Suppressive effect of RNA interference on BCSG-1 gene expression in MCF-7 cell line
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Objective To investigate the suppressive effects of siRNA on the BCSG-1 gene expression in breast cancer MCF-7 cells. Methods The four-pair designed and chemically synthesized in vitro of small interfering RNA (siRNA) sequences ( BCSG-1-siRNA-1 ,-2,-3 and 4) for BCSG-1, and one pair of non-specific siRNA sequence were transducted into plasmid vectors, following by transfection into into MCF-7 cells. Blank control group (empty vector transfection) and normal control group (no transfection) were set up. After 48 h, the mRNA and protein expression levels of BCSG-1 in transfected cells were detected by using reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry respectively. Results The expression of BCSG1 protein was 4.27 ±0. 12, 4.19 ±0.22, 4.15 ± 0.14,4.17 ±0.13, 7.92 ±0.22, 8.02 ±0.13, 8.02 ±0.13, and that of BCSG1 mRNA was 0.624 ±0.010,0.626±0.013, 0.634 ±0.008, 0.631 ±0.010, 0.976±0.076, 0.983 ±0.052, 1.014±0.034 respectively. As compared with control groups, there was significant difference in the expression of BCSG-1 mRNA and protein among the above groups (P ＜0.01 ). The expression level of BCSG-1 mRNA and protein in the groups of MFC-1 cells transfected with BCSG-1-siRNA was lower than that in control groups.Conclusion Expression of BCSG-1 gene can be effectively inhibited by siRNA in MCF7 cells.
Key words:
BCSG1; RNA-interference; Transfection; InhibitionKeywords:
MCF-7
RNA polymerase III
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Objective: To investigate the effectiveness of human breast cancer cell line MDA-MB-231 proliferation and apoptosis activity to △Np73 small interfering RNA expression plasmid transfection. Methods: △Np73 small interfering expression plasmids were constructed and transfected into human breast cancer cell line MDA-MB-231. Transfection efficiency was calculated by counting green fluorescent protein expressing cells under fluorescence microscope. △Np73 and TAp73 mRNA expression was measured by realtime PCR. After pcDNA3-HA / DNp73α expression plasmids were co-transfected with △Np73 small interfering plasmid,Western blot test was used to detect △Np73 and TAp73 expression level. Cell growth inhibition rate was detected by CCK-8 assay. Caspase-3 activity spectrophotometry of △Np73 transfected and doxorubicin treated cells was examined to determine its apoptosis level. Results:△Np73 small interfering expression plasmids were successfully constructed and transfection efficiency reached 60%. △Np73 siRNA plasmid transfection effectively inhibited endogeneous △Np73 mRNA expression and exogenous △Np73 protein expression,showing no significant inhibitory effects on TAp73 mRNA and protein expression. △Np73 siRNA plasmid transfection significantly decreased proliferative activity of MDA-MB-231 compared with blank control group. △Np73 siRNA plasmid transfection apparently increased doxorubicin-induced apoptosis level of MDA-MB-231,with significantly difference from negative control group. Conclusion: △Np73 siRNA expression plasmids can significantly down-regulate △Np73 mRNA and protein expression in MDA-MB-231 cells,inhibit cell growth and proliferation,enhance its level of apoptosis induced by doxorubicin,indicating that targeted silencing △Np73 is likely a hopeful strategy for treatment of breast cancer in the future.
Growth inhibition
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MCF-7
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【Objective】 Observing the effect of RNA interference(RNAi) technique to suppress the expression of G250 gene in G250 positive renal cell carcinoma cell line 786-0,in order to establish stable RNAi technique and use this technique for further gene research.【Methods】 Four small fragments of siRNA against the G250 gene and one negative control were sequenced and cloned into vector pRNAT-U6.1/Neo,The recombinant plasmids were then transfected into renal cell carcinoma cell line 786-0 cells respectively,by positive liposomal transfection assay.G250 mRNA expression was measured using real-time fluorescence quantitative RT-PCR,G250 protein expression was analyzed by Western-blot.【Results】 DNA sequencing and enzyme digestion confirmed that the siRNA expression vectors targeting G250 gene were successfully constructed.35.56%,49.27%,45.88% and 65.13% silencing of G250 mRNA expression were observed respectively.The corresponding decrease of G250 protein expression was confirmed.【Conclusions】 These results demonstrated that siRNA against G250 gene could effectively down regulate G250 gene expression in 786-0 cell line,it can be used for next experiment.
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OBJECTIVE:To explore the blocking effect of siRNA on the expression of Survivin in gene in osteosarcoma cell line MG63.METHODS:According to Survivin cDNA coding sequence,the specific RNA interference(RNAi)fragments targeting Survivin gene were designed and synthesized,which were cloned into pSilencer 3.0-H1 neo plasmid vector,and the shRNA eukaryotic expression vector siRNA Survivin targeting Survivin gene was constructed.After the vector was constructed,MG63 cells were transfected with negative control vector siRNA neg or RNAi vectors and selected by G418.Expression of mRNA and protein of Survivin in the stable transfected cells was investigated seperately by RT-PCR,Western blot,immunofluorescence microscopy,and flow cytometry.RESULTS:The specific siRNA eukaryotic expression vector PsvA and PsvB targeting Survivin gene were constructed successfully.The stable transfectants containing negative control vector siRNA neg,PsvA and PsvB were obtained.Expression of mRNA and protein of Survivin was inhibited significantly in MG63/PsvB cells.Whereas Survivin gene expression levels were hardly changed in the other groups.Otherwise,MG63/PsvB had significant decreases in cell number compared with other transfectants.Analysis of DNA content in PsvB transfectants revealed a 7-fold increase in the fraction of sub-G1 peak(apoptotic peak)as compared with vector control and PsvA transfectants,under the same experimental conditions,P0.01.CONCUSION:Survivin gene expression can be suppressed markedly by specific shRNA in MG63 cells,which establishes the experimental foundation for further studying the biological functions and its mechanisms of Survivin in MG63 cells.
Survivin
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Objective:To investigate the interfering effect of siRNA on the expression of VEGF in Hela cells.Methods:Three pairs of siRNA were designed according to the 1-5 extrons sequence of VEGF and synthesized by T7 RNA ploymerase transcription in vitro. To evaluate the inhibition activity of siRNA, Hela cells were transfected via siPORT Lipid. The interfering effect of siRNAs in hRPE cells was examined by semi-quantitative RT-PCR and immunofluorescence technique.Results:The results showed that the three pairs of siRNA could effectively inhibit gene expression of VEGF in Hela cells with the rates of 88.7%, 94.2% and 80.3%. But the 1-base mismatched siRNA and ssRNA(+) didn′t show significant interfering effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect(5-10 pmol).Conclusion:The siRNAs synthesized by T7 RNA ploymerase in vitro could effectively and specifically interfere the expression of VEGF in Hela cells, providing a novel approach for gene therapy of tumor.
HeLa
Transcription
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Objective To study the inhibition effect of siRNA on the survivin expression in HepG2 cells. Methods One pair of 21bp reverse repeated motifs of survivin target sequence with 9 spacer were synthesized and inserted into plasmid Psilencer2.1 to generate siRNA eukaryotic expression vector. After stable transfection into HepG2 cells , the survivin mRNA and protein expression inhibition was detected by use of RT-PCR and flow cytometer. Results The recombinant plasmid P Silencer(+)-survivin was successfully constructed. By use of RT-PCR and flow cytometer detection , survivin mRNA and protein expression inhibition ratio reached 73% and 75% respectively. Conclusion siRNA targeting survivin gene can specially suppress its expression in HepG2 cells. This provides a new method and material to the biological therapy of cancer.
Survivin
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Objective To construct small interfering RNA(siRNA)expression vector and to investigate its inhibitory effect on vascular endothelial growth factor-C(VEGF-C) expression in human breast cancer cell line MCF-7.Methods A pair of hairpin-like oligonucleotide sequences specific for VEGF-C gene were designed and synthesized.The annealed oligonucleotide fragments were subcloned into plasmid pRNAT-U6.1/Neo to construct VEGF-C siRNA expressing vector.Then VEGF-C siRNA expression vectors were confirmed by PCR and sequencing.Transfection of VEGF-C siRNA expressing vector into MCF-7 cells was performed using liposome transfection reagents.VEGF-C mRNA and protein expression were examined by RT-PCR and immunocytochemistry respectively.Results PCR and DNA sequencing showed that siRNA expression vector targeting VEGF-C was successfully constructed.VEGF-C expression in the cells transfected by VEGF-C siRNA expression vectors was inhibited significantly at both mRNA and protein levels.VEGF-C mRNA and protein expression were decreased 61.8% and 78.3% compared with control plasmid respectively(P0.05).Conclusion VEGF-C siRNA expression vector can effectively inhibit the expression of VEGF-C in transfected MCF-7 cells,and provides a basis for breast cancer therapy targeting VEGF-C gene.
MCF-7
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Objective To explore the inhibitory effect of RNA interference(RNAi) targeting human telomerase reverse transcriptase(hTERT) gene of cervical carcinoma Hela cells.Methods Small interfering RNAs(siRNAs) targeting hTERT gene was synthesized with chemical technique,and siRNAs were transfected into Hela cells by cationic lipofection.Cervical carcinoma Hela cells were divided into six groups,blank group(untransfected Hela cells group),negative group(negative control siRNA group),siRNA 1,2,3 and 4 groups(RNA interference groups).The telomerase hTERT mRNA and hTERT protein were detected with reverse transcriptase polymerase chain reaction and Western blotting technique,respectively.The proliferation of the Hela cell was analyzed with MTS,and flow cytometry was used to observe apoptosis of the Hela cell mediated by siRNA.Results Transfection of siRNA1-3 and the hTERT mRNA and protein expressions were significantly lower,and the apoptosis rate increased obviously,which were significantly different from those in blank group(P0.01),and there was no significant difference between blank group and negative group(P0.05).Conclusion RNAi can specifically silence the hTERT gene of cervical carcinoma Hela cells by down-regulating the expression levels of the hTERT mRNA and its protein.Therefore,RNAi can inhibit the proliferation of the cancer cells and induce their apoptosis.The experimental evidence of gene therapy for cervical carcinoma cells might be expected.
HeLa
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Objective To investigate the effects of cyclin-dependent kinase 2(CDK2) expression suppression induced by small interfering RNAs(siRNAs) on mRNA expression of cell cycle related genes RB,CyclinE and E2F1 in hepatic carcinoma cells SMMC7721.Methods The siRNA eukaryotic expression plasmids of CDK2 gene were constructed firstly and then were transfected into SMMC7721 cells with the Lipofectmine TM 2000 liposome.The transfected cells were divided into six groups: recombinant plasmid 190 group,recombinant plasmid 191 group,SMMC7721 group,CDK2-siRNA transfection group,negative control group,and blank vector group.The expression of CDK2 gene was detected with Western blot method.Real-time fluorescent quantitation polymerase chain reaction(PCR) method was utilized to detect the mRNA expression of RB,Cyclin E and E2F1 which were related to CDK2 gene,and then the effective siRNA sequence of CDK2 gene was screened.Results After the siRNA eukaryotic expression plasmids of CDK2 gene was transfected into SMMC7721 cells,mRNA expression of RB was up-regulated and the mRNA expression of CyclinE and E2F1 was down-regulated.Conclusion CDK2 gene expression suppression can up-regulate the mRNA expression of RB in SMMC7721,and down-regulate the mRNA expression of CyclinE and E2F1,indicating that the mRNA expression of RB,Cyclin E and E2F1 genes is correlated with CDK2 gene expression.
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