组织特异性CXCR4-siRNA对前列腺癌骨转移影响的实验研究
|
摘要
㈠CXCR4/SDF-1通路在前列腺癌靶向性转移转移中作用的初步研究
目的初步探讨趋化因子CXC受体4/基质细胞衍生因子1(CXCR4/SDF-1)通路在前列腺癌(PCa)靶向性转移中的作用。方法逆转录-链式聚合酶反应(RT-PCR)法和蛋白质斑迹法(Western Blot)法分别检测PCa细胞系LNCaP和PC-3m中CXCR4 mRNA和蛋白表达,免疫组化检测正常前列腺组织和前列腺癌组织CXCR4蛋白的表达。RT-PCR法检测并比较成人骨、淋巴结、肺、脑、肝组织中SDF-1 mRNA表达,酶联免疫吸附法(ELISA)检测人成骨细胞(HOB)条件培养基上清中SDF-1蛋白水平。观察外源性SDF-1对PCa细胞体外黏附、侵袭和迁移的影响及CXCR4抗体对其效应。结果两种PCa细胞系均有CXCR4 mRNA和蛋白表达,前列腺癌组织高表达CXCR4蛋白。成人骨、淋巴结、肺、脑、肝组织均有SDF-1 mRNA表达,半定量分析显示淋巴结和骨组织表达量最高;HOB细胞培养上清中可测及较高浓度的SDF-1蛋白。外源性SDF-1可促进两种PCa细胞黏附、迁移和侵袭,呈剂量依赖关系,原代HOB培养72小时收集的条件培养基亦可促进PCa细胞体外迁移能力;CXCR4抗体则可抑制上述效应。结论PCa细胞高表达具有活化功能状态的CXCR4,PCa常见的转移靶器官则高表达SDF-1;SDF-1可促进PCa细胞体外转移过程,且可被CXCR4抗体阻断,CXCR4/SDF-1通路可能在PCa靶向性转移中发挥重要作用。
㈡携带报告基因EGFP的CXCR4 RNA干扰质粒的构建及其对前列腺癌细胞CXCR4基因表达的抑制作用
目的研究RNA干扰(RNA interference, RNAi)表达载体对前列腺癌细胞趋化因子CXC受体4(CXCR4)基因的抑制作用。方法CXCR4特异性小干扰RNA(small interfering RNA, siRNA)转入带有增强型绿色荧光蛋白(EGFP)和启动子U6的Pgensil-1质粒构建针对CXCR4基因的RNAi质粒表达载体,脂质体法转染前列腺癌PC-3m、LNCaP细胞株,应用RT-PCR、Western Blot检测其对CXCR4 mRNA及蛋白表达的影响。结果构建的表达质粒在前列腺癌PC-3m、LNCaP细胞株中均抑制了CXCR4 mRNA及蛋白表达。与空白载体组细胞作对照,前者小干扰RNA (siRNA)在对CXCR4 mRNA的抑制率24小时、48小时分别为87.81±10.20%、56.10±9.32%,后者为56.93±8.78%、49.24±11.23% ,蛋白抑制率24小时前者为64.71±6.68%,后者为58.66±11.56%。结论携带有siRNA-CXCR4序列的RNAi表达载体可以有效地抑制前列腺癌细胞CXCR4基因的表达,为后续实验奠定基础。
㈢hTERT启动子调控的CXCR4-siRNA逆转录病毒载体的构建及其体外靶向抑制hTERT+前列腺癌细胞生物学效应研究
目的:构建人端粒酶逆转录酶(hTERT)启动子调控的趋化因子受体4(CXCR4)的RNA干扰逆转录病毒载体,并研究其在hTERT+前列腺癌细胞、乳腺癌细胞中的表达。方法:用PCR扩增CXCR4特异性小干扰RNA(small interfering RNA, siRNA),转入带有增强型绿色荧光蛋白(EGFP)和启动子U6的Pgensil-1质粒,以hTERT启动子代替U6启动子,再将重组基因片段导入到逆转录病毒真核表达载体PLXSN中,ERFP基因片段代替EGFP基因片段,行限制性酶切鉴定及测序。转染PA317病毒包装细胞,收获病毒上清分别转染前列腺癌细胞PC-3m、LNCaP及人胚肺成纤维细胞MRC5和人乳腺癌细胞MCF7,RT-PCR、Western Blot检测CXCR4 mRNA及蛋白表达。观察外源性SDF-1对CXCR4基因表达抑制后PCa细胞体外黏附、侵袭和迁移的影响。结果:限制性酶切、测序鉴定该重组质粒,成功构建了PLXSN/ERFP-hTERT-siCXCR4。与空载体组比较,MCF7、PC-3m、LNCaP细胞中CXCR4-siRNA对CXCR4 mRNA的48小时抑制率分别为(88.31±9.20)%、(87.20±10.34)%、(76.40±9.77)%,CXCR4蛋白的48小时抑制率分别为(82.60±7.26)%、(80.23±4.54)%、(84.09±3.37)%。MRC5细胞中PLXSN/ERFP-hTERT-siCXCR4对CXCR4 mRNA及蛋白不抑制。CXCR4基因敲低后,外源性SDF-1促进两种PCa细胞黏附、迁移和侵袭的能力下降。结论:该逆转录病毒系统中hTERT启动子调控的下游RNA干扰序列可选择性地在hTERT+前列腺癌细胞、乳腺癌细胞中表达,在hTERT- MRC5细胞不表达,具有显著的靶向性。敲低CXCR4基因表达可抑制前列腺癌细胞体外黏附、侵袭、转移。
㈣hTERT启动子调控的CXCR4-siRNA逆转录病毒载体抑制前列腺癌细胞骨转移的体内实验研究
目的:构建可荧光示踪的前列腺癌骨转移动物模型,并探讨抑制CXCR4基因表达后对前列腺癌体内骨转移的影响。方法:选用6-8周龄体重为20-22克NOD/SCID雄性小鼠,利用外科植入术将成人骨组织(HAB)植入小鼠皮下。2-3周后,将携有ERF(PDsRED2)基因及干扰序列CXCR4-siRNA的重组逆转录病毒转染前列腺癌细胞PC-3m与LNCaP,收获稳定表达红色荧光基因(ERFP)的前列腺癌细胞并配置成细胞悬液,通过小鼠尾静脉注入小鼠体内。2周后开始通过整体荧光显像系统观察小鼠体内移植人骨组织处前列腺癌转移瘤细胞ERFP的表达。对比CXCR4基因敲低组和对照组前列腺癌骨转移瘤的骨转移率、体积、湿重、存活时间。HE染色镜检转移瘤组织切片。结果:两种前列腺癌细胞CXCR4基因敲低组骨转移瘤的骨转移率、体积、湿重均低于对照组,而存活时间高于对照组(基因未敲低组)。组织切片显示干扰CXCR4基因表达后前列腺癌骨转移瘤较基因未敲低组肿瘤组织分化程度高。结论:成功构建了可体外无创性动态观察的稳定的前列腺癌骨转移动物模型,证实敲低CXCR4基因表达可抑制体内前列腺癌骨转移并抑制前列腺癌细胞恶性分化。
⒈The effect of CXCR4/SDF-1 pathway on prostate cancer targeted metastasis
Objective To investigate preliminarily the role of CXCR4/SDF-1 in prostate cancer (PCa) targeted metastasis. Methods CXCR4 mRNA and protein expression in two PCa cancer cell lines, LNCaP and PC-3m, were examined by reverse transcript-PCR、western bloting and prostate cancer tissue was detected by immunohistochemistry. SDF-1 mRNA expressions in human bone, lymph node, lung, brain and liver tissues were determined by RT-PCR and SDF-1 secretary level in the culture medium of primary human osteoblasts (HOB) was detected by in ELISA. The effect of exogenous recombinant SDF-1 on PCa cells adhesion, transendothelial migration and invasion, and the effect of anti-CXCR antibody in this procedure were observed using an in vitro Transwell and Matrigel models.
Results Both CXCR4 mRNA and protein expressed in LNCaP and PC-3m. CXCR4 protein expressed highly in prostate cancer tissues. Meanwhile, SDF-1 mRNA expressed in divers human tissues and semi-quantitative analysis illustrated that most highly in lymph node and bone. It is also demonstrated that SDF-1 were detected in the culture medium of HOB cells. In addition, pretreatment of exogenous SDF-1 enhanced significantly the in vitro adhesion, invasion and migration of both cell lines with a dose-dependent manner, and 50% primary osteoblast conditioned medium collected at 72h increased as well in vitro PCa cell invasion. Such performances could be blocked by administration of anti-CXCR4 antibody.
Conclusion PCa Cells highly express functional CXCR4 and the most frequent metastasis organs intensively express SDF-1. SDF-1 can obviously increase the in vitro adhesion, migration and invasion of PCa cells while anti-CXCR4 antibody can block such effect. CXCR4/SDF-1 pathway may therefore play a of very importance role in PCa targeted metastasis.
⒉The Inhibitory Effects of siRNA Expression Vector on CXCR4 Expression of Prostate carcinoma cell lines
Objective To investigate the inhibitory effects of RNAi (RNA interference, RNAi) expression vector on CXCR4 expression of Prostate carcinoma cell lines. Methods CXCR4 targeting siRNA gene was inserted into the Pgensil-1 plasmid containing U6 promoter and EGFP and siRNA expression vectors were constructed to be aimed directly at CXCR4 gene. The recombinants were transfected into Prostate carcinoma cell line, PC-3m and LNCaP, with liposomes.The expression of CXCR4 was detected by RT-PCR and Western Blot. Results Expression vectors could reduced the expressions of CXCR4 mRNA and protein in the PC-3m and LNCaP cells. Compared with NC, the former, the ratio of inhibition of the expression of CXCR4 mRNA was 87.81±10.20%、56.10±9.32% in 24th hour and 48th hour, the latter,was 56.93±8.78%、49.24±11.23% , respectively. The ratio of inhibitory of the expression of CXCR4 protein was 64.71±6.68%、58.66±11.56% in 24th hour respectively . Conclusion The RNAi expression vectors containing CXCR4-siRNA sequence can effectively inhibit the expression of CXCR4 gene, which lays a foundation for post study.
⒊Establishment of RNA interfering retrovirus vector targeting CXCR4 gene driven by hTERT promoter and its biological effects on hTERT+ prostate cancer cells in vitro
Objective: To construct RNA interfering retrovirus vector targeting CXCR4 gene driven by human telomerase reserve transcriptase gene promoter and investigate CXCR4 expression in hTERT+ carcinoma cells. Methods: To clone the CXCR4 targeting siRNA gene by PCR. The PCR products were inserted into the Pgensil-1 plasmid containing U6 promoter and EGFP.U6 promoter was replaced by hTERT promoter. Then, the recombinant EGFP-hTERT-siCXCR4 fragment was sub-cloned into PLXSN and EGFP was replaced by ERFP.Then the recombinant was evaluated by restriction enzyme and sequencing. The virus obtained from transfected PA317 cells was transfected respectively into PC-3m、LNCaP、MCF7、MRC5 cell. The expression of CXCR4 mRNA and protein was detected by RT-PCR and Western Blot. The effect of exogenous recombinant SDF-1 on adhesion, transendothelial migration and invasion of PCa cells interferenced by CXCR4-siRNA were observed using an in vitro Transwell and Matrigel models. Results: The expression of CXCR4 mRNA and protein in MCF7、PC-3m and LNCaP were reduced by the recombinant PLXSN-DsRED2-hTERT-siCXCR4 constructed successfully. The inhibitory rates of the expression of CXCR4 mRNA and protein were(88.31±9.20)%、(87.20±10.34)%、(76.40±9.77)% and (82.60±7.26)%、(80.23±4.54)%、(84.09±3.37)% at 48th hour respectively. CXCR4 mRNA and protein can not be inhibited by PLXSN-hTERT-siCXCR4 in MRC5 cell. The ability of exogenous SDF-1 enhancing adhesion, invasion and migration of prostate cancer cells in vitro descended. Conclusion: The downstream CXCR4-siRNA controlled by hTERT promoter in retrovirus system can be expressed selectively in hTERT+ carcinoma cells, but not in hTERT- cells, which showed the obvious targeting character. Knocking down CXCR4 gene can inhibit significantly PCa adhesion, invasion and migration of prostate cancer cells in vitro.
4. The study on inhibition of prostate carcinoma metastasis to bone by using retrovirus-mediated CXCR4-siRNA driven by hTERT promoter in vivo
Objective: To establish a stable prostatic carcinoma osseous metastasis NOD/SCID mouse model expressing enhanced red fluorescent protein and investigate effect of prostate carcinoma metastasis to bone by inhibiting CXCR4 expression. Methods: 6-8 monthsold, male mice were selected and implanted with human adult bone (HAB) by surgical implantation subcutaneously. Then about 2-3 weeks later, human prostatic carcinoma cell lines PC-3m and LNCaP, transfected by retroviral containing ERFP gene and interference sequence, were harvested and injected into the NOD/SCID mice through the lateral tail vein.2 weeks after injection, all NOD/SCID mice were started to be observed under the whole-body fluorescent imaging system to look for the sites with the expression of ERFP. Then, to 2 prostate carcinoma cell line, ratio of osseous metastasis、humid weight、volume and time of surviving were contrasted between interferenced group and uninterferenced group. Tissue slices of metastasic tumor used HE dyeing were detected. Results: To 2 prostate cancer cell lines PC-3m、LNCaP interferenced by CXCR4-siRNA,ratio of osseous metastasis、humid weight、volume were all lower than those of uninterferebnced prostate cancer lines, but time of surviving was longer and histodifferentiation degree of osseous metastasic tumor was superer. Conclusion: A stable prostate cancer osseous metastasis model expressing enhanced red fluorescent protein (ERFP) observed dynamically in vitro in a noninvasive way was established. Knocking down expression of CXCR4 gene can inhibit prostate cancer metastasis to bone and inhibit cancer cell maligdifferentiation in vivo.
目的初步探讨趋化因子CXC受体4/基质细胞衍生因子1(CXCR4/SDF-1)通路在前列腺癌(PCa)靶向性转移中的作用。方法逆转录-链式聚合酶反应(RT-PCR)法和蛋白质斑迹法(Western Blot)法分别检测PCa细胞系LNCaP和PC-3m中CXCR4 mRNA和蛋白表达,免疫组化检测正常前列腺组织和前列腺癌组织CXCR4蛋白的表达。RT-PCR法检测并比较成人骨、淋巴结、肺、脑、肝组织中SDF-1 mRNA表达,酶联免疫吸附法(ELISA)检测人成骨细胞(HOB)条件培养基上清中SDF-1蛋白水平。观察外源性SDF-1对PCa细胞体外黏附、侵袭和迁移的影响及CXCR4抗体对其效应。结果两种PCa细胞系均有CXCR4 mRNA和蛋白表达,前列腺癌组织高表达CXCR4蛋白。成人骨、淋巴结、肺、脑、肝组织均有SDF-1 mRNA表达,半定量分析显示淋巴结和骨组织表达量最高;HOB细胞培养上清中可测及较高浓度的SDF-1蛋白。外源性SDF-1可促进两种PCa细胞黏附、迁移和侵袭,呈剂量依赖关系,原代HOB培养72小时收集的条件培养基亦可促进PCa细胞体外迁移能力;CXCR4抗体则可抑制上述效应。结论PCa细胞高表达具有活化功能状态的CXCR4,PCa常见的转移靶器官则高表达SDF-1;SDF-1可促进PCa细胞体外转移过程,且可被CXCR4抗体阻断,CXCR4/SDF-1通路可能在PCa靶向性转移中发挥重要作用。
㈡携带报告基因EGFP的CXCR4 RNA干扰质粒的构建及其对前列腺癌细胞CXCR4基因表达的抑制作用
目的研究RNA干扰(RNA interference, RNAi)表达载体对前列腺癌细胞趋化因子CXC受体4(CXCR4)基因的抑制作用。方法CXCR4特异性小干扰RNA(small interfering RNA, siRNA)转入带有增强型绿色荧光蛋白(EGFP)和启动子U6的Pgensil-1质粒构建针对CXCR4基因的RNAi质粒表达载体,脂质体法转染前列腺癌PC-3m、LNCaP细胞株,应用RT-PCR、Western Blot检测其对CXCR4 mRNA及蛋白表达的影响。结果构建的表达质粒在前列腺癌PC-3m、LNCaP细胞株中均抑制了CXCR4 mRNA及蛋白表达。与空白载体组细胞作对照,前者小干扰RNA (siRNA)在对CXCR4 mRNA的抑制率24小时、48小时分别为87.81±10.20%、56.10±9.32%,后者为56.93±8.78%、49.24±11.23% ,蛋白抑制率24小时前者为64.71±6.68%,后者为58.66±11.56%。结论携带有siRNA-CXCR4序列的RNAi表达载体可以有效地抑制前列腺癌细胞CXCR4基因的表达,为后续实验奠定基础。
㈢hTERT启动子调控的CXCR4-siRNA逆转录病毒载体的构建及其体外靶向抑制hTERT+前列腺癌细胞生物学效应研究
目的:构建人端粒酶逆转录酶(hTERT)启动子调控的趋化因子受体4(CXCR4)的RNA干扰逆转录病毒载体,并研究其在hTERT+前列腺癌细胞、乳腺癌细胞中的表达。方法:用PCR扩增CXCR4特异性小干扰RNA(small interfering RNA, siRNA),转入带有增强型绿色荧光蛋白(EGFP)和启动子U6的Pgensil-1质粒,以hTERT启动子代替U6启动子,再将重组基因片段导入到逆转录病毒真核表达载体PLXSN中,ERFP基因片段代替EGFP基因片段,行限制性酶切鉴定及测序。转染PA317病毒包装细胞,收获病毒上清分别转染前列腺癌细胞PC-3m、LNCaP及人胚肺成纤维细胞MRC5和人乳腺癌细胞MCF7,RT-PCR、Western Blot检测CXCR4 mRNA及蛋白表达。观察外源性SDF-1对CXCR4基因表达抑制后PCa细胞体外黏附、侵袭和迁移的影响。结果:限制性酶切、测序鉴定该重组质粒,成功构建了PLXSN/ERFP-hTERT-siCXCR4。与空载体组比较,MCF7、PC-3m、LNCaP细胞中CXCR4-siRNA对CXCR4 mRNA的48小时抑制率分别为(88.31±9.20)%、(87.20±10.34)%、(76.40±9.77)%,CXCR4蛋白的48小时抑制率分别为(82.60±7.26)%、(80.23±4.54)%、(84.09±3.37)%。MRC5细胞中PLXSN/ERFP-hTERT-siCXCR4对CXCR4 mRNA及蛋白不抑制。CXCR4基因敲低后,外源性SDF-1促进两种PCa细胞黏附、迁移和侵袭的能力下降。结论:该逆转录病毒系统中hTERT启动子调控的下游RNA干扰序列可选择性地在hTERT+前列腺癌细胞、乳腺癌细胞中表达,在hTERT- MRC5细胞不表达,具有显著的靶向性。敲低CXCR4基因表达可抑制前列腺癌细胞体外黏附、侵袭、转移。
㈣hTERT启动子调控的CXCR4-siRNA逆转录病毒载体抑制前列腺癌细胞骨转移的体内实验研究
目的:构建可荧光示踪的前列腺癌骨转移动物模型,并探讨抑制CXCR4基因表达后对前列腺癌体内骨转移的影响。方法:选用6-8周龄体重为20-22克NOD/SCID雄性小鼠,利用外科植入术将成人骨组织(HAB)植入小鼠皮下。2-3周后,将携有ERF(PDsRED2)基因及干扰序列CXCR4-siRNA的重组逆转录病毒转染前列腺癌细胞PC-3m与LNCaP,收获稳定表达红色荧光基因(ERFP)的前列腺癌细胞并配置成细胞悬液,通过小鼠尾静脉注入小鼠体内。2周后开始通过整体荧光显像系统观察小鼠体内移植人骨组织处前列腺癌转移瘤细胞ERFP的表达。对比CXCR4基因敲低组和对照组前列腺癌骨转移瘤的骨转移率、体积、湿重、存活时间。HE染色镜检转移瘤组织切片。结果:两种前列腺癌细胞CXCR4基因敲低组骨转移瘤的骨转移率、体积、湿重均低于对照组,而存活时间高于对照组(基因未敲低组)。组织切片显示干扰CXCR4基因表达后前列腺癌骨转移瘤较基因未敲低组肿瘤组织分化程度高。结论:成功构建了可体外无创性动态观察的稳定的前列腺癌骨转移动物模型,证实敲低CXCR4基因表达可抑制体内前列腺癌骨转移并抑制前列腺癌细胞恶性分化。
⒈The effect of CXCR4/SDF-1 pathway on prostate cancer targeted metastasis
Objective To investigate preliminarily the role of CXCR4/SDF-1 in prostate cancer (PCa) targeted metastasis. Methods CXCR4 mRNA and protein expression in two PCa cancer cell lines, LNCaP and PC-3m, were examined by reverse transcript-PCR、western bloting and prostate cancer tissue was detected by immunohistochemistry. SDF-1 mRNA expressions in human bone, lymph node, lung, brain and liver tissues were determined by RT-PCR and SDF-1 secretary level in the culture medium of primary human osteoblasts (HOB) was detected by in ELISA. The effect of exogenous recombinant SDF-1 on PCa cells adhesion, transendothelial migration and invasion, and the effect of anti-CXCR antibody in this procedure were observed using an in vitro Transwell and Matrigel models.
Results Both CXCR4 mRNA and protein expressed in LNCaP and PC-3m. CXCR4 protein expressed highly in prostate cancer tissues. Meanwhile, SDF-1 mRNA expressed in divers human tissues and semi-quantitative analysis illustrated that most highly in lymph node and bone. It is also demonstrated that SDF-1 were detected in the culture medium of HOB cells. In addition, pretreatment of exogenous SDF-1 enhanced significantly the in vitro adhesion, invasion and migration of both cell lines with a dose-dependent manner, and 50% primary osteoblast conditioned medium collected at 72h increased as well in vitro PCa cell invasion. Such performances could be blocked by administration of anti-CXCR4 antibody.
Conclusion PCa Cells highly express functional CXCR4 and the most frequent metastasis organs intensively express SDF-1. SDF-1 can obviously increase the in vitro adhesion, migration and invasion of PCa cells while anti-CXCR4 antibody can block such effect. CXCR4/SDF-1 pathway may therefore play a of very importance role in PCa targeted metastasis.
⒉The Inhibitory Effects of siRNA Expression Vector on CXCR4 Expression of Prostate carcinoma cell lines
Objective To investigate the inhibitory effects of RNAi (RNA interference, RNAi) expression vector on CXCR4 expression of Prostate carcinoma cell lines. Methods CXCR4 targeting siRNA gene was inserted into the Pgensil-1 plasmid containing U6 promoter and EGFP and siRNA expression vectors were constructed to be aimed directly at CXCR4 gene. The recombinants were transfected into Prostate carcinoma cell line, PC-3m and LNCaP, with liposomes.The expression of CXCR4 was detected by RT-PCR and Western Blot. Results Expression vectors could reduced the expressions of CXCR4 mRNA and protein in the PC-3m and LNCaP cells. Compared with NC, the former, the ratio of inhibition of the expression of CXCR4 mRNA was 87.81±10.20%、56.10±9.32% in 24th hour and 48th hour, the latter,was 56.93±8.78%、49.24±11.23% , respectively. The ratio of inhibitory of the expression of CXCR4 protein was 64.71±6.68%、58.66±11.56% in 24th hour respectively . Conclusion The RNAi expression vectors containing CXCR4-siRNA sequence can effectively inhibit the expression of CXCR4 gene, which lays a foundation for post study.
⒊Establishment of RNA interfering retrovirus vector targeting CXCR4 gene driven by hTERT promoter and its biological effects on hTERT+ prostate cancer cells in vitro
Objective: To construct RNA interfering retrovirus vector targeting CXCR4 gene driven by human telomerase reserve transcriptase gene promoter and investigate CXCR4 expression in hTERT+ carcinoma cells. Methods: To clone the CXCR4 targeting siRNA gene by PCR. The PCR products were inserted into the Pgensil-1 plasmid containing U6 promoter and EGFP.U6 promoter was replaced by hTERT promoter. Then, the recombinant EGFP-hTERT-siCXCR4 fragment was sub-cloned into PLXSN and EGFP was replaced by ERFP.Then the recombinant was evaluated by restriction enzyme and sequencing. The virus obtained from transfected PA317 cells was transfected respectively into PC-3m、LNCaP、MCF7、MRC5 cell. The expression of CXCR4 mRNA and protein was detected by RT-PCR and Western Blot. The effect of exogenous recombinant SDF-1 on adhesion, transendothelial migration and invasion of PCa cells interferenced by CXCR4-siRNA were observed using an in vitro Transwell and Matrigel models. Results: The expression of CXCR4 mRNA and protein in MCF7、PC-3m and LNCaP were reduced by the recombinant PLXSN-DsRED2-hTERT-siCXCR4 constructed successfully. The inhibitory rates of the expression of CXCR4 mRNA and protein were(88.31±9.20)%、(87.20±10.34)%、(76.40±9.77)% and (82.60±7.26)%、(80.23±4.54)%、(84.09±3.37)% at 48th hour respectively. CXCR4 mRNA and protein can not be inhibited by PLXSN-hTERT-siCXCR4 in MRC5 cell. The ability of exogenous SDF-1 enhancing adhesion, invasion and migration of prostate cancer cells in vitro descended. Conclusion: The downstream CXCR4-siRNA controlled by hTERT promoter in retrovirus system can be expressed selectively in hTERT+ carcinoma cells, but not in hTERT- cells, which showed the obvious targeting character. Knocking down CXCR4 gene can inhibit significantly PCa adhesion, invasion and migration of prostate cancer cells in vitro.
4. The study on inhibition of prostate carcinoma metastasis to bone by using retrovirus-mediated CXCR4-siRNA driven by hTERT promoter in vivo
Objective: To establish a stable prostatic carcinoma osseous metastasis NOD/SCID mouse model expressing enhanced red fluorescent protein and investigate effect of prostate carcinoma metastasis to bone by inhibiting CXCR4 expression. Methods: 6-8 monthsold, male mice were selected and implanted with human adult bone (HAB) by surgical implantation subcutaneously. Then about 2-3 weeks later, human prostatic carcinoma cell lines PC-3m and LNCaP, transfected by retroviral containing ERFP gene and interference sequence, were harvested and injected into the NOD/SCID mice through the lateral tail vein.2 weeks after injection, all NOD/SCID mice were started to be observed under the whole-body fluorescent imaging system to look for the sites with the expression of ERFP. Then, to 2 prostate carcinoma cell line, ratio of osseous metastasis、humid weight、volume and time of surviving were contrasted between interferenced group and uninterferenced group. Tissue slices of metastasic tumor used HE dyeing were detected. Results: To 2 prostate cancer cell lines PC-3m、LNCaP interferenced by CXCR4-siRNA,ratio of osseous metastasis、humid weight、volume were all lower than those of uninterferebnced prostate cancer lines, but time of surviving was longer and histodifferentiation degree of osseous metastasic tumor was superer. Conclusion: A stable prostate cancer osseous metastasis model expressing enhanced red fluorescent protein (ERFP) observed dynamically in vitro in a noninvasive way was established. Knocking down expression of CXCR4 gene can inhibit prostate cancer metastasis to bone and inhibit cancer cell maligdifferentiation in vivo.
引文
1. Taichman RS, Cooper C, Keller ET, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone [J]. Cancer Res, 2002, 62(6):1832-1837.
2. Müller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis[J]. Nature, 2001, 410(21):50-56.
3. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411(6836):494-498.
4. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002, 296(44):550-553.
5. Xia HB, Mao QW, Paulson HL, et al. siRNA-mediated gene silencing in vitro and in vivo [J]. Nat Biotechnol, 2002, 20(21):1006-1010.
6. Yang M, Baranov E, Wang JW, et al. Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model [J]. Proc Natl Acad Sci USA, 2002, 99(8):3824-3829.
1. Taichman RS, Cooper C, Keller ET, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone [J]. Cancer Res, 2002, 62(6):1832-1837.
2. Mochizuki H, Matsubara A, Teishima J, et al. Interaction of ligand-receptor system between stromal-cell-derived factor-1 and CXC chemokine receptor 4 in human prostate cancer: a possible predictor of metastasis [J]. Biochem Biophys Res Commun, 2004, 320(3):656-663.
3. Libura J, Drukala J, Majka M, Tomescu O, et al. CXCR4-SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion [J]. Blood, 2002, 100(7):2597-2606.
4. Muller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis [J]. Nature, 2001, 410(21):50-56.
5. Gazitt Y. Homing and mobilization of hematopoietic stem cells and hematopoietic cancer cells are mirror image processes, utilizing similar signaling pathways and occurring concurrently: circulating cancer cells constitute an ideal target for concurrent treatment with chemotherapy and antilineage-specific antibodies [J]. Leukemia. 2004,18(1):1-10.
6. Roudire MP, Ture LD, Higano CS, et al. Phenotypic heterogeneity of end-stage prostate carcinoma metastatic to bone [J]. Hum Pathol, 2003, 34(5):646-653.
7. Bubendorf L, Schopfer A, Wagner U, et al. Metastatic patterns of prostate cancer: an autopsy study of 1,589 pateints [J]. Hum Pathol, 2000, 31(5):578-583.
8. Shah RB, Mehra R, Chinnaiyan AM, et al. Androgen-independent prostate cancer is a heterogenous group os diseases: lessons from a rapid autopsy program [J]. Cancer Res, 2004, 64(24): 9209-9216.
9. Scher HI. Prostate carcinoma defining therapeutic objectives and improving overall outcomes [J]. Cancer, 2003, 97(11):758-771.
1. Gayle G, Vaday, Shao-Bing Hua, et al. CXCR4 and CXCL12 (SDF-1)in prostate cancer inhibitory effects of human single chain fv Antibodies [J]. Clinical Cancer Research, 2004, 10(6):5630-5639.
2. Ui-Teik, Naito Y, Takahashi F,et,al. Guidelines for the selection of highly effectively siRNA sequence for mammalian and chick RNA interference [J]. Nucleic Acids Res, 2004, 32(3):936-948.
3. Horuk R, Zlotnik A, Devora R, et al. The biology of chemokine and their receptors and chemokine beyond inflamation [J]. Nature, 1998, 393(6685): 524.
4. Schrader AJ, Lechner O, Templin M, el al. CXCR4/CXCl12 expression and signaling in kidney cancer [J]. Br J Cancer, 2002, 86(8):1250-1256.
5. Bertolini F,Dells Agnola C, Mancuso P,et al. CXCR4 neutralization, a novel therapeutic approach for non-Hodgkin’s lymphoma [J]. Cancer Res, 2002, 62(11):3112.
6. Kijiama T, Maμlik G, ma PC, et al.Regulation of cellular proliferation, cytoskeletal function, and signal transduction through CXCR4 and c-Kit in small cell lung cells [J]. Cancer Res, 2002, 62(21): 6304-6311。
7. Shibuta K, Mori M, Shimoda K, et al. Regional expression of CXCL12/CXCR4 in liver and hepatocellular carcinoma and cell cycle variation during in vitro differentiation [J]. Jnp J cancer Res, 2002, 93(7):789-797.
8. Libura J, Drukala J, Majka M, el al. CXCR4-SDF-1 signaling is active inrhabdomyosarcoma cell and regulates locomotion, chemotaxis, and adhesion [J]. Blood, 2002, 100(7):2597-2606.
9. Muller A ,Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis[J]. Nature, 2001, 410(21):50-56.
10. Taichman RS, Cooper C, Keller ET, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone [J]. Cancer Res, 2002,62(6):1832-1837.
11. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21 nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411(6836):494-498.
12. Hannon G J, Conklin DS. RNA interference by short hairpin RNAs expressed in vertebrate cells [J]. Methods Mol Biol, 2004, 25(7):255-266.
13. Yoshinari K, Miyagishi M, Taira K. Effects on RNAi of the tight structure, sequence and position of the targeted region. [J]. Nucleic Acids Res, 2004, 32(2):691-699.
14. Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNA) induce sequence-specific silencing in mammalian cell [J]. Genes Dev, 2002, 16(8):981- 984.
1. Ohira S, Sasaki M, Harada K, et al. Possible regulation od migration of intrahepatic cholangiocarcinoma cells by interaction of CXCR4 expressed in carcinoma cells with tumor necrosis factor -{alpha} and Stromal-Derived Factor-1 Released in Stroma [J]. Am J Pathol, 2006, 168(4):1155-1168.
2. Libura J, Drukala J, Majka M, et al. CXCR4-SDF-1 signaling is active inrhabdomyosarcoma cell and regulates locomotion, chemotaxis, and adhesion [J]. Blood, 2002, 100(7):2597-2606.
3. Payne AS, Cornelius LA. The role of chemokines in melanoma tumor growth andmetastasis [J]. J Invest Dermatol, 2002, 118(6):915-922.
4. Schrader AJ, Lechner O, Templin M, et al. CXCR4/CXCl12 expression and signaling in kidney cancer [J]. Br J Cancer, 2002, 86(8):1250-1256.
5. Bertolini F, Dell’s Agnola C, Mancuso P, et al. CXCR4 neutralization, a novel therapeutic approach for non-Hodgkin’s lymphoma [J]. Cancer Res, 2002, 62(11):3112.
6. Kijiama T, Maulik G, Ma PC, et al. Regulation of cellular proliferation, cytoskeletal function, and signal transduction through CXCR4 and c-Kit in small cell lung cells [J]. Cancer Res, 2002, 62(21):6304-6311.
7. Shibuta K, Mori M, Shimoda K, et al. Regional expression of CXCL12/CXCR4 in liver and hepatocellular carcinoma and cell cycle variation during in vitro differentiation [J]. Jnp J cancer Res, 2002, 93(7):789-797.
8. Majumdar AS, Hughes DE, Lichtstciner SP, et al. The telomerase reserve transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoter [J]. Gene Ther, 2001, 8(7):568-578
9. Ui-Teik, Naito Y, Takahashi F, et al. Guidelines for the selection of highly effectively siRNA sequence for mammalian and chick RNA interference [J]. Nucleic Acids Res, 2004, 32(3):936-948.
10.刘苏虎,张王刚,张镁,等. Fas靶向RNA干扰质粒的构建及生物活性的鉴定[J]。西安交通大学学报(医学版), 2005, 26(2):115-118.
11. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411(6836):494-498.
12. Bertolini F, Dell’Agnola C, Mancuso P, et al. CXCR4 neutralization, a novel therapeutic approach for mon-Hodgkin’s lymphoma[J]. Cancer Res, 2002, 62(11):3160-3163.
13. Muller A ,Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis [J]. Nature, 2001, 410(21):50-56.
14.苏丽萍,张进平,徐焕宾,等.趋化因子受体CXCR4在人肺癌高转移细胞株的表达及意义[J].中国免疫学杂志,2004,20(9):603-605.
15.魏立,孔佩艳,陈幸华,等.抑制SDF-1活性对人急性髓性白血病HL-60细胞系增殖的影响[J].中国实验血液学杂志,2004,12(2):154-156.
16. Ohuchida k, Mizumoto K, Yamada D, et al. Quantitative analysis of human telomerase reverse transcriptase in pancreatic cancer [J]. Clin Cancer Res, 2006, 12(7):2066-2069.
1. Wu TT, Sikes RA, Cui Q , et al . Establishing human prostate cancer cell xenografts in bone: induction of osteoblastic reac2 tion by prostate - specific antigen - producing tumors in athymic and SCID/ bg mice using LNCaP and lineage - derived metastatic sublines [J]. Int J Cancer. 1998, 77(6):887-94.
2. Jeffrey A, Nemeth, John F, et al. Severe Com2 bined immunodeficient - hu model of human prostate cancer metastasis to human bone [J]. Cancer Res, 1999, 59(8):1987-93.
3. Glinskii AB, Smith BA, Jiang P, et al. Viable circulating metastatic cells produced in orthotopic but not ectopic prostate cancer models [J]. Cancer Res, 2003, 63 (14):4239-4243.
4. Wetterwald A, van der PG, Que I, et al. Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease [J]. AmJ Pathol, 2002, 160(22):1143-1153.
5. Adams JY, Johnson M, Sato M, et al. Visualization of advanced hu2 man prostate cancer lesions in living mice by a targeted gene transfer vector and potical imaging [J]. Nat Med, 2002, 8(8):891-897.
6. Sato N, Gleve ME, Bruchovsky N, et al. A metastatic and androgen-sensitive human prostate cancer model using intraprostatic inoculation of LNCaP cells in SCID mice [J]. Cancer Res, 1997, 57(8):1584-1589.
7. Rembrink K, Romijin JC, Rubben H, et al. Orthotopic implantation of human prostate cancer cell lines: a clinically relevant animal model for metastatic prostate cancer [J]. Prostate, 1997, 31(3):168-174.
8. Yang M, Jiang P, Sun FX, et al. A Fluorescent Orthotopic Bone Metastasis Model of Human Prostate Cancer [J]. Cancer Res, 1999, 59(21):781-786.
9. Yang M, Luiken G, Baranov E, et al. Facile whole-body imaging of internal fluorescenttumors in mice with an LED flashlight [J]. Benchmarks, 2005, 39(2):170-172.
10. Hsieh JT. Derivation of androgen-indepent cancer progression and bone metastasis in the LNCaP model of human prostate cancer [J]. Int J Cancer, 1994, 57(13):406-412.
1. Tuschl T, Zamore PD, LehmannR, et al. Targeted mRNA degradation by double-stranded RNA in vitro [J]. Genes Dev, 1999, 13(24):3191.
2. Guo S, Kemphues K J. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed [J]. Cell, 1995, 81(4):611-620.
3. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans [J]. Nature, 1998, 391(6669):806-811.
4. Ngo H, Tschudi C, Gull K, et al. Double-stranded RNA induce mRNA degradation in Trypanosoma brucei [J]. Proc Natl Acad Sci USA, 1998, 95(8):14687-14692.
5. Kennerdell JR, Carthew RW. Heritable gene silencing in Drosophila using double-stranded RNA [J]. Natural Biotechnology, 2000, 17(6):896-898.
6. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411(13):494-498.
7. Mette MF, Matzke AJ, Matzke MA. Resistance of RNA-mediated TGS to HC-Pro, a viral suppressor of PTGS, suggests alternative pathways for dsRNA processing [J]. Curr Biol, 2001, 11(14):1119-1123.
8. Tamaru H, Selker EU. A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature, 2001, 414(6861):277-283.
9. Ramaswamy G, Slack F J. siRNA: A guide for RNA silencing [J]. Chem Biol, 2002, 9(10):1053-1055.
10. Hutvagner G, Zamore PD. A microRNA in a multiple turn-over RNAi enzyme complex [J]. Science, 2002, 297(5589):2002-2003.
11. Elbashir SM, Lendeckel W, Tuschl T. RNA is mediated by 21 and 22 nucleotide RNAs [J]. Gene Dev, 2001, 15(2):188-200.
12. Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs [J]. Science, 2001, 294(5543):853-858.
13. Nishikura K. A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst [J]. Cell, 2001, 107(4):415~418.
14. Montgomery MK, Xu S, Fire A. RNA as a target of double-stranded RNA mediated genetic interference in Caeanorhabditis elegans [J]. Proc Natl Acad Sci USA, 1998, 95(25):15502-15507.
15. Harborth J, Elbashir SM, Bechert K, et al. Identification of essential genes in cultured mammalian cells using small interfering RNAs [J]. J Cell Sci, 2001, 114(24): 4557-4565.
16. Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs [J]. Cell, 2001, 107(3):297-307.
17. Irie N, Sakai N, Ueyama T, et al. Subtype and species-specific knockdown of PKC using short interfering RNA [J]. Biochemical and Biophysical Research Communications, 2002, 298(5):738-743.
18. Gan L, Anton KE, Masterson BA, et al. Specific interference with gene expression and gene function mediated by long double-stranded RNA in neural cells [J]. J Neurosci Methods, 2002, 121(2):151-157.
19. Salgado PS, koivunen MR, Makeyev EV, et al. The structure of an RNAi polymerase links RNA silencing and transcription [J]. PLoS Biol. 2006, 4(12):434.
20. Clemens JC, Worby CA, Simonson Leff N, et al. Use of double-stranded RNA interference in Drosophila cells lines to dessect signal transduction pathways [J]. Proc Natl Acad Sci USA, 2000, 97(12):6499-6503.
21. Sabine B. Antisense-RNA regulation and RNA interference [J]. Biochem Biophys Acta, 2002, 1575(1-3):15-25.
22. Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells [J]. Nature, 2002, 418(13):430-434.
23. Miyagishi M, Taira K. U6 promotor-driven siRNAs with four uridine 3'overhangs efficiently suppress targeted gene expression in mammalian cells [J]. Nat-Biotechnol, 2002, 2(5):497-500.
24. Sui G, Soohoo C, Affar EIB, et al. A DNA vector based RNAi technology to suppress gene expression in mammalian cells [J]. Proc Natl Acad Sci, 2002, 99(8):5515-5520.
25. Liu XD, Ma SM, Liu Y, et al. Short hairpin RNA and retroviralvector-mediated silencing of p53 in mammalian cells [J]. BiochemBiophys Res Commun, 2004, 324(4):1173-1178.
26. Zhang L, Yang N, Mohamed-Hadley A, et al. Vector-based RNAi, a noveltool for isoform specific knock-down of VEGF and anti-angiogenesisgene therapy of cancer [J]. Biochem Biophys Res Commun, 2003, 303(4):169-1178.
27. Takabatake Y, Isaka Y, Mizui M, et al. Exploring RNA interference as a therapeutic strategy for renal disease [J]. Gene Ther, 2005, 27(3):984-987.
28. Aoki Y, Cioca DP, Oidaira H, et al. RNA interference may be more potent than antisense RNA in human cancer cell lines [J]. Clin Exp Pharmacol Physiol, 2003, 30(1-2): 96-102.
29. Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemia cells with a BCR/ABL fusion gene by RNA interference (RNAi) [J]. Oncogene, 2002, 21(37):5716-5724.
30. Cioca DP, Aoki Y, Kijosawa K. RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines [J]. Cancer Gene Ther, 2003, 10(2):125-133.
31. Aoki Y, Cioca DP, Oidaioa H, et al. RNA interference may be more potent than antisense RNA in human cancer cell lines [J]. Clin Exp Pharmacol Physiol, 2003, 30(1-2):96-102.
32. Schramme A, Sers, Techernites, et al. Functional dissection of signal transduction pathways in mammalian cells using RNA interference reveals specific roles of individual effecttor kinase isoforms in Ras-induced transformation [J]. European Journal of Biochemistry, 2003, 270(suppl 1): 204.
33. Hemann MT, Fridman JS, Zilfou JT, et al. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo [J]. Nat Genet, 2003, 33(3):396-400.
34. Brummel Kamp, Thijn R, Bernards Rene, et al. A system for stable expression of short interfering RNAs in mammalian cells [J]. The American Association for the Advanced of Science, 2002, 296(5567):550-553.
35. Jiang M, Milner J. Bcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cancer cells [J]. Genes Dev, 2003, 17(7):832-837.
36. Williams NS, Gaynor RB, Scoggin S, et al. Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNAinterference [J]. Clin Cancer Res, 2003, 9(3):931-946.
37. Paddison PJ, Caudy AA, Hannon GJ, et al. Stable suppression of gene expression by RNAi in mammalian cells [J]. Proc Natl Acad Sci U S A, 2002, 99(3):1443-1448.
38. Sohail M, Doran G, Riedemann J, et al. A simple and cost-effective method for producing small interfering RNAs with high efficacy [J]. Nucleic Acids Res, 2003, 31(7):334-338.
39. Nagy P, Arndt-Jouin DJ, Jouin TM. Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbB1-over expressing cells [J]. Exp Cell Res, 2003, 285(1):39-49.
40. De Schrijver E, Brusselmans K, Heyns W, et al. RNA Interference-mediated Silencing of the Fatty Acid Synthase Gene Attenuates Growth and Induces Morphological Changes and Apoptosis of LNCaP Prostate Cancer Cells [J]. Cancer Res, 2003, 63(13): 3799-804.
41. Milner J. RNA interference for treating cancers caused by viral infection [J]. Expert Opin BioTher, 2003, 3(3):459-467.
42. Wengenmayer Tobias, Plmann Tobias, Market UR. Successful inhibition of HLA-G production in JEG-3 Chonio carcinoma cells by RNA interference [J]. American Journal of Reproductive Immunology, 2003, 49(6):364.
43. Czauderna F, Fechtner M, Aygun H, et al. Functional studies of the PI(3)-kinase signaling pathway employing synthetic and expressed siRNA [J]. Nucleic Acid Res, 2003, 31(7):670-682.
44. Stege A, Priebsch A, Nieth C, et al. Stable and complete overcoming of MDR1/P- glycoprotein-mediated multidrug resistance in human gastriccarcinoma cells by RNA interference [J]. Cancer Gene Ther, 2004, 11(4):699-706.
45. Yague E, Higgins CF, Raguz S. Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1 [J]. Gene Ther, 2004, 11(6): 1170-1174.
46. Park WS, Miyano-Kurosaki N, Hayafune M, et al. Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference. Nucleic Acids Res, 2002, 30(22):4830-4835.
47. Coburn GA, Cullen BR. Potent and specific inhibition of human immunodeficiencyvirus type 1 replication by RNA interference [J]. J Virol, 2002, 76(18):9225-9231.
48. Das AT, Brummelkamp TR, Westerhout EM, et al. Human immunodeficiency virus Type 1 escapes from RNA interference-mediated inhibition [J]. J Virol, 2004, 78(5): 2601-2605.
49. Novina CD, Murray MF, Dykxhoorn DM, et al. siRNA-directed inhibition of HIV-1 infection [J]. Nat Med, 2002, 8(7):681-686.
50. Martinez MA, Gutierrez A, Armand-Ugon M, et al. Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication [J]. AIDS, 2002, 16(18):2385-2390.
51. Chiu YL, Cao H, Jacque JM, et al. Inhibition of human immunodeficiency virus type 1 replication by RNA interference directed against human transcription elongation factor P-TEFb (CDK9/CyclinT1) [J]. J Virol, 2004, 78(5):2517-2529.
52. Wilson JA, Jayasena S, Khvorova A. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells [J]. Proc Natl Acad Sci U S A, 2003, 100(5):2783-8.
53. Yokota T, Sakamoto N, Enomoto N. Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs [J]. EMBO Rep, 2003, 4(6):602-608.
54. Milner J. RNA interference for treating cancers caused by viral infection [J]. Expert Opin Biol Ther, 2003, 3(3):459-467.
1. Bubendorf L, Schopfer A, Wagner U, et al. Metastatic patterns of prostate cancer:An autopsy study of 1589 patients [J]. Hum Pathol, 2000, 31(5):578-583.
2. Jacob K, Webber M, Benayahu D, et al. Osteonectin promotes prostate cancer cell migration and invasion: a possible mechanism for metastasis to bone [J]. Cancer Res, 1999, 59(17):4453-4457.
3. Lehr J E, Pienta KJ. Preferential adhesion of prostate cancer cells to a human bone marrow endothelial cell line [J]. J Natl Cancer Inst, 1998, 90(2): 118-123.
4. Cooper CR, Mclean L, Walsh M, et al. Perferential adhesion of prostate cancer cells to bone is mediated by binding to bone marrow endothelial cells as compared to extracellular matrix components in vitro[J]. Clin Cancer Res, 2000, 6(12): 4839-4847.
5. 8Kierszenbaum AL, Rivkon E, Chang PL, et al. Galactosyl receptor, a cell surface C2type lectin of normal and tumoral prostate epithe2 lial cells with binding affinity to endothelial cells [J]. Prostate, 2000, 43(3):175-183.
6. Bryden AA, Hoyland JA, Freemont AJ, et al. Parathyroid hormone related peptide and receptor expression in paired primary prostate cancer and bone metastases [J]. Br J Cancer, 2002, 86(3):322-325.
7. Shen X, Falzon M. PTH-related protein modulates PC-3 prostate cancer cell adhesion and integrin subunit profile [J]. Mol Cell Endocrinol, 2003, 199(1-2):165-177.
8. Shen X, Falzon M. Parathyroid hormone-related protein upregulates integrin expression via an intracrine pathway in PC-3 prostate cancer cells [J]. Regul Pept, 2003, 113(1-3):17-29.
9. Lee Y, Schwarz E, Davies M, et al. Differences in the cytokine profiles associated with prostate cancer cell induced osteoblastic and osteolytic lesions in bone [J]. J Orthop Res, 2003, 21(1):62-72.
10. Guise TA, Yin JJ, Mohammad KS. Role of endothelin-1 in osteoblastic bone metastases [J]. Cancer, 2003, 97(3 Suppl):779-784.
11. Grande M, Carlstrom K, Stege R, et al. Estrogens affect endothelin-1 mRNA expression in LNCaP human prostate carcinoma cells [J]. Eur Urol, 2002, 41(5):568-72; discussion 573-4.
12. Chua CC, Chua BH, Chen Z, et al. TGF-beta1 inhibits multiple caspases induced by TNF-alpha in murine osteoblastic MC3T3-E1 cells [J]. Biochim Biophys Acta, 2002, 1593(1):1-8.
13. Shah AH, Tabayoyong WB, Kundu SD, et al. Suppression of tumor metastasis by blockade of transforming growth factor beta signaling in bone marrow cells through a retroviral-mediated gene therapy in mice [J]. Cancer Res, 2002, 62(24):7135-7138.
14. Bello-DeOcampo D, Tindall DJ. TGF-betal/Smad signaling in prostate cancer [J]. Curr Drug Targets, 2003, 4(3):197-207.
15. Ibanez-Tallon I, Ferrai C, Longobardi E, et al. Binding of Sp1 to the proximal promoter links constitutive expression of the human uPA gene and invasive potential of PC3 cells [J]. Blood, 2002, 100(9):3325-3332.
16. Pakneshan P, Xing RH, Rabbani SA. Methylation status of uPA promoter as a molecular mechanism regulating prostate cancer invasion and growth in vitro and in vivo [J]. FASEB J, 2003, 17(9):1081-1088.
17. Aguirre-Ghiso JA, Estrada Y, Liu D, et al. ERK(MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38(SAPK) [J]. Cancer Res, 2003, 63(7):1684-1695.
18. Xiao G, Jiang D, Gopalakrishnan R, et al. Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2 [J]. J Biol Chem, 2002, 277(39):36181-36187.
19. Kim HJ, Kim JH, Bae SC, et al. The protein kinase C pathway plays a central role in the fibroblast growth factor-stimulated expression and transactivation activity of Runx2 [J]. J Biol Chem, 2003, 278(1):319-326.
20. Sobue T, Gravely T, Hand A, et al. Regulation of fibroblast growth factor 2 and fibroblast growth factor receptors by transforming growth factor beta in human osteoblastic MG-63 cells [J]. J Bone Miner Res, 2002, 17(3):502-512.
21. Okada Y, Montero A, Zhang X, et al. Impaired osteoclast formation in bone marrow cultures of fgf2 null mice in response to parathyroid hormone [J]. J Biol Chem, 2003, 278(23):21258-21266.
22. Tokuda H, Hirade K, Wang X, et al. Involvement of SAPK/JNK in basic fibroblast growth factor-induced vascular endothelial growth factor release in osteoblasts [J]. JEndocrinol, 2003, 177(1):101-107.
23. Shimoaka T, Ogasawara T, Yonamine A, et al. Regulation of osteoblast, chondrocyte, and osteoclast functions by fibroblast growth factor (FGF)-18 in comparison with FGF-2 and FGF-10 [J]. J Biol Chem, 2002, 277(9):7493-7500.
24. Gruber R, Varga F, Fischer MB, et al. Platelets stimulate proliferation of bone cells: involvement of platelet-derived growth factor, microparticles and membranes [J]. Clin Oral Implants Res, 2002, 13(5):529-535.
25. Tanaka H, Wakisaka A, Ogasa H, et al. Effect of IGF-I and PDGF administered in vivo on the expression of osteoblast-related genes in old rats [J]. J Endocrinol, 2002, 174(1):63-70.
26. Uehara H, Kim SJ, Karashima T, et al. Effects of blocking platelet-derived growth factor-receptor signaling in a mouse model of experimental prostate cancer bone metastases [J]. J Natl Cancer Inst, 2003, 95(6): 458-470.
27. Kubota K, Sakikawa C, Katsumata M, et al. Platelet-derived growth factor BB secreted from osteoclasts acts as an osteoblastogenesis inhibitory factor [J]. J Bone Miner Res, 2002, 17(2):257-265.
28. Nemeth JA, Harb JF, Barroso UJ, et al. Severe combined immun2 odeficient2hu model of human prostate cancer metastasis to human bone [J]. Cancer Res, 1999, 59 (8):1987-1993.
29. Yonou H, Yokose T, Kamijo T, et al. Establishment of a novel species2 and tissue2specific metastasis model of human prostate cancer in humanized non2obese diabetic/ severe combined immunodeficient mice engrafted with human adult lung and bone [J]. Cancer Res, 2001, 61 (5): 2177-2182.
30. Zhau HE, Li CL, Chung LW, et al. Establishment of human prostate carcinoma skeletal metastasis models [J]. Cancer , 2000 , 88(12 Suppl): 2995-3001.
31. 14Boissier S, Ferreras M, peyruchaud O, et al. Bisphosphonates in2 hibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastasis [J]. Cancer Res, 2000, 60(11): 2949-2954.
32. Heidenreich A, Hofmann R, Engelmann UH. The use of bisphos2phonate for the palliative treatment of painful bone metastasis due to hormone refractory prostate cancer [J]. J Urol, 2001, 165(1):136-140.
33. Tamada T, Sone T, Tomomitsu T , et al. Biochemical markers for the detection of bone metastasis in patients with prostate cancer: di2 agnostic efficacy and the effect of hormonal therapy [J]. J Bone Min2 er Metab, 2001, 19(1):45-51.
34. Drobnjak M, Osman I, Scher HI, et al. Overexpression of cy2 clinD1 is associated with metastatic prostate cancer to bone [J]. Clin Cancer Res, 2000, 6(5):1891-1895.
35. Iddon J, Bundred NJ, Hoyland J, et al. Expression of parathyroid hormone2related protein and its receptor in bone metastases from prostate cancer [J]. J Pathol, 2000, 191 (2):170-174.
36. Reiter RE, Gu Z, Watabe T, et al. Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer [J]. Proc Natl Acad Sci USA, 1998, 95(4):1735-1740.
37. Gu Z, Thomas G, Yamashiro J, et al. Prostate stem cell antigen (PSCA) expression increases with high Gleason score, advanced stage and bone metastasis in prostate cancer [J]. Oncogene, 2000, 19(10):1288-1296.
2. Müller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis[J]. Nature, 2001, 410(21):50-56.
3. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411(6836):494-498.
4. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002, 296(44):550-553.
5. Xia HB, Mao QW, Paulson HL, et al. siRNA-mediated gene silencing in vitro and in vivo [J]. Nat Biotechnol, 2002, 20(21):1006-1010.
6. Yang M, Baranov E, Wang JW, et al. Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model [J]. Proc Natl Acad Sci USA, 2002, 99(8):3824-3829.
1. Taichman RS, Cooper C, Keller ET, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone [J]. Cancer Res, 2002, 62(6):1832-1837.
2. Mochizuki H, Matsubara A, Teishima J, et al. Interaction of ligand-receptor system between stromal-cell-derived factor-1 and CXC chemokine receptor 4 in human prostate cancer: a possible predictor of metastasis [J]. Biochem Biophys Res Commun, 2004, 320(3):656-663.
3. Libura J, Drukala J, Majka M, Tomescu O, et al. CXCR4-SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion [J]. Blood, 2002, 100(7):2597-2606.
4. Muller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis [J]. Nature, 2001, 410(21):50-56.
5. Gazitt Y. Homing and mobilization of hematopoietic stem cells and hematopoietic cancer cells are mirror image processes, utilizing similar signaling pathways and occurring concurrently: circulating cancer cells constitute an ideal target for concurrent treatment with chemotherapy and antilineage-specific antibodies [J]. Leukemia. 2004,18(1):1-10.
6. Roudire MP, Ture LD, Higano CS, et al. Phenotypic heterogeneity of end-stage prostate carcinoma metastatic to bone [J]. Hum Pathol, 2003, 34(5):646-653.
7. Bubendorf L, Schopfer A, Wagner U, et al. Metastatic patterns of prostate cancer: an autopsy study of 1,589 pateints [J]. Hum Pathol, 2000, 31(5):578-583.
8. Shah RB, Mehra R, Chinnaiyan AM, et al. Androgen-independent prostate cancer is a heterogenous group os diseases: lessons from a rapid autopsy program [J]. Cancer Res, 2004, 64(24): 9209-9216.
9. Scher HI. Prostate carcinoma defining therapeutic objectives and improving overall outcomes [J]. Cancer, 2003, 97(11):758-771.
1. Gayle G, Vaday, Shao-Bing Hua, et al. CXCR4 and CXCL12 (SDF-1)in prostate cancer inhibitory effects of human single chain fv Antibodies [J]. Clinical Cancer Research, 2004, 10(6):5630-5639.
2. Ui-Teik, Naito Y, Takahashi F,et,al. Guidelines for the selection of highly effectively siRNA sequence for mammalian and chick RNA interference [J]. Nucleic Acids Res, 2004, 32(3):936-948.
3. Horuk R, Zlotnik A, Devora R, et al. The biology of chemokine and their receptors and chemokine beyond inflamation [J]. Nature, 1998, 393(6685): 524.
4. Schrader AJ, Lechner O, Templin M, el al. CXCR4/CXCl12 expression and signaling in kidney cancer [J]. Br J Cancer, 2002, 86(8):1250-1256.
5. Bertolini F,Dells Agnola C, Mancuso P,et al. CXCR4 neutralization, a novel therapeutic approach for non-Hodgkin’s lymphoma [J]. Cancer Res, 2002, 62(11):3112.
6. Kijiama T, Maμlik G, ma PC, et al.Regulation of cellular proliferation, cytoskeletal function, and signal transduction through CXCR4 and c-Kit in small cell lung cells [J]. Cancer Res, 2002, 62(21): 6304-6311。
7. Shibuta K, Mori M, Shimoda K, et al. Regional expression of CXCL12/CXCR4 in liver and hepatocellular carcinoma and cell cycle variation during in vitro differentiation [J]. Jnp J cancer Res, 2002, 93(7):789-797.
8. Libura J, Drukala J, Majka M, el al. CXCR4-SDF-1 signaling is active inrhabdomyosarcoma cell and regulates locomotion, chemotaxis, and adhesion [J]. Blood, 2002, 100(7):2597-2606.
9. Muller A ,Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis[J]. Nature, 2001, 410(21):50-56.
10. Taichman RS, Cooper C, Keller ET, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone [J]. Cancer Res, 2002,62(6):1832-1837.
11. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21 nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411(6836):494-498.
12. Hannon G J, Conklin DS. RNA interference by short hairpin RNAs expressed in vertebrate cells [J]. Methods Mol Biol, 2004, 25(7):255-266.
13. Yoshinari K, Miyagishi M, Taira K. Effects on RNAi of the tight structure, sequence and position of the targeted region. [J]. Nucleic Acids Res, 2004, 32(2):691-699.
14. Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNA) induce sequence-specific silencing in mammalian cell [J]. Genes Dev, 2002, 16(8):981- 984.
1. Ohira S, Sasaki M, Harada K, et al. Possible regulation od migration of intrahepatic cholangiocarcinoma cells by interaction of CXCR4 expressed in carcinoma cells with tumor necrosis factor -{alpha} and Stromal-Derived Factor-1 Released in Stroma [J]. Am J Pathol, 2006, 168(4):1155-1168.
2. Libura J, Drukala J, Majka M, et al. CXCR4-SDF-1 signaling is active inrhabdomyosarcoma cell and regulates locomotion, chemotaxis, and adhesion [J]. Blood, 2002, 100(7):2597-2606.
3. Payne AS, Cornelius LA. The role of chemokines in melanoma tumor growth andmetastasis [J]. J Invest Dermatol, 2002, 118(6):915-922.
4. Schrader AJ, Lechner O, Templin M, et al. CXCR4/CXCl12 expression and signaling in kidney cancer [J]. Br J Cancer, 2002, 86(8):1250-1256.
5. Bertolini F, Dell’s Agnola C, Mancuso P, et al. CXCR4 neutralization, a novel therapeutic approach for non-Hodgkin’s lymphoma [J]. Cancer Res, 2002, 62(11):3112.
6. Kijiama T, Maulik G, Ma PC, et al. Regulation of cellular proliferation, cytoskeletal function, and signal transduction through CXCR4 and c-Kit in small cell lung cells [J]. Cancer Res, 2002, 62(21):6304-6311.
7. Shibuta K, Mori M, Shimoda K, et al. Regional expression of CXCL12/CXCR4 in liver and hepatocellular carcinoma and cell cycle variation during in vitro differentiation [J]. Jnp J cancer Res, 2002, 93(7):789-797.
8. Majumdar AS, Hughes DE, Lichtstciner SP, et al. The telomerase reserve transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoter [J]. Gene Ther, 2001, 8(7):568-578
9. Ui-Teik, Naito Y, Takahashi F, et al. Guidelines for the selection of highly effectively siRNA sequence for mammalian and chick RNA interference [J]. Nucleic Acids Res, 2004, 32(3):936-948.
10.刘苏虎,张王刚,张镁,等. Fas靶向RNA干扰质粒的构建及生物活性的鉴定[J]。西安交通大学学报(医学版), 2005, 26(2):115-118.
11. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411(6836):494-498.
12. Bertolini F, Dell’Agnola C, Mancuso P, et al. CXCR4 neutralization, a novel therapeutic approach for mon-Hodgkin’s lymphoma[J]. Cancer Res, 2002, 62(11):3160-3163.
13. Muller A ,Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis [J]. Nature, 2001, 410(21):50-56.
14.苏丽萍,张进平,徐焕宾,等.趋化因子受体CXCR4在人肺癌高转移细胞株的表达及意义[J].中国免疫学杂志,2004,20(9):603-605.
15.魏立,孔佩艳,陈幸华,等.抑制SDF-1活性对人急性髓性白血病HL-60细胞系增殖的影响[J].中国实验血液学杂志,2004,12(2):154-156.
16. Ohuchida k, Mizumoto K, Yamada D, et al. Quantitative analysis of human telomerase reverse transcriptase in pancreatic cancer [J]. Clin Cancer Res, 2006, 12(7):2066-2069.
1. Wu TT, Sikes RA, Cui Q , et al . Establishing human prostate cancer cell xenografts in bone: induction of osteoblastic reac2 tion by prostate - specific antigen - producing tumors in athymic and SCID/ bg mice using LNCaP and lineage - derived metastatic sublines [J]. Int J Cancer. 1998, 77(6):887-94.
2. Jeffrey A, Nemeth, John F, et al. Severe Com2 bined immunodeficient - hu model of human prostate cancer metastasis to human bone [J]. Cancer Res, 1999, 59(8):1987-93.
3. Glinskii AB, Smith BA, Jiang P, et al. Viable circulating metastatic cells produced in orthotopic but not ectopic prostate cancer models [J]. Cancer Res, 2003, 63 (14):4239-4243.
4. Wetterwald A, van der PG, Que I, et al. Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease [J]. AmJ Pathol, 2002, 160(22):1143-1153.
5. Adams JY, Johnson M, Sato M, et al. Visualization of advanced hu2 man prostate cancer lesions in living mice by a targeted gene transfer vector and potical imaging [J]. Nat Med, 2002, 8(8):891-897.
6. Sato N, Gleve ME, Bruchovsky N, et al. A metastatic and androgen-sensitive human prostate cancer model using intraprostatic inoculation of LNCaP cells in SCID mice [J]. Cancer Res, 1997, 57(8):1584-1589.
7. Rembrink K, Romijin JC, Rubben H, et al. Orthotopic implantation of human prostate cancer cell lines: a clinically relevant animal model for metastatic prostate cancer [J]. Prostate, 1997, 31(3):168-174.
8. Yang M, Jiang P, Sun FX, et al. A Fluorescent Orthotopic Bone Metastasis Model of Human Prostate Cancer [J]. Cancer Res, 1999, 59(21):781-786.
9. Yang M, Luiken G, Baranov E, et al. Facile whole-body imaging of internal fluorescenttumors in mice with an LED flashlight [J]. Benchmarks, 2005, 39(2):170-172.
10. Hsieh JT. Derivation of androgen-indepent cancer progression and bone metastasis in the LNCaP model of human prostate cancer [J]. Int J Cancer, 1994, 57(13):406-412.
1. Tuschl T, Zamore PD, LehmannR, et al. Targeted mRNA degradation by double-stranded RNA in vitro [J]. Genes Dev, 1999, 13(24):3191.
2. Guo S, Kemphues K J. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed [J]. Cell, 1995, 81(4):611-620.
3. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans [J]. Nature, 1998, 391(6669):806-811.
4. Ngo H, Tschudi C, Gull K, et al. Double-stranded RNA induce mRNA degradation in Trypanosoma brucei [J]. Proc Natl Acad Sci USA, 1998, 95(8):14687-14692.
5. Kennerdell JR, Carthew RW. Heritable gene silencing in Drosophila using double-stranded RNA [J]. Natural Biotechnology, 2000, 17(6):896-898.
6. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411(13):494-498.
7. Mette MF, Matzke AJ, Matzke MA. Resistance of RNA-mediated TGS to HC-Pro, a viral suppressor of PTGS, suggests alternative pathways for dsRNA processing [J]. Curr Biol, 2001, 11(14):1119-1123.
8. Tamaru H, Selker EU. A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature, 2001, 414(6861):277-283.
9. Ramaswamy G, Slack F J. siRNA: A guide for RNA silencing [J]. Chem Biol, 2002, 9(10):1053-1055.
10. Hutvagner G, Zamore PD. A microRNA in a multiple turn-over RNAi enzyme complex [J]. Science, 2002, 297(5589):2002-2003.
11. Elbashir SM, Lendeckel W, Tuschl T. RNA is mediated by 21 and 22 nucleotide RNAs [J]. Gene Dev, 2001, 15(2):188-200.
12. Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs [J]. Science, 2001, 294(5543):853-858.
13. Nishikura K. A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst [J]. Cell, 2001, 107(4):415~418.
14. Montgomery MK, Xu S, Fire A. RNA as a target of double-stranded RNA mediated genetic interference in Caeanorhabditis elegans [J]. Proc Natl Acad Sci USA, 1998, 95(25):15502-15507.
15. Harborth J, Elbashir SM, Bechert K, et al. Identification of essential genes in cultured mammalian cells using small interfering RNAs [J]. J Cell Sci, 2001, 114(24): 4557-4565.
16. Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs [J]. Cell, 2001, 107(3):297-307.
17. Irie N, Sakai N, Ueyama T, et al. Subtype and species-specific knockdown of PKC using short interfering RNA [J]. Biochemical and Biophysical Research Communications, 2002, 298(5):738-743.
18. Gan L, Anton KE, Masterson BA, et al. Specific interference with gene expression and gene function mediated by long double-stranded RNA in neural cells [J]. J Neurosci Methods, 2002, 121(2):151-157.
19. Salgado PS, koivunen MR, Makeyev EV, et al. The structure of an RNAi polymerase links RNA silencing and transcription [J]. PLoS Biol. 2006, 4(12):434.
20. Clemens JC, Worby CA, Simonson Leff N, et al. Use of double-stranded RNA interference in Drosophila cells lines to dessect signal transduction pathways [J]. Proc Natl Acad Sci USA, 2000, 97(12):6499-6503.
21. Sabine B. Antisense-RNA regulation and RNA interference [J]. Biochem Biophys Acta, 2002, 1575(1-3):15-25.
22. Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells [J]. Nature, 2002, 418(13):430-434.
23. Miyagishi M, Taira K. U6 promotor-driven siRNAs with four uridine 3'overhangs efficiently suppress targeted gene expression in mammalian cells [J]. Nat-Biotechnol, 2002, 2(5):497-500.
24. Sui G, Soohoo C, Affar EIB, et al. A DNA vector based RNAi technology to suppress gene expression in mammalian cells [J]. Proc Natl Acad Sci, 2002, 99(8):5515-5520.
25. Liu XD, Ma SM, Liu Y, et al. Short hairpin RNA and retroviralvector-mediated silencing of p53 in mammalian cells [J]. BiochemBiophys Res Commun, 2004, 324(4):1173-1178.
26. Zhang L, Yang N, Mohamed-Hadley A, et al. Vector-based RNAi, a noveltool for isoform specific knock-down of VEGF and anti-angiogenesisgene therapy of cancer [J]. Biochem Biophys Res Commun, 2003, 303(4):169-1178.
27. Takabatake Y, Isaka Y, Mizui M, et al. Exploring RNA interference as a therapeutic strategy for renal disease [J]. Gene Ther, 2005, 27(3):984-987.
28. Aoki Y, Cioca DP, Oidaira H, et al. RNA interference may be more potent than antisense RNA in human cancer cell lines [J]. Clin Exp Pharmacol Physiol, 2003, 30(1-2): 96-102.
29. Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemia cells with a BCR/ABL fusion gene by RNA interference (RNAi) [J]. Oncogene, 2002, 21(37):5716-5724.
30. Cioca DP, Aoki Y, Kijosawa K. RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines [J]. Cancer Gene Ther, 2003, 10(2):125-133.
31. Aoki Y, Cioca DP, Oidaioa H, et al. RNA interference may be more potent than antisense RNA in human cancer cell lines [J]. Clin Exp Pharmacol Physiol, 2003, 30(1-2):96-102.
32. Schramme A, Sers, Techernites, et al. Functional dissection of signal transduction pathways in mammalian cells using RNA interference reveals specific roles of individual effecttor kinase isoforms in Ras-induced transformation [J]. European Journal of Biochemistry, 2003, 270(suppl 1): 204.
33. Hemann MT, Fridman JS, Zilfou JT, et al. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo [J]. Nat Genet, 2003, 33(3):396-400.
34. Brummel Kamp, Thijn R, Bernards Rene, et al. A system for stable expression of short interfering RNAs in mammalian cells [J]. The American Association for the Advanced of Science, 2002, 296(5567):550-553.
35. Jiang M, Milner J. Bcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cancer cells [J]. Genes Dev, 2003, 17(7):832-837.
36. Williams NS, Gaynor RB, Scoggin S, et al. Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNAinterference [J]. Clin Cancer Res, 2003, 9(3):931-946.
37. Paddison PJ, Caudy AA, Hannon GJ, et al. Stable suppression of gene expression by RNAi in mammalian cells [J]. Proc Natl Acad Sci U S A, 2002, 99(3):1443-1448.
38. Sohail M, Doran G, Riedemann J, et al. A simple and cost-effective method for producing small interfering RNAs with high efficacy [J]. Nucleic Acids Res, 2003, 31(7):334-338.
39. Nagy P, Arndt-Jouin DJ, Jouin TM. Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbB1-over expressing cells [J]. Exp Cell Res, 2003, 285(1):39-49.
40. De Schrijver E, Brusselmans K, Heyns W, et al. RNA Interference-mediated Silencing of the Fatty Acid Synthase Gene Attenuates Growth and Induces Morphological Changes and Apoptosis of LNCaP Prostate Cancer Cells [J]. Cancer Res, 2003, 63(13): 3799-804.
41. Milner J. RNA interference for treating cancers caused by viral infection [J]. Expert Opin BioTher, 2003, 3(3):459-467.
42. Wengenmayer Tobias, Plmann Tobias, Market UR. Successful inhibition of HLA-G production in JEG-3 Chonio carcinoma cells by RNA interference [J]. American Journal of Reproductive Immunology, 2003, 49(6):364.
43. Czauderna F, Fechtner M, Aygun H, et al. Functional studies of the PI(3)-kinase signaling pathway employing synthetic and expressed siRNA [J]. Nucleic Acid Res, 2003, 31(7):670-682.
44. Stege A, Priebsch A, Nieth C, et al. Stable and complete overcoming of MDR1/P- glycoprotein-mediated multidrug resistance in human gastriccarcinoma cells by RNA interference [J]. Cancer Gene Ther, 2004, 11(4):699-706.
45. Yague E, Higgins CF, Raguz S. Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1 [J]. Gene Ther, 2004, 11(6): 1170-1174.
46. Park WS, Miyano-Kurosaki N, Hayafune M, et al. Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference. Nucleic Acids Res, 2002, 30(22):4830-4835.
47. Coburn GA, Cullen BR. Potent and specific inhibition of human immunodeficiencyvirus type 1 replication by RNA interference [J]. J Virol, 2002, 76(18):9225-9231.
48. Das AT, Brummelkamp TR, Westerhout EM, et al. Human immunodeficiency virus Type 1 escapes from RNA interference-mediated inhibition [J]. J Virol, 2004, 78(5): 2601-2605.
49. Novina CD, Murray MF, Dykxhoorn DM, et al. siRNA-directed inhibition of HIV-1 infection [J]. Nat Med, 2002, 8(7):681-686.
50. Martinez MA, Gutierrez A, Armand-Ugon M, et al. Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication [J]. AIDS, 2002, 16(18):2385-2390.
51. Chiu YL, Cao H, Jacque JM, et al. Inhibition of human immunodeficiency virus type 1 replication by RNA interference directed against human transcription elongation factor P-TEFb (CDK9/CyclinT1) [J]. J Virol, 2004, 78(5):2517-2529.
52. Wilson JA, Jayasena S, Khvorova A. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells [J]. Proc Natl Acad Sci U S A, 2003, 100(5):2783-8.
53. Yokota T, Sakamoto N, Enomoto N. Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs [J]. EMBO Rep, 2003, 4(6):602-608.
54. Milner J. RNA interference for treating cancers caused by viral infection [J]. Expert Opin Biol Ther, 2003, 3(3):459-467.
1. Bubendorf L, Schopfer A, Wagner U, et al. Metastatic patterns of prostate cancer:An autopsy study of 1589 patients [J]. Hum Pathol, 2000, 31(5):578-583.
2. Jacob K, Webber M, Benayahu D, et al. Osteonectin promotes prostate cancer cell migration and invasion: a possible mechanism for metastasis to bone [J]. Cancer Res, 1999, 59(17):4453-4457.
3. Lehr J E, Pienta KJ. Preferential adhesion of prostate cancer cells to a human bone marrow endothelial cell line [J]. J Natl Cancer Inst, 1998, 90(2): 118-123.
4. Cooper CR, Mclean L, Walsh M, et al. Perferential adhesion of prostate cancer cells to bone is mediated by binding to bone marrow endothelial cells as compared to extracellular matrix components in vitro[J]. Clin Cancer Res, 2000, 6(12): 4839-4847.
5. 8Kierszenbaum AL, Rivkon E, Chang PL, et al. Galactosyl receptor, a cell surface C2type lectin of normal and tumoral prostate epithe2 lial cells with binding affinity to endothelial cells [J]. Prostate, 2000, 43(3):175-183.
6. Bryden AA, Hoyland JA, Freemont AJ, et al. Parathyroid hormone related peptide and receptor expression in paired primary prostate cancer and bone metastases [J]. Br J Cancer, 2002, 86(3):322-325.
7. Shen X, Falzon M. PTH-related protein modulates PC-3 prostate cancer cell adhesion and integrin subunit profile [J]. Mol Cell Endocrinol, 2003, 199(1-2):165-177.
8. Shen X, Falzon M. Parathyroid hormone-related protein upregulates integrin expression via an intracrine pathway in PC-3 prostate cancer cells [J]. Regul Pept, 2003, 113(1-3):17-29.
9. Lee Y, Schwarz E, Davies M, et al. Differences in the cytokine profiles associated with prostate cancer cell induced osteoblastic and osteolytic lesions in bone [J]. J Orthop Res, 2003, 21(1):62-72.
10. Guise TA, Yin JJ, Mohammad KS. Role of endothelin-1 in osteoblastic bone metastases [J]. Cancer, 2003, 97(3 Suppl):779-784.
11. Grande M, Carlstrom K, Stege R, et al. Estrogens affect endothelin-1 mRNA expression in LNCaP human prostate carcinoma cells [J]. Eur Urol, 2002, 41(5):568-72; discussion 573-4.
12. Chua CC, Chua BH, Chen Z, et al. TGF-beta1 inhibits multiple caspases induced by TNF-alpha in murine osteoblastic MC3T3-E1 cells [J]. Biochim Biophys Acta, 2002, 1593(1):1-8.
13. Shah AH, Tabayoyong WB, Kundu SD, et al. Suppression of tumor metastasis by blockade of transforming growth factor beta signaling in bone marrow cells through a retroviral-mediated gene therapy in mice [J]. Cancer Res, 2002, 62(24):7135-7138.
14. Bello-DeOcampo D, Tindall DJ. TGF-betal/Smad signaling in prostate cancer [J]. Curr Drug Targets, 2003, 4(3):197-207.
15. Ibanez-Tallon I, Ferrai C, Longobardi E, et al. Binding of Sp1 to the proximal promoter links constitutive expression of the human uPA gene and invasive potential of PC3 cells [J]. Blood, 2002, 100(9):3325-3332.
16. Pakneshan P, Xing RH, Rabbani SA. Methylation status of uPA promoter as a molecular mechanism regulating prostate cancer invasion and growth in vitro and in vivo [J]. FASEB J, 2003, 17(9):1081-1088.
17. Aguirre-Ghiso JA, Estrada Y, Liu D, et al. ERK(MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38(SAPK) [J]. Cancer Res, 2003, 63(7):1684-1695.
18. Xiao G, Jiang D, Gopalakrishnan R, et al. Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2 [J]. J Biol Chem, 2002, 277(39):36181-36187.
19. Kim HJ, Kim JH, Bae SC, et al. The protein kinase C pathway plays a central role in the fibroblast growth factor-stimulated expression and transactivation activity of Runx2 [J]. J Biol Chem, 2003, 278(1):319-326.
20. Sobue T, Gravely T, Hand A, et al. Regulation of fibroblast growth factor 2 and fibroblast growth factor receptors by transforming growth factor beta in human osteoblastic MG-63 cells [J]. J Bone Miner Res, 2002, 17(3):502-512.
21. Okada Y, Montero A, Zhang X, et al. Impaired osteoclast formation in bone marrow cultures of fgf2 null mice in response to parathyroid hormone [J]. J Biol Chem, 2003, 278(23):21258-21266.
22. Tokuda H, Hirade K, Wang X, et al. Involvement of SAPK/JNK in basic fibroblast growth factor-induced vascular endothelial growth factor release in osteoblasts [J]. JEndocrinol, 2003, 177(1):101-107.
23. Shimoaka T, Ogasawara T, Yonamine A, et al. Regulation of osteoblast, chondrocyte, and osteoclast functions by fibroblast growth factor (FGF)-18 in comparison with FGF-2 and FGF-10 [J]. J Biol Chem, 2002, 277(9):7493-7500.
24. Gruber R, Varga F, Fischer MB, et al. Platelets stimulate proliferation of bone cells: involvement of platelet-derived growth factor, microparticles and membranes [J]. Clin Oral Implants Res, 2002, 13(5):529-535.
25. Tanaka H, Wakisaka A, Ogasa H, et al. Effect of IGF-I and PDGF administered in vivo on the expression of osteoblast-related genes in old rats [J]. J Endocrinol, 2002, 174(1):63-70.
26. Uehara H, Kim SJ, Karashima T, et al. Effects of blocking platelet-derived growth factor-receptor signaling in a mouse model of experimental prostate cancer bone metastases [J]. J Natl Cancer Inst, 2003, 95(6): 458-470.
27. Kubota K, Sakikawa C, Katsumata M, et al. Platelet-derived growth factor BB secreted from osteoclasts acts as an osteoblastogenesis inhibitory factor [J]. J Bone Miner Res, 2002, 17(2):257-265.
28. Nemeth JA, Harb JF, Barroso UJ, et al. Severe combined immun2 odeficient2hu model of human prostate cancer metastasis to human bone [J]. Cancer Res, 1999, 59 (8):1987-1993.
29. Yonou H, Yokose T, Kamijo T, et al. Establishment of a novel species2 and tissue2specific metastasis model of human prostate cancer in humanized non2obese diabetic/ severe combined immunodeficient mice engrafted with human adult lung and bone [J]. Cancer Res, 2001, 61 (5): 2177-2182.
30. Zhau HE, Li CL, Chung LW, et al. Establishment of human prostate carcinoma skeletal metastasis models [J]. Cancer , 2000 , 88(12 Suppl): 2995-3001.
31. 14Boissier S, Ferreras M, peyruchaud O, et al. Bisphosphonates in2 hibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastasis [J]. Cancer Res, 2000, 60(11): 2949-2954.
32. Heidenreich A, Hofmann R, Engelmann UH. The use of bisphos2phonate for the palliative treatment of painful bone metastasis due to hormone refractory prostate cancer [J]. J Urol, 2001, 165(1):136-140.
33. Tamada T, Sone T, Tomomitsu T , et al. Biochemical markers for the detection of bone metastasis in patients with prostate cancer: di2 agnostic efficacy and the effect of hormonal therapy [J]. J Bone Min2 er Metab, 2001, 19(1):45-51.
34. Drobnjak M, Osman I, Scher HI, et al. Overexpression of cy2 clinD1 is associated with metastatic prostate cancer to bone [J]. Clin Cancer Res, 2000, 6(5):1891-1895.
35. Iddon J, Bundred NJ, Hoyland J, et al. Expression of parathyroid hormone2related protein and its receptor in bone metastases from prostate cancer [J]. J Pathol, 2000, 191 (2):170-174.
36. Reiter RE, Gu Z, Watabe T, et al. Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer [J]. Proc Natl Acad Sci USA, 1998, 95(4):1735-1740.
37. Gu Z, Thomas G, Yamashiro J, et al. Prostate stem cell antigen (PSCA) expression increases with high Gleason score, advanced stage and bone metastasis in prostate cancer [J]. Oncogene, 2000, 19(10):1288-1296.