AIM: To study the inhibitory effects of siRNAs targeting different hTERT sequences and to screen the effective siRNA sequence.METHODS: Five double-stranded siRNAs targeting coding and non-coding regions of hTERT gene ...AIM: To study the inhibitory effects of siRNAs targeting different hTERT sequences and to screen the effective siRNA sequence.METHODS: Five double-stranded siRNAs targeting coding and non-coding regions of hTERT gene were designed and synthesized by T7 transcription system in vitro. siRNA4sequence was screened by full length gene targeting technique and the rest of the siRNA sequences were selected randomly. After being purified by ethanol precipitation, the siRNAs were transfected to the human hepatocellular carcinoma cell (HepG2) by Lipofectamine 2000TM. At 48-72 h after siRNAs transfection, MTT assay,RT-PCR and Western-blot were applied to evaluate the effects of siRNAs on cell growth, mRNA and protein expression level of hTERT gene, respectively.RESULTS: Compared to the control cells, the cells treated with the five double-stranded siRNAs exhibited different degrees of inhibition of cell proliferation in a dose-dependent manner. siRNA2 and siRNA4, exhibited obvious effects of inhibiting hTERT mRNA and protein expression in HepG2cells.CONCLUSION: siRNAs targeting different hTERT sequences have significantly various inhibitory effects on hTERT gene expression. The siRNA sequence screened by full length gene targeting technique has comparable inhibitory effect with the rest siRNA sequences screened by random selection, suggesting that siRNAs and antisense oligonucleic acids may have the same effective target sites. Compared with chemical synthesis method,synthesizing double-stranded siRNA by T7 transcription system in vitro is a rapid, simple, and inexpensive method suitable for screening high-effect siRNA targeting site for specific gene.展开更多
Oncogenic H-Ras G12V and its variants have been shown to inhibit muscle differentiation. However, the role of proto-oncogenic Ras （c-Ras） in muscle differentiation remains unclear. The active GTP-bound form of Ras h...Oncogenic H-Ras G12V and its variants have been shown to inhibit muscle differentiation. However, the role of proto-oncogenic Ras （c-Ras） in muscle differentiation remains unclear. The active GTP-bound form of Ras has been known to associate with diverse effectors including Raf, phosphatidylinositol 3-kinase （PI3K）, RaI-GDS, and other molecules to transmit downstream signals. We hypothesize that c-Ras may stimulate muscle differentiation by selectively activating PI3K, an important mediator for muscle differentiation. In our experiments, inhibition of c-Ras by farnesyltransferase inhibitors and a dominant negative form of H-Ras （Ras S17N） suppressed muscle differentiation. Consistently, individual knockdown of H-Ras, K-Ras, and N-Ras by siRNAs all blocked muscle differentiation. Interestingly, we found that c-Ras preferentially interacts with PI3K rather than its major binding partner c-Raf, during myogenic differentiation, with total c-Ras activity remaining unchanged. PI3K and its downstream myogenic pathway, the Nox2/NF-kB/inducible nitric oxide synthase （iNOS） pathway, were found to be suppressed by inhibition of c-Ras activity during differentiation. Furthermore, expression of a constitutively active form of PI3K completely rescued the differentiation block and reactivated the Nox2/NF-kB/iNOS pathway in c-Ras-inhibited cells. On the ba- sis of our results, we conclude that contrary to oncogenic Ras, proto-oncogenic H-Ras, K-Ras, and N-Ras are directly involved in the promotion of muscle differentiation via PI3K and its downstream signaling pathways. In addition, PI3K pathway activation is associated with a concurrent suppression of the otherwise predominantly activated Raf/ Mek/Erk pathway.展开更多
Antisense oligonucleotides(ASONs) and siRNAs have been applied extensively for the regulation of cellular and viral gene expression,and RNAi is currently one of the most promising new approaches for anti-tumor and ant...Antisense oligonucleotides(ASONs) and siRNAs have been applied extensively for the regulation of cellular and viral gene expression,and RNAi is currently one of the most promising new approaches for anti-tumor and anti-viral therapy.In order to improve bioactivity properties and physicochemical properties of siRNA,we synthesized a novel class of ASONs II-VII incorporated with amino-isonucleoside(isoA1 and isoA2) for investigation on basic physicochemical properties.Then we designed amino-isonucleoside(isoA1,isoA2 and isoT1) incorporated siRNA 2-7.Some meaningful results have been obtained from the physicochemical property experiments in ASONs.In RNAi potency experiments,we investigated RNAi potency of each strand of the siRNA.These amino-isonucleosides incorporated siRNAs showed promising bioactivity properties and had position specificity.Reduced off target effect from sense strand loading in siRNA application was observed.展开更多
基金Supported by the National Natural Science Foundation of China, No. 30371662
文摘AIM: To study the inhibitory effects of siRNAs targeting different hTERT sequences and to screen the effective siRNA sequence.METHODS: Five double-stranded siRNAs targeting coding and non-coding regions of hTERT gene were designed and synthesized by T7 transcription system in vitro. siRNA4sequence was screened by full length gene targeting technique and the rest of the siRNA sequences were selected randomly. After being purified by ethanol precipitation, the siRNAs were transfected to the human hepatocellular carcinoma cell (HepG2) by Lipofectamine 2000TM. At 48-72 h after siRNAs transfection, MTT assay,RT-PCR and Western-blot were applied to evaluate the effects of siRNAs on cell growth, mRNA and protein expression level of hTERT gene, respectively.RESULTS: Compared to the control cells, the cells treated with the five double-stranded siRNAs exhibited different degrees of inhibition of cell proliferation in a dose-dependent manner. siRNA2 and siRNA4, exhibited obvious effects of inhibiting hTERT mRNA and protein expression in HepG2cells.CONCLUSION: siRNAs targeting different hTERT sequences have significantly various inhibitory effects on hTERT gene expression. The siRNA sequence screened by full length gene targeting technique has comparable inhibitory effect with the rest siRNA sequences screened by random selection, suggesting that siRNAs and antisense oligonucleic acids may have the same effective target sites. Compared with chemical synthesis method,synthesizing double-stranded siRNA by T7 transcription system in vitro is a rapid, simple, and inexpensive method suitable for screening high-effect siRNA targeting site for specific gene.
文摘Oncogenic H-Ras G12V and its variants have been shown to inhibit muscle differentiation. However, the role of proto-oncogenic Ras （c-Ras） in muscle differentiation remains unclear. The active GTP-bound form of Ras has been known to associate with diverse effectors including Raf, phosphatidylinositol 3-kinase （PI3K）, RaI-GDS, and other molecules to transmit downstream signals. We hypothesize that c-Ras may stimulate muscle differentiation by selectively activating PI3K, an important mediator for muscle differentiation. In our experiments, inhibition of c-Ras by farnesyltransferase inhibitors and a dominant negative form of H-Ras （Ras S17N） suppressed muscle differentiation. Consistently, individual knockdown of H-Ras, K-Ras, and N-Ras by siRNAs all blocked muscle differentiation. Interestingly, we found that c-Ras preferentially interacts with PI3K rather than its major binding partner c-Raf, during myogenic differentiation, with total c-Ras activity remaining unchanged. PI3K and its downstream myogenic pathway, the Nox2/NF-kB/inducible nitric oxide synthase （iNOS） pathway, were found to be suppressed by inhibition of c-Ras activity during differentiation. Furthermore, expression of a constitutively active form of PI3K completely rescued the differentiation block and reactivated the Nox2/NF-kB/iNOS pathway in c-Ras-inhibited cells. On the ba- sis of our results, we conclude that contrary to oncogenic Ras, proto-oncogenic H-Ras, K-Ras, and N-Ras are directly involved in the promotion of muscle differentiation via PI3K and its downstream signaling pathways. In addition, PI3K pathway activation is associated with a concurrent suppression of the otherwise predominantly activated Raf/ Mek/Erk pathway.
基金supported by the National Natural Science Foundation of China(20932001)the Ministry of Science and Technology of China (2006AA02Z144,2009ZX09503)
文摘Antisense oligonucleotides(ASONs) and siRNAs have been applied extensively for the regulation of cellular and viral gene expression,and RNAi is currently one of the most promising new approaches for anti-tumor and anti-viral therapy.In order to improve bioactivity properties and physicochemical properties of siRNA,we synthesized a novel class of ASONs II-VII incorporated with amino-isonucleoside(isoA1 and isoA2) for investigation on basic physicochemical properties.Then we designed amino-isonucleoside(isoA1,isoA2 and isoT1) incorporated siRNA 2-7.Some meaningful results have been obtained from the physicochemical property experiments in ASONs.In RNAi potency experiments,we investigated RNAi potency of each strand of the siRNA.These amino-isonucleosides incorporated siRNAs showed promising bioactivity properties and had position specificity.Reduced off target effect from sense strand loading in siRNA application was observed.
