[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 construct the small interfering RNA(siRNA) expression cassettes (SECs) targeting activated K-ras gene sequence and investigate the effects of SECs on K-ras gene in human pancreatic cancer cell line MIAPaCa-2. Methods: Three different sites of SECs were constructed by PCR. The K1/siRNA, K2/siRNA and K3/siRNA were located at the site 194, 491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of site 194, 491, we observed the cytopathic effect of confluent MiaPaCa-2 cells after they were incubated for 48 hours, and detected the apoptosis in cells by FACS, then we tested the alternation of K-ras gene in confluent MiaPaCa-2 cells by RT-PCR, immunofluorescence and western blot, respectively. Results: Introductions of the K1/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells led to cytopathic effect, slower proliferation and increased apoptosis, while the appearances of control MiaPaCa-2 cells remained well. The number of apoptotic cells increased compared with control cells. RT-PCR, immunofluorescence and western blot showed the effects of inhibited expression of activated K-ras gene by RNA interference in the K1/siRNA and K2/siRNA groups. We also found that the introduction of K3/siRNA had no effect on MiaPaCa-2 cells. Conclusion: K1/siRNA and K2/siRNA can inhibit the expression of activated K-ras and decrease the growth of MiaPaCa-2 cells, while K3/siRNA has no such effect, demonstrating that the suppression of tumor growth by siRNA is sequence-specific. We conclude that K-ras is involved in maintenance of tumor growth of human pancreatic cancer, and SECs against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.
Immunofluorescence
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Objective:To study the inhibitory effects of c-myc antisense RNA on human hepatocarcinoma cells HepG2 in vitro.Methods:Three days after the four element complex with c-myc antisense RNA for transient transfection,the expression of c-myc protein,cell cycle and cell apoptosis were assayed by flow cytometry.Established hepato carcinoma cell line which could express c-myc antisense RNA stably.By growth curve, to study the biological effects on HepG2/as-c-myc cells which could express c-myc antisense RNA stably.Results:c-myc antisense RNA caused the expression of c-myc protein of HepG2 cells diminish;induced cell apoptosis and effected cell cycle pause at G0/G1 phase.Study of HepG2/as-c-myc cells,which could express c-myc antisense RNA stably,revealed that c-myc antisene RNA could inhibit cell growth.Conclusion:c-myc antisene RNA could effectively reduce the expression of c-myc protein on human hepatocarcinoma cells HepG2,induce cell cycle pause at G0/G1 phase and inhibit the growth of hepatocarcinoma cells.
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To construct recombinant expression vectors of small interfering RNA (siRNA) targeting survivin and investigate apoptosis of glioma cell line U251 mediated by the survivin-targeting siRNA.According to the sequence of the coding region of survivin gene, two strings of 19 nucleotides of inverted sequence flanking the loop sequence of two complementary 9-base oligonucleotides were designed and synthesized to form hairpin construct as the DNA templates for the target siRNA. The siRNA templates were cloned into siRNA expression vector pGenesil-1, and the resulted vector pGenesil-1/survivin was transfected into U251 cells using Metafectene following the standard protocols. Real-time PCR and Western blotting were performed to evaluate survivin gene silencing induced by siRNA transfection at the RNA and protein levels, respectively. Flow cytometry analysis with Annexin-V/PI double staining was used to determine the cell apoptosis.Real-time RT-PCR and Western blotting revealed significantly lowered survivin expression at both RNA and protein levels in transfected U251 cells, which exhibited a significantly higher apoptosis rate after transfection as shown by flow cytometry analysis.RNA interference mediated by the siRNA expression vector pGenesi-l/survivin can significantly reduce survivin expression and induce remarkable apoptosis in U251 cells.
Survivin
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OBJECTIVE To explore the influence of miRNA-224-5p gene silencing on cell proliferation,apoptosis and invasion in human laryngeal cancer cell line Hep-2 by lentiviral vector-mediated RNA interference.METHODS miRNA-224-5p specific siRNA gene was synthesized and cloned into the recombinant lentiviral vector.Hep-2 cells were infected by miRNA-224-5p-siRNA recombinant lentivirus(miRNA-224-5p-siRNA-LV3).The Hep-2 cells were divided into SI group,NC group and BC group in vitro.The expression of the targets of miRNA-224-5p was detected by RT-PCR.Cell proliferation was detected by CCK8 kit.Cell cycle and apoptosis were detected by flow cytometry.The invasion was detected by Transwell test.RESULTS The expression level of miRNA-224-5p was inhibited significantly by miRNA224-5p-siRNA-LV3.The proliferation of Hep-2 was also markedly suppressed.The number of invasive cells that migrated through the chamber decreased.The cell cycle and apoptosis had no obvious effect.CONCLUSION MiRNA-224-5p gene silencing by RNAi can suppress the proliferation and reduce the invasiveness of Hep-2 cells.
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Breast cancer resistance to therapy can result from expression of antiapoptotic genes. Survivin is an antiapoptotic gene that is over expressed in most human tumors. RNA interference using short interfering RNA (siRNA) can be used to specifically inhibit survivin expression. A novel siRNA targeting survivin was used to process MCF-7 cells. Cellular survivin mRNA and protein levels were determined by real-time qRT-PCR and Western blot, respectively. Cellular morphology and cell cycle were determined by fluorescence microscopy and flow cytometry. Cell proliferation was measured by MTT assay. Our data showed that the novel survivin-targeted siRNA could efficiently knockdown the expression of survivin, inhibit cell proliferation and cell cycle, especially at the G2/M checkpoint. These data suggest that the siRNA has potential for therapeutic applications.
Survivin
MTT assay
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Objective To study the inhibition of the expression of bcl-xL gene induced by RNA interference in CNE-2Z cell line in addition to the inhibition of its proliferation and apoptotic induction. Methods Small interfering RNAs targeting bcl-xL gene were synthesized by using web design software provided by Ambion and the silencer short interfering RNA (siRNA) construction kit; fluorescein-labeled siRNAs were done by FAM-silencer TM siRNA labeling kit; siRNAs were transfected into CNE-2Z cells by using lipofectamine TM 2000 reagent; siRNA transfection efficiencies were analyzed by fluorescent microscopy; down-regulation of bcl-xL was detected by RT-PCR; thiazolyl blue(MTT) assay was used to assess the cell growth; apoptosis of CNE-2Z cells was analyzed by flow cytometry. Results Green fluorescence in the cells was seen clearly in FAM-labeled siRNA transfected group under the fluorescent microscope while none in the untransfected group. Different down-regulations of bcl-xL mRNA expression were found in the transfected groups. The expression of bcl-xL mRNA decreased by 10%-70% in the siRNAs transfected CNE-2Z by RT-PCR scan analysis. The inhibitory rate of cell proliferation depended on time and concentrations to some extent. Different cell apoptosis could be induced by different concentrations of siRNA4. Conclusions The synthesized siRNAs in vitro were able to down-regulate the expression of bcl-xL. There were different capabilities of the specific siRNAs down-regulation. The transient transfected bcl-xL siRNA4 could effectively inhibit the growth of the cancer cells and induce theirs apoptosis. It was suggested that the siRNA technique provide not only an extremely powerful tool for the functional analysis of genome but also a new method for anti- nasopharyngeal carcinoma gene therapy.
Bcl-xL
Lipofectamine
Viability assay
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Objective:To study the antitumor activity of short interfering RNA (siRNA). Methods: The growth inhibitory effect of siRNA was measured by MTT assay. The cytomorphosis and DNA fragmentation of siRNA were respectively observed by HE staining and TUNEL labeling method. The protein level and changes of cell cycle distribution were detected respectively by western blot assay and flow cytometry. Results:Data indicated that the growth of MCF-7 cells was obviously inhibited by siRNA-Bcl2, siRNA-MDM2, siRNA-CDK2 and siRNA-HRas and their chromatin conden-sation and DNA fragmentation could be induced by siRNA-MDM2 and siRNA-Bcl2; target gene ex-pression level could be significantly reduced by siRNA-Bcl2, siRNA-MDM2, siRNA-CDK2 or siRNA-HRas as well as GI phase arrest of MCF-7 cells could be blocked.Conclusion:That siRNA can inhibit the growth of tumor cells, suppress expression level of target genes, induce apoptosis and GI arrest of tumor cells may become a cancer gene therapy.
MTT assay
Fragmentation
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OBJECTIVE To study the influence of small interfering RNA(siRNA)silencing cyclin D1 gene on cell proliferation and apoptosis in Hep-2 cell line. METHODS The special siRNA was transfected into Hep-2 cell line using liposomal transfection reagent. The inhibitory effects on the growth of Hep-2 cells were assessed by the MTT assay. Cell cycle analysis and quantization of apoptosis were valued by flow cytometry. RESULTS Silencing cyclin D1 could significantly suppress the growth of the Hep-2 cell in a time-dependent manner. The cell cycle arrest and apoptosis was detected. CONCLUSION Transfecting the special siRNA of cyclin D1 could effectively inhibit the growth of the canner cells, influence the cell cycle and induce their apoptosis.
Cyclin B
Cyclin A
Cyclin E1
Cyclin D
Cyclin B1
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Objective To study the effect of small interfering RNA(siRNA) targeting CD47 gene mediated by lentivirus vectors on proliferation and apoptosis of human laryngocarcinoma Hep-2 cells. Methods After building lentivirus vector, a siRNA directed against CD47 gene was transfected into Hep-2 cells. Cell morphological changes were observed by fluorescence microscopy. The changes in CD47 mRNA expression were detected by semi-quantitative RT-PCR, and the protein of CD47 was evaluated byWestern blotting. Suppression of proliferation and apoptosis of Hep-2 cells were observed by MTT assay. Results After the laryngocarcinoma Hep-2 cells were transfected by CD47-siRNA lentiviral plasmid, it was showed by fluorescence microscopy that the CD47 siRNA was able to effectively suppress the Hep-2 cell proliferation, the cells were deformed and diminished in size obviously, and necrosis and apoptosis were observed. Semi-quantitative RT-PCR andWestern blotting revealed that the expression of CD47 mRNA and protein decreased by 76%–82% and 77%, respectively(P<0.05). Forty-eight hours after the lentivirus vectors transfection, MTT assay revealed that cell apoptosisi ncreased significantly(P<0.01). Conclusion Small interfering RNA targeting CD47 gene mediated by lentivirus vectors can significantly inhibit CD47 gene expression in Hep-2cells and induce cell apoptosis in vitro.
CD47
MTT assay
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