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AIM: To investigate the effect of knockdown of Forkhead box M1 (FoxM1) on the proliferation and invasion capacities of human gallbladder carcinoma (GBC)-SD cells. METHODS: Four FoxM1 shRNAs were transfected into GBC-SD cells with Lipofectamine 2000 to select the appropriate shRNA for down-regulation of FoxM1. A recombinant lentivirus for shFoxM1 (Lv-shFoxM1), which expresses FoxM1-specific shRNA, and a negative control carrying green fluorescent protein, which expresses a scrambled RNA, were constructed. After transfection with the recombinant adenovirus and screened with puromycin, RT-PCR and Western blot were utilized to evaluate the inhibition efficiency. Cell viability was evaluated by MTT assay, and cell migration and invasion were assessed using the Transwell system. Cells were suspended in serum-free medium and seeded into Transwell inserts either uncoated (for migration assay) or coated (for invasion assay) with growth factor-reduced Matrigel. To verify the involvement of FoxM1 in the senescence of tumor cells, staining of senescence β-galactosidase (SA β-gal), the widely used biomarker of cellular senescence, was also performed. RESULTS: After successful transfection of four FoxM1 small interfering RNAs (shRNAs) with Lipofectamine 2000, the shF1822 was selected as the most appropriate shRNA according to its obvious inhibitory effect. The recombinant adenovirus was then constructed with the shF1822 and successfully transfected into the GBC-SD cells, resulting in the significant inhibition of FoxM1 expression at both the mRNA and protein levels, compared with the negative control (P < 0.05). After transfection, down-regulation of FoxM1 significantly inhibited cell viability according to the MTT assay (P < 0.05). In addition, Transwell migration and invasion assays also suggested the suppression of invasion ability of the transfected cells. SA β-gal staining showed that down-regulation of FoxM1 could induce more senescent GBC cells (P < 0.05), suggesting the possible involvement of the senescence process of the FoxM1-deficient cells in GBC. CONCLUSION: FoxM1 is functionally involved in viability of GBC cells, partially dependent on the inducement of cellular senescence, and is a potential target for GBC therapy.

AIM: To investigate the growth-inhibiting and apoptosis-inducing effects of the gene MOB2 on human hepatic carcinoma cell line SMMC-7721. METHODS: The full-length cDNA of the MOB2 gene was amplified from human umbilical vein endothelial cells. The correct full-length MOB2 cDNA was subcloned into the eukaryotic expression vector pEGFP-C1. After lipofection of the MOB2 gene into cancer cells, the levels of MOB2 protein in the cancer cells were detected by immunoblotting. To transfect the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells, the cells were cultured in Dulbecco’s Modified Eagle’s Medium with 10% fetal calf serum and glutamine, and then mixed with liposomes, Lipofectamine 2000 and the plasmid vector pEGFP-CI-MOB2. RESULTS: We observed the growth and proliferation of SMMC-7721 cells containing pEGFP-CI-MOB2 and analyzed their apoptosis and growth cycle phases by flow cytometry. We successfully transfected the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells and screened for a single clone cell containing MOB2. After transfection, MOB2 enhanced growth suppression, induced apoptosis, increased the ratio of G0/G1, significantly inhibited the advance of cell cycle phase, and arrested cells in G0/G1 phase. CONCLUSION: MOB2 overexpression induces apoptosis and inhibits the growth of human hepatic cancer cells, which may be useful in gene therapy for hepatic carcinoma.

AIM: To investigate the expression of vascular endothelial cell growth factor (VEGF) and its receptors Fms-like tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1) in colorectal carcinoma (CRC), and the blocking effects of small interfering RNAs (siRNAs) on VEGF expression in human colorectal cancer HCT116 cells. METHODS: Immunohistochemical staining for VEGF, FLT-1 and FLK-1 proteins was performed in 82 cases of CRC and 14 normal colorectal mucosae. A siRNA targeting VEGF was synthesized and transfected into HCT116 cells using lipofectamine 2000. Immunocytochemical staining and Western blotting analyses were performed to detect the expression of VEGF protein. The suppressive effect of the siRNA on cell proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay. Cellular apoptosis was detected using flow cytometry (FCM). RESULTS: The expression of VEGF, FLT-1 and FLK-1 in tumor tissues was significantly higher than that in normal tissues (P = 0.008, P = 0.000, P = 0.000). The expression of VEGF was positively correlated with both lymph node metastasis and clinical stage (P = 0.009 and P = 0.025, respectively). Immunocytochemistry showed that the expression of VEGF was weakly positive and Western blotting indicated a significant reduction in VEGF-siRNA cell protein levels. VEGF-siRNA cell growth inhibition was assessed by the MTT assay, and the tumor cell proliferation rate was significantly different at 24, 48, and 72 h after transfection. FCM results showed that the VEGF-siRNA group had an apparent aneuploid peak. CONCLUSION: VEGF, FLT-1 and FLK-1 are associated with colorectal carcinogenesis. siRNA silencing of the VEGF gene suppresses proliferation, and induces apoptosis in HCT116 cells. The results suggest that VEGF may be a new gene therapy target for colorectal cancer.

AIM: To investigate the effects of interferon-alpha (IFN-α) to restrain the growth and invasive potential of hepatocellular carcinoma (HCC) induced by hepatitis B virus (HBV) X protein. METHODS: The pcDNA3.1-HBx plasmid was transfected into Chang cells by Lipofectamine in vitro, and Chang/HBx was co-cultured with IFN-α. Cell survival growth curve and clonogenicity assay were used to test the growth potential of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vitro. Growth assay in nude mice was used to detect the growth potential of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vivo. Wound healing and transwell migration assays were used to detect the invasive ability of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx. RESULTS: Compared with CCL13 cells transfected with pcDNA3.1, CCL13 with stable expression of hepatitis B virus X protein showed the characteristics of malignant cells with high capability of growth and invasion by detecting their growth curves, colony forming efficiency, wound healing , transwell migration assays and growth assays in nude mice. Its capability of growth and invasion could be controlled by IFN-α. CONCLUSION: IFN-α can restrain the growth and invasive potential of HCC cells induced by HBx protein, which has provided an experimental basis for IFN-α therapy of HCC.

AIM: To observe the growth inhibitory effect of wild-type Krasl gene on a colonic adenocarcinoma cell line Caco-2.METHODS: Recombinant plasmid pCI-neo-Kras2 with wild type Krasl open reading frame was constructed. The Caco-2 cells were transfected with either pCI-neo or pCl-neo-Kras2 using Lipofectamine 2000. The expression of wild type Krasl was examined by Northern blot analysis. And the expression of wild type Kras2 protein was examined by Western blot analysis. The effects of wild-type Krasl on cell proliferation were analyzed by monotetrazolium (MTT) assay, meanwhile analyses of cell cycle and spontaneous apoptosis rate were carried out by flow cytometry (FCM).RESULTS: The plasmid of pCl-neo-Kras2 was successfully established. The growth rate of cells transfected with pCl-neo-Kras2 was significantly lower than the control cells transfected with the empty pCI-neo vector (P < 0.05). Cell cycle analysis revealed arrest of the pCI-neo-Kras2 transfected cells in Go/G1 phases, decreased DNA synthesis and decreased fractions of cells in S phase. The proliferative index of cells transfected with pCI-neo-Kras2 was decreased compared with the control cells (49.78% vs 64.21%), while the apoptotic rate of Caco-2 cells with stable Krasl expression increased (0.30% vs 0.02%).CONCLUSION: The wild-type Krasl gene effectively inhibits the growth of the colonic adenocarcinoma cell line Caco-2.

AIM: To investigate the effect of deleted pancreatic cancer locus 4 (DPC4) gene transfection on biological behaviors of human colorectal carcinoma cells and the role of DPC4 gene in colorectal carcinogenesis. METHODS: PcDNA3.1-DPC4 plasmid was re-constructed by gene-recombination technology. SW620 cells, a human colorectal carcinoma cell line, were transfected with PcDNA3.1-DPC4 plasmid using lipofectamine transfecting technique. Transfected cells were selected with G418. Expression of Smad4 protein was detected in cells transfected with DPC4 gene by immunohistochemistry and Western blot. Biological characteristics of transfected cells were evaluated by population-doubling time and cloning efficiency. Alterations of percentage of S phage cells (S%) and apoptosis rate were determined by flow- cytometry. RESULTS: PcDNA3.1-DPC4 plasmid was constructed successfully. SW620 cells transfected with PcDNA3.1-DPC4 plasmid (DPC4+-SW620 cells) showed a strong intracellular expression of Smad4 protein, and the positive signal was localized in cytoplasm and nuclei, mainly in cytoplasm, where the expressions of Smad4 protein in SW620 cells transfected with PcDNA3.1 plasmid (PcDNA3.1-SW620 cells) and non-transfected SW620 cells (SW620 cells) were weaker than those in DPC4+-SW620 cells. The population- doubling time in DPC4+-SW620 cells (116 h) was significantly longer than that in SW620 cells (31 h) and PcDNA3.1-Sw620 cells (29 h) (P

To construct a pEgr-IFNgamma plasmid and to investigate its expression properties of interferon-gamma (INF-gamma) induced by irradiation and the effect of gene-radiotherapy on the growth of melanoma.A recombined plasmid, pEgr-IFNgamma, was constructed and transfected into B16 cell line with lipofectamine. The expression properties of pEgr-IFNgamma were investigated by ELISA. Then, a B16 melanoma-bearing model was established in mice, and the plasmid was injected into the tumor tissue. The tumor received 20 Gy X-ray irradiation 36 h after injection, and IFN-gamma expression was detected from the tumor tissue. A tumor growth curve at different time points was determined.The eukaryotic expression vector, pEgr-IFNgamma, was successfully constructed and transfected into B16 cells. IFN-gamma expression was significantly increased in transfected cells after X-ray irradiation in comparison with 0 Gy group (77.73-94.60 pg/mL, P0.05-0.001), and was significantly higher at 4 h and 6 h than that of control group after 2 Gy X-ray irradiation (78.90-90.00 pg/mL, P0.01-0.001). When the transfected cells were given 2 Gy irradiation 5 times at an interval of 24 h, IFN-gamma expression decreased in a time-dependent manner. From d 3 to d 15 after IFNgamma gene-radiotherapy, the tumor growth was significantly slower than that after irradiation or gene therapy alone.The anti-tumor effect of pEgr-IFNgamma gene-radiotherapy is better than that of genetherapy or radiotherapy alone for melanoma. These results may establish an important experimental basis for gene-radiotherapy of cancer.

AIM: To construct and identify the recombinant vectors carrying herpes simplex virus thymidine kinase (HSV-TK) and tumor necrosis factor alpha (TNF-α) or interleukin-2 (IL-2) genes expressed in gastric carcinoma cell line SGC7901. METHODS: The fragments of HSV-TK, internal ribosome entry sites (IRES) and TNF-α or IL-2 genes were inserted in a TK-IRES-TNF-α or TK-IRES-IL-2 order into pEGFP-N3 and pLXSN to generate the therapeutic vectors pEGFP-TT, pEGFP-TI, pL(TT)SN and pL(TI)SN respectively, which were structurally confirmed by the digestion analysis of restriction endonuclease. The former two plasmids were used for the transient expression of recombinant proteins in the target cells while pL(TT)SN and pL(TI)SN were transfected into SGC7901 cells by lipofectamine for the stable expression of objective genes through G418 selection. The protein products expressed transiently and stably in SGC7901 cells by the constructed vectors were confirmed by fluorescent microscopy and Western blot respectively. RESULTS: The inserted fragments in all constructed plasmids were structurally confirmed to be consistent with that of the published data. In the transient expression, both pEGFP-TT and pEGFP-TI were shown expressed in nearly 50% of the transfected SGC7901 cells. Similarly, the G418 selected vectors PL(TT)SN and PL(TI)SN were confirmed to be successful in the stable expression of the objective proteins in the target cells. CONCLUSION: The constructed recombinant vectors in the present study that can express the suicide gene TK in combination with cytokines genes may serve as the potential tools to perform more effective investigations in future for the gene therapy of gastric carcinoma.

AIM: To study the effect of hepatitis C virus nonstructural protein 3 c-terminal deleted protein (HCV NS3-5’) on hepatocyte transformation and tumor development. METHODS: QSG7701 cells were transfected with plasmid pRcHCNS3-5’ (expressing HCV NS3 c-terminal deleted protein) by lipofectamine and selected in G418. The expression of HCV NS3 gene and protein was determined by PCR and immunohistochemistry respectively. Biological behavior of transfected cells was observed through cell proliferation assay, anchorage-independent growth and tumor development in nude mice. The expression of HCV NS3 and c-myc proteins in the induced tumor was evaluated by immunohistochemistry. RESULTS: HCV NS3 was strongly expressed in QSG7701 cells transfected with plasmid pRcHCNS3-5’ and the positive signal was located in cytoplasm. Cell proliferation assay showed that the population doubling time in pRcHCNS3-5’ transfected cells was much shorter than that in pRcCMV and non-transfected cells (24 h, 26 h, 28 h respectively). The cloning ratio of cells transfected with pRcHCNS3-5’, pRcCMV and non-transfected cells was 33%, 1.46%, 1.11%, respectively, the former one was higher than that in the rest two groups (P < 0.01). Tumor development was seen in nude mice inoculated with pRcHCNS3-5’ transfected cells after 15 days. HE staining showed its feature of hepatocarcinoma, and immunohistochemistry confirmed the expressions of HCV NS3 and c-myc proteins in tumor tissue. The positive control group inoculated with HepG2 also showed tumor development, while no tumor developed in the nude mice injected with pRcCMV and non-transfected cells after 40 days. CONCLUSION: 1.HCV NS3 c-terminal deleted protein has transforming and oncogenic potential. 2. Human liver cell line QSG7701 may be used as a good model to study HCV NS3 pathogenesis.

AIM: To investigate the effect of antisense RNA to vascular endothelial growth factor165 (VEGF165) on human esophageal squamous cell carcinoma cell line EC109 and the feasibility of gene therapy for esophageal carcinoma. METHODS: By using subclone technique, the full length of VEGF165 amino acid cDNA, which was cut from pGEM-3Zf(+), was cloned inversely into the eukaryotic expression vector pCEP4.The recombinant plasmid pCEP-AVEGF165 was transfected into EC109 cell with lipofectamine. After a stable transfection, dot blot, enzyme-linked immunosorbent assay (ELISA), laser confocal imaging system analysis, transmission electron microscopy and flow cytometry were performed to determine the biological characteristics of EC109 cell line before and after transfection in vitro and whether there was a reversion in the tumorigenic properties of the EC109 cell in vivo. RESULTS: The eukaryotic expression vector pCEP-AVEGF165 was successfully constructed and transfected into EC109 cells. The expression of VEGF165 was significantly decreased in the transfected cells while the biological characteristics of the cells were not influenced by the expression of antisense gene. The tumorigenic and angiogenic capabilities were greatly reduced in nude mice, as demonstrated by reduced tumor end volume (820 ± 112.5) mm3 vs (7930 ± 1035) mm3 and (7850 ± 950) mm3,P£¼0.01£½ and microvessel density(8.5 ± 1.2) mm-2 vs (44.3 ± 9.4) mm-2 and (46.4 ± 12.6) mm-2,P < 0.01) in comparison between experimental groups empty vector transfected group and control group. CONCLUSION: The angiogenesis and tumorigenicity of human esophageal squamous cell carcinoma were effectively inhibited by VEGF165 antisense RNA. Antisense RNA to VEGF165 can potentially be used as an adjuvant therapy for solid tumors.

AIM: To determine whether the human giant larvae homolog 1 gene (Hugl-1/Llg1/Lgl1) exerts tumor suppressor effects in esophageal cancer. METHODS: We constructed a Hugl-1 expression plasmid, pEZ-M29-Hugl1, for gene transfection. We transfected the pEZ-M29-Hugl1 plasmid into Eca109 esophageal cancer cell lines with Lipofectamine 2000 to overexpress Hugl-1. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the effects of the plasmid on Hugl-1 expression. In vitro cell proliferation and apoptosis were examined separately by cell counting Kit-8 (CCK-8) assay, flow cytometry, and Western blotting before and after the transfection of the plasmid into Eca109 cells. Cell cycle distribution was assessed with flow cytometry. The effect of Hugl-1 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice. Expression of Hugl-1 in xenograft tumor was analyzed by immunohistochemistry. The transferase-mediated dUTP nick end-labeling (TUNEL) technique was performed to detect and quantitate apoptotic cell. RESULTS: The transfection efficiency was confirmed with real-time RT-PCR and Western blotting. Our results show that compared with control groups the mRNA levels and protein levels of Hugl-1 in pEZ-M29-Hugl1-treated group were remarkably increased (P < 0.05). The CCK-8 assay demonstrated that the growth of cells overexpressing Hugl-1 was significantly lower than control cells. Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing Hugl-1 (64.09% ± 3.14% vs 50.32% ± 4.60%, 64.09% ± 3.14% vs 49.13% ± 2.24%). Annexin V-fluorescein isothiocyanate revealed that apoptosis was significantly increased in cells overexpressing Hugl-1 compared with control group (17.33% ± 4.76% vs 6.90% ± 1.61%, 17.33% ± 4.76% vs 6.27% ± 0.38%). Moreover, we found that Hugl-1 changes the level of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax and the activation of both caspase-3 and caspase-9. With a TUNEL assay, we found that Hugl-1 markedly increased the apoptosis rate of Eca109 cells in vivo (60.50% ± 9.11% vs 25.00% ± 12.25%). It was shown that Hugl-1 represents a significantly more effective tumor suppressor gene alone in a xenograft tumor mouse model. This data suggest that Hugl-1 inhibited tumor growth and induced cell apoptosis in vivo. CONCLUSION: These results suggest that Hugl-1 induces growth suppression and apoptosis in a human esophageal squamous cell carcinoma cell line both in vitro and in vivo.

AIM: To investigate the regulative effect of miRNA (miR)-221 on colorectal carcinoma (CRC) cell radiosensitivity and the underlying mechanisms. METHODS: A human CRC-derived cell line was cultured conventionally and exposed to different doses of X-rays (0, 2, 4, 6 and 8 Gy). The total RNA and protein of the cells were extracted 24 h after irradiation, and the alteration of miR-221 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene mRNA expression was detected by real-time reverse transcriptase polymerase chain reaction (PCR). The protein alteration of PTEN in the cells was detected by Western blotting. Caco2 cells were pretreated with or without anti-PTEN-siRNA prior to the addition of pre-miR-221 or anti-miR-221 using Lipofectamine 2000. Colony formation assay and flow cytometry analysis were used to measure the surviving cell fraction and the sensitizing enhancement ratio after irradiation. Additionally, PTEN 3′-untranslated region fragment was PCR amplified and inserted into a luciferase reporter plasmid. The luciferase reporter plasmid construct was then transfected into CRC cells together with pre-miR-221 or anti-miR-221, and the luciferase activity in the transfected cells was detected. RESULTS: The X-ray radiation dose had a significant effect on the expression of miR-221 and PTEN protein in human Caco2 cells in a dose-dependent manner. The miR-221 expression level improved gradually with the increase in irradiation dose, while the PTEN protein expression level reduced gradually. miR-221 expression was significantly reduced in the anti-miR-221 group compared with the pre-miR-221 and negative control groups (P < 0.01). Anti-miR-221 upregulated expression of PTEN protein and enhanced the radiosensitivity of Caco2 cells (P < 0.01). Moreover, the inhibitory effect was dramatically abolished by pretreatment with anti-PTEN-siRNA, suggesting that the enhancement of radiosensitivity was indeed mediated by PTEN. A significant increase of luciferase activity was detected in CRC cells that were cotransfected with the luciferase reporter plasmid construct and anti-miR-221 (P < 0.01). CONCLUSION: Anti-miR-221 can enhance the radiosensitivity of CRC cells by upregulating PTEN.

AIM: To investigate whether uncoupling protein 2 (UCP2) affects oleic acid-induced secretion of glucagon-like peptide-1 (GLP-1) in L-cells. METHODS: mRNA and protein expression of UCP2 were analyzed in human NCI-H716 cells, which serve as a model for enteroendocrine L-cells, by quantitative reverse transcription-polymerase chain reaction and Western blotting before and after treatment with oleic acid. Localization of UCP2 and GLP-1 in NCI-H716 cells was assessed by immunofluorescence labeling. NCI-H716 cells were transiently transfected with a small interfering RNA (siRNA) that targets UCP2 (siUCP2) or with a non-specific siRNA using Lipofectamine 2000. The concentrations of bioactive GLP-1 in the medium were measured by enzyme linked immunosorbent assay. RESULTS: Both GLP-1 and UCP2 granules were expressed mainly in the cytoplasm of NCI-H716 cells. NCI-H716 cells that secreted GLP-1 also expressed UCP2. Time-course experiments revealed that release of GLP-1 from NCI-H716 cells into the medium reached a maximum at 120 min and remained stable until at least 180 min after treatment with oleic acid (the level of GLP-1 increased about 2.3-fold as compared with the level of GLP-1 in the control cells, P < 0.05). In an experiment to determine dose dependence, stimulation of NCI-H716 cells with ≤ 8 mmol oleic acid led to a concentration-dependent release of GLP-1 into the medium; 10 mmol oleic acid diminished the release of GLP-1. Furthermore, GLP-1 secretion induced by oleic acid from NCI-H716 cells that were transfected with siUCP2 decreased to 41.8%, as compared with NCI-H716 cells that were transfected with a non-specific siRNA (P < 0.01). CONCLUSION: UCP2 affected GLP-1 secretion induced by oleic acid. UCP2 plays an important role in L-cell secretion that is induced by free fatty acids.

AIM: To identify differentially expressed microRNAs (miRNAs) in human colon cancer stem cells (SW1116csc) and study their function in SW1116csc proliferation. METHODS: SW1116csc were isolated from the human colon cancer cell line, SW1116 and cultured in serum-free medium. A miRNA microarray was used to detect differential expression profiles of miRNAs in SW1116csc and SW1116 cells. Real-time quantitative polymerase chain reaction (PCR) was performed to verify the differential expression of candidate miRNAs obtained from the microarray. Target mRNAs of differentially expressed miRNAs were predicted with target prediction tools. miRNA expression plasmids were transfected into SW1116csc using Lipofectamine 2000 reagent. Cell proliferation curves were generated with trypan blue staining, and the colony formation rate of transfected cells was measured with the soft agar colony formation assay. Expression of target mRNAs and proteins from differentially expressed miRNAs were detected using reverse transcription (RT)-PCR and western blotting. RESULTS: Compared with expression in SW1116 cells, 35 miRNAs (including hsa-miR-192, hsa-miR-29b, hsa-miR-215, hsa-miR-194, hsa-miR-33a and hsa-miR-32) were upregulated more than 1.5-fold, and 11 miRNAs (including hsa-miR-93, hsa-miR-1231, hsa-miRPlus-F1080, hsa-miR-524-3p, hsa-miR-886-3p and hsa-miR-561) were downregulated in SW1116csc. The miRNA microarray results were further validated with quantitative RT-PCR. miR-93 was downregulated, and its predicted mRNA targets included BAMBI, CCND2, CDKN1A, HDAC8, KIF23, MAP3K9, MAP3K11, MYCN, PPARD, TLE4 and ZDHHC1. Overexpressed miR-93 significantly inhibited cell proliferation and colony formation by SW1116csc. Furthermore, miR-93 negatively regulated the mRNA and protein levels of HDAC8 and TLE4. CONCLUSION: Some miRNAs were differentially expressed during differentiation of SW1116csc into SW1116 cells. miR-93 may inhibit SW1116csc proliferation and colony formation.

AIM: To construct pcDNA3.1-Egr.1p-p16 recombinant plasmid and investigate the expression of p16 in pancreatic cancer JF305 cells induced by radiation and the feasibility of gene radiotherapy for pancreatic carcinoma. METHODS: Human p16 cDNA was ligated to the downstream of Egr-1 promotor to construct pcDNA3.1-Egr.1p-p16 plasmid by restriction enzyme digested. The recombined plasmids were transfected into pancreatic cancer JF305 cells with lipofectamine. p16 mRNA level was detected by RT-PCR. The expression of p16 after different doses of X-ray radiation was detected by Western blot technique. Cell survival was assessed by clonogenic assays and cell viability was analysed by trypen blue exclusion. Flow cytometry was performed to study the apoptosis of JF305 cells. RESULTS: Restriction enzyme digestion showed the correctly constructed pcDNA3.1-Egr.1p-p16. The p16 expression in cells transfected with pcDNA3.1-Egr.1p-p16 induced by different doses of radiation was higher than that in the control group (P < 0.05). Eight hours after 2 Gy X-ray radiation, the expression reached its peak (87.00 ng/L), and was significantly higher than that in the control group (P < 0.0.5). Clonogenic analysis and trypan blue extraction test showed that the pcDNA3.1-Egr.1p-p16 transfer enhanced radiation-induced cell killing in p16-null JF305 cell lines. The induction of apoptosis was lower in combined transfection and irradiation group than that in irradiation alone. CONCLUSION: X-ray can induce the recombinant plasmid pcDNA3.1-Egr.1p-p16 expression in JF305 cells. The detection of dose and time provides an experimental basis for in vivo study in future.

AIM: To explore the influence of angiostatin up-regulation on the biologic behavior of gallbladder carcinoma cells in vitro and in vitro, and the potential value of angiostatin gene therapy for gallbladder carcinoma. METHODS: A eukaryotic expression vector of pcDNA3.1(+) containing murine angiostatin was constructed and identified by restriction endonuclease digestion and sequencing. The recombinant vector pcDNA3.1-angiostatin was transfected into human gallbladder carcinoma cell line GBC-SD with Lipofectamine 2000, and paralleled with the vector and mock control. The resistant clone was screened by G418 filtration. Angiostatin transcription and protein expression were examined by RT-PCR, immunofluorescence and Western-blot. The supernatant was collected to treat endothelial cells. Cell proliferation and growth in vitro were observed under microscope. RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). After 14 d of transfection and selection with G418, macroscopic resistant cell cloning was formed in the experimental group transfected with pcDNA 3.1(+)-angiostatin and vector control. But untreated cells died in the mock control. Angiostatin was detected by RT-PCR and protein expression was detected in the experimental group by immunofluorescence and Western-blot. Cell proliferation and growth in vitro in the three groups were observed respectively under microscope. No significant difference was observed in the growth speed of GBC-SD cells between groups that were transfected with and without angiostatin. After treatment with supernatant, significant differences were observed in endothelial cell (ECV-304) growth in vitro. The cell proliferation and growth were inhibited. CONCLUSION: Angiostatin does not directly inhibit human gallbladder carcinoma cell proliferation and growth in vitro, but the secretion of angiostatin inhabits endothelial cell proliferation and growth.

AIM: To investigate the contribution of HBV in the development of hepatocarcinoma by examining the effects of HBV on p53 function in SMMU-7721 cell line. METHODS: Plasmid pCMVp53 was transfected or cotransfected with pCMVHBVa (wild-type HBV) or PCMVHBVb (mutation type HBV) into the hepatoma cell line SMMU-7721 by lipofectamine. Apoptosis cells were labeled with annexin V-FITC and confirmed by flow cytometry. Reporter plasmid PG13-CAT or p21-luc was cotransfected, respectively, into each group to determine the transactivation activity of p53 and its effect on p21 promoter. Western blot was performed to observe p53 expression in hepatoma cell line of each group. RESULTS: The group transfected with pCMVp53 alone exhibited higher luciferase activity and higher apoptosis rate, otherwise, the p53 expression and reporter activity of PG13-CAT or P21-luc as well as cell apoptosis rate were obviously higher in the group cotransfected of pCMVp53 with pCMVHBVa, but not in the other cotransfected group. CONCLUSION: Transient transfection of HBV into the SMMU-7721 cell line can enhance p53 expression and its effects on development of hepatocarcinoma.

AIM: To investigate the therapeutic effect of somatostatin receptor type 2 (SSTR2) gene transfection on pancreatic carcinoma xenografts in vivo in experimental cancers. METHODS: Human pancreatic cancer cell line Panc-1 was inoculated subcutaneously into the back of nude mice. When tumor nodules were grown as large as about 5 mm×5 mm days after inoculation, the mice were randomly divided into 3 groups (6 mice in each group). Group I served as untreated control group. Group II received an intratumoral injection of a combination of human cytomegalovirus promoter-6C (pCMV-6C) and lipofectamine 2000. Group III received an intratumoral injection of a combination of pCMV-6C-SSTR2 and lipofectamine 2000. The rate of tumor growth was compared among these three groups. The expression of SSTR2 in these tumors was detected by immunohistochemistry and Western-blot. Apoptosis index (AI) in these tumors was examined by using TUNEL in situ. RESULTS: Intratumoral injection of a combination of pCMV-6C-SSTR2 and lipofectamine 2000 resulted in the expression of SSTR2 protein. The tumor size and weight in group III (0.318±0.098 cm3, and 0.523±0.090 g, respectively) were significantly lower than those in group I (2.058±0.176 cm3, and 1.412±0.146 g, respectively) and group II (2.025±0.163 cm3, and 1.365±0.116 g, respectively) (P

AIM: To study the function of a-fetoprotein (AFP) in SMMC-7721 hepatoma cells. METHODS: A hairpin siRNA expressing plasmid pSilencer3.0-H1-afp was constructed andtransfected into SMMC-7721 cells with Lipofectamine 2000. The expression of AFP was monitored by real-time RT-PCR and immunoassays, its effect on SMMC-7721 cell proliferation and cell death was detected by MTT and fluorescence- activated cell sorter (FACS). RESULTS: The AFP-siRNA expressing plasmid downregulated the expression of AFP obviously (about 34%), and inhibited SMMC-7721 cell proliferation, but did not induce apoptosis. CONCLUSION: Downregulation of AFP siRNA inhibits proliferation of SMMC-7721 cells, but cannot cause apoptosis.

To study the cell cycle alterations of human hepatoma cell line HepG(2) in vitro after (60)Co gamma-irradiation and further to examine the mechanisms underlying the enhancement of radiosensitivity to gamma-irradiation in HepG(2) transiently transfected with wild type p27(kip1).The proliferation of HepG(2) cells was evaluated with MTT assay, and the cell cycle profile and apoptosis were assessed by cell morphology, DNA fragmentation analysis and flow cytometry. HepG(2) cells were transfected with p27(kip1) wild type by using Lipofectamine (LF2000), and the expression and subcellular localization of p27(kip1) in HepG(2) were detected by immunocytochemistry.(60)Co gamma-irradiation inhibited the growth of HepG(2) cells in a dose-dependent manner. Apoptosis of HepG(2) cells was induced 48 h after gamma ray exposure. Furthermore research was carried out to induce exogenous expression of p27(kip1) in HepG(2). The expression of p27(kip1) induced G(0)/G(1) phase arrest in HepG(2) cells. The overexpression of p27(kip1) enhanced (60)Co gamma-irradiation-induced radiosensitivity in HepG(2) cells.Overexpression of p27(kip1) is a rational approach to improve conventional radiotherapy outcomes, which may be a possible strategy for human hepatoma therapy.

AIM: Recombined plasmid pETNF-P16 was constructed to investigate its expression properties in esophageal squamous carcinoma cell line EC9706 induced by X-ray irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma. METHODS: Recombined plasmid pETNF-P16 was constructed and transfected into EC9706 cells with lipofectamine. ELISA, Western blot, and immunocytochemistry were performed to determine the expression properties of pETNF-P16 in EC9706 after transfection induced by X-ray irradiation. RESULTS: Eukaryotic expression vector pETNF-P16 was successfully constructed and transfected into EC9706 cells. TNFα expressions were significantly increased in the transfected cells after different doses of X-ray irradiation than in those after 0Gy irradiation (1192.330-2026.518 pg/mL, P < 0.05-0.01), and the TNFα expressions and P16 were significantly higher 6-48 h after 2 Gy X-ray irradiation (358.963-585.571 pg/mL, P < 0.05-0.001). No P16 expression was detected in normal EC9706 cells. However, there was strong expression in the transfected and irradiation groups. CONCLUSION: X-ray irradiation induction could significantly enhance TNFα and P16 expression in EC9706 cells transfected with pETNF-P16 plasmid. These results may provide important experimental data and therapeutic potential for gene-radiotherapy of esophageal carcinoma.

AIM: To investigate the inhibitory effect of hepatitis C virus internal ribosome entry site (HCV IRES) specific inhibitor RNA (IRNA) on gene expression mediated by HCV IRES in vivo. METHODS: By using G418 screening system, hepatoma cells constitutively expressing IRNA or mutant IRNA (mIRNA) were established and characterized, and HCV replicons containing the 5’ untranslated region (5’UTR) were constructed by using the same method. Cotransfection of pCMVNCRluc containing HCV 5’UTR-luc fusion genes and eukaryotic vector of IRNA into human hepatic carcinoma cells (HepG2) was performed and the eukaryotic expression plasmid of IRNA was transfected transiently into HCV replicons. pCMVNCRluc or pCDNA-luc was cotransfected with pSV40-β Gal into IRNA expressing hepatoma cells by using lipofectamine 2000 in vitro. Then the reporting gene expression level was examined at 48 h after transfection by using a luminometer and the expressing level of HCV C antigen was analysed with a confocal microscope. RESULTS: Transient expression of IRES specific IRNA could significantly inhibit the expression of reporter gene and viral antigen mediated by HCV IRES by 50% to 90% in vivo, but mIRNA lost its inhibitory activity completely. The luciferase gene expression mediated by HCV IRES was blocked in the HHCC constitutively expressing IRNA. At 48 h after transfection, the expression level of reportor gene descreased by 20%, but cap-dependent luciferase gene expression was not affected. IRNA could inhibit the HCV replicon expression 24 h after transfection and the highest inhibitory activity was 80% by 72 h, and the inhibitory activity was not increased until 7d after transfection. CONCLUSION: IRNA can inhibit HCV IRES mediated gene expression in vivo.

AIM: To investigate the modification of baculovirus vector and the feasibility of delivering exogenous genes into mammalian cells with the culture supernatant of Spodoptera frugiperta (Sf9) cells infected by recombinant baculoviruses. METHODS: Two recombinant baculoviruses (BacV-CMV-EGFPA, BacV-CMV-EGFPB) containing CMV-EGFP expression cassette were constructed. HepG2 cells were directly incubated with the culture supernatant of Sf9 cells infected by recombinant baculoviruses, and reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM). The optimal transduction conditions were investigated by FCM assay in HepG2 cells. Gene-transfer and expression efficiencies in HepG2 or CV1 cells by baculovirus vectors were compared with lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Twenty different mammalian cell lines were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the culture supernatant of infected Sf9 cells. RESULTS: CMV promoter could directly express reporter genes in Sf9 cells with a relatively low efficiency. Target cells incubated with the 1:1 diluted culture supernatant (moi = 50) for 12 h at 37 °C could achieve the highest transduction and expression efficiencies with least impairment to cell viability. Under similar conditions the baculovirus vector could achieve the highest gene-transfer and expression efficiency than lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Most mammalian cell lines could be transduced with recombinant baculovirus. In primate adherent culture cells the recombinant baculovirus could arrive the highest infection and expression efficiencies, but it was not very satisfactory in the cell lines from mice and suspended culture cells. CONCLUSION: Mammalian cells incubated with the culture supernatant of infected Sf9 cells could serve as a very convenient way for rapid and efficient expression of foreign genes in mammalian cells, but it might be more suitable for primate adherent culture cells.

AIM: To construct a gene modified hepatocellular carcinoma (HCC) specific EGFP expression vector regulated by abbreviated cis-acting element of AFP gene. METHODS: The minimal essential DNA segments of AFP gene enhancer and promoter were synthesized through PCR from Genome DNA of HepG2 cells. Gene fragments were then cloned into the multiple cloning site of non-promoter EGFP vector pEGFP-1. Recombinant plasmid was transferred into positive or negative AFP cell lines by means of lipofectamine. The expression of EGFP was tested by fluorescence microscope and flow cytometry. The effect of all-trans retinoic acid (ATRA) on the expression of EGFP was tested in different concentrations. RESULTS: By the methods of restriction digestion and sequence analyses we confirmed that the length, position and orientation of inserted genes of cis-acting element of AFP were all correct. The transcription of EGFP was under the control of AFP cis-acting element. The expressing EGFP can only been detected in AFP producing hepatoma cells. The expression rate of EGFP in G418 screened cell line was 34.9% ± 4.1%. 48 h after adding 1 × 10-7 M retinoic acid, EGFP expression rate was 14.7% ± 3.5%. The activity of AFP gene promoter was significantly suppressed by addition of 1 × 10-7 M retinoic acid (P < 0.05, P = 0.003, t = 6.488). CONCLUSION: This recombinant expression vector can be used as a gene therapy vector for HCC. The expression of tumor killing gene will be confined within the site of tumor and the activity of which can be regulated by retinoic acid.

AIM: To investigate the expression level of ZNRD1 gene in gastric cancer cells SGC7901 and gastric cancer MDR (multidrug resistant) cells SGC7901/VCR, and to observe the drug sensitizing and proliferation effect of ZNRD1 antisense nucleic acid transduction on SGC7901/VCR cells. METHODS: Amplification of sequences encoding ZNRD1 from SGC7901/VCR cDNA by PCR. The levels of ZNRD1 mRNA expression were demonstrated using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Eukaryotic expression vector pcDNA3.1-anti ZNRD1 was constructed and transfected into SGC7901/VCR cells by lipofectamine. Immunochemical method was used to detect the expression of protein in SGC7901/VCR cells and transfectants. The cell cycle alteration and the intracellular adriamycin (ADM) accumulation were observed by FACS. Growth curve and drug sensitization of cells for vincristine (VCR) were analyzed with MTT assay. RESULTS: We cloned the open reading frame of full-length ZNRD1. The expression of ZNRD1 showed higher in SGC7901/VCR than in SGC7901 cells. The antisense ZNRD1 drug-resistant clones were selected after gene transfection. Immunochemical results showed that the expression level of ZNRD1 protein was lower in anti ZNRD1-SGC7901/VCR cells than that in non-transfectants. Comparing to SGC7901/VCR and pcDNA3.1-SGC7901/VCR, anti ZNRD1-SGC7901/VCR showed gradually accumulated in G1 phase, with a concomitant decrease of cell population in S phase. FACS also suggested intracellular ADM accumulation increased 2fold in SGC7901/VCR cells after transfected with antisense ZNRD1. MTT assay showed that transfectants cells proliferation was lagged and more sensitive to VCR than non-transfectants. CONCLUSION: ZNRD1 gene displayed highly expression in VCR resistant gastric cancer cells. Expression of ZNRD1 protein was effectively blocked in anti ZNRD1-SGC7901/VCR cells by gene transfection. ZNRD1 antisense nucleic acid transfection sensitized drug resistant gastric cancer cells to VCR, increased ADM accumulation and inhibited the cells proliferation. ZNRD1 antisense RNA transduction could reverse the MDR of human drug-resistant gastric cancer cell SGC7901/VCR to a degree.

AIM: To construct the recombinant pEgr-P16 plasmid for the investigation of its expression properties in esophageal squamous cell carcinoma induced by ionizing irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma. METHODS: The recombinant pEgr-P16 plasmid was constructed and transfected into EC9706 cells with lipofectamine. Western blot, quantitative RT-PCR and flow cytometry were performed to study the expression of pEgr-P16 in EC9706 cells and the biological characteristics of EC9706 cell line after transfection induced by ionizing irradiation. RESULTS: The eukaryotic expression vector pEgr-P16 was successfully constructed and transfected into EC9706 cells. The expression of P16 was significantly increased in the transfected cells after irradiation while the transfected cells were not induced by ionizing irradiation. The induction of apoptosis in transfection plus irradiation group was higher than that in plasmid alone or irradiation alone. CONCLUSION: The combination of pEgr-P16 and irradiation could significantly enhance the P16 expression property and markedly induce apoptosis in EC9706 cells. These results may lay an important experimental basis for gene radiotherapy for esophageal carcinoma.

AIM: To observe the growth suppression effect of exogenous introduction of early growth response gene-1 (Egr-1 gene) on esophageal carcinoma tissue as well as on esophageal carcinoma cell line Eca109 and to explore the potential application of Egr-1 gene in gene therapy of tumor. METHODS: Eukaryotic expression vector of PCMV-Egr-1 plasmid was introduced into Eca109 cell line which expressed no Egr-1 protein originally with lipofectamine transfection method. The introduction and expression of PCMV-Egr-1 plasmid into Eca109 cell line was confirmed by G418 selection culture, PCR amplification of neogene contained in the vector, Western blot analysis and immunocytochemical analysis. The cell growth curve, soft agar colony formation rate and tumorigenicity in SCID mice were examined to demonstrate the growth suppression effect of exogenous Egr-1 gene on Eca109 cell line. The Egr-1 mRNA and Egr-1 protein were also detected in 50 surgical specimens of esophageal carcinoma by in situ hybridization and immunohistochemistry. RESULTS: Exogenous Egr-1 gene was introduced successfully into Eca109 cell line and expressed Egr-1 protein stably. The transfected Eca109 cell line grew more slowly than control Eca109 as shown by cell growth curves, the soft agar colony formation rate (4.0% vs 6.9%, P < 0.01) and the average growth rate of tumor in SCID mice (35.5 ± 7.6 vs 65.8 ± 7.6, P < 0.05). The expression level of Egr-1 mRNA and protein significantly increased in dysplastic epithelia adjacent to cancer rather than in cancer tissues (65.8% vs 20.0% by ISH and 57.9% vs 14.0% by IHC, P < 0.01). CONCLUSION: Exogenous Egr-1 gene shows the strong effect of growth inhibition in Eca109 cell line. Egr-1 in the cancer tissue shows down-regulated expression that supports the inhibited function of Egr-1 in cancer growth and suggests Egr-1 may have an important role in gene therapy of esophageal carcinoma.