Transfection and expression of murine interleukin-12 gene in B16F10 melanoma cells

AIM:To study the expression of murine interleukin-12(mIL-12) gene in B16F10 melanoma cells.METHODS:mIL-12 gene was inserted into pcDNA3.1 eukaryotic expression vector by DNA recombination and then transfected into B16F10 melanoma cells by electroporation. The integration and expression of mIL-12 gene in B16F10 melanoma cells were detected by PCR, RT-PCR and Western blot after positive cell clones had been screened.RESULTS:mIL-12 gene was confirmed to have been transfected and expressed in B16F10 cells on three levels of DNA, mRNA and protein.CONCLUSION:mIL-12 gene can successfully be transfected and expressed in B16F10 melanoma cells in vitro, which lays a foundation for the further investigation of mIL-12 gene tumor vaccine.     

Inhibition of ICAM-1 expression on endothelial cells in hypoxia/reoxygenation by antisense oligodeoxynucleotides

AIM: To investigate the effect of antisense oligodeoxynucleotides (AS-ODN) on the intercellsular adhesion molecule-1(ICAM-1) expression on endothelial cells in hypoxia/reoxygenation(H/R). METHODS: With cultured glomerular vascular endothelial cell in H/R, the positive percentage of ICAM-1 expression was measured by flow cytometry before and after giving AS-ODN. RESULTS: The ICAM-1 expression did not increase on glomerular vascular endothelial cell in 10 hours hypoxia compared to control group, it increased in 6 hours reoxygenation, and decreased by 40.6% after giving AS-OND. CONCLUSION: AS-ODN may decrease the expression of ICAM-1 on endothelial cells in H/R.     

The antisense block of CROC-1 gene expression makes cells grow slower

AIM:To establish FL-CROC- 1 ^- cell line in which CROC- 1 gene expression was blocked and study the role of CROC- 1 gene in cell growth. METHODS:The appropriate length cDNA fragment of the recently identified human gene CROC- 1 which encodes ubiquitin-conjugating enzyme like protein(Ubc-like protein) was cloned into the reconstructed eukaryotic expression vector pMAMneo-amp^- by antisense strategy. The recombinant plasmid which can express CROC- 1 antisense RNA was selected by restriction enzyme map analysis. The antisense expression recombinant plasmid pMAM-antiCROC- 1 was then transfected into human amnion FL cells by a modified calcium phosphate-mediated transfection procedure and selected with MEM medium containing 400 μg/mL geneticin. Finally ,the growth rate of the G418 resistant FL-CROC- 1 ^- cell line was determined.RESULTS:When the antisense inhibition of CROC-1 gene expression was induced by dexamethasone,the growth rate of the FL-CROC-1^- cel line was obviously slower as compared with that of the control FL cell and FL-MAMneo cell which was established by transfection of plasmid pMAMneo-amp^-(P<0.05).CONCLUSIONS:FL-CROC- 1 ^- cell line was successfully established in this study and the result of FL-CROC- 1 ^- cell growth suppression suggests that CROC- 1 gene encoded Ubc-like protein plays a positive regulation role in cell growth.     

Evaluation of tyrosinase gene's expression in HEK293 cells by magnetic resonance imaging

AIM:Tyrosinase gene was transfected into HEK293 cell as a reporter gene, it's property of synthesizing melanin, which can be examined by magnetic resonance imaging(MRI), is used to evaluate the tyrosinase gene's expression. The aim of this study was to search a way to evaluate the results of gene expression by MRI in vitro.METHODS:The plasmid of pcDNA3tyr which carried the full-length cDNA of tyrosinase gene was transfected into HEK293 cell by lipofectin. To observe the MRI signals of expressed melanin, the transfected cells were scanned by MRI sequences of T₁WI, T₁WI/SPIR and T₂WI. On the other hand, fontana stain was used to search for melanin granules in transfected cells, RT-PCR method was used to search for cDNA of tyrosinase gene.RESULTS:(1) Plasmids of pcDNA3tyr could be transfected into HEK293 cells and could synthesize a large amount of melanin. The synthetic melanins of 10⁶ cells, which had been transfected 5μg, 10μg, 20μg plasmids of pcDNA3tyr separately, were all sufficient to be detected by MRI and appeared high signal in MRI T₁WI、T₁WI/SPIR、T₂WI sequences. The signal intensities of MRI imaging were related to the amounts of transfected plasmids positively. (2) The melanin granules could be found in HEK293 cells by Fontana stain. (3) The cDNA fragment of tyrosinase gene could be detected in transfected HEK293 cells by RT-PCR.CONCLUSION:The fact that MRI could detect the synthet ic melanin of HEK293 cells, which controlled by expression of exogenous gene, demonstrates that medical imaging connecting with molecular biology technology can evaluate the result of gene expression in vitro.     

Macrophage migration inhibitory factor gene expression is suppressed by using antisense RNA mediated by expression vector

AIM:To study the potential of using antisense RNA mediated by expression vector to suppress MIF expression. METHODS:MIF gene was sub-cloned into plasmid pcDNA3 to construct MIF antisense RNA expression vector, pcDNA3-antiMIF, which was identified by restriction enzyme digestion and DNA sequencing. By using lipofectamine 2000, plasmid pcDNA3 and pcDNA3-antiMIF were transfected into MIF expression cells, 293-MIF, separately. 60 h later, the 293-MIF cells were collected and used to determine the MIF mRNA expression by real-time quantitive PCR. Plasmid pcDNA3-antiMIF was transformed into HUVECs, named HUVECs-antiMIF, to express MIF antisense RNA. HUVECs-antiMIF was screened by sulfate G418 and identified by PCR and RT-PCR analysis. Then the MIF expression vector, pSecTag-MIF, was transfected into HUVECs-antiMIF, and the MIF mRNA expression in HUVECs-antiMIF was determined by quantitative PCR. RESULTS:Restriction enzyme digestion and DNA sequencing analysis showed that MIF antisense RNA expression vector, pcDNA3-antiMIF, was constructed correctly. The results of quantitative PCR showed that MIF mRNA expression was suppressed by MIF antisense RNA at level about 32% (P<0.05) in 293-MIF cells. The HUVECs-antiMIF, which expressed MIF antisense RNA, was obtained and identified by PCR and RT-PCR assay. The results of quantitative PCR revealed that MIF mRNA expression was also down-regulated by about 40% (P<0.05) in HUVECs-antiMIF cells. CONCLUSION:MIF expression was suppressed efficiently by MIF antisense RNA mediated by expression vector, and the HUVECs-antiMIF was established to express MIF antisense RNA.     

Inhibition of survivin expression in BGC-823 cells by RNA interference

AIM: To investigate the feasibility and specificity of gastric carcinoma gene therapy by utilizing RNA interference (RNAi) to inhibit survivin expression in vitro and in vivo. METHODS: Small interference RNA (siRNA) homologous to survivin was designed. pTZU6+1-siRNA-survivin vector was constructed and transfected into BGC-823 cells. The transplanted BGC-823 tumor in nude mice was established to induce RNAi. The changes of survivin gene expression, tumor cell cycle and cell apoptosis were detected by flow cytometry, RT-PCR, Western blotting, immunochemistry and TUNEL. RESULTS: The expression of survivin was obviously inhibited by RNAi in vitro. The phase of cell cycle indicated the reduction of S phase, while G1/G0 phase increased. Cell apoptosis was obvious. Both the mRNA level and the protein expression of survivin decreased obviously. The tumor size reduced after treated with pTZU6+1-siRNA-survivin vector in vivo. The expression of survivin decreased in siRNA treatment group. In contrast, little change in control group in vitro and in vivo was observed. CONCLUSION: RNA interference down-regulates survivin gene expression, inhibits BGC-823 cell proliferation and induces cell apoptosis with good specificity, which may be a possible new approach for neoplasm gene therapy.     

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA on the proriferation and collagen Ⅰ gene expression in cultured hepatic stellate cells(HSC) in rats.METHODS:The c-myb antisense gene recombinant retroviral vector(pDOR-myb) was constructed, and then was transfected into retroviral package cell line PA 317 by means of N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate(DOTAP) liposomal transfection reagent. The pseudoviruses produced from the resistant PA317 cells selected with G418 were collected, with which HSCs isolated from rat liver were infected. The cell proliferation was measured by MTT method, c-myb, α₁-Ⅰ collagen mRNA expression and c-myb protein in HSCs were detected with semi-quantitative RT-PCR and western-blot, respectively.RESULTS:HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and α₁-Ⅰ collagen mRNA expression were repressed significantly.CONCLUSIONS:c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and α₁-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.     

Inhibition of 293T cell proliferation by blocking miR-20 and miR-106 expression with antisense RNA

AIM: To inhibit 293T cell proliferation by reducing miR-20 and miR-106 expression with antisense RNA. METHODS: Antisense RNA (or antisense oligonucleotides, ASO) specific to miR-20 and miR-106 was synthesized, and the 293T cells were treated with these antisense RNA. Then, the cell proliferation derived from suppression of antisense RNA was studied by microscopy and fluorescence-activated cell sorter. The expression of miRNA and its target gene Rb were analyzed by real-time PCR and ELISA, respectively. RESULTS: Our results showed that ASO could inhibit the expression of miR-20 and miR-106 effectively and inhibit 293T cell proliferation. The results also demonstrated that ASO specific to miR-106 could up-regulate anti-oncogene Rb expression. CONCLUSION: ASO could inhibit 293T cell proliferation by inhibiting miRNA expression and up-regulating Rb expression.     

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.     

Inhibition of ET-1 mRNA expression by hammerhead ribozyme in ECV304 cells

AIM:To find out effective hammerhead ribozyme from the designed those in order to inhibit endothelin-1(ET-1) mRNA expression in ECV304 cells.METHODS:The sequences of hammerhead ribozyme were determined by computer. The eukaryotic expression vector pEGFP-R including ribozyme DNA was constructed and delivered into ECV304 cells by liposomes. The expression of ET-1 mRNA was examined by RT-PCR.RESULTS:Ri bozyme 145 decreased the expression of ET-1 mRNA in ECV304 cells.CONCLUSION:ET-1 mRNA in ECV304 cells was degraded by R145.     

Inhibition of HMGB1 expression and release by nicotine in RAW264.7 cells

AIM: To investigate that nicotine inhibits HMGB1 expression and release in RAW264.7 cells.METHODS: (1) RAW264.7 cells were cultured in 6 wells plate, treated with 250 μg/L LPS and 1 μmol/L or 10 μmol/L nicotine, in which the cells treated with or without 250 μg/L LPS were regarded as nicotine 1 group (N1), nicotine 2 group (N2), LPS group (LPS) and control group (C), respectively. HMGB1 protein in the cell culture media and in cell nuclear was examined by Western blotting and the cellular HMGB1 mRNA level was detected by RT-PCR. (2) Transfected with antisense RNA or sense RNA of α7 subunit-containing nicotinic receptor (α7nAChR), RAW264.7 cells were treated with 250 μg/L LPS and 10 μmol/L nicotine, HMGB1 protein in the culture media was also tested by Western blotting.RESULTS: (1) HMGB1 mRNA level in C group was low (1 659.20±121.05) and no significant statistical difference among groups of N1, N2 and LPS was observed (P>0.05). (2) Higher HMGB1 accumulation in the cell culture media was detected in LPS group (445.34±28.52) than that in C group. Compared to LPS group, both N1 and N2 groups distinctly attenuated HMGB1 accumulation in culture media (P<0.05). (3) Nuclear HMGB1 accumulation was lower in LPS group than that in C group, and two different nicotine concentrations markedly increased the nuclear HMGB1 accumulation compared to LPS group (P<0.05). (4) No significant difference of HMGB1 levels in culture media between antisense RNA group and LPS group was observed (P>0.05). In sense RNA group, however, HMGB1 level was observably reduced compared to antisense group (P<0.05).CONCLUSION: The present results suggest that nicotine dramatically inhibits RAW264.7 cell nuclear HMGB1 translocation and extracellular release, and this effect relies on α7nAch receptor expression.     

Comparative study on intraocular transplatation of three B16 melanoma cell lines in mice

AIM:To establish an animal model for studying the development and metastasis of melanoma.METHODS:C57BL/6 mice were used as host to receive melanoma cell transplantation. Three kinds of melanoma cell lines, B16F0, B16F1 and B16F10, cultured to prepare the cell suspensions, were transplanted into the mouse anterior chamber (AC) of the eye. The time of eyeball diabross, time of survival and metastasis of lymph node and lung were observed.RESULTS:The time of eyeball diabross in F10 group was earlier than that in other groups. The time of eyeball diabross was no difference between F0 and F1 groups. Metastasis was developed 18 days after transplantation in F1 and F10 groups, where the tumor cells was found in ipsilateral cervical lymph nodes. The melanoma cells metastasized to lung in all three groups 28 days after transplantation. The survival time in F0 group was longer than F1 and F10 groups. There was no difference in survival times between F1 and F10 group.CONCLUSION:The differences of three kinds of melanoma cell lines in tumor development and metastasis provided the evidence that was useful for choosing suitable animal model further to study the eye melanima.     

Gene silencing of indoleamine 2,3-dioxygenase 2 influences proliferation,migration and invasion of B16-BL6 melanoma cells

AIM: To investigate the effects of indoleamine 2, 3-dioxygenase 2 (IDO2) silencing on proliferation, migration and invasion of B16-BL6 melanoma cells.METHODS: IDO2-siRNA was transfected into the B16-BL6 melanoma cells in vitro. The expression of IDO2 or IDO1 at mRNA and protein levels was detected by real-time PCR and Western blot. Colony formation assay was performed to analyze the proliferation of IDO2-silencing tumor cells. The migration ability of B16-BL6 cells after silencing of IDO2 was measured by wound healing assay and Transwell cell migration assay. The invasion ability of the tumor cells was detected by Transwell cell invasion assay.RESULTS: IDO2-siRNA signi-ficantly down-regulated IDO2 expression in B16-BL6 melanoma cells, and did not affect IDO1 expression. Compared with control group, the colony formation ability, the migratory distance measured by wound healing assay, and the migration and the invasion cell numbers detected by Transwell assay all remarkably decreased in the IDO2-silencing cells.CONCLUSION: IDO2 silencing affects the proliferation, migration and invasion abilities of the B16-BL6 melanoma cells.     

Radiation-induced expression of IL-18 and B7.1 genes in transfected B16 cells and antitumor effect of Egr-IL18-B7.1 in vivo

AIM: Plasmid Egr-IL18-B7.1 was constructed to explore its expression characteristics induced by different doses of radiation and its suppressive effect on melanoma under radiotherapy. METHODS: The plasmid containing both IL-18 and B7.1 genes downstream of Egr-1 was constructed using gene recombination technique. The in vitro expression of IL-18 and B7.1 induced by ionizing radiation was measured with ELISA and flow cytometry, respectively. The effect of gene radiotherapy on malignant melanoma was assayed by observing the growth rate of B16 cells implanted into C57BL/6J mice. RESULTS: Effective expression of IL-18 and B7.1 by plasmid Egr-IL18-B7.1 treated with different doses of X-irradiation were observed and in vivo experiments showed significant inhibition of tumor growth after combined gene-radiotherapy. CONCLUSION: Data presented in this paper implicate that gene radiotherapy with plasmid containing double genes might be one of the effective anticancer therapeutic measures.