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Objective To explore the effects of zinc finger E-box binding protein (ZEB)2 3′UTR gene transfection on proliferation, invasion and migration in human gastric epithelial cell line GES-1. Methods The synthetic ZEB2 3′UTR and miR-200b micmics were transfected into GES-1 cell line by lipofectamine 2000. We set up control grop, the mutation group and ZEB2 3′UTR group. Real-time quantitative PCR was performed to evaluate the expression levels of miR-200a/b/c and ZEB1/ZEB2 mRNAs after transfection.And then we set up control group, ZEB2 3′UTR group, ZEB2 3′UTR+negative control group and ZEB2 3′UTR+miR-200b micmics group. The protein expression levels of ZEB1, ZEB2, matrix metallopro-teinases (MMP) 2/9 and proliferating cell nuclear antigen (PCNA) were detected by Western blot assay. The invasion and mi-gration capability were analyzed by transwell assay and wound healing test. MTT assay was used to detect the proliferation ability. Results Compared with control group and mutation group, the expressions of miR-200a/b/c were significantly de-creased, especially for miR-200b. And the expressions of ZEB1/ZEB2 were significantly increased at both mRNA and pro-tein levels after transfected with the ZEB2 3′UTR, enhancing the capability of migration,invasion,and proliferation (P <0.05). Compared with ZEB2 3′UTR group, the capabilities of proliferation,invasion and migration were significantly lower in combined group. Conclusion ZEB2 3′UTR can increase the ability of cell proliferation, invasion and metastasis through regulating the levels of miR-200a/b/c, and then influence the regulation of transcription of the target gene, which could lead to malignant transformation of GES-1 cells.
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To determine the effects of Von Hippel-Lindau (VHL) on the invasion and migration of glioma U251 cells. Methods:U251 GBM cells were transfected using VHL expression plasmid. Real-time polymerase chain reaction was conducted to de-tect VHL mRNA expression after transfection. Western blot assay was used to measure protein (VHL, MMP-2, and MMP-9) expres-sion. Tumor invasion and migration were examined by the Transwell and wound-healing experimental methods after VHL up-regula-tion. The intracranial model of nude mouse was developed using U251 cells transfected by VHL expression plasmid, and immunohisto-chemical staining was used to measure protein (VHL, MMP-2, and MMP-9) expression in the tissue sections. Results: In the U251 cells transfected by VHL expression plasmid, the expression of VHL mRNA and VHL proteins increased, and the expression of MMP-2 and MMP-9 protein decreased. Meanwhile, the invasion and migration of glioma U251 cells were also inhibited. Immunohistochemical staining results showed that the expression of MMP-2 and MMP-9 proteins decreased, and the VHL protein expression increased after transfection. Conclusion:VHL can inhibit the invasion and migration of glioma U251 cells. Thus, VHL gene can be used as a target for the gene therapy of gliomas.
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Objective:To explore the effect and mechanism of miRNA sponge on the epithelial-mesenchymal transition (EMT) of gastric carcinoma cell lines SGC7901. Methods:Synthetic ZEB2 3'UTR plasmid and siRNA targeting ZEB2 were transfected into the SGC7901 cell line by Lipofectamine 2000. Real-time quantitative polymerase chain reaction was performed to evaluate the expres-sion levels of miR-200a/b/c. Finally, the migratory, invasive, and proliferative activities of the gastric carcinoma cells in vitro were ana-lyzed by the scratch test, the Transwell cell invasion, and the cell cloning assay. The expression of the target protein was detected by Western blot. Results:Compared with the control group, the expressions of miR-200a/b/c significantly decreased, and their migration, invasion, and proliferation capabilities were considerably higher after they were transfected with ZEB2 3'UTR. Although the expres-sions of miR-200a/b/c significantly increased, the migratory, invasive, and proliferative activities of SGC7901 cells also degraded after they were transfected with siRNA targeting ZEB2. The expression of ZEB2 increased, and that of E-cadherin decreased at the protein level after they were transfected with ZEB2 3'UTR. The protein expression of Vimentin in SGC7901 cells significantly increased. The indicators show the opposite trend when cells were transfected with siZEB2, and the differences between the control and mutation groups were insignificant. Conclusion:ZEB2 3'UTR can regulate EMT course by regulating the miR-200a/b/c expression in gastric car-cinoma, consequently regulating the invasion and migration of carcinoma cells.
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Objective To investigate the relationship between phosphatidylinositol 3-kinase catalytic subunit α(PIK3CA) expression and the incidence and different pathological grade of gastric cancer, and the mechanism thereof. Meth-ods The expressions of PIK3CA, serine/threonine protein kinase (pAkt) and cell proliferation associated nuclear antigen (ki-67) in gastric carcinoma and adjacent tissues and normal gastric mucosa were detected by immunohistochemical method. The relationship between expressions of PIK3CA, pAkt and ki-67 and tumorigenesis was analyzed. The expressions of PIK3CA, pAkt and ki-67 in different pathological conditions of gastric tissues were analyzed. The relationship between tu-mor pathologic classification and differentiation were analyzed too. Results There were significantly higher expressions of PIK3CA, pAkt and ki-67 in gastric cancer (P＜0.05), which were the highest in the poorly differentiated gastric adenocarci-noma (P＜0.05). There were a positive correlation between expressions of PIK3CA, pAkt and ki-67 and different pathologi-cal levels of gastric carcinoma and adjacent tissues and normal gastric tissues (P＜0.05). Conclusion PIK3CA may be the initiating factor of PI3K/Akt signaling pathway, which induced phosphorylation of Akt and activation PI3K/Akt signaling pathway, promoting the proliferation, invasion and metastasis of gastric adenocarcinoma.
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There has been increasing evidence that transcription of non-coding genome sequence is important to life:Relative to the protein coding sequence and a variety of small molecule RNA.IncRNA research is still only in its infancy.Its function and regulation need to be of further studied.In this paper,the current relationship between the tumor and the IncRNA is reviewed.IncRNA may provide new basis and targets for cancer diagnosis and treatment.
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Objective Increasing evidence suggest that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer therapy. The study is to evaluate the effects of combined therapy of PI3K inhibitor (LY294002) and Ad-PTEN in athymic mice xenogeneic transplant model of human glioma and to reveal the possible mechanisms involved.Methods Twenty-four athymic mice were randomly divided into 4 groups (DMSO、Ad-vector plus DMSO、LY294002 alone and Ad-PTEN plus LY294002), and were treated, respectively. Athymic mice xenogeneic transplant model was established by inoculation (sc) with LN229 glioma cells. Body mass (BM) and diameter of tumor mass were measured. Furthermore, The protein expressions of PTEN、p-Akt、CyclinD1、Caspase-3、MMP-2、p-FAK in tumor tissues were analyzed with immunohistochemistry.Results The tumor-inhibiting rate of was significantly higher in Ad-PTEN plus LY294002 than in the LY294002 alone (92.46 vs 65.59%)( P <0.05).The protein expressions of PTEN and Caspase-3 were significantly higher, while PCNA、CyclinD1、bcl-2 and MMP-2、p-FAK was significantly lower in Ad-PTEN plus LY294002 group than in the other three groups ( P <0.05).Conclusions LY294002 plus Ad-PTEN achieve better outcome than either alone in treating glioma possibly through enhancement of the inhibitory action of PI3K/Akt pathway and Ad-PTEN pathway.
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Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting P85 and protein kinase B1 (PKB1/Akt1) and study its effects on the growth of SGC-7901 human gastric adenocareinoma cells. Methods P85 and Aktl shRNA expression frames were subcloned to pGSadeno adenovirus vector with homologous recombination technology to construct pGSadeno-P85 + Akt1 (rAd5-P + A) vector. After screening and amplification, the recombinant adenovirus vector was digested with PacI and transfected into SGC-7901 cells and then its titer and transfection efficiency were detected with fluorescent microscope. P85 and Akt1 mRNA protein expression was identified with real-time PCR and Western blot. The proliferative activity of tumor cells was evaluated with MTr assay and flow cytometry in vitro, rAd5-HK and rAd5-P + A mediated by adenovirus were injected into the established subcutancous SGC-7901 gastric adenocarcinoma in nude mice. During the observation period of 21 days, tumor volume was measured every 3 days to further testify the anti-tumor effect of rAd5-P + A on the SGC-7901 gastric adenocarcinoma cells and cell in situ apoptosis was detected with TUNEL assay. Results The adenovirus vector rAd5-P + A was successfully constructed and it dramatically downregulated P85 and Akt1 mRNA expression in SGC-7901 gastric adenocarcinoma cells. Compared with a control group of SGC-7901 cells and cells transfected with general adenovirus rAd5-HK as control, P85 and Akt1 protein expression 48 h and 72 h after rAd5-P + A transfection was decreased by 57.5% and 63. 7%, 67. 8% and 75.6% with statistical significance(P = 0. 005, P = 0. 003). Cell proliferative activity in rAd5-P + A transfected cells was suppressed from the second day (P <0. 001) and the decreased P85 and Akt1 expression was accompanied by 5.9% -7. 1% decrease of S phase fraction and 12. 1% - 13.7% increase of G0/G1 phase. The tumor volume of rAd5-P + A treated group was smaller than that of the control and rAd.5-HK group with statistical significance (F = 9. 871, P = 0. 025) . Moreover, rAd5-P + A could induce cell in situ apoptosis. Conclusions Adenovirus-mediated targeting P85 and Akt1 shRNA can inhibit the growth of SGC-7901 human gastric adenocarcinoma cells and this may provide a new strategy of combination gene therapy in gastric adenocarcinoma.
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cells, the expression of PTEN was up-regulated while pAkt down-regulated. Conclusions AS-miR-221 and AS-miR-222 may enhance the radiosensitivity of MCF-7 breast cancer cells by up-regulating the expression of PTEN.
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Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting Aktl (protein kinase B1, PKBI/Aktl) and cyclooxygenase-2 (COX-2) and study its effects on the invasion and metastasis of SGC-7901 human gastric adenocarcinoma cells. Methods Aktl and COX-2 shRNA expression frames were subcloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl+COX-2 (rAdS-A+C) vector. After screening and amplification, the recombinant adenovirus vector was digested with PacI and transfected into SGC-7901 cells, the titer and transfection efficiency were detected by fluorescent microscopy. Aktl and COX-2 mRNA and protein expression was identified by real-time PCR and Western blot. MMP-2 and MMP-9 contents in control group SGC-7901、rAd5-HK and treatment group rAdS-A+C were detected by ELISA assay and transwell assay analyzed cell invasion and metastasis ability. Results Adenovirus vector rAdS-A+C was successfully constructed and it dramatically down-regulated Aktl and COX-2 mRNA and protein expression in SGC-7901 gastric cancer cells. MMP-2 and MMP-9 contents in treatment group rAd5-A+C were respectively (39.7± 1.7) ng/ml, (31.3±3.6) ng/ml, and they were lower than those in control group SGC-7901 (278.4± 15.5) ng/ml, (225.4±15.1) ng/ml and rAd5-HK (275.5±2.1) ng/ml, (226.0±23.3) ng/ml (P= 0.01, P=0.021). Transwell assay showed treatment group rAd5-A+C significantly inhibited the invasion and metastasis of SGC-7901 gastric adenocacinoma cells. Conclusions Adenovirus-mediated targeting Aktl and COX-2 shRNA can inhibit the invasion and metastasis of SGC-7901 human gastric adenocarcinoma cells.
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<p><b>OBJECTIVE</b>To study the radiation-sensitizing effect of up-regulating p27(kip1) expression by knocking down miR-221/222 in the U251 human glioblastoma cell line.</p><p><b>METHODS</b>By bioinformatic analysis, we searched the miRNA-221/222 sequence and found the relationship between p27(kip1) and miRNA-221/222. miRNA-221/222 antisense oligonucleotides were transfected into U251 human glioblastoma cells. Northern blot analysis was conducted to detect the expression of miR-221/222 in control, scrambled oligonucleotide (ODN) transfected and anti-mi-221/222ODNs transfected cell groups. The cell cycle kinetics was detected by flow cytometry. Clonogenic assay was used to measure the mitotic cell death and p27(kip1) expression was examined by Western blot analysis.</p><p><b>RESULTS</b>Based on bioinformatic analysis, we found that the seed sequences of miR-221 and miR-222 coincide with each other, and p27(kip1) is a target for miRNA-221/222. The expression level of miR-221/222 was significantly knocked down in cells transfected with antimiR-221/222 as compared to the parental cells or cells transfected with scrambled ODN. Cell cycle was arrested in G0 or G1 phase in the anti-miR-221/222 group. When combined with irradiation, S phase fraction in the anti-miR-221/222 cell group is lower than that in the other two control groups. Anti-miR-221/222 combined with irradiation could synergistically enhance mitotic cell death. The expression of p27(kip1) was up regulated in the anti-miR-221/222 group revealed by Western blot analysis.</p><p><b>CONCLUSION</b>Anti-miR-221/222 may enhance the radiosensitivity of U251 human glioblastoma through upregulation of p27(kip1).</p>
Subject(s)
Humans , Base Sequence , Cell Cycle , Radiation Effects , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p27 , Genetics , Metabolism , Glioblastoma , Genetics , Metabolism , MicroRNAs , Genetics , Metabolism , Molecular Sequence Data , Oligonucleotides, Antisense , Genetics , Metabolism , Radiation Tolerance , Sequence Alignment , Up-Regulation , Radiation Effects , X-RaysABSTRACT
Objective To investigate abnormal protein expression of matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) in human gastric adenoearcinoma, and further reveal the clinical significance. Method The MMP-9, VEGF and PCNA proteins expression was determined by immunohistochemistry staining in 45 gastric adenocarcinoma tissues, 45 adjacent specimens and 10 normal gastric mucosa tissues via tissue arrays accordingly. The relationship of these protein expression with differentiation degree, development and progression of gastric adenocarcinoma were also analyzed. Results Positive rates of MMP-9, VEGF and PCNA in gastric adenocarcinoma, adjacent specimens and gastric normal mucosa were as follows: MMP-9, 82.2%(37/45), 64.4% (29/45), 30.0% (3/10) (P=0.019); VEGF, 73.3% (33/45), 62.2% (28/45), 30. 0% (3/10) (P=0.029); PCNA,84.4% (38/45), 71.1% (32/45), 10.0% (1/10) ,there were statistically significant difference (P = 0. 001). The positive rates of MMP-9, VEGF and PCNA in well-differentiated adenocarcinoma, moderately differentiated adenocarcinoma and poorly differentiated adenocarcinoma were as follows: MMP-9,70.0%(7/10), 80. 0% (8/10), 88.0%(22/25), there were statistically significant difference (P=0.015);VEGF, 50.0%(5/10), 60.0% (6/10), 88.0% (22/25), there were statistically significant difference (P =0.000);PCNA, 60.0% (6/10) ,90.0% (9/10) ,92.0% (23/25) ,the difference is significant statistically (P = 0.004). The expression of MMP-9, VEGF and PCNA showed positive relationship with each other by rank correlation analysis (P < 0. 05). Conclusion Tissue arrays technology is effective tool to analyze the expression of cancer related proteins in gastric adenocarcinoma. The expression of MMP-9, VEGF and PCNA proteins participates in the tumorigenesis and development process of gastric adenocarcinoma, and these can be used as indexes to evaluate prognosis in clinical.
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objective To observe the cyclooxygenase-2(COX-2)expression and the cell proliferation and apoptosis of gastric adenocarcinoma cells after transiently transfecting gastric cancer cells using specific COX-2 small interfering RNA(siRNA)and discuss the role of COX-2 in gastric tumorigenesis and the effect of RNA interference(RNAi)as anti-cancer gene therapy.Methods Three groups were included in the study,i.e.a COX-2 siRNA group,a non-sense siRNA group and a control group.Gastric adenocarcinoma cells SGC7901 were cuhured at 37℃ in an atmosphere containing 5%CO2.72 hours after transfecting SGC7901 cells with specific COX-2 siRNA or non-sense siRNA,RT-PCR was used to detect COX-2 mRNA,immunohistochemistry and Western blot were taken to detect the expression of COX-2 protein and flow cytometry was taken to detect the cell cycle and apoptosis.MTT method was used to detect the proliferation and activity of the cells every day for one week after transfection.Results The expression of COX-2 mRNA and protein in the COX-2 siRNA group was obviously suppressed as compared with non-sense siRNA group and control group.No change was found between the non-sense siRNA group and the control group.The reduced expression of COX-2 could inhibit SGC7901 cells proliferation and induce cell apoptosis,but had no effect on cancer cell cycle.Conclusions The experimental results suggest that effectively inhibiting the expression of COX-2 can suppress the proliferation of gastric adenocarcinoma cell and promote the process of cancer cell apoptosis.so RNAi is a powerful tool of gene therapeutic method.
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<p><b>OBJECTIVE</b>To study the anti-invasion effect of SEPT7 gene on U251MG glioma cells and its possible molecular mechanism.</p><p><b>METHODS</b>Recombinant adenovirus vector carrying SEPT7 gene (rAd5-SEPT7) was transduced to human glioma cell line U251MG, and empty adenovirus vector was used as control. Tumor invasion was examined by Transwell method and 3 D-Matrigel assay, and tumor cell migration by wound-healing method and 2 D-Matrigel assay. Three major molecular events associated with cell motility and migration, including changes of expression in MMP2, MMP9, MT1-MMP, TIMP1 and TIMP2, the alteration of integrin alpha(v)beta(3) expression, and the structural change of cytoskeleton protein, tubulin-alpha, in U251 cells transduced with rAd5-SEPT7 were studied by Western blotting, immunofluorescence and laser scanning confocal microscope, respectively.</p><p><b>RESULTS</b>The invasive and migratory capabilities of cells transduced with rAd5-SEPT7 were inhibited. The expression of extracellular matrix metalloproteinases MMP-2, MMP-9, MT1-MMP and integrin alpha(v)beta(3) was significantly decreased, while the expression of matrix metalloproteinase inhibitor TIMP1, TIMP2 was upregulated. Intracellular cytoskeleton protein-tubulin-alpha in U251 cells exhibited prominent morphological changes which including the appearance of distortion and aggregation resulting from redistribution of tubulin-alpha, and this feature of alteration was similar to the tubulin-alpha structure in normal non-tumor cells.</p><p><b>CONCLUSION</b>SEPT7 gene can inhibit the invasion and migration ability of U251 glioma cells. Its molecular mechanism may include that SEPT7 gene reverses the imbalanced state of MMPs/TIMPs, downregulates the expression of integrin alpha(v)beta(3) and alters the structure of tubulin-alpha of U251MG glioma cells. It is suggested that SEPT7 gene could be a good candidate for gene therapy of gliomas.</p>
Subject(s)
Humans , Adenoviridae , Genetics , Blotting, Western , Cell Cycle Proteins , Genetics , Physiology , Cell Line, Tumor , Cell Movement , Genetics , Genetic Vectors , Genetics , Glioma , Metabolism , Pathology , Integrin alphaVbeta3 , Metabolism , Matrix Metalloproteinase 14 , Metabolism , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Microscopy, Confocal , Neoplasm Invasiveness , Genetics , Septins , Tissue Inhibitor of Metalloproteinase-1 , Metabolism , Tissue Inhibitor of Metalloproteinase-2 , MetabolismABSTRACT
Background Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to have the ability of migrating after transplantation into rat model of traumatic brain injury. In this study, MSCs'characteristic of tropism for intracranial glioma was explored following being labeled and transplanted into brain and blood of gliomabearing rats.Methods Cellulae medullares were cultured to get pure MSCs. The cell surface antigen and cell cycle of MSCs were detected to confirm their identity by flow cytometry. MSCs were marked with BrdU and then were injected into contralateral brain or collateral internal carotid artery of rats bearing glioma. 2 weeks later, the brains were resected and pathological sections were made. Immunohistochemistry and immunofluorescence technology were performed to detect MSCs.Results MSCs displayed extensive tropism for intracranial glioma. MSCs which were injected in the contralateral brain of the glioma scattered densely in the juncture between brain tissue and tumor and there were a few MSCs in the glioma; MSCs which were injected into the collateral internal carotid artery scattered widespread in the glioma and there were a few MSCs in the juncture area.Conclusions MSCs have the ability to migrating toward glioma and penetrating blood brain barrier. MSCs may be ideal gene therapy vehicles against glioma.
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<p><b>OBJECTIVE</b>To construct the small interfering RNA (siRNA) expression constructs targeting epidermal growth factor receptor(EGFR) and express them in TJ905 human malignant cells.</p><p><b>METHODS</b>Two target sequences from Receptor L domain and catalytic domain were selected to create two expression constructs using psiRNA-NeoG2. Furthermore, the siRNA constructs were transfected into TJ905 cells as mediated by Lipofectamin. Meanwhile, an antisense EGFR construct p-anti-hEGFR was set as control. Immunofluorescence and Western blot were performed to detect EGFR expression.</p><p><b>RESULTS</b>With the successful construction of the two siRNA expression plasmids and the stable transfection to TJ905 cells, the expression of EGFR was down-regulated to 90% and 92% respectively, but to 82% in the anti-sense EGFR group.</p><p><b>CONCLUSION</b>The siRNA expression constructs targeting EGFR could specifically inhibit EGFR expression, and should be a new strategy in glioma gene therapy targeting EGFR.</p>
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Humans , Blotting, Western , Cell Line, Tumor , Fluorescent Antibody Technique , Models, Genetic , Plasmids , Genetics , RNA Interference , RNA, Small Interfering , Genetics , ErbB Receptors , Genetics , Metabolism , Transfection , MethodsABSTRACT
<p><b>OBJECTIVE</b>To study the gene expression profiles of oligodendrogliomas with gene cDNA array.</p><p><b>METHODS</b>(32)P tagged cDNA probes converted from the total RNA, which had been extracted from 2 fresh samples of oligodendroglioma and 1 of normal brain tissue, were hybridized with the Atlas array. After washing the membranes, the autoradiography was performed and the autoradiograms were analyzed through the special software.</p><p><b>RESULTS</b>As compared to the normal brain tissue, there were 63 co-upregulated genes and 4 co-downregulated genes in these 2 tumor samples. However, a significant quantitative difference existed between them. The expression trend of some genes differed from the known information.</p><p><b>CONCLUSION</b>cDNA array is effective for studying the gene expression profiles of oligodendrogliomas and provides new information for the further research on their molecular mechanisms.</p>