[Inhibition of growth and proliferation of Hep-2 cells by targeting c-myc gene using small interference RNA technology].
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OBJECTIVE To investigate the effect of small interfering RNA (siRNA) targeting c-myc gene in Hep-2 cells. METHOD siRNA targeting c-myc mRNA was designed and synthesized. In vitro cultured Hep-2 cells were transfected with lipofectamine 2000 and the inhibitory effect was detected by MTT, morphology, real time PCR assay. RESULT 1) The MTT result showed the c-myc siRNA to be able effectively to suppress the Hep-2 cell multiplication; 2) The real time PCR result showed c-myc at mRNA level inhibition ratio at 94% in group S3; 3) The morphology result showed the c-myc siRNA to be able effectively to suppress the Hep-2 cell multiplication, the cell heteromorphism was diminished. CONCLUSION siRNA targeting c-myc can remarkably suppress the Hep-2 cell growth and multiplication.Keywords:
Lipofectamine
MTT assay
Growth inhibition
Hep G2
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Objective To evaluate the effect of small interferring RNA(siRNA) targeting c-Myc gene on the proliferation and apoptosis of MCF-7cells.Methods SiRNA for c-Myc gene was transfected into MCF-7 cells by LipofectimineTM2000,c-Myc gene expression was assessed by real-time quantitive PCR,and c-Myc protein expression was tested by Western Blotting.MTT assay was used to assess the proliferation of the cells.Fluorescence-activated cell sorter analysis was used to determine apoptosis of the cells.Results Our data showed that the expression of c-Myc gene in MCF-7 cells can be effectively suppressed by siRNA,and the growth rate of MCF-7 cells is decreased markedly.We also found that depeletion of c-Myc in this matter promoted apoptosis of MCF-7 cells.Conclusion The expression of c-Myc in MCF-7 cells can be decreased by RNAi,which can inhibit MCF-7 cells growth and promote the apoptosis of the cells.
MCF-7
Growth inhibition
MTT assay
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To investigate the effect of MAT2A on growth and apoptosis of hepatoma cells by small interfering RNA (siRNA) targeting the gene, the expression vector pSilence-2.1-U6-siRNA were constructed and short interfering RNA (siRNA) against MAT2A gene was expressed, effects of siRNAs on the expression of MAT2A gene and protein and on the enzymatic activity of the protein were determined by RT-PCR, Western blot, and activity assays, respectively. Effects of siRNAs on cell growth and apoptosis of hepatoma cell lines Bel-7402, HepG2, and Hep3B were evaluated by MTT method, FACS analysis, and fluorescent microscopy examination. The changes in SAM level and the ratio of SAM∶SAH were measured by reverse-phase high performance liquid chromatography(RP_HPLC). Effects of siRNAs on the expression of MAT1A gene were determined by RT-PCR. The effective siRNA molecules targeting the MAT2A gene were identified that could specific knock-downed MAT2A expression. The MAT activity was decreased after siRNA but not control siRNA treatment in hepatoma cells at 48 hours after transfection and lasted for at least 3 days. This resulted in a great increased SAM content and the ratio of SAM to SAH in hepatoma cells. A switch in the gene expression from MAT2A to MAT1A has been found in hepatoma cells with siRNA. Furthermore, silencing of the MAT2A gene by RNAi significantly inhibited hepatoma cell growth and eventually lead to induction of apoptosis. A greater apoptosis response was observed in siRNA transfected cells than control cells (P0.05). Staining of the nuclear DNA of effective siRNA transfected cells with DAPI reveled nuclear fragmentation and chromatin condensation; otherwise, typical hallmarks of apoptosis were not detected in control cells. The results demonstrated that RNA interference-mediated silencing of MAT2A gene inhibited growth and induced apoptosis of human hepatoma cells and suggested that MAT2A was a suitable target for RNA interference, which holds great potentials for specific gene therapy of liver cancer.
Trans-acting siRNA
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AIM: To inhibit c-Myc gene expression in human rectal cancer cell line Colo320 by small interfering RNA (siRNA), and to provide a new method to study the role of c-Myc in Colo320 cell. METHODS: The c-Myc gene specific siRNA was designed according to its sequence and made by in vitro transcription. The c-Myc siRNA was transfected into Colo320 cell, and the cell was cultured for 48 to 96 hours before harvesting. c-Myc mRNA and protein level were monitored by using fluorescence real time reverse transcription polymerase chain reaction. Cellular proliferation activities were assayed by tetrazolium bromide (MTT) colorimetry and clone test. RESULTS: Compared with control group, there was a significant decrease in the c-Myc mRNA. Colo320 cells transfected with c-Myc siRNA had lower cellular proliferation than no-transfected Colo320 cells. CONCLUSION: Our results suggest that there exist RNA interference mechanism in Colo320 cell line, and the c-Myc siRNA can specifically inhibit the expression of c-Myc gene in Colo320 cells. RNA interference method provides a new way to study the role of c-Myc in cancer cell.
Transcription
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Aim To study the interference role of Bcl-2 siRNA on HL-60 cells. Methods Bcl-2 siRNA was synthesized in vitro transcription with silencer siRNA construction kit. The synthesized siRNA was transfected into HL-60 cells with lipid siPORT transfection. Forty-eight hours after the transfection, we used MTT and immunofluorescence to detect cell proliferation and apoptosis,and used RT-PCR and immunofluorescence to detect the level of Bcl-2 mRNA and Bcl-2 protein expression. Result Bcl-2 siRNA reduced the level of Bcl-2 mRNA and Bcl-2 protein expression in HL-60 cells and induced cell apoptosis. There was no difference on the effect of other groups compared with the control. Effective Bcl-2 siRNA specifically degraded Bcl-2 expression in the levels of mRNA and protein and induced HL-60 cells apoptosis.Conclusion These results indicate that siRNA is a highly specific tool for targeted gene knockdown. siRNA-mediated gene silencing is a reliable approach for large-scale screening of gene function and drug target validation.
Immunofluorescence
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Objective To investigate the inhibition of c-myc gene and protein in medulloblastoma cell line D341 by means of RNA interference in vitro,and investigate the cell cycle and apoptosis difference after interference. To explore the gene therapeutic possibility of siRNA for medulloblastoma. Methods The siRNA for c-myc gene was designed and transfected into D341 cells line. Real time PCR was carried out to detect the expression of target gene mRNAs,Immunoblots were carried out to detected the expression of c-myc protein. The cell cycle and apoptosis were detected by FCM. Results After transfection siRNA into D341 cells,c-myc gene was inhibited. C-myc gene was inhibited greatest at RNA level 24 hours after transfecting. At the 48th hour,there was the greatest inhibition effect for the protein level. With the increasing of concentration,the inhibition effect of c-myc gene raised. Silencing the targeted gene can induce the G1 phase arrest,which was confirmed by FCM. Conclusion The siRNA targeting c-myc gene can inhibit proliferation of D341 cells by inhibiting the expression of c-myc mRNA and protein and might be a new therapeutic modalities in treatment of medulloblastoma.
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Abstract We investigated the effects of RNA interference-mediated silencing of the c-myc gene on celluar proliferation and apoptosis in human colon cancer HT-29 cells in vitro and in vivo. A small interfering RNA (siRNA) targeting c-myc was designed, the DNA template was synthesized, and the siRNA was obtained by in vitro transcription. After siRNA transfection into HT-29 and human neuroblastoma IMR-32 cells with Lipofectamine 2000™, the proliferation of the HT-29 and IMR-32 cells was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and Hoechst 33258 staining was used to observe cell apoptosis. Following gene transfer to HT-29 cells, the expression of c-myc mRNA was examined via reverse transcription polymerase chain reaction, and the level of the protein via Western blot assay. Growth curves were constructed and in vivo experiments were performed on nude mice to assess the effects of c-myc silencing on tumor growth. The c-myc expression in the tumor tissue was measured by reverse transcription polymerase chain reaction and subsequently by immunohistochemistry. Our paper demonstrates that the delivery of siRNA directed against c-myc not only efficiently down-regulated the expression of c-myc, inhibited the proliferation of HT-29 cells and induced apoptosis in vitro, but also suppressed the growth of colon cancer cells in vivo.
Lipofectamine
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OBJECTIVE To investigate the effects of plasmid-based siRNA targeting to oncogene c-myc on c-myc/ c-Myc expressions and cells proliferation in MCF-7 breast cancer cells. METHODS siRNA eukaryotic expression plasmid p-Mat01-1 targeting to the sequence 589-609 of oncogene c-myc and its mismatch plasmid p-Mis09-1 were constructed, and transiently transfected MCF-7 cells using Lipo2000. Semi-quantitative RT-PCR and Western blot were used to analyze the expressions of c-myc/c-Myc in MCF-7 cells, and cells proliferation was detected by MTT assay. RESULTS p-Mat01-1 inhibited the expressions of c-myc mRNA (24 h: P < 0.01) and c-Myc protein (5 d. P < 0.01) in MCF-7 cells as compared with pEGFP-C1 and p-Mis09-1 controls, and suppressed the proliferation of MCF-7 cells significantly (3 d: P < 0.05, 5, 7 d: P < 0. 01). CONCLUSION Plasmid-based siRNA targeting to oncogene c-myc could inhibit the expressions of c-myc/c-Myc in MCF-7 breast cancer cells efficiently, suggesting that the downregulation of c-myc/c-Myc could suppress the proliferation of MCF-7 cells in vitro.
MCF-7
MTT assay
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To study the inhibitory effect of small interfering RNA (siRNA) targeting c-myc gene in K562 cells.siRNAs targeting the site 1357 of c-myc mRNA was designed and synthesized. In vitro cultured K562 cells were transfected with lipofectamine 2000 and the inhibitory effect was detected by reverse transcriptase (RT)-PCR, cell count, MTT assay and fluorescence-activated cell sorting.Compared with the negative and blank control group, the transfection group showed marked decrease in the c-myc expression and the K562 cells exhibited increased apoptosis rate.RNA interference can effectively inhibit c-myc expression and induce apoptosis in K562 cells.
K562 cells
Lipofectamine
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Objective To construct recombinant small interfering RNA (siRNA) plasmid vector targeting VEGF-C, and observe its impact on the growth and apoptosis of human breast cancer cell line MDA-MB-435. Methods Three small fragments of siRNA and one negative control were sequenced and cloned into vector pSilencer3.0-H1. The recombinant plasmids were then transfected into breast cancer cell line MDA-MBA-435, by positive liposomal transfection assay. The transcription and translation level of VEGF-C expression were detected by RT-PCR and Western Blot. Meanwhile, the proliferation and apoptosis of transfected tumor cells were determined by MTT and flow-cytometry assay. Results The recombinant plasmids containing VEGF-C siRNA were successfully constructed. VEGF-C expression was significantly knocked-down after siRNA plasmid transfection. The maximum inhibitory rate of transfected breast cancer cells reached 85.4%, significantly higher that observed in the control group. The apoptosis rate was 60% 72hr after transfection. Conclusions VEGF-C gene silenced by siRNA can inhibit the proliferation and induce the apoptosis of human breast cancer cell line MDA-MBA-435. The process might serve as a potential approach for cancer gene therapy.
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