STAT3特异性短干涉RNA抑制神经胶质瘤细胞增殖的研究
|
摘要
目的:探讨在STAT3基因不同的结构域选择干涉靶点制备的siRNA对大鼠神经胶质瘤细胞增殖的影响。
方法:针对STAT3编码区的三个不同结构域SH2、CCD和TAD各分别选择一段序列,在体外构建短发夹结构,连到人U6启动子后,克隆到T载体中,构建重组体,再对重组质粒进行酶切鉴定,阳性的质粒DNA测序分析后再克隆到穿梭质粒PDC316中,通过Lipofectamine2000的介导和腺病毒包装质粒PBHG共转染至人胚肾HEK293细胞株中,经同源重组后获得重组腺病毒rAdUshSTAT3,应用PCR鉴定重组腺病毒,空斑传代纯化病毒并反复冻融扩增病毒,以50%组织培养感染剂量法(TCID50)测定病毒滴度。以阴性为对照,转染到大鼠神经胶质瘤(C6)细胞,MTT法检测C6细胞增殖情况,吖啶橙法检测C6细胞形态的改变,RT-PCR法和Western-blot法观察STAT3基因表达改变,用免疫组织化学卵白素-生物素-过氧化酶(ABC)检测C6细胞中增殖细胞核抗原(PCNA)的表达。
结果:获得了带有U6启动子及STAT3反向互补靶序列的重组腺病毒载体rAdUshSTAT3(1-4),病毒滴度分别为108.6 TCID50/ml, 109.2 TCID50/ml,108 TCID50/ml, 108.4 TCID50/ml;
未转导病毒的细胞相比,rAdSTAT3(1)、rAdSTAT3(2)和rAdSTAT3(3)对C6细胞增殖抑制率在24h、48h和72h分别为20.16%、56.25%、73.18%; 19.76%、52.06%、70.2%;12.93%、49.59%、60.39%。rAdUshSTAT3(1)转导的C6细胞的STAT3 mRNA相对表达量下降了68.82% (p<0.01),蛋白相对表达量下降了68% (p<0.005);rAdUshSTAT3(2)转导的C6细胞的STAT3 mRNA相对表达量下降了56.93% (p<0.01),蛋白相对表达量下降了44% (p<0.01); rAdUshSTAT3(3)转导的C6细胞的STAT3 mRNA相对表达量下降了56.95% (p<0.01),蛋白相对表达量下降了38%;与rAdSTAT3(4)相比,rAdSTAT3(1)对C6细胞增殖抑制率在24h、48h和72h分别为18.76%、41.22%和62.35%;rAdSTAT3(2)对C6细胞增殖抑制率在24h、48h和72h分别为18.34%、35.58%和58.16%;rAdSTAT3(3)对C6细胞增殖抑制率在24h、48h和72h分别为11.4%、31.7%和48.33%。rAdUshSTAT3(1)转导的C6细胞的STAT3 mRNA相对表达量下降了65.24% (p<0.01),蛋白相对表达量下降了56.25% (p<0.01);rAdUshSTAT3(2)转导的C6细胞的STAT3 mRNA相对表达量下降了51.98% (p<0.01),蛋白相对表达量下降了37% (p<0.05); rAdUshSTAT3(3)转导的C6细胞的STAT3 mRNA相对表达量下降了50.95% (p<0.01),蛋白相对表达量下降了31% (p<0.05)。
吖啶橙染色法结果显示rAdUshSTAT3(1-3)转染后的细胞数量明显减少,细胞质萎缩,细胞核相对较大,并且出现凋亡小体。尤其是rAdUshSTAT3(1)转染后最为明显。免疫组化法显示rAdUshSTAT3(1)转染C6细胞后的PCNA表达量明显低于rAdUshSTAT3(4)转染C6细胞后的PCNA表达量(P<0.05)
结论:在SH2结构域上选择的干涉靶点上制备的SiRNA抑制神经胶质瘤细胞增殖的效果最强,在CCD和TAD结构域上选择的干涉靶点上制备的SiRNA抑制神经胶质瘤细胞增殖的效果虽然也很好,但弱于在SH2结构域上选择的干涉靶点制备的SiRNA,这为今后针对STAT3的RNA干涉的再深入研究奠定了坚实基础。
Signal transducer and activator of transcription (STAT3) is known to be oncogene which is connected with many tumors closely.The content of STAT3 mRNA (5kb) is high in the brain, heart, liver, testes and thoracic gland thymus, it also can be detected in spleen. STAT3 is expressed in different types of cells and tissues widely, and its signal transduction pathway is connected with cell proliferation, differentiation and apoptosis closely. It has been found that STAT3 has a high expression in leukemia, multiple myeloma, squamous cell carcinoma on head and neck, melanoma, breast cancer, prostate cancer and lung cancer. Study shows that STAT3 expression has positive correlation with the high degree of tumor malignancy and poor prognosis. Therefore, a variety of strategies including antisense oligonucleotides, ribozymes was taken to reduce the expression of STAT3 so as to weaken the founction of promting desintegration and transfer to tumor cells of JAK-STATs.It is a promising strategy to inhibit tumor cell proliferation and induce tumor cell apoptosis by inhibiting the expression of STAT3.
There are many ways to inhibiting the ptoteinum repression of STAT3 ,but they generally have some disadvantages of low transduction efficiency, short fonuction time,etc.The discovery and application of RNA interference (RNA interference, RNAi) phenomenon provide us a new thinking way to inhibit the expression of genes. RNAi is a RNA-induced post- transcriptional regulation and control method with a high degree of sequence-specific which can make specific gene silencing and functional incapacitation .RNAi has become an effective way to study gene function and cure tumor gene .
In this experiment,we select the adenovirus vector as the tool to transport siRNA.We look torward to inhibit the proliferation of glioma cell and induce apoptosis of cell by inhibiting the expression of STAT3 by viral vector-mediated RNAi technology, make theoretical foundation for gene therapy for tumor.
First of all, in accordance with the principle of RNA interference, we design the targeting point of RNA interference, make synthetic primers containing shSTAT3, gain Human U6 promoter and reverse complementary target sequence fragments HU6shSTAT3 with STAT3 by PCR method, construct recombinant adenovirus vector (rAdUSTAT3) containing the short hairpin RNA of STAT3 under the supervision of fluorescent protein adenovirus vector(Ad eGFP) through Microbix adenovirus AdMaxTM the company's packaging systems and transfer it to the C6 cells. Thenchoose the cells of non transduction virus as basic reference, monitor efficiency of transduction by GFP, choose the cells transducted by the virus vector with STAT3 irrelevant shRNA as non-specific inhibiting reference ,choose P-actin as a inner reference, detect the levels of STAT3 mRNA by RT - PCR, detect the levels of protein of STAT3 by Western Blot. Finally, assay the proliferation capacity of STAT3 cells by MTT. Detect transfected cell morphology changes and apoptosis by acridine orange staining.Detect the levels of PCNA expression of transfected cell by immunohistochemical.
The results showed that: the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3(l) decreased by 41.36% (p <0.01) at 48h compared to rAdUshSTAT3 (4),the relative expression of mRNA of C6 transducted by rAdUshSTAT3(2) decreased by 20.51% (p <0.01), the relative expression of mRNA of C6 transducted by rAdUshSTAT3(3) decreased by 15.6% (p <0.01); Compared to the cells of untransduced by virus, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 46.77% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 27.85% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 33.38% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (4) decreased by 3.6% (p> 0.05). Compared to rAdUshSTAT3 (4) at 72h, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 65.24% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 51.98% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 50.95% (p <0.01); Compared to the cells of untransducted by virus, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 68.82% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 56.93% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 56.95% (p <0.01); the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (4) decreased by 10.29% (p <0.05).
The relative expression of STAT3 protein did not change (p> 0.05) 48h after recombinant adenovirusvector transducting C6 cells ; after 72h the expression of STAT3 of the cells untransducted the virus has no significant difference (p> 0.05) compared to the negative control group .Compared to the negative control group rAdUshSTAT3 (4) , the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 31% (p <0.05), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 37% (p <0.05), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 56.25% (p <0.01). Compared to the cells of untransduced by virus, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 68% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 44% (p <0.01). the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 38% (p <0.01).
Compared to the cells of untransduced by virus, the inhibiting rate of AdUshSTAT3 (1-3)to the proliferation of C6 cells at 24h, 48h, 72h were 20.16%, 56.25%, 73.18%; 19.76%, 52.06%, 70.2%; 12.93% , 49.59%, 60.39%; compared to rAdUSTAT3 (4). The results show that, the inhibiting rate of rAdUSTAT3 (4)to the proliferation of C6 cells at 24h, 48h, 72h were 18.76%, 41.22% and 62.35%; 18.34%, 35.58% and 58.16%; 11.4%, 31.7% and 48.33%.
The results of acridine orange staining showed: the quantity of transfected cells by rAdUshSTAT3 (1-3) decieased significantly, cytoplasmic shrinkage, cell nucleus enlarged and apoptotic bodies appeared. Especially the cell transfected by rAdUshSTAT3 (1) is the most obviously.
The results of immunohistochemistry showed: the level of PCNA expression of transfected cells by rAdUshSTAT3 (1) was significantly lower than the level of PCNA expression of transfected cells by rAdUshSTAT3(4)(P <0.05).
We can draw the following conclusions from this experiment: adenovirus vector transduction can efficiently transduct C6 cells, is an effective carrier for gene therapy to glioma; the short hairpin RNA to STAT3 by adenovirus vector can effectively inhibit the expression of STAT3 of neural glioma cells, block JAK-STAT3 signaling pathway, thus inhibiting the proliferation of glioma cells. The secondary structure of mRNA should be fully taken into account in choosing target of the RNAi-specific interference, try to avoid too much stem-loop structure. At the same time, consider the domain of structure of STAT3 protein in choosing the target sequence ,we found the effect of interference in SH2 is superior than the other domains.the effect of inhibition to C6 cell by SiRNA of SH2 domain was the most obviously. The effect of inhibition to C6 cell by SiRNA of the CCD and TAD domains was obviously, but weaker than the effect of inhibition to C6 cell by SiRNA of SH2 domain.These estabilish solid foundation for the further resarch of RNAi against STAT3-depth study
To sum up, the next step is to make effective siRNA by the selection of specific and effective target to SH2 domain of STAT3,and enhance the role of aging by transforming the adenovirus vector; stuff on JAK-STAT3 signaling pathway and other tumor-related genes by the strategy of multi-genes combined application of RNAi ,eventually develop a specific and efficient drug of gene therapy to inhibiting the cell proliferation of glioma.
方法:针对STAT3编码区的三个不同结构域SH2、CCD和TAD各分别选择一段序列,在体外构建短发夹结构,连到人U6启动子后,克隆到T载体中,构建重组体,再对重组质粒进行酶切鉴定,阳性的质粒DNA测序分析后再克隆到穿梭质粒PDC316中,通过Lipofectamine2000的介导和腺病毒包装质粒PBHG共转染至人胚肾HEK293细胞株中,经同源重组后获得重组腺病毒rAdUshSTAT3,应用PCR鉴定重组腺病毒,空斑传代纯化病毒并反复冻融扩增病毒,以50%组织培养感染剂量法(TCID50)测定病毒滴度。以阴性为对照,转染到大鼠神经胶质瘤(C6)细胞,MTT法检测C6细胞增殖情况,吖啶橙法检测C6细胞形态的改变,RT-PCR法和Western-blot法观察STAT3基因表达改变,用免疫组织化学卵白素-生物素-过氧化酶(ABC)检测C6细胞中增殖细胞核抗原(PCNA)的表达。
结果:获得了带有U6启动子及STAT3反向互补靶序列的重组腺病毒载体rAdUshSTAT3(1-4),病毒滴度分别为108.6 TCID50/ml, 109.2 TCID50/ml,108 TCID50/ml, 108.4 TCID50/ml;
未转导病毒的细胞相比,rAdSTAT3(1)、rAdSTAT3(2)和rAdSTAT3(3)对C6细胞增殖抑制率在24h、48h和72h分别为20.16%、56.25%、73.18%; 19.76%、52.06%、70.2%;12.93%、49.59%、60.39%。rAdUshSTAT3(1)转导的C6细胞的STAT3 mRNA相对表达量下降了68.82% (p<0.01),蛋白相对表达量下降了68% (p<0.005);rAdUshSTAT3(2)转导的C6细胞的STAT3 mRNA相对表达量下降了56.93% (p<0.01),蛋白相对表达量下降了44% (p<0.01); rAdUshSTAT3(3)转导的C6细胞的STAT3 mRNA相对表达量下降了56.95% (p<0.01),蛋白相对表达量下降了38%;与rAdSTAT3(4)相比,rAdSTAT3(1)对C6细胞增殖抑制率在24h、48h和72h分别为18.76%、41.22%和62.35%;rAdSTAT3(2)对C6细胞增殖抑制率在24h、48h和72h分别为18.34%、35.58%和58.16%;rAdSTAT3(3)对C6细胞增殖抑制率在24h、48h和72h分别为11.4%、31.7%和48.33%。rAdUshSTAT3(1)转导的C6细胞的STAT3 mRNA相对表达量下降了65.24% (p<0.01),蛋白相对表达量下降了56.25% (p<0.01);rAdUshSTAT3(2)转导的C6细胞的STAT3 mRNA相对表达量下降了51.98% (p<0.01),蛋白相对表达量下降了37% (p<0.05); rAdUshSTAT3(3)转导的C6细胞的STAT3 mRNA相对表达量下降了50.95% (p<0.01),蛋白相对表达量下降了31% (p<0.05)。
吖啶橙染色法结果显示rAdUshSTAT3(1-3)转染后的细胞数量明显减少,细胞质萎缩,细胞核相对较大,并且出现凋亡小体。尤其是rAdUshSTAT3(1)转染后最为明显。免疫组化法显示rAdUshSTAT3(1)转染C6细胞后的PCNA表达量明显低于rAdUshSTAT3(4)转染C6细胞后的PCNA表达量(P<0.05)
结论:在SH2结构域上选择的干涉靶点上制备的SiRNA抑制神经胶质瘤细胞增殖的效果最强,在CCD和TAD结构域上选择的干涉靶点上制备的SiRNA抑制神经胶质瘤细胞增殖的效果虽然也很好,但弱于在SH2结构域上选择的干涉靶点制备的SiRNA,这为今后针对STAT3的RNA干涉的再深入研究奠定了坚实基础。
Signal transducer and activator of transcription (STAT3) is known to be oncogene which is connected with many tumors closely.The content of STAT3 mRNA (5kb) is high in the brain, heart, liver, testes and thoracic gland thymus, it also can be detected in spleen. STAT3 is expressed in different types of cells and tissues widely, and its signal transduction pathway is connected with cell proliferation, differentiation and apoptosis closely. It has been found that STAT3 has a high expression in leukemia, multiple myeloma, squamous cell carcinoma on head and neck, melanoma, breast cancer, prostate cancer and lung cancer. Study shows that STAT3 expression has positive correlation with the high degree of tumor malignancy and poor prognosis. Therefore, a variety of strategies including antisense oligonucleotides, ribozymes was taken to reduce the expression of STAT3 so as to weaken the founction of promting desintegration and transfer to tumor cells of JAK-STATs.It is a promising strategy to inhibit tumor cell proliferation and induce tumor cell apoptosis by inhibiting the expression of STAT3.
There are many ways to inhibiting the ptoteinum repression of STAT3 ,but they generally have some disadvantages of low transduction efficiency, short fonuction time,etc.The discovery and application of RNA interference (RNA interference, RNAi) phenomenon provide us a new thinking way to inhibit the expression of genes. RNAi is a RNA-induced post- transcriptional regulation and control method with a high degree of sequence-specific which can make specific gene silencing and functional incapacitation .RNAi has become an effective way to study gene function and cure tumor gene .
In this experiment,we select the adenovirus vector as the tool to transport siRNA.We look torward to inhibit the proliferation of glioma cell and induce apoptosis of cell by inhibiting the expression of STAT3 by viral vector-mediated RNAi technology, make theoretical foundation for gene therapy for tumor.
First of all, in accordance with the principle of RNA interference, we design the targeting point of RNA interference, make synthetic primers containing shSTAT3, gain Human U6 promoter and reverse complementary target sequence fragments HU6shSTAT3 with STAT3 by PCR method, construct recombinant adenovirus vector (rAdUSTAT3) containing the short hairpin RNA of STAT3 under the supervision of fluorescent protein adenovirus vector(Ad eGFP) through Microbix adenovirus AdMaxTM the company's packaging systems and transfer it to the C6 cells. Thenchoose the cells of non transduction virus as basic reference, monitor efficiency of transduction by GFP, choose the cells transducted by the virus vector with STAT3 irrelevant shRNA as non-specific inhibiting reference ,choose P-actin as a inner reference, detect the levels of STAT3 mRNA by RT - PCR, detect the levels of protein of STAT3 by Western Blot. Finally, assay the proliferation capacity of STAT3 cells by MTT. Detect transfected cell morphology changes and apoptosis by acridine orange staining.Detect the levels of PCNA expression of transfected cell by immunohistochemical.
The results showed that: the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3(l) decreased by 41.36% (p <0.01) at 48h compared to rAdUshSTAT3 (4),the relative expression of mRNA of C6 transducted by rAdUshSTAT3(2) decreased by 20.51% (p <0.01), the relative expression of mRNA of C6 transducted by rAdUshSTAT3(3) decreased by 15.6% (p <0.01); Compared to the cells of untransduced by virus, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 46.77% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 27.85% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 33.38% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (4) decreased by 3.6% (p> 0.05). Compared to rAdUshSTAT3 (4) at 72h, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 65.24% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 51.98% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 50.95% (p <0.01); Compared to the cells of untransducted by virus, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 68.82% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 56.93% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 56.95% (p <0.01); the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (4) decreased by 10.29% (p <0.05).
The relative expression of STAT3 protein did not change (p> 0.05) 48h after recombinant adenovirusvector transducting C6 cells ; after 72h the expression of STAT3 of the cells untransducted the virus has no significant difference (p> 0.05) compared to the negative control group .Compared to the negative control group rAdUshSTAT3 (4) , the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 31% (p <0.05), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 37% (p <0.05), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 56.25% (p <0.01). Compared to the cells of untransduced by virus, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 68% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 44% (p <0.01). the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 38% (p <0.01).
Compared to the cells of untransduced by virus, the inhibiting rate of AdUshSTAT3 (1-3)to the proliferation of C6 cells at 24h, 48h, 72h were 20.16%, 56.25%, 73.18%; 19.76%, 52.06%, 70.2%; 12.93% , 49.59%, 60.39%; compared to rAdUSTAT3 (4). The results show that, the inhibiting rate of rAdUSTAT3 (4)to the proliferation of C6 cells at 24h, 48h, 72h were 18.76%, 41.22% and 62.35%; 18.34%, 35.58% and 58.16%; 11.4%, 31.7% and 48.33%.
The results of acridine orange staining showed: the quantity of transfected cells by rAdUshSTAT3 (1-3) decieased significantly, cytoplasmic shrinkage, cell nucleus enlarged and apoptotic bodies appeared. Especially the cell transfected by rAdUshSTAT3 (1) is the most obviously.
The results of immunohistochemistry showed: the level of PCNA expression of transfected cells by rAdUshSTAT3 (1) was significantly lower than the level of PCNA expression of transfected cells by rAdUshSTAT3(4)(P <0.05).
We can draw the following conclusions from this experiment: adenovirus vector transduction can efficiently transduct C6 cells, is an effective carrier for gene therapy to glioma; the short hairpin RNA to STAT3 by adenovirus vector can effectively inhibit the expression of STAT3 of neural glioma cells, block JAK-STAT3 signaling pathway, thus inhibiting the proliferation of glioma cells. The secondary structure of mRNA should be fully taken into account in choosing target of the RNAi-specific interference, try to avoid too much stem-loop structure. At the same time, consider the domain of structure of STAT3 protein in choosing the target sequence ,we found the effect of interference in SH2 is superior than the other domains.the effect of inhibition to C6 cell by SiRNA of SH2 domain was the most obviously. The effect of inhibition to C6 cell by SiRNA of the CCD and TAD domains was obviously, but weaker than the effect of inhibition to C6 cell by SiRNA of SH2 domain.These estabilish solid foundation for the further resarch of RNAi against STAT3-depth study
To sum up, the next step is to make effective siRNA by the selection of specific and effective target to SH2 domain of STAT3,and enhance the role of aging by transforming the adenovirus vector; stuff on JAK-STAT3 signaling pathway and other tumor-related genes by the strategy of multi-genes combined application of RNAi ,eventually develop a specific and efficient drug of gene therapy to inhibiting the cell proliferation of glioma.
引文
[1]Darnell，J．E．Jr．STATs and gene regulation．Science．1997，277(5332)：1630-1635．
[2]Klampfer，L．，2008．The role of signal transducers and activators of transcription incolon cancer．Front．Biosci．13，2888-2899．
[3]PELLEGRINI S，DUSANTER-FI．The structure，re-gulation and function of the Janus kinase(JAKs)and the signal transducers and activators of transcriptions rSTATs)[J]．Eur JBiochem，1997，248：615-633．
[4]Kim，D．J．，Chan，K．S．，Sano，S．，Digiovanni，J．，2007．Signal transducer and activatorof transcription 3(Stat3)in epithelial carcinogenesis．Mol．Carcinog．46，725-731．
[5]Murray，P．J．，2007．The JAK-STAT signaling pathway：input and output integration．J．Immunol．178．2623-2629．
[6]HOU XS，MELNICK MB，PARRIMON N．Marrele acts downstreamof the dDrosophilaHOP／Jak kinase and encodes a protein similar to the mammalian STATs．Cell，1996，84：41 1—420．
[7]高丽芳．RNA沉默STAT3抗前列腺癌及黑色素瘤作用的实验研究[D]．吉林：吉林大学，2004．
[8]Lassmann，S．，Schuster，I．，Walch，A．，Gobel，H．，Jutting，U．，Makowiec，F．，Hopt，U．，Werner，M．，2007．STAT3 mRNA and protein expression in colorectal cancer：effects onSTAT3-inducible targets linked to cell survival and proli-feration．J．Clin．Path01．60．173—179．
[9]Akira S，NishioY，Inoue M，et al．Molecular cloning of APRF，a novel IFN-stimulatedgene factor 3 p91-related transcription factor involved in the gp 130-mediated signalingpathway．Cell．1994，77(1)：63-71．
[10]Mitsuyama，K．，Matsumoto，S．，Masuda，J．，Yamasakii，H．，Kuwaki，K．，Takedatsu，H．，Sata，M．，2007．Therapeutic strategies for targeting the IL-6／STAT3 cytokine signalingpathway in inflammatory bowel disease．Anticancer Res．27，3749-3756．
[11]Zhu，B．R．，Cai，J．M．，Tang，G．S．，Li，B．L．，Gao，F．，Cui，J．G．，Liu，H．C．，2007．Effectsof STAT3 antisense oligonucleotide on proliferation and apoptosis of non-small cell lungcancer cell line A549．Ai Zheng 26，820-827．
[12]IHLE YN，WITTHUHN BA，QUELLE FW，et al．Signal through the hematopoieticcytokine receptors[J]．Annu Rev Immunol，1995，13：369-398．
[13]PELLEGRINI S，DUSANTER-FI．The structure，re-gulation and function of the Januskinase(JAKs)and the signal transducers and activators of transcriptions(STATs)[J]．Eur JBiochem,1997,248:615-633.
[14]IHLE JN. STATs: Signal transducers and activators of transcription [J].Cell,1996,84: 331-334.
[15]STAHL N,FARRUGGELLA JT,BOULTON GT,et al.Choice of STATs and other substances specified by modular tyrosine-based motifs in cytokine receptors [J].Science, 1995,267:1349-1353.
[16]YAMASAKA Y,NAKAJIMA K,FUKADA T,et al.Differentiation and growth arrest signals are generated through the cytoplasmic region of gpl30 that is essential for stat3 activation[J].EMBOJ,1996,15:1557-1565.
[17]HEMMANN U,GERHARTZ C,HEESEL B,et al.Differential activation of acute phase response factor/STAT3 and STAT1 via the cytoplasmic domain of the IL-b singal transducer gpl30[J]JBiol Chem, 1996,271:12991-12998.
[18]PARK OK,SCHAEFER TS,NATHANS D.In vitro activation of STAT3 by epidermal growth factor receptor kinase[J].Proc Natl Acad Sci USA,1996,93:13704-13708.
[19]ZH0NG Z,ZILONG W,DARNELL J.STAT3:A STAT family number activated by tyrosine phosphorylation in res-ponse to epidermal growth factor and IL-6[J]. Science, 1994,264:95-98.
[20]SCHAPER F,SIEWERT E,GOMEZ-LECHON JM,et al.Hepatocyte growth factor/ scatter factor(HGF/SF) signals via the STAT3/APRF transcription factor in human he-patoma cells and hepatocytes[J].FEBS Lett, 1997,405:99-103.
[21]BHAT JG,BAKER MK.Angiotensin II stimulates tapid serine phosphorylation of transcription factor STAT3[J].Mol Cel Biol,1997,170:171-176.
[22]N Carlesso, DA Frank and JD Griffin Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. Journal of Experi mental Medicine, 1996,183: 811-820.
[23]Pfeffer, L. M., J. E. Mullersman, S. R. Pfeffer, A. Murti, W. Shi, C. H. Yang. STAT3 as an adapter to couple phosphatidylinositol-3 kinase to the IFNAR-1 chain of the type I IFN receptor. Science 1997.276: 1418-1420
[24]HoeyT, Grusby MJ STATs as mediators of cytokine-induced response J Adv immunol.1998, 71(8): 145-160.
[25]Raz, R., Durbin, J.E., and Levy, D.E. 1994. Acute phase response factor and additional members of the interferon-stimulated gene factor 3 family integrate diverse signals from cytokines, interferons, and growth factors. J Biol Chem. 1994,269(39): 24391-5.
[26]Takeda K , Clausen BE, Kaisho T et al. Enhanced Thl activity and development of chronic enterocolitis in mice devoid of STAT3 in macrophages and neutrophils .Immunity. 1999, 10(1): 39-49.
[27]McLemore M L,Grewal S,Liu F,et al. STAT-3activation is required for normal G-CSF-dependent proliferation and granulocytic differentiation. Immunity,2001,14 (2) :193.
[28]Boccaccio C,Ando M,Tamagnone L,et al. Induction of epithelial tubules by growth factor HGF depends on the STAT pathway. Nature, 1998,391 (6664) :285.
[29]Riley J K,Takeda K,Akira S,et al. Interleukin-10 receptor signaling through the JAK-STAT pathway. Requirement for two distinct receptor-derived signals for anti-inflammatory action. J Biol Chem, 1999,274 (23 ) : 16513.
[30]Hirano T,Ishihara K,Hibi M. Roles of STAT3in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene,2000,19 (21) :2548.
[31]Alonzi T,Maritane D,Gorgoni B,et al. Essential Role of STAT3 in the control of the acute-phase response as revealed by inducible gene inactivation in the liver. Mol Cell Biol,2001,21 (5) :1621.
[32]Hirano, T., Ishihara, K., and Hibi, M. . Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene. 2000,19(21): 2548-2556.
[33]Alonzi T, Maritano D, Gorgoni B, et al. 2001. Essential Role of STAT3 in the control of the acutephase response as revealed by inducible gene inactivation in the liver. Mol Cell Biol. 2001 ,21(5): 1621-32.
[34]Heinrich P.C., Behrmann I, Miiller-Newen G.,et al. Interleukin-6-type cytokine signalling through the gpl30/Jak/STAT pathwayl. Biochem J. 1998 ,1; 334 ( Pt 2):297-314.
[35]Karihtala, P., Soini, Y., 2007. Reactive oxygen species and antioxidant mechanisms inhuman tissues and their relation to malignancies. APMIS 115, 81-103.
[36]Bromberg JF, Wrzeszczynska MH, Devgan G, et al. STAT3 as an oncogene. Cell.1999, 98(3): 295-303.
[37]Yu CL, Meyer DJ, Campbell GS et al 1995. Enhanced DNA-binding activity of a STAT3 related protein in cells transformed by the Src oncoprotein. Science. 1995,269(5220) : 81-3.
[38]Levy DE, Gilliland DG Divergent role s of STAT1 and STAT5 malignancy asrevealed by gene disruptions in mice. On cogene. 2000 ,19(21): 2505-10
[39]Ning Z Q,Li J,McGuinness M,et al. Stat3A ctivation is required for Asp (816) mutant c-kit induced tumorigenicity. Oncogene,2001,20 (33) :4528.
[40]Burke WM,Jin X,Lin HJ,et al. Inhibition of constitutively active Stat3 suppresses growth of human ovarian and breast cancer cells. Oncogene,2001,20 (55 ) :7925-7934.
[41]Bromberg J.Stat proteins and oncogenesis. J Clin Invest,2002,109 (9) :1139.
[42]Fjose A., Ellingsen S., Wargelius A.,et al. RNA interference: mechanisms and applications. Biotech Ann Rev, 2001; 7: 31-57.
[43]Hannon G. J. RNA interference. Nature, 2002; 418:244-251.
[44]Agami R. RNAi and related mechanisms and their potential use for therapy. Curr Opin Chem Biol, 2002; 6 (6) :829-834.
[45]Ramaswam G, Slack FJ. SiRNA , A guidd for RNA silencing. J Chem Biol, 2002; 9 (10):1053-1055.
[46]Napoli C, Lemienx C, Jorgensen R, Introduction of a Chimeric chalcone synthase gene into Petumia results in reversible co-suppression of homologous genes in trans. Plant cell, 1990, 2(4):279-289.
[47]Cogoni C, Romano N, Macino G. Suppressionof gene expression by homologus trans genes[J]AntonieVan Leeuwenhoek ,1994,65(3):205-209.
[48]Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 1995;81:611-620.
[49]Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806-811.
[50]Fire A. RNA-triggered gene silencing. Trends Genet, 1999; 15: 358-363.
[51]Cox DN, Chao A, Baker J, et al. A novel class of evolutionarily conserved genes defined by piwi are essentioal for stem cell self-renewal. Genes Dev, 1998; 12: 3715-3727.
[52]Bernsein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001; 409: 363-366.
[53]Gregory J, Hannon. RNA interference. Nature, 2002,418: 244-251.
[54]Martubez J, Patkaniowska A, Urlaub H, et al. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell, 2002, 110(5): 563-574.
[55]Elabshir J, Harborth SM, Endeckel W, et al. Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells. Nature, 2001,411:494-498.
[56]Chiu YL, Rana TM. RNAi in human cell, basic structural and functional features of small interfering RNA. Mol Cell, 2002, 10(3): 549-561.
[57]Lipardi C , Wei Q, Paterson BM. et al . RNAi as random degradative PCR:siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell, 2001, 107 (3) : 297-307.
[58]Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science, 2001, 294: 862-864.
[59]Billy E, Brondani V, Zhang HD, Muller U, Filipowicz W. Specific interference with gene expression induced by long, doublestranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci USA 2001;98: 14428- 14433.
[60]Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411: 494-498.
[61]Sui GH, Soohoo C, Affarel B, Gay F, Shi Y, Forrester WC, Shi Y. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002; 99:5515-5520.
[62]Xia HB, Mao QW, Paulson HL, Davidson BL. Si-RNA mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002; 20:1006-1010.
[63]Tavernarakis N , Wang SL , Dorovkov M , et al. Heritable and inducible genetic interference by double2stranded RNA encoded by transgenes.Nat Genet, 2000 , 24 (2): 180-183
[64]Fire A, Xu S, Montgomery MK, et al. potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans.Nature,1998; 391(6669):806-811.
[65]Parrish S , Fleenor J , Xu S , et al. Functional anatomy of a dsRNA trigger : differential requirement for the two trigger strands in RNA interference. Mol Cell, 2000, 6 (5) : 1077-1087.
[66]Elbashir SM , Martinez J , Patkaniowska A , et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J, 2001 ,20 (23) : 6877-6888.
[67]Brummelkamp T.R, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002; 296: 550-553.
[68]Miyagishi M, Taira K. U6 promoter-driven siRNA with four uridines 3' overhangs efficiently suppress targeted gene expression in mammalian cell. Nat Biotechnol, 2002; 20:497-500.
[69]Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol, 2002; 20: 500-505.
[70]Xia H, Mao Q, Paulson HL, et al. SiRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol, 2002; 20: 1006-1010.
[71]Hiroaki K, Kazunari T. Short haipin type of dsRNAs that are controlled by tRNAval promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Research, 2003, 31(2): 700-707.
[72]Sioud M. Therapeutic siRNAs. TRENDS in Pharmacological Sciences, 2004; 25(1): 22-28.
[73]Castanotto D, Li H, Rossi JJ. Functional siRNA expression from transfected PCR products. RNA. 2002; 8(1 I ): 1454-60.
[74]Zeng Y, Wagner EJ, Cullen BR. Both natural and designed microRNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell, 2002; 9: 1327-1333.
[75]Hannon G. J. RNA interference. Nature, 2002; 418:244-251.
[76]Shen C, Buck AK, Liu X, et al. Gene silencing by adenovirus-delivered siRNA. FEBS Lett, 2003: 539:111-114.
[77]Gregory MB, Ruslan M. Retroviral delivery of small interfering RNA into primary cells. PNAS, 2002; 99(12): 14943-14945.
[78]Tomar RS, Matta H, Chaudhary PM. Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene, 2003; 22: 5712-5715.
[79]McLemore M L,Grewal S,Liu F,et al. STAT-3activation is required for normal G-CSF-dependent proliferation and granulocytic differentiation. Immunity,2001,14 (2) :193.
[80]Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cell. Nat Biotech, 2002; 20: 500-505.
[81]Crowe S. Suppression of chemokine receptor expression by RNA inter- ference slows for inhibition of HIV-1 replication, by Martinez et al. AIDS, 2003; 17 Suppl 4: sl03-105.
[82]Capodici J, Kariko K, Weissman D, et al. Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference. J Immunol, 2002; 169: 5196-5201.
[83]Cobum GA, Cullen BR. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J Virol, 2002;76: 9225-9231.
[84]Jacque JM, Triques K, Stevenson M, et al. Modulation of HIV-1 replication by RNAinterference．Nature．2002；418：435-438
[85]Zhang Y，Li T，Fu L，et al．Silencing SARS-CoV Spike protein expression in culturedcells by RNA interference．FEBS Lett，2004；560：141-146．
[86]Elbashir SM，Martinez J，Patkaniowska A，et al．Functional anatomy of siRNAs formediating efficient RNAi in Drosophila melanogaster embryo lysate．EMBO J，2001，20(23)：6877-6888．
[87]Wilda M，Fuchs U，Wossmann W，et al．Killing of leukemic cells with a BCR／ABLfusion geng by RNA interference(RNAi)．Oncogene，2002；21：5716-5724．
[88]Tuschl T，Borkhardt A．Small interfering RNAs：a revolutionary tool for the analysisofgene function and gene therapy．Molecular Interventions，2002；2(3)：158-167．
[89]Wesley SV，Hwlliqwll CA，Smih NA，et al．Construct design for efficient，effectiveand high-throughput gene silencing in plants．Plant J．2001；27：581-590．
[90]Pekarik V，Bouurikas D，Miglino N，et al．Screening for gene function in chickenembryo using RNAi and electroporation．Nat Biotechnol，2003；21(1)：93-96．
[91]赵慧．利用RNA干扰抑制乳腺癌细胞C-Met表达的研究[D]．吉林：吉林大学，2004．
[92]尹一恒：章翔等．STAT3与脑胶质细胞瘤[J]．中华神经外科疾病研究杂志．2008．7(5)：472-474
[93]KonnikovaL，KoteckiM，KrugerMM，et al．Knockdown of STAT3 expression byRNAi induces apoptosis in astrocytoma cells[J]．BMC Cancer，2003，3：23．
[94]Rahaman SO，Harbor PC，Chemova O，et al．Inhibition of constitutively activeSTAT3 suppresses proliferation and inducesapoptosis in glioblastoma multiforme cells[J]．Oncogene，2002，21(55)：8404-8413．
[95]WeissenbergerJ，Loefier S，Kappeler A，et al．IL-6 is required for glioma developmentin amousemodel[J]．Oncogene，2004，23(19)：3308-3316．
[96]Schaefer LK，Ren ZY，Fuller GN，et al．Constitutive activation of Stat3 alpha in braintumors：localization to tumor endothelial cells and activation by the endothelial tyrosine kinasereceptor(VEGFR-2)[J]．Oncogene，2002，2113：2058-2065
[97]Turkson J．STAT proteins as novel targets for cancer drug discoveryJ．STAT proteinsas novel targets for cancer drug di scovery[J]．Expert Opin Ther Targets，2004，8(5)：409-422．
[98]Colot V，Maloisel L，Rossignol JL．Interchromosomal transfer of epigenetic states inascobolus：Transfer of DNA methylation is mechanistically related to homologousrecombination．Cell，2006；86：855-864．
[99]马宁．腺病毒介导干扰hTGF-β1基因表达的shRNA抑制瘢痕疙瘩成纤维细胞生长的实验研究[D]．吉林：吉林大学，2007
[100]Miyagishi M，Taira K．U6 P romoter-driven siRNAs with foururidine overhangsefciently suppress targeted gene expression in mammalian cells[J]．Nat Biotechnol，2002，20497-500
[101]Lee NS，Dohj ima T，Bauer G，et al．Exp ression of small interfering RNAs targetedagainst H IV-1 rev transcrip ts in human cells[J]．NatBiotechnol，2002，20：500-505
[102]Holen T，Amarzguioui M，Wiiger MT，et al．Positionaleffects of short interferingRNAs targeting the human coagu21ation trigger tissue factor[J]．Nucleic Acids Res，2002，301757-1766
[103]Schwarz D S，Hutva gner G，Du T，et al．Asymmetry in the assembly of the RNAienzyme comp lex[J]．Cell，2003，115，199-208 [104]燕翔，马文丽，宋艳斌，等．目标序列二级结构对RNA干扰效果的影响[J]．基础与临床，2006，26(6)：570-574．
[105]Hyung Gyun Kim，Yong Pil Hwang，Hye Gwang Jeong Kahweol blocks STAT3phosphorylation and induces apoptosis in human lung adenocarcinoma A549 cells ToxicologyLetters，187(2009)28-34
[106]Leeman RJ，Lui VW，Grandis JR．STAT3 as a therapeutic target in head and neckcancer[J]．Expert Opin Biol Ther，2006，6(3)：231-241
[2]Klampfer，L．，2008．The role of signal transducers and activators of transcription incolon cancer．Front．Biosci．13，2888-2899．
[3]PELLEGRINI S，DUSANTER-FI．The structure，re-gulation and function of the Janus kinase(JAKs)and the signal transducers and activators of transcriptions rSTATs)[J]．Eur JBiochem，1997，248：615-633．
[4]Kim，D．J．，Chan，K．S．，Sano，S．，Digiovanni，J．，2007．Signal transducer and activatorof transcription 3(Stat3)in epithelial carcinogenesis．Mol．Carcinog．46，725-731．
[5]Murray，P．J．，2007．The JAK-STAT signaling pathway：input and output integration．J．Immunol．178．2623-2629．
[6]HOU XS，MELNICK MB，PARRIMON N．Marrele acts downstreamof the dDrosophilaHOP／Jak kinase and encodes a protein similar to the mammalian STATs．Cell，1996，84：41 1—420．
[7]高丽芳．RNA沉默STAT3抗前列腺癌及黑色素瘤作用的实验研究[D]．吉林：吉林大学，2004．
[8]Lassmann，S．，Schuster，I．，Walch，A．，Gobel，H．，Jutting，U．，Makowiec，F．，Hopt，U．，Werner，M．，2007．STAT3 mRNA and protein expression in colorectal cancer：effects onSTAT3-inducible targets linked to cell survival and proli-feration．J．Clin．Path01．60．173—179．
[9]Akira S，NishioY，Inoue M，et al．Molecular cloning of APRF，a novel IFN-stimulatedgene factor 3 p91-related transcription factor involved in the gp 130-mediated signalingpathway．Cell．1994，77(1)：63-71．
[10]Mitsuyama，K．，Matsumoto，S．，Masuda，J．，Yamasakii，H．，Kuwaki，K．，Takedatsu，H．，Sata，M．，2007．Therapeutic strategies for targeting the IL-6／STAT3 cytokine signalingpathway in inflammatory bowel disease．Anticancer Res．27，3749-3756．
[11]Zhu，B．R．，Cai，J．M．，Tang，G．S．，Li，B．L．，Gao，F．，Cui，J．G．，Liu，H．C．，2007．Effectsof STAT3 antisense oligonucleotide on proliferation and apoptosis of non-small cell lungcancer cell line A549．Ai Zheng 26，820-827．
[12]IHLE YN，WITTHUHN BA，QUELLE FW，et al．Signal through the hematopoieticcytokine receptors[J]．Annu Rev Immunol，1995，13：369-398．
[13]PELLEGRINI S，DUSANTER-FI．The structure，re-gulation and function of the Januskinase(JAKs)and the signal transducers and activators of transcriptions(STATs)[J]．Eur JBiochem,1997,248:615-633.
[14]IHLE JN. STATs: Signal transducers and activators of transcription [J].Cell,1996,84: 331-334.
[15]STAHL N,FARRUGGELLA JT,BOULTON GT,et al.Choice of STATs and other substances specified by modular tyrosine-based motifs in cytokine receptors [J].Science, 1995,267:1349-1353.
[16]YAMASAKA Y,NAKAJIMA K,FUKADA T,et al.Differentiation and growth arrest signals are generated through the cytoplasmic region of gpl30 that is essential for stat3 activation[J].EMBOJ,1996,15:1557-1565.
[17]HEMMANN U,GERHARTZ C,HEESEL B,et al.Differential activation of acute phase response factor/STAT3 and STAT1 via the cytoplasmic domain of the IL-b singal transducer gpl30[J]JBiol Chem, 1996,271:12991-12998.
[18]PARK OK,SCHAEFER TS,NATHANS D.In vitro activation of STAT3 by epidermal growth factor receptor kinase[J].Proc Natl Acad Sci USA,1996,93:13704-13708.
[19]ZH0NG Z,ZILONG W,DARNELL J.STAT3:A STAT family number activated by tyrosine phosphorylation in res-ponse to epidermal growth factor and IL-6[J]. Science, 1994,264:95-98.
[20]SCHAPER F,SIEWERT E,GOMEZ-LECHON JM,et al.Hepatocyte growth factor/ scatter factor(HGF/SF) signals via the STAT3/APRF transcription factor in human he-patoma cells and hepatocytes[J].FEBS Lett, 1997,405:99-103.
[21]BHAT JG,BAKER MK.Angiotensin II stimulates tapid serine phosphorylation of transcription factor STAT3[J].Mol Cel Biol,1997,170:171-176.
[22]N Carlesso, DA Frank and JD Griffin Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. Journal of Experi mental Medicine, 1996,183: 811-820.
[23]Pfeffer, L. M., J. E. Mullersman, S. R. Pfeffer, A. Murti, W. Shi, C. H. Yang. STAT3 as an adapter to couple phosphatidylinositol-3 kinase to the IFNAR-1 chain of the type I IFN receptor. Science 1997.276: 1418-1420
[24]HoeyT, Grusby MJ STATs as mediators of cytokine-induced response J Adv immunol.1998, 71(8): 145-160.
[25]Raz, R., Durbin, J.E., and Levy, D.E. 1994. Acute phase response factor and additional members of the interferon-stimulated gene factor 3 family integrate diverse signals from cytokines, interferons, and growth factors. J Biol Chem. 1994,269(39): 24391-5.
[26]Takeda K , Clausen BE, Kaisho T et al. Enhanced Thl activity and development of chronic enterocolitis in mice devoid of STAT3 in macrophages and neutrophils .Immunity. 1999, 10(1): 39-49.
[27]McLemore M L,Grewal S,Liu F,et al. STAT-3activation is required for normal G-CSF-dependent proliferation and granulocytic differentiation. Immunity,2001,14 (2) :193.
[28]Boccaccio C,Ando M,Tamagnone L,et al. Induction of epithelial tubules by growth factor HGF depends on the STAT pathway. Nature, 1998,391 (6664) :285.
[29]Riley J K,Takeda K,Akira S,et al. Interleukin-10 receptor signaling through the JAK-STAT pathway. Requirement for two distinct receptor-derived signals for anti-inflammatory action. J Biol Chem, 1999,274 (23 ) : 16513.
[30]Hirano T,Ishihara K,Hibi M. Roles of STAT3in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene,2000,19 (21) :2548.
[31]Alonzi T,Maritane D,Gorgoni B,et al. Essential Role of STAT3 in the control of the acute-phase response as revealed by inducible gene inactivation in the liver. Mol Cell Biol,2001,21 (5) :1621.
[32]Hirano, T., Ishihara, K., and Hibi, M. . Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene. 2000,19(21): 2548-2556.
[33]Alonzi T, Maritano D, Gorgoni B, et al. 2001. Essential Role of STAT3 in the control of the acutephase response as revealed by inducible gene inactivation in the liver. Mol Cell Biol. 2001 ,21(5): 1621-32.
[34]Heinrich P.C., Behrmann I, Miiller-Newen G.,et al. Interleukin-6-type cytokine signalling through the gpl30/Jak/STAT pathwayl. Biochem J. 1998 ,1; 334 ( Pt 2):297-314.
[35]Karihtala, P., Soini, Y., 2007. Reactive oxygen species and antioxidant mechanisms inhuman tissues and their relation to malignancies. APMIS 115, 81-103.
[36]Bromberg JF, Wrzeszczynska MH, Devgan G, et al. STAT3 as an oncogene. Cell.1999, 98(3): 295-303.
[37]Yu CL, Meyer DJ, Campbell GS et al 1995. Enhanced DNA-binding activity of a STAT3 related protein in cells transformed by the Src oncoprotein. Science. 1995,269(5220) : 81-3.
[38]Levy DE, Gilliland DG Divergent role s of STAT1 and STAT5 malignancy asrevealed by gene disruptions in mice. On cogene. 2000 ,19(21): 2505-10
[39]Ning Z Q,Li J,McGuinness M,et al. Stat3A ctivation is required for Asp (816) mutant c-kit induced tumorigenicity. Oncogene,2001,20 (33) :4528.
[40]Burke WM,Jin X,Lin HJ,et al. Inhibition of constitutively active Stat3 suppresses growth of human ovarian and breast cancer cells. Oncogene,2001,20 (55 ) :7925-7934.
[41]Bromberg J.Stat proteins and oncogenesis. J Clin Invest,2002,109 (9) :1139.
[42]Fjose A., Ellingsen S., Wargelius A.,et al. RNA interference: mechanisms and applications. Biotech Ann Rev, 2001; 7: 31-57.
[43]Hannon G. J. RNA interference. Nature, 2002; 418:244-251.
[44]Agami R. RNAi and related mechanisms and their potential use for therapy. Curr Opin Chem Biol, 2002; 6 (6) :829-834.
[45]Ramaswam G, Slack FJ. SiRNA , A guidd for RNA silencing. J Chem Biol, 2002; 9 (10):1053-1055.
[46]Napoli C, Lemienx C, Jorgensen R, Introduction of a Chimeric chalcone synthase gene into Petumia results in reversible co-suppression of homologous genes in trans. Plant cell, 1990, 2(4):279-289.
[47]Cogoni C, Romano N, Macino G. Suppressionof gene expression by homologus trans genes[J]AntonieVan Leeuwenhoek ,1994,65(3):205-209.
[48]Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 1995;81:611-620.
[49]Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806-811.
[50]Fire A. RNA-triggered gene silencing. Trends Genet, 1999; 15: 358-363.
[51]Cox DN, Chao A, Baker J, et al. A novel class of evolutionarily conserved genes defined by piwi are essentioal for stem cell self-renewal. Genes Dev, 1998; 12: 3715-3727.
[52]Bernsein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001; 409: 363-366.
[53]Gregory J, Hannon. RNA interference. Nature, 2002,418: 244-251.
[54]Martubez J, Patkaniowska A, Urlaub H, et al. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell, 2002, 110(5): 563-574.
[55]Elabshir J, Harborth SM, Endeckel W, et al. Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells. Nature, 2001,411:494-498.
[56]Chiu YL, Rana TM. RNAi in human cell, basic structural and functional features of small interfering RNA. Mol Cell, 2002, 10(3): 549-561.
[57]Lipardi C , Wei Q, Paterson BM. et al . RNAi as random degradative PCR:siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell, 2001, 107 (3) : 297-307.
[58]Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science, 2001, 294: 862-864.
[59]Billy E, Brondani V, Zhang HD, Muller U, Filipowicz W. Specific interference with gene expression induced by long, doublestranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci USA 2001;98: 14428- 14433.
[60]Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411: 494-498.
[61]Sui GH, Soohoo C, Affarel B, Gay F, Shi Y, Forrester WC, Shi Y. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002; 99:5515-5520.
[62]Xia HB, Mao QW, Paulson HL, Davidson BL. Si-RNA mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002; 20:1006-1010.
[63]Tavernarakis N , Wang SL , Dorovkov M , et al. Heritable and inducible genetic interference by double2stranded RNA encoded by transgenes.Nat Genet, 2000 , 24 (2): 180-183
[64]Fire A, Xu S, Montgomery MK, et al. potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans.Nature,1998; 391(6669):806-811.
[65]Parrish S , Fleenor J , Xu S , et al. Functional anatomy of a dsRNA trigger : differential requirement for the two trigger strands in RNA interference. Mol Cell, 2000, 6 (5) : 1077-1087.
[66]Elbashir SM , Martinez J , Patkaniowska A , et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J, 2001 ,20 (23) : 6877-6888.
[67]Brummelkamp T.R, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002; 296: 550-553.
[68]Miyagishi M, Taira K. U6 promoter-driven siRNA with four uridines 3' overhangs efficiently suppress targeted gene expression in mammalian cell. Nat Biotechnol, 2002; 20:497-500.
[69]Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol, 2002; 20: 500-505.
[70]Xia H, Mao Q, Paulson HL, et al. SiRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol, 2002; 20: 1006-1010.
[71]Hiroaki K, Kazunari T. Short haipin type of dsRNAs that are controlled by tRNAval promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Research, 2003, 31(2): 700-707.
[72]Sioud M. Therapeutic siRNAs. TRENDS in Pharmacological Sciences, 2004; 25(1): 22-28.
[73]Castanotto D, Li H, Rossi JJ. Functional siRNA expression from transfected PCR products. RNA. 2002; 8(1 I ): 1454-60.
[74]Zeng Y, Wagner EJ, Cullen BR. Both natural and designed microRNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell, 2002; 9: 1327-1333.
[75]Hannon G. J. RNA interference. Nature, 2002; 418:244-251.
[76]Shen C, Buck AK, Liu X, et al. Gene silencing by adenovirus-delivered siRNA. FEBS Lett, 2003: 539:111-114.
[77]Gregory MB, Ruslan M. Retroviral delivery of small interfering RNA into primary cells. PNAS, 2002; 99(12): 14943-14945.
[78]Tomar RS, Matta H, Chaudhary PM. Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene, 2003; 22: 5712-5715.
[79]McLemore M L,Grewal S,Liu F,et al. STAT-3activation is required for normal G-CSF-dependent proliferation and granulocytic differentiation. Immunity,2001,14 (2) :193.
[80]Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cell. Nat Biotech, 2002; 20: 500-505.
[81]Crowe S. Suppression of chemokine receptor expression by RNA inter- ference slows for inhibition of HIV-1 replication, by Martinez et al. AIDS, 2003; 17 Suppl 4: sl03-105.
[82]Capodici J, Kariko K, Weissman D, et al. Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference. J Immunol, 2002; 169: 5196-5201.
[83]Cobum GA, Cullen BR. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J Virol, 2002;76: 9225-9231.
[84]Jacque JM, Triques K, Stevenson M, et al. Modulation of HIV-1 replication by RNAinterference．Nature．2002；418：435-438
[85]Zhang Y，Li T，Fu L，et al．Silencing SARS-CoV Spike protein expression in culturedcells by RNA interference．FEBS Lett，2004；560：141-146．
[86]Elbashir SM，Martinez J，Patkaniowska A，et al．Functional anatomy of siRNAs formediating efficient RNAi in Drosophila melanogaster embryo lysate．EMBO J，2001，20(23)：6877-6888．
[87]Wilda M，Fuchs U，Wossmann W，et al．Killing of leukemic cells with a BCR／ABLfusion geng by RNA interference(RNAi)．Oncogene，2002；21：5716-5724．
[88]Tuschl T，Borkhardt A．Small interfering RNAs：a revolutionary tool for the analysisofgene function and gene therapy．Molecular Interventions，2002；2(3)：158-167．
[89]Wesley SV，Hwlliqwll CA，Smih NA，et al．Construct design for efficient，effectiveand high-throughput gene silencing in plants．Plant J．2001；27：581-590．
[90]Pekarik V，Bouurikas D，Miglino N，et al．Screening for gene function in chickenembryo using RNAi and electroporation．Nat Biotechnol，2003；21(1)：93-96．
[91]赵慧．利用RNA干扰抑制乳腺癌细胞C-Met表达的研究[D]．吉林：吉林大学，2004．
[92]尹一恒：章翔等．STAT3与脑胶质细胞瘤[J]．中华神经外科疾病研究杂志．2008．7(5)：472-474
[93]KonnikovaL，KoteckiM，KrugerMM，et al．Knockdown of STAT3 expression byRNAi induces apoptosis in astrocytoma cells[J]．BMC Cancer，2003，3：23．
[94]Rahaman SO，Harbor PC，Chemova O，et al．Inhibition of constitutively activeSTAT3 suppresses proliferation and inducesapoptosis in glioblastoma multiforme cells[J]．Oncogene，2002，21(55)：8404-8413．
[95]WeissenbergerJ，Loefier S，Kappeler A，et al．IL-6 is required for glioma developmentin amousemodel[J]．Oncogene，2004，23(19)：3308-3316．
[96]Schaefer LK，Ren ZY，Fuller GN，et al．Constitutive activation of Stat3 alpha in braintumors：localization to tumor endothelial cells and activation by the endothelial tyrosine kinasereceptor(VEGFR-2)[J]．Oncogene，2002，2113：2058-2065
[97]Turkson J．STAT proteins as novel targets for cancer drug discoveryJ．STAT proteinsas novel targets for cancer drug di scovery[J]．Expert Opin Ther Targets，2004，8(5)：409-422．
[98]Colot V，Maloisel L，Rossignol JL．Interchromosomal transfer of epigenetic states inascobolus：Transfer of DNA methylation is mechanistically related to homologousrecombination．Cell，2006；86：855-864．
[99]马宁．腺病毒介导干扰hTGF-β1基因表达的shRNA抑制瘢痕疙瘩成纤维细胞生长的实验研究[D]．吉林：吉林大学，2007
[100]Miyagishi M，Taira K．U6 P romoter-driven siRNAs with foururidine overhangsefciently suppress targeted gene expression in mammalian cells[J]．Nat Biotechnol，2002，20497-500
[101]Lee NS，Dohj ima T，Bauer G，et al．Exp ression of small interfering RNAs targetedagainst H IV-1 rev transcrip ts in human cells[J]．NatBiotechnol，2002，20：500-505
[102]Holen T，Amarzguioui M，Wiiger MT，et al．Positionaleffects of short interferingRNAs targeting the human coagu21ation trigger tissue factor[J]．Nucleic Acids Res，2002，301757-1766
[103]Schwarz D S，Hutva gner G，Du T，et al．Asymmetry in the assembly of the RNAienzyme comp lex[J]．Cell，2003，115，199-208 [104]燕翔，马文丽，宋艳斌，等．目标序列二级结构对RNA干扰效果的影响[J]．基础与临床，2006，26(6)：570-574．
[105]Hyung Gyun Kim，Yong Pil Hwang，Hye Gwang Jeong Kahweol blocks STAT3phosphorylation and induces apoptosis in human lung adenocarcinoma A549 cells ToxicologyLetters，187(2009)28-34
[106]Leeman RJ，Lui VW，Grandis JR．STAT3 as a therapeutic target in head and neckcancer[J]．Expert Opin Biol Ther，2006，6(3)：231-241
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |