Effect of PTEN gene overexpression on cell cycle and tumor invasion in a human ovarian carcinoma SKOV3 cell line

AIM:To investigate the role of phosphatase and tensin homology deleted on chromosome(PTEN) gene in the cell cycle and invasion ability of human ovarian carcinoma SKOV3 cell line in vitro. METHODS:Human ovarian carcinoma SKOV3 cells were transfected with a eukaryotic expression plasmid vector containing PTEN gene in vitro,and then the positive cell clones were selected and amplified. MTT method was used to observe the inhibitory rate,flow cytometry was used to detect the cycle of transfected PTEN cells and apoptosis level. Western blotting analysis was used to determine PTEN gene expression. The invasiveness of transfected cells were measured quantitatively by Matrigel invasion assays (Transwell chamber). RESULTS:The expression of PTEN mRNA in SKOV3 cells increased after transfection with PTEN gene. Flow cytometry showed that the percentage of cells in S phase increased,but that in G₂/M phase decreased. Invasiveness of SKOV3 was significantly decreased. CONCLUSION:The transfection of PTEN gene into SKOV3 cells can inhibit human ovarian carcinoma SKOV3 cell proliferation,invasion and induce SKOV3 cell apoptosis.     

Diversity of angiogenesis in ovarian carcinoma

The growth and spread of malignant neoplasms largely depend on angiogenesis. Recent studies demonstrate that a various types of neovascularization including angiogenesis, vasculogenesis and vasculogenic mimicry exist in a few highly aggressive tumors. Ovarian carcinoma is the leading cause of death in gynecologic malignancy. Here, we review the effect of angiogenesis, vasculogenesis and vasculogenic mimicry in development and spread of ovarian carcinoma.     

Inhibitory effects of recombinant human Mullerian inhibiting substance on cell proliferation in two human ovarian carcinoma cell lines

AIM: To investigate the inhibitory effects of recombinant human Mullerian inhibiting substance on cell proliferation in human ovarian carcinoma cells (OVCAR8 and SKOV3 cell lines). METHODS: The expression of MISIIR protein and the localization of MISIIR protein were analyzed by Western blotting and confocal spectral microscopy, respectively. Cell apoptosis and cell cycle were detected by flow cytometry (FCM). Cell viability was determined via MTT method. Clone formation test was used to detect oncogenicity in vitro.RESULTS: The MISIIR protein expression in OVCAR8 cells but not in SKOV3 cells was observed. MISIIR expression was seen on the OVCAR8 cell surface and in the cytoplasm with both antibodies. After treated with rhMIS for 48 h, the cell viability was significantly decreased in OVCAR8 cells. rhMIS inhibited the oncogenicity of OVCAR8 cells greatly. The cell apoptosis of OVCAR8 cell exposed to 10 mg/L rhMIS was (31.3±2.1)%, and OVCAR8 cells in the G1 phase were increased by (70.4±3.0)%. Compared to SKOV3 cells the differences were significant (P<0.01). CONCLUSION: Recombinant human Mullerian inhibiting substance suppresses the growth of MISIIR-positive ovarian cancer cells by inducing apoptosis and cell cycle arrest. We predict that rhMIS might be a new target to treat human ovarian malignancies.     

Effect of 3 isoforms of Ikaros on proliferation of human ovarian cancer SKOV3 cells

AIM: To investigate the effect of Ikaros isoforms on the proliferation of human ovarian cancer SKOV3 cells. METHODS: Three isoforms of Ikaros, IK1, IK2 and IK6, were transfected into ovarian cancer SKOV3 cells. CCK-8 assay and cell counting were used to detect the effects of Ikaros isoforms on the proliferation of SKOV3 cells. The cell cycle was analyzed by flow cytometry. The cell cycle-related proteins were detected by Western blot. RESULTS: IK1 and IK2 expression inhibited SKOV3 cells proliferation. Flow cytometry analysis indicated that IK1 and IK2 induced SKOV3 cell cycle arrest at the G₁ phase. IK6 isoform exerted no obvious effect on the proliferation or cell cycle of SKOV3 cells. Compared with control EV group, IK1 group and IK2 group showed a dramatic elevation in the expression of the cell cycle inhibitor p21, along with a substantial decrease in the expression of the cell cycle inducers cyclin D1 and cyclin D2, which did not change in IK6 group. CONCLUSION: IK1 and IK2 significantly inhibit the proliferation of ovarian cancer SKOV3 cells and induce cell cycle arrest at G₁ phase by regulation of cell cycle-related proteins cyclin D1, cyclin D2 and p21, while IK6 isoform exerts no obvious effect on the proliferation and cell cycle of SKOV3 cells.     

Overexpression of N-terminal amino acids of p55γ regulatory subunit of PI3K inhibits cell adhesion of human gastric carcinoma MGC803 cells

AIM: To investigate the effect of the N-terminal 24-amino acid(N24) overexpression in p55γ re-gulatory subunit of phosphatidylinositiol 3-kinase(PI3K) on the cell adhesion of human gastric carcinoma cell MGC803.METHODS: MGC803 cells, which stably expressed GFP-N24 fusion protein and GFP alone, were generated by transfection with pEGFPN-24 plasmid and control plasmid pEGFP-C1, respectively. The morphological change of the cells was observed under inverted microscope. The expression of GFP-N24 fusion protein was detected by Western blot. The adhesion of the cells was determined by cell adhesion assay. The effects of GFP-N24 fusion protein on the expression of E-cadherin and β-catenin were analyzed by Western blot. The expression and secretion of matrix metalloproteinase 9(MMP9) and urokinase-type plasminogen activator(uPA) in the MGC803 cells were detected by gelatin zymography.RESULTS: MGC803/GFP-N24 cell line steadily expressed GFP-N24 fusion protein and MGC803/GFP cell line steadily expressing GFP were successfully established, but the expression of fusion protein GFP-N24 was lower than that of the control protein GFP. The morphological changes of the transfected cells from paving stone to fibroblast cell form after gene transfection, and the cytoplasm secretory granules were increased significantly. The cell adhesion to fibronectin and collagen decreased after GFP-N24 transfection. GFP-N24 fusion protein increased the expression of cell adhesion molecule E-cadherin and decreased the wnt signal pathway molecule β-catenin in the MGC803 cells. However, it did not affect the expression and secretion of tumor metastasis-related proteins MMP9 and uPA.CONCLUSION: Overexpression of N24p55γ inhibits cell adhesion by influencing the expression of E-cadherin and β-catenin in the MGC803 cells.     

Effect of quercetin on proliferation of human ovarian cancer SKOV-3 cells

AIM：To study the inhibitory effect of quercetin on the proliferation and apoptosis of human ovarian cancer SKOV-3 cells. METHODS：SKOV-3 cells were treated with different doses of quercetin. The inhibitory effect of quercetin on the proliferation was detected by MTT assay. The cell apoptosis was determined by immunocytochemistry. The cell cycle and apoptosis were assessed by flow cytometry. RESULTS：Quercetin inhibited the proliferation of ovarian cancer SKOV-3 cells in a time- and dose-dependent manner. Quercetin induced apoptosis of SKOV-3 cells. The proportion of S phase and the apoptotic rate were significantly increased, and the proportion of G2/M phase was reduced after treatment with quercetin. CONCLUSION：Quercetin inhibits the growth of ovarian cancer SKOV-3 cells in vitro, and promotes apoptosis through S phase arrest.     

Expression of Glypican-3 gene and its regulating mechanism in hepatocellular carcinoma

AIM:To investigate the expression of Glypican-3 gene and its regulating mechanism in hepatocellular carcinoma. METHODS:The expression of GPC3 mRNA and its gene mutation in 48 hepatocellular carcinoma tissues, 39 paracarcinomatous tissues and 31 normal liver tissues were detected by using RT-PCR and PCR-SSCP. The expressions of GPC3 protein, P53 and PCNA protein were detected by using immunohistochemistry S-P. RESULTS:The positive expressive rate of GPC3 mRNA was 77.1% in hepatocellular carcinoma tissue. No expression of GPC3 mRNA in paracarcinomatous tissues and normal liver tissues was observed. No gene mutation of GPC3 in hepatocellular carcinoma tissue was found. No correlation was found between GPC3 and P53 (r=-0.12574, P>0.05). The mean index of proliferating cell nuclear antigen(PCNA) in positive and negative GPC3 expression were (46.32±27.54)% and (39.83±21.47)%, respectively (P>0.05). CONCLUSION:The high expression of GPC3 mRNA in hepatocellular carcinoma is independent to the gene mutation, and to the expression of P53 and PCNA protein.     

Effect of antisense VEGF gene on VEGF expression in human renal cell carcinoma

AIM: To construct eukaryotic expression vector carrying human antisense VEGF gene and to study its effect on VEGF expression and growth of renal cell carcinoma. METHODS: VEGF gene was cloned. Antisense VEGF gene was inserted into eukaryotic expression vector pcDNA3.1(-), then using restrict enzyme to confirm the result. The vector was transfected into renal cell carcinoma and positive clone was selected by using G418. The VEGF expression was detected by using RT-PCR and immunohistochemical method. The growth of cells was measured by MTT and cell cycle by FCM. RESULTS: Antisense VEGF gene eukaryotic expression vector was successfully constructed. Compared with empty vector group and control group, the amount of VEGF mRNA expression and VEGF protein were decreased in the antisense VEGF group, the difference was significant. There was no significant difference between empty vector group and control group. The cell growth in the antisense VEGF group became slower, and the percent of G1 phase increased and the percent of S phase decreased. CONCLUSION: Antisense VEGF gene decreases the VEGF mRNA and protein expression in renal cell carcinoma and inhibits the growth of renal carcinoma. Antisense VEGF gene may be used in gene therapy of renal carcinoma.     

Resveratrol induces apoptosis in human ovarian cancer SKOV3 cells via Sirt3-SOD2-ROS pathway

AIM: To investigate whetier resveratrol induces apoptosis of human ovarian cancer SKOV3 cells through Sirt3-SOD2-ROS pathway. METHODS: SKOV3 cells were cultured in vitro and treated with resveratrol at 0, 2.5, 5, 10, 20, 40 and 80 mg/L for 24 h. The inhibitory effect of resveratrol on the viability of SKOV3 cells was measured by MTT assay. SKOV3 cells were randomly divided into blank control group, 10 mg/L resveratrol group, 20 mg/L resveratrol group and 40 mg/L resveratrol group. After 24 h of treatment, Hoechst 33342 staining and confocal microscopy were used to observe the nuclear changes. The protein levels of silent mating type information regulation 2 homolog 3 (Sirt3), superoxide dismutase 2 (SOD2), Bcl-2, Bax and cleaved caspase-3 were determined by Western blot. RESULTS: Treatment with resveratrol at 2.5, 5, 10, 20, 40 and 80 mg/L for 24 h significantly reduced the viability of SKOV3 cells. The observation by confocal microscopy showed that the nucleus of SKOV3 cells was markedly condensed and heavily stained with the increase in the concentration of resveratrol. Compared with blank control group, the red fluorescence intensity of ROS in different concentrations of resveratrol groups was significantly reduced. The results of Western blot showed that the protein levels of Sirt3, SOD2, Bax and cleaved caspase-3 in resveratrol groups were significantly higher than those in control group, while the protein expression of Bcl-2 was significantly lower than that in control group (P<0.05). CONCLUSION: Resveratrol induces apoptosis of SKOV3 cells by regulating Sirt3-SOD2-ROS pathway.     

Effect of berberine on endoplasmic reticulum stress-autophagy pathway in human ovarian cancer SKOV3 cells

AIM: To investigate the regulatory effect of berberine on the endoplasmic reticulum stress-auto-phagy pathway in human ovarian cancer SKOV3 cells. METHODS: Human ovarian cancer SKOV3 cells were cultured in vitro, and berberine at doses of 12.5, 25, 50, 100, 200 and 400 μmol/L were added. After exposure for 12 h, 24 h and 48 h, the viability of the SKOV3 cells was measured by MTT assay. The cells were divided into control group, berberine (50 μmol/L) group, berberine (100 μmol/L) group, and berberine (200 μmol/L) group. After treatment with berberine for 24 h, the effects of berberine on the morphological changes of SKOV3 cells were observed under inverted phase-contrast microscope. The protein expression of microtubule-associated protein 1 light chain 3 (LC3) and ubiquitin-binding protein p62 was observed by indirect immunofluorescence method under laser confocal microscope. The protein expression of beclin-1,LC3,p62, CCAAT/lenhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) was determined by Western blot. RESULTS: Berberine at 12.5, 25, 50, 100, 200 and 400 μmol/L significantly decreased the viability of SKOV3 cells at 12 h, 24 h and 48 h, and the IC₅₀ values of 12 h, 24 h and 48 h were (764.7±0.3) μmol/L, (231.6±0.1) μmol/L and (96.2±0.1) μmol/L, respectively. Laser confocal microscopy showed that the LC3 and p62 proteins were scattered and the fluorescence intensity was increased, while the point-like aggregation was also observed. Berberine at 200 μmol/L obviously enhanced the co-localization of LC3 and p62 proteins. Compared with control group, the expression of endoplasmic reticulum stress-related proteins GRP78 and CHOP, and autophagy-related proteins beclin-1, LC3 and p62 in berberine (200 μmol/L) group was increased significantly (P<0.05). CONCLUSION: Berberine may promote endoplasmic reticulum stress in SKOV3 cells by regulating autophagy.     

Effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells

AIM: To observe the effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells. METHODS: Both hepatocellular carcinoma cells (H2M) and epidermoid carcinoma cells (A431) with methylation of TIMP-3 promoter gene were treated with 5-Aza-2'-deoxycytidine (5Aza-dc). Invasion ability and motility of the cells were detected by Transwell experiments. Expressions of TIMP-3 protein and mRNA were detected by Western blotting and RT-PCR, respectively. TIMP-3 gene promoter methylation was detected by methylation-specific PCR (MSP). RESULTS: ① Invasion ability and motility of H2M and A431 cells were declined after treatment with 5Aza-dc; ② After treatment with 5Aza-dc, the expression of TIMP-3 protein and mRNA were increased in H2M and A431 cells; ③ After treatment with 5Aza-dc, methylation of TIMP-3 promoter gene was not detectable in the cell lines. CONCLUSIONS: 5Aza-dc induces demethylation in TIMP-3 promoter gene, restores TIMP-3 gene and protein expression, and inhibits invasion ability and motility of the carcinoma cells.     

Cholesterol depletion up-regulates expression of prohibitin in human prostate carcinoma PC-3 cells

AIM: To study how cholesterol delpetion affects prohibitin expression and the development of prostate cancer. METHODS: Human prostate carcinoma PC-3 cells were cultured in normal medium (NM) and cholesterol delpetion medium (CDM) for 48 h.The mRNA expression of prohibitin was detected by real-time PCR. Prohibitin promoter was cloned and inserted into PGL3-Basic to reconstruct plasmid. The plasmid was transiently transfected into PC-3 cells. The cells were cultured in the medium with different concentrations of cholesterol for 48 h and luciferase expression was detected.RESULTS: The results of real-time PCR showed that the mRNA level of prohibitin increased about 19 times in PC-3 cells in the presence of CDM. After transfected with prohibitin promoter plasmid for 48 h, PC-3 cells cultured in CDM had higher luciferase expression than the cells cultured in NM or in CDM with cholesterol. CONCLUSION: Cholesterol depletion up-regulates prohibitin expression in PC-3 cells, which may be one of the self-prophylactic and regulatory mechanisms to protect PC-3 cells from apoptosis caused by cholesterol insufficiency.     

Sinomenine inhibits viability,migration and invasion of human ovarian cancer SKOV3 cells

AIM:To investigate the effect of sinomenine on the viability, migration and invasion of human ovarian cancer SKOV3 cells and its possible mechanism. METHODS:The SKOV3 cells were treated with sinomenine at different concentrations for 12 h, 24 h and 48 h. CCK-8 assay was employed to detect the effects of sinomenine on the viability of the SKOV3 cells. Flow cytometry was used to analyze the cell cycle distribution. The cell migration and invasion abilities were measured by Transwell assay. Western blot was used to determine the protein levels of cyclin A, cyclin D1, E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS:Sinomenine remarkably inhibited the viability of SKOV3 cells and IOSE80 cells in a time-dependent and dose-dependent manner (P<0.05), and the IC₅₀ values of 48 h were 2.12 mmol/L and 17.35 mmol/L, respectively. In a dose-dependent manner, sinomenine induced G₀/G₁ and S phase arrest in SKOV3 cells (P<0.05), suppressed the migration and invasion abilities of SKOV3 cells (P<0.05), down-regulated the protein levels of cyclin A, cyclin D1 and MMP-9 (P<0.05), and up-regulated the protein level of E-cadherin (P<0.05). CONCLUSION:Sinomenine inhibits the viability, migration and invasion of human ovarian cancer SKOV3 cells most likely via down-regulation of the protein levels of cyclin A, cyclin D1 and MMP-9, and up-regulation of the protein level of E-cadherin.     

Progress in pulmonary carcinoma gene therapy

The high morbidity and mortality of pulmonary carcinoma, especially in male patient, are still the unsolved problems. Recently, more and more reports showed that gene therapy could have potential application in the treatment of the disease. In this article, some progresses in the gene therapy of pulmonary carcinoma, such as tumor suppressor gene, drug sensitive gene, antisense gene, multi-drug resistance gene, immune gene as well as anti-angiogenesis gene, were discussed.     

Effect of biological clock gene Timeless silencing on apoptosis and invasion of ovarian cancer SKOV3 cells

AIM:To evaluate the effect of biological clock gene Timeless (TIM) silencing on the apoptosis and invasion ability of human ovarian cancer SKOV3 cells. METHODS:The protein expression of TIM in the ovarian cancer tissues and normal ovarian tissues was detected by immunohistochemistry, and the correlation between the protein expression of TIM in ovarian cancer tissues and the pathological features was analyzed. The ovarian cancer SKOV3 cells were transfected with PBS (blank control group), control siRNA (siRNA control group) or TIM siRNA (TIM siRNA group). The protein expression of TIM, Bcl-2, Bax, MMP-2, MMP-9, caspase-3 and caspase-9 was determined by Western blot. The apoptosis was detected by flow cytometry. The invasion ability was measured by Transwell chamber test. RESULTS:The positive expression rate of TIM in the ovarian cancer tissues (84.0%) was significantly higher than that in the normal ovarian tissues (10.0%; P<0.01). TIM expression was associated with ovarian cancer differentiation, depth of invasion, lymph node metastasis and TNM stage (P<0.05), but was not associated with age and pathological type (P>0.05). The protein expression levels of TIM, MMP-2, MMP-9 and Bcl-2 in TIM siRNA group were significantly decreased as compared with control group and siRNA control group (P<0.01), and the protein expression of Bax, caspase-3 and caspase-9 in TIM siRNA group was significantly increased as compared with blank control group and siRNA control group (P<0.01). No significant difference of the protein expression of TIM, MMP-2, MMP-9, Bcl-2, Bax, caspase-3 and caspase-9 between blank control group and siRNA control group was observed (P>0.05). The apoptotic rate in TIM siRNA group was significantly higher than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). The penetrated cell number in TIM siRNA group was significantly less than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). CONCLUSION:Silencing of TIM gene in ovarian cancer SKOV3 cells by siRNA promotes apoptosis, and inhibits cell invasion.     

Effects of recombinant human CD40 ligand on biological behavior of ovarian cancer SKOV3 cells in vitro

AIM: To investigate the effect of recombinated human CD40 ligand (rhCD40L) on the biological behavior of ovarian cancer SKOV3 cell line in vitro.METHODS: After the SKOV3 cells were incubated with different concentrations of rhCD40L for various times, the cell proliferation was determined by MTT assay.The expression of the co-stimulatory molecules or adhesion molecules on SKOV3 cells and the changes of tumor necrosis factor receptor associated factor (TRAFs) inside the cells were measured by flow cytometry and direct immunofluorescence.Annexin V and PI dual color label assay were used to detect cell apoptosis or death in culture contained with rhCD40L.RT-PCR assay was employed to determine the change of apoptosis related gene c-myc, bcl-2 and bcl-xl expression in SKOV3 cells.RESULTS: rhCD40L inhibited proliferation of SKOV3 cells at concentration of 100 μg/L (0.65±0.10 vs 0.81±0.05) and reached a peak at concentration of 10 mg/L (0.13±0.12 vs 0.83±0.15, P＜0.01).The inhibitory effects showed a dose dependent manner.Cell cycle analysis showed that cell division was blocked in G1 phase.Increasing proportion of apoptosis of SKOV3 cells was related to up-regulation of CD95 expression (42.4% vs 59.2%, P＜0.05) and down-regulation of anti-apoptosis genes such as bcl-2 and bcl-xl expressions after incubation with rhCD40L.TRAF 2, 5 and 6 expressed highly in SKOV3 cells.The expression of TRAF 2 (81.3%±9.2% vs 50.4%±5.3%，P＜0.05), TRAF5 (47.2%±7.2% vs 7.2%±2.1%, P＜0.01) and TRAF6 (44.5%±6.3% vs 5.1%±1.1%, P＜0.01) was down-regulated and expression of TRAF 3 (25.2%±6.2% vs 68.8%±5.3%, P＜0.01) was up-regulated after co-culture with rhCD40L, but there was no effects found on the expression of TRAF 1 (4.3%±1.2% vs 5.1%±1.4%) and TRAF4 (7.4%±1.2% vs 8.1%±1.4%).CONCLUSION: By down-regulating expression of bcl-2, bcl-xl and changing expression profile of TRAF, rhCD40L inhibits the growth of SKOV3 cells by blocking the cell cycle progress in G1 and promotes the cells to apoptosis.     

Overexpression of TAP1 up-regulates HLA-Ι in human glioma U251 cells

AIM：To investigate the relationship between the overexpression of transporter associated with antigen processing 1 (TAP1) and the human leukocyte antigen I (HLA-I).METHODS：The full length of TAP1 gene was obtained from the cDNA library. The lentiviral vector pSIN-EF2-IRES-GFP-puro was digested by BamH I and EcoR I, and the full length of TAP1 gene was inserted into the vector by T4 DNA ligase. Subsequently, the recombinant plasmid was transformed into Escherichia coli DH5α cells and the correct transformant was selected. The recombinant plasmid and the Lenti-X HTX packaging mixture were co-transfected into 293T cells, and the virus particle was acquired. Human glioma U251 cells were transfected with the lentivirus. The expression of TAP1 and HLA-I was determined by real-time fluorescence quantitative PCR, Western blotting and flow cytometric analysis.RESULTS：TAP1 gene was successfully transfected into the U251 cells and stably expressed in the cell line. The expression of TAP1 in U251 cells at mRNA and protein levels increased by (8.73±1.07) and (11.71±0.83) folds, respectively. As a result, the mRNA expression of HLA-A, HLA-B, HLA-C (heavy chain) and β2-microglobulin (light chain) was up-regulated by (3.51±0.36), (4.78±0.85), (2.94±0.28) and (3.23±0.24) folds, respectively. The protein expression of HLA-I also increased to (3.14±0.53) fold. The surface expression of HLA-I on the U251 cells transfected with TAP1 gene was largely enhanced as well.CONCLUSION：Overexpression of TAP1 up-regulates the expression of HLA-I. TAP1 plays an important role in HLA-I processing pathway.     

Construction of a lentiviral vector for RNA interference of human EGFL7 gene and its stable expression in human laryngeal carcinoma cells

AIM：To investigate the role of epidermal growth factor-like domain 7 (EGFL7) in the pathogenesis and progress of laryngeal carcinoma via constructing a lentiviral expression vector for RNA interference (RNAi) of human EGFL7 gene and assessing the gene-silencing effect of the vector in human laryngeal epidermoid carcinoma (HEp-2) cells. METHODS：Specific RNAi target sequences were designed focused on human EGFL7 gene sequence. The double-stranded oligonucleotides were cloned into the pcDNA6.2-GW/EmGFP-miR plasmid after synthesis and annealing. A positive clone was subcloned into the pLenti6.3-MCS/V5-DEST vector after sequence analysis. The recombinant lentivirus was harvested from 293T cells co-transfected with the positive recombinant plasmid and lentiviral packing materials. HEp-2 cells were infected with the recombinant lentivirus and the cells with stable EGFL7 knockdown were screened by blasticidin selection. EGFL7 mRNA expression in the cells was determined by real-time fluorescence quantitative PCR. RESULTS：A recombinant lentiviral vector expressing short hairpin RNAs (shRNAs) against EGFL7 gene was obtained and confirmed by DNA sequencing. The virus titer was 5×1011 TU/L, and the silencing efficiency was 97%. CONCLUSION：A lentiviral vector targeting human EGFL7 gene, capable of stable EGFL7 gene knockdown in HEp-2 cells, has been successfully constructed, which provides a basis for further study of the relationship between human laryngeal carcinoma and EGFL7 protein.     

Effect of bFGF on glucose regulated protein 78 expression and apoptosis in human ovarian cancer CAOV3 cells

AIM: To evaluate the effect of bFGF on the apoptosis and the expression of glucose regulated protein 78 (GRP78) in ovarian cancer CAOV3 cells.METHODS: Starvation induced cell apoptosis was conducted. After treatment with bFGF, the cell cycle and apoptosis were determined by FACS analysis and Annexin V/PI staining, respectively. The expression of protein kinase B (PKB) and GRP78 were detected by Western blotting and RT-PCR. RESULTS: Compared to starvation group, the cells treated with bFGF were still viable and increased activation of PKB and high expression of GRP78 were observed, which were prevented by PKB inhibitor wortmannin effectively (P<0.01). CONCLUSION: bFGF plays a critical role in anti-apoptosis and proliferation in human ovarian cancer through PKB signal transduction pathway, partly by upregulating the expression of GRP78.