靶向OPN基因的RNA干扰对膀胱癌T24细胞生物学行为的影响
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摘要
第一部分RNA干扰OPN基因的慢病毒载体的构建及鉴定
目的:构建并制备携带靶向shRNA-OPN基因的慢病毒载体。
方法:根据OPN的基因序列设计四条siRNA作为RNA干扰靶点,分别构建4个shRNA质粒表达载体并通过PCR和测序进行鉴定。经鉴定正确后分别转染293T细胞,采用Western blot检测目的蛋白的表达情况,进而判断不同靶点的干扰效果。选取最有效的靶点包装OPN-RNAi慢病毒。对所获病毒载体进行鉴定分析和滴度测定。
结果:构建的质粒表达载体经PCR鉴定均可扩增出预期条带,测序证明质粒构建成功。Western blot显示KD1,KD2,KD3,KD4靶点对目的基因的表达都有显著敲减作用,逐孔稀释滴度测定法测定shRNA-OPN病毒滴度约为2×109 TU/ml。
结论:成功制备了OPN-shRNA慢病毒载体。
第二部分OPN-shRNA对人膀胱癌(T24细胞)生物学行为影响的体外研究
目的:观察由慢病毒携带的OPN-shRNA对体外培养的人膀胱癌的抑制作用。
方法:将OPN-shRNA转染入人膀胱癌T24细胞株中,并测定转染效率。PCR、Western blot检测OPN mRNA和蛋白表达,MTT法检测干扰组(OPN-RNAi组)增殖能力,流式细胞仪检测周期, Annexin V-FITC/PI双染检测细胞凋亡,Transwell小室检测其侵袭能力。
结果: OPN-shRNA被成功地转染入T24细胞中,转染效率在90%以上。与阴性对照组和空白对照组比较,干扰组(实验组)OPN细胞的mRNA表达明显受到抑制,同样的结果见于蛋白表达的检测中。同时,干扰组细胞增殖能力下降,凋亡率增高、侵袭能力下降。
结论:在体外,OPN-shRNA能抑制人膀胱癌T24细胞株的OPN基因表达,并能抑制细胞增殖,促进凋亡,抑制侵袭转移能力。
第三部分OPN-shRNA对人膀胱癌(T24细胞)生物学行为影响的体内研究
目的:在整体水平研究OPN-shRNA对T24生长、转移和浸润行为的抑制作用。
方法:建立人膀胱癌裸鼠皮下移植瘤模型。在皮下模型中,按500ug标准在瘤内分别注射OPN-shRNA,阴性对照shRNA病毒,等量生理盐水观察抑瘤效果, S-P法检测OPN、VEGF蛋白表达。
结果:干扰组皮下移植瘤生长明显受抑制,体积和重量明显小于阴性对照组和空白对照组。OPN、VEGF蛋白表达弱于对照组(OPN, H= 11.2049,P=0.0037; VEGF, H=9.5697, P=0.0084)。
结论在体内实验中,OPN-shRNA能明显抑制人膀胱癌的生长,减少新生血管形成,显示一定的抑瘤效应。
PartⅠConstruction and identification of a Lentivirus expression vector coding for OPN-RNAi
Objective: To construct a Lentivirus expression vector coding for the short hairpin RNA (shRNA) targeting OPN mRNA.
Methods: Four plasmid expression vectors coding for siRNA targeting OPN gene sequence were constructed.The recombinant plasmids were identified by PCR and sequencing, and then transfected stably into 293T cells respectively. The targeting OPN gene silencing effect was detected by Western blotting, then judging effectiveness of RNAi . We choose target point of which OPN-RNAi effectively silences OPN gene the most in 293T cells for the package.pGC-LV ,pHelper 1.0 and pHelper 2.0 were used for the package of OPN-shRNA vector.The viral purity,identity,titer of Lentivirus viral stock were analyzed.
Results :The expected bands were amplified from the plasmids coding for shRNA by PCR,then sequencing confirmed that the OPN-shRNA plasmids were successfully constructed. Transfection of 293T cells with shRNA plasmids resulted in an inhibition of OPN protein expressions respectively. The target point of KD1,KD2,KD3 and KD4 have been successfully knockdown, The titer of OPN -shRNA was approximately 2×109 TU/ml.
Conclusions : The Lentivirus viral vector of OPN-shRNA is successfully constructed and prepared.
PartⅡStudy of the impact of biological behaviors on human bladdle cancer cell line T24 by OPN-shRNA in vitro
Objective: To observe the inhibitory effects of OPN-siRNA mediated by Lentivirus on human pancreatic carcinoma cells cultured in vitro.
Methods: OPN-shRNA was transfected into human bladdle cancer cell line T24 and the efficiency of transfection was detected. Then RT-PCR, and Western blotting were used to detect OPN mRNA and protein expression. While MTT assay was used to determine proliferation rate of OPN. Detection of apoptosis was manipulated by the means of Annexin V-FITC/PI double staining. Detection of invasion was manipulated by the means of Transwell cabin.
Results: OPN-shRNA gene has been integrated into T24 cells and the transfection efficiency detected was above 90%.The characteristics of the OPN-RNAi cells were studied.Compared with the Negative Control cells and Control cell,the OPN-shRNA cells showed that: (1) the growth rate of OPN-shRNA cells was diminished; (2) The ability of OPN-shRNA cell’s invasion and migration diminished; (3) Western blot analysis also showed that the products of OPN gene have decreased in OPN-RNAi cells.
Conclusions:OPN-shRNA can dramatically inhibit products of OPN, growth ,invasion and promote apoptosis of human bladdle cancer in vivo.
PartⅢresearch of the impact of biological behaviors on human bladdle cancer cell line T24 by OPN-shRNA in vivo
Objective: To research inhibition of biological behaviors of human bladdle cancer such as growth,metastasis and invasion by OPN-shRNA in vivo.
Methods Human T24 carcinoma transplanted subcutaneously in nude mouse were established firstly. OPN-shRNA,negative-shRNA and physiological saline were intratumorally injected with 500ug in subcutaneous xenograft model, inhibitory effect of tumor growth was observed,OPN protein expressions as well as value of VEGF were detected by immunohistochemical SP method.
Results:The inhibitory effect of tumor growth and metastasis of tumor were observed. Growth rate of subcutaneous xenograft significantly decreased in OPN-RNAi group,the tumor’s size and weight of experimental group were significantly lesser than those of negative control group and blank control group.For experimental group, expressions of OPN and VEGF proteins were weaker than those of control groups (for OPN, H= 11.2049,P=0.0037; for VEGF, H=9.5697, P=0.0084).
Conclusions: OPN-shRNA can dramatically inhibit growth, and reduce neovascularization of human bladdle cance carcinoma in vivo.
目的:构建并制备携带靶向shRNA-OPN基因的慢病毒载体。
方法:根据OPN的基因序列设计四条siRNA作为RNA干扰靶点,分别构建4个shRNA质粒表达载体并通过PCR和测序进行鉴定。经鉴定正确后分别转染293T细胞,采用Western blot检测目的蛋白的表达情况,进而判断不同靶点的干扰效果。选取最有效的靶点包装OPN-RNAi慢病毒。对所获病毒载体进行鉴定分析和滴度测定。
结果:构建的质粒表达载体经PCR鉴定均可扩增出预期条带,测序证明质粒构建成功。Western blot显示KD1,KD2,KD3,KD4靶点对目的基因的表达都有显著敲减作用,逐孔稀释滴度测定法测定shRNA-OPN病毒滴度约为2×109 TU/ml。
结论:成功制备了OPN-shRNA慢病毒载体。
第二部分OPN-shRNA对人膀胱癌(T24细胞)生物学行为影响的体外研究
目的:观察由慢病毒携带的OPN-shRNA对体外培养的人膀胱癌的抑制作用。
方法:将OPN-shRNA转染入人膀胱癌T24细胞株中,并测定转染效率。PCR、Western blot检测OPN mRNA和蛋白表达,MTT法检测干扰组(OPN-RNAi组)增殖能力,流式细胞仪检测周期, Annexin V-FITC/PI双染检测细胞凋亡,Transwell小室检测其侵袭能力。
结果: OPN-shRNA被成功地转染入T24细胞中,转染效率在90%以上。与阴性对照组和空白对照组比较,干扰组(实验组)OPN细胞的mRNA表达明显受到抑制,同样的结果见于蛋白表达的检测中。同时,干扰组细胞增殖能力下降,凋亡率增高、侵袭能力下降。
结论:在体外,OPN-shRNA能抑制人膀胱癌T24细胞株的OPN基因表达,并能抑制细胞增殖,促进凋亡,抑制侵袭转移能力。
第三部分OPN-shRNA对人膀胱癌(T24细胞)生物学行为影响的体内研究
目的:在整体水平研究OPN-shRNA对T24生长、转移和浸润行为的抑制作用。
方法:建立人膀胱癌裸鼠皮下移植瘤模型。在皮下模型中,按500ug标准在瘤内分别注射OPN-shRNA,阴性对照shRNA病毒,等量生理盐水观察抑瘤效果, S-P法检测OPN、VEGF蛋白表达。
结果:干扰组皮下移植瘤生长明显受抑制,体积和重量明显小于阴性对照组和空白对照组。OPN、VEGF蛋白表达弱于对照组(OPN, H= 11.2049,P=0.0037; VEGF, H=9.5697, P=0.0084)。
结论在体内实验中,OPN-shRNA能明显抑制人膀胱癌的生长,减少新生血管形成,显示一定的抑瘤效应。
PartⅠConstruction and identification of a Lentivirus expression vector coding for OPN-RNAi
Objective: To construct a Lentivirus expression vector coding for the short hairpin RNA (shRNA) targeting OPN mRNA.
Methods: Four plasmid expression vectors coding for siRNA targeting OPN gene sequence were constructed.The recombinant plasmids were identified by PCR and sequencing, and then transfected stably into 293T cells respectively. The targeting OPN gene silencing effect was detected by Western blotting, then judging effectiveness of RNAi . We choose target point of which OPN-RNAi effectively silences OPN gene the most in 293T cells for the package.pGC-LV ,pHelper 1.0 and pHelper 2.0 were used for the package of OPN-shRNA vector.The viral purity,identity,titer of Lentivirus viral stock were analyzed.
Results :The expected bands were amplified from the plasmids coding for shRNA by PCR,then sequencing confirmed that the OPN-shRNA plasmids were successfully constructed. Transfection of 293T cells with shRNA plasmids resulted in an inhibition of OPN protein expressions respectively. The target point of KD1,KD2,KD3 and KD4 have been successfully knockdown, The titer of OPN -shRNA was approximately 2×109 TU/ml.
Conclusions : The Lentivirus viral vector of OPN-shRNA is successfully constructed and prepared.
PartⅡStudy of the impact of biological behaviors on human bladdle cancer cell line T24 by OPN-shRNA in vitro
Objective: To observe the inhibitory effects of OPN-siRNA mediated by Lentivirus on human pancreatic carcinoma cells cultured in vitro.
Methods: OPN-shRNA was transfected into human bladdle cancer cell line T24 and the efficiency of transfection was detected. Then RT-PCR, and Western blotting were used to detect OPN mRNA and protein expression. While MTT assay was used to determine proliferation rate of OPN. Detection of apoptosis was manipulated by the means of Annexin V-FITC/PI double staining. Detection of invasion was manipulated by the means of Transwell cabin.
Results: OPN-shRNA gene has been integrated into T24 cells and the transfection efficiency detected was above 90%.The characteristics of the OPN-RNAi cells were studied.Compared with the Negative Control cells and Control cell,the OPN-shRNA cells showed that: (1) the growth rate of OPN-shRNA cells was diminished; (2) The ability of OPN-shRNA cell’s invasion and migration diminished; (3) Western blot analysis also showed that the products of OPN gene have decreased in OPN-RNAi cells.
Conclusions:OPN-shRNA can dramatically inhibit products of OPN, growth ,invasion and promote apoptosis of human bladdle cancer in vivo.
PartⅢresearch of the impact of biological behaviors on human bladdle cancer cell line T24 by OPN-shRNA in vivo
Objective: To research inhibition of biological behaviors of human bladdle cancer such as growth,metastasis and invasion by OPN-shRNA in vivo.
Methods Human T24 carcinoma transplanted subcutaneously in nude mouse were established firstly. OPN-shRNA,negative-shRNA and physiological saline were intratumorally injected with 500ug in subcutaneous xenograft model, inhibitory effect of tumor growth was observed,OPN protein expressions as well as value of VEGF were detected by immunohistochemical SP method.
Results:The inhibitory effect of tumor growth and metastasis of tumor were observed. Growth rate of subcutaneous xenograft significantly decreased in OPN-RNAi group,the tumor’s size and weight of experimental group were significantly lesser than those of negative control group and blank control group.For experimental group, expressions of OPN and VEGF proteins were weaker than those of control groups (for OPN, H= 11.2049,P=0.0037; for VEGF, H=9.5697, P=0.0084).
Conclusions: OPN-shRNA can dramatically inhibit growth, and reduce neovascularization of human bladdle cance carcinoma in vivo.
引文
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1. Tuck AB,Chambers AF,Allan AL , et al.Osteopontin over expression inbreast cancer:Knowledge gained and possible implications for clinical management[J].J Cell Biochem,2007, 102(4):859-868.
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4. Wai PY,Mi Z,Guo H,et al. Osteopontin silencing by small interfering RNA suppresses in vitro and in vivo CT26 murine colon adenocarcinoma metastasis[J]. Carcinogenesis. 2005,26(4):741-751.
5.段波,梁培禾,吴刚等,骨桥蛋白在膀胱移行细胞癌中的表达及临床意义[J].重庆医学,2007, 36(14),1372-1374.
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10. Catalano C,Azzalin G, Macino G ,et al.Gene silencing in worms and fungi [J]. Nature, 2000, 404(6775): 245-250.
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17. Smith A E.Viral vector in gene therapy[J].Annu Rev Microbiol,1995,49:807- 838.
18. Chartier C,Gantzer D M.Methali M,et al.Efficient gene ration of recombinant adenovirus vectors by homologous recombination in Escherichiacoli.Journal of virology[J],1996,70:4805-4810.
19. Rubinson D A, Dillon C P, Kwiatkowski A V,et al. A lentivirus-based system to functionally silence genes in primarymammalian cells,stem cells and transgenicmice by RNA interference [J]. Nat Genet,2003, 33 (3): 401-406.
20. Dann C T. New technology for an old favorite: lentiviral transgenesis and RNAi in rats[J]. Transgenic Res, 2007, 16(5): 571-580.
21. Chang,A.H.and Sadelain,M.The genetic engineering of hematopoietic stem cells:the rise of lentiviral vectors,the conundrum of the LTR,and the promise of lineage- restricted vectors[J].Mol.Ther,2007,15:445-456.
1. Coppola D,Szabo M,Boulware D. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies[J]. Clin Cancer Res,2004,10(1):184-190.
2. Hass HG, Nehls O, Jobst J, et al.Identification of osteopontin as the most consistently over-expressed gene in intrahepatic cholangiocarcinoma:Detection by oligonucleotide microarray and real-time PCR Analysis [J]. World J Gastroenterol, 2008,14(16):2501-2510.
3. Shevde LA, Samant RS, Paik JC,Osteopontin knockdown suppresses tumorigenicity of human metastatic breast carcinoma, MDA-MB-435 [J]. Clin Exp Metastasis. 2006,23(2):123-133.
4. Denhardt DT,Giachelli CM, Rittling SR, et al,Role of osteopontin in cellular signaling and toxicant injury[J].Annu Rev Pharmacol Toxicol,2001,41:723-749.
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