.1-7 cells was detectable. CONCLUSION: Asynchronization of HLA-E expression between mRNA and protein levels was found in hepatocellular carcinoma cell lines.     

Regulation of octreotide on SSTR2 expression in hepatocellular carcinoma

AIM: To observe the regulation of octreotide (OCT) on the expression of somatostatin receptor 2 (SSTR2) in Bel7402 hepatocellular carcinoma (HCC) cells, and the inhibition effect of OCT on the growth of HCC. METHODS: The effect of OCT on proliferative ability of Bel7402 cells was observed by MTT assay. The cell form was observed by light invert microscope. The adhesive and invasive ability was detected by cell adhesion and migration experiments. The cell cycle, SSTR2 expression of 7402 cells were determined by immunofluorescence flow cytometry. Nude mice bearing xenografts in situ were treated with OCT or saline control for 7 weeks since tumor implantation. The immunohistochemistry for SSTR2 was performed. SSTR2 mRNA expression in cell line and xenografts was measured by semi-quantitative RT-PCR. RESULTS: After OCT treatment, the proliferative ability and cell form of 7402 cells didn't change significantly. The adhesive and invasive ability decreased significantly. The ratio of cells in resting state (G₀/G₁) increased, but no apoptosis peak was observed. The SSTR2 expression on 7402 cell membranes decreased significantly. SSTR2 expression in cell line of OCT group was higher than control group, but there was no significant difference between them. The mean tumor weight in mice given OCT was significantly lower than that in control group. SSTR2 immunostaining in tumor cells of treatment group showed stronger positivity, compared with control group. SSTR2 mRNA expression in xenografts after OCT treatment was significantly higher than that in control group. CONCLUSIONS: OCT inhibits the growth of HCC through SSTR2. SSTR2 is regulated by its ligand, the long-term OCT treatment increases the SSTR2 expression and enhances the effect of inhibiting HCC, however, short-term treatment may induces its desensitization and the decrease in anti-tumor effect.     

Cross-species comparison of Smad3 expression in hepatocellular carcinoma tissues

AIM： To study the expression of Smad3 and its significance in hepatocellular carcinoma (HCC) in different species including human, rat and tree shrew, and to verify the feasibility of cross-species oncogenomic approach. METHODS：Real-time fluorescent quantitative polymerase chain reaction and Western blotting were applied to detect the expression of Smad3 at mRNA and protein levels in HCC tissues, corresponding HCC adjacent liver tissues and normal liver tissues collected from different species including human, rat and tree shrew. RESULTS：The mRNA expression of Smad3 in HCC tissues of human, rat and tree shrew was lower than those in the corresponding HCC tissues adjacent liver tissues. The mRNA level of Smad3 in HCC tissues of rat was lower than those in its normal liver tissues, while that in HCC adjacent tissues of tree shrew appeared higher than those in the normal liver tissues. No significant difference of Smad3 expression in other tissues was observed. The protein levels of Smad3 in HCC of human and rat were lower than those in the corresponding HCC adjacent liver tissues and the normal liver tissues. However, the protein expression of Smad3 was at a low level in HCC tissues of tree shrew and was lower than that in the HCC adjacent liver tissues and the normal liver tissues, although without statistical difference. The differences of Smad3 expression between HCC adjacent liver tissues and normal liver tissues in all the 3 species were not statistically significant. CONCLUSION：Smad3 is lowly expressed in HCC tissues of different species, suggesting that it might play a pivotal role in hepatocarcinogenesis and be applied as a key molecular target in HCC prevention and treatment.     

Prognostic significance of CUEDC2 expression in hepatocellular carcinoma

AIM: To investigate the expression of CUE domain-containing 2 (CUEDC2) in hepatocellular carcinoma (HCC) and to analyze its clinical prognostic significance. METHODS: Total 186 formalin-fixed paraffin-embedded tissues obtained from surgical HCC with detailed clinicopathological and follow-up data were used. The expression of CUEDC2 was detected by immunohistochemistry. The relationships between the expression of CUEDC2 and clinicopathological characteristics and prognosis were analyzed. RESULTS: The positive rate of CUEDC2 in HCC was 85.5% (159/186), among which, the low expression was 52.2% (97/186) and the high expression was 47.8% (89/186). CUEDC2 expression was correlated with serum alpha-fetal protein (AFP) level, tumor size, tumor number, tumor differentiation and TNM stage (P<0.05). Kaplan-Meier survival curves showed that the patients with high expression of CUEDC2 were associated with significantly shorter overall survival and recurrence-free survival than those with low CUEDC2 expression (P<0.05). Multivariate Cox regression analysis revealed 3 independent prognostic factors including CUEDC2 expression, serum AFP and tumor number. CONCLUSION: CUEDC2 was expressed in most HCC tissues, which was relevant to tumor growth, tumor differentiation and prognosis. CUEDC2 could be a novel valuable molecular marker to predict the HCC prognosis.     

Effects of changing PKC activity on proliferation and telomerase expression in human hepatocellular carcinoma cells

AIM: To investigate whether protein kinase C (PKC) is involved in the proliferation and the telomerase expression in human hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cells (BEL-7402) were treated with exogenous phorbol-12-myristate-13-acetate (PMA, PKC activator) and staurosporine (SP, PKC inhibitor) for 48 hours. The techniques of cell culture and the telomeric repeat amplification protocol silver staining in combination with computer image scanning system in vitro were used to observe the variations of the growth and the telomerase expression. RESULTS: The proliferative potential of BEL-7402 cells was decreased by the action of PMA as well as SP, and the telomerase expression was also inhibited by PMA and SP. CONCLUSION: Our findings suggest that the proliferation of human hepatocellular carcinoma cells and the telomerase expression may be related to PKC.     

Activated NK cells against human hepatocellular carcinoma cell lines in vitro and in vivo

AIM: To evaluate the role of natural killer(NK) cells against a variety of human hepatocellular carcinoma (HCC) cell lines in vitro and in vivo, and to investigate the expression of MHC class I chain-related protein (MIC protein) in these HCC cell lines. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 50 mL peripheral blood donored by a healthy volunteer and cultured in the NK cell kit followed by amplification and cytokine activation. The release of lactate dehydrogenase (LDH) was detected by lysis experiment. Animal experiments were performed following vaccination with HCC cell lines (BEL7402, HepG2 and SMMC7721) in BALB/c-nu/nu mice. The diameters of the tumors were measured and the growth curves of difference cell lines were delineated. Three weeks later, these mice were sacrificed and the weight of the transplanted tumors was also detected. Furthermore, the expression of MIC protein was determined. RESULTS: The obvious differences of lysis effects of NK cells on different cell lines were observed, in which the lysis effect of NK cells on K562 cells was the strongest, the effect on BEL7402 cells was in the middle and the effect on SMMC7721 cells was the weakest. In animal experiments, NK cells also showed obvious and different restraining effects on a variety of HCC cell line-transplanted tumors in nude mice. The tumor inhibitory rates of NK cells were 43.5% in BEL-7402 cell-transplanted tumor, 40.7% in HepG2 cell-transplanted tumor and 36.0% in SMMC-7721 cell-transplanted tumor. The protein expression of MIC was 48.7%, 32.8% and 0.9% in the 3 cell lines,respectively. CONCLUSION: The lysis effects of NK cells on a variety of human HCC cell lines are different. The expression of MIC in the tumor cells may play distinct role in evaluating these differences.     

Relationship of Ki-67 expression,microvessel density and therapeutic effect of sorafenib on patients with tumor recurrence after liver transplantation

AIM: To study the therapeutic effect of sorafenib on patients with tumor recurrence after liver transplantation and its relation with the expression of Ki-67 and microvessel density(MVD). METHODS: Thirty patients with tumor recurrence after liver transplantation were treated with sorafenib. The therapeutic effect of sorafenib was observed. The expression of Ki-67 and CD34 in the tissue samples of liver cancer were examined by immunohistochemistry. The MVD was also calculated according to the expression of CD34. RESULTS: In 30 patients treated with sorafenib, none of them achieved complete response (CR), 13 achieved partial response (PR), 8 had stable disease (SD), and 9 had progressive disease (PD). The therapeutic effect of sorafenib was associated significantly with Ki-67 expression .The mean counts of MVD were 356.45±156.13 in PR patients, and 99.39±49.88 in PD patients. A significant difference of MVD between PR and PD patients was observed. CONCLUSION: There is a better therapeutic effect of sorafenib in treating tumor recurrence after liver transplantation in the patients with Ki-67-positive expression and high MVD.     

Expression of adhesion molecules in hepatocellurlar carcinoma and its clinical significance

AIM: To investigate the expression of adhesion molecules in hepatocellular carcinoma (HCC), and analyze its clinical significance. METHODS: The expressions of adhesion molecules of tumor tissues of 64 cases and adjacent tissues of 12 cases of HCC were detected with RT-PCR. RESULTS: ①The expression rates of E-cadherin, ICAM-1, CD44, CD44V, α₅, β₁ were 90.62%, 93.75%, 50.00%, 96.88%, 100%, 100%, respectively, and there was a significant difference between CD44 and other adhesion molecules. ②The expression level of E-cadherin, ICAM-1, CD44, CD_44V, α₅,β₁ in liver cancer tissues were 1.24±0.54, 0.96±0.37, 0.62±0.73, 0.86±0.33, 0.97±0.49, 1.41±0.24, respectively, and there was a significant difference between CD44 and E-cadherin, β₁. ③The expression level of E-cadherin and CD44 mRNA declined as HCC stage become higher, and there was a statistical difference in the expression level of CD44 mRNA between Ⅰ-Ⅱ stage and Ⅳ stage. The expression level of ICAM-1, α₅, β₁ had a trend to rise as HCC stage become higher, and there was a statistical difference in the expression level of ICAM-1 between Ⅰ-Ⅱ stage and Ⅳ stage. ④The expression level of ICAM-1,CD_44V, α₅, β₁ had positive correlation with tumor volume, tumor nodules, tumor metastasis, and had negative correlation with tumor encapsulation. E-cadherin and CD44 had negative correlation with tumor volume, tumor nodules, tumor metastasis, and had positive correlation with tumor encapsulation. All showed no significant correlation with the level of AFP , the degree of cirrhosis and the function of liver. CONCLUSION: There was a significant difference in the expression level of adhesion molecule mRNA in HCC, and their expression had Spearman correlation with each other. The expression level of adhesion molecule mRNA is associated with tumor volume, tumor nodules and tumor metastasis.     

Effects of RNA interference of FABP5 on subcutaneous tumor of human hepatocellular carcinoma cell line HepG 2 transplanted in nude mice

AIM: To investigate the effect of recombinant lentiviral vector for RNA interference (RNAi) on the expression of fatty acid-binding protein 5 (FABP5) gene in hepatocellular carcinoma HepG2 cells and tumor formation in nude mice.METHODS: RNAi lentiviral vector was used in the experiment. Human hepatocellular carcinoma HepG2 cells were divided into 3 groups:the HepG2 cells in experimental group were transfected with the recombinant lentivirirus vector LV-shRNA-FABP5, the cells in negative control group were transfected with a control lentiviral vector LV-shRNA-NC, and the cells in normal control group were without any treatment. The nude mice were randomly divided into 3 groups. The growth of the transplanted tumor cells in the nude mice was observed. The tumor growth curve, volume and weight were determined 4 weeks after the cell inoculation. The expression of FABP5 was detected by real-time PCR, Western blot and immunohistochemical staining.RESULTS: Transfection of the lentiviral vector FABP5-shRNA obviously reduced FABP5 expression in the HepG2 cells. Tumor formation was all positive in the 3 groups of the nude mice inoculated with the tumor cells. Compared with normal control group and negative control group, the tumor growth slowed significantly in experimental group with smaller volume and weight. FABP5 expression in the transplanted tumor tissues was significantly down-regulated at mRNA and protein levels in experimental group as compared with normal control group and negative control group.CONCLUSION: RNAi-induced down-regulation of FABP5 effectively inhibits the growth of transplanted hepatocellular carcinoma, suggesting that FABP5 gene may be an effective target for gene therapy in treating liver cancer.     

and laminin on adhesion and proliferation of human hepatocellular carcinoma cell

AIM:To explore the effects of PMA(phorbol-12-myristate-13-acetate, a tumor promoter, mimicking the action of diacylglycerol on PKC)and laminin on the adhesion and the proliferation of human hepatocellular carcinoma cells, and provide a new clue to liver cancer treatment.METHODS:Human hepatocellular carcinoma cell line(BEL-7402)was used to identify the endogenous laminin and protein kinase C-α(PKC-α) expression, and the effects of laminin and PMA on the adhesion and the proliferation were also investigatedin vitro.RESULTS:By the effect of exogenous laminin, human hepatocellular carcinoma cell (BEL-7402) possessed endogenous laminin expression and increased the adhesion and the proliferation, which was showed the synergistic action by the effect of PMA in combination. By the action of PMA alone, the proliferation and the PKC-α expression increased by exogenous laminin were decreased, and the adhesion and the endogenous laminin expression were increased.CONCLUSIONS:The finding suggested that the adhesion and the proliferation of human hepatocellular carcinoma cell were closely related to the effects of endogenous or exogenous laminin, which were associated with cPKC-α activity. Therefore, the application of anti-laminin antibody in combination with PKC antagonist might be a new clue to find out the therapy for liver cancer.     

Effect of sorafenib on liver regeneration after partial hepatoectomy in liver cirrhotic rats

AIM: To study the effect of sorafenib on the liver regeneration after partial hepatectomy (PH) in cirrhotic rats. METHODS: Thirty Wistar rats with liver cirrhosis induced successfully with diethylnitrosamine (DEN) underwent 30% PH and then were randomly divided into 2 groups (n=15). The rats in experimental group were fed with sorafenib at dose of 30 mg·kg^-1·d^-1 from the 1st day to the 10th day after PH, while those in control group were fed with vehicle by gavage. The blood and liver tissues of the rats were collected after PH and at the end of the experiment. Liver regeneration rate (LRR) and proliferating cell nuclear antigen (PCNA) expression were assessed for determining the hepatocyte proliferation. The content of alanine transaminase (ALT), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), angiogenesis related factors including vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR-2), platelet-derived growth factor receptor β (PDGFR-β) and micro-vessel density (MVD) were measured in both groups. RESULTS: LRRs on day 10 after PH were 45.43%±3.36% and 44.21%±2.77% in experimental group and control group, respectively (P>0.05), and the expression of PCNA in hepatic tissues of the rats was not found by the method of immunohistochemistry in both groups. Liver function index had no significant difference between the 2 groups (P>0.05). However, other than VEGF, sorafenib resulted in inhibition of VEGFR-2 and PDGFR-β expression and reduction of MVD in experiment group, and significant difference between the 2 groups was observed (P<0.01). CONCLUSION: Sorafenib does not influence live regeneration after PH in liver cirrhotic rats.     

Role of multidrug resistance gene 1 polymorphism in prognosis of hepatocellular carcinoma patients treated with liver transplantation

AIM: To evaluate the relationship between three multidrug resistance gene 1 (MDR1) polymorphisms (C1236T, G2677A/T, C3435T) and the prognosis of hepatocellular carcinoma (HCC) in Chinese liver transplantation (LT) patients.METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was applied to determine the genotypes of MDR1 gene in 50 HCC patients treated with LT. The tumor-free survival and overall survival were compared among these patients according to the polymorphisms of MDR1 by Kaplan-Meier method, multivariate regression analysis was also performed.RESULTS: No significant association was found between C1236T, G2677T, C3435T and prognosis of these patients. But interestingly, 2677A carrier group had significantly higher tumor-free survival rate than 2677A noncarrier group (P<0.05). The multivariate regression analysis revealed that 2677A carrier genotype was one of the independent factors for predicting tumor-free survival (RR=0.143, P<0.01).CONCLUSION: MDR1 2677A carrier genotype is correlated with the tumor-free survival. MDR1 2677A carrier genotype may be a useful independent prognostic factor in HCC patients treated with LT.     

Inhibitory effect of sorafenib on collagen synthesis in human hepatic stellate cells

AIM: To investigate the effects of sorafenib on collagen synthesis in human hepatic stellate cells (HSCs). METHODS: HSC cell line LX-2 was used in vitro in this study. -proline incorporation assay was performed to measure the collagen synthesis. Immunocytochemistry was applied to detect type I collagen and real-time PCR was used to determine the mRNA expression of collagen α1 (I). RESULTS: Stimulation with platelet-derived growth factor (PDGF) induced the increase in type I collagen synthesis, while treatment with sorafenib (10.0 μmol/L) for 24 h markedly decreased the collagen synthesis. Sorafenib resulted in dose-dependent and time-dependent decrease in collagen synthesis in LX-2 cells in the absence or presence of PDGF by -proline incorporation assay. The inhibition rates were 22.69%, 37.52% and 71.74%, respectively, when LX-2 cells was treated with sorafenib at 10.0 μmol/L for 12 h, 24 h and 48 h. Sorafenib dose-dependently blocked the mRNA expression of collagen α1 (I) in LX-2 cells stimulated with PDGF. Sorafenib at the concentrations of 2.5 μmol/L, 5.0 μmol/L and 10.0 μmol/L down-regulated the mRNA expression of collagen α1 (I) in LX-2 cells by 58.66%, 67.06% and 81.64%, respectively. CONCLUSION: Sorafenib inhibits the collagen synthesis and blocks the expression of type I collagen at mRNA and protein levels in vitro in LX-2 cells. Therefore, sorafenib may be a potential therapeutic agent in the treatment of liver fibrosis.