RNA干扰下调MBP-1对骨肉瘤Saos-2细胞生物学的影响

Effects of RNA Interfering of MBP-1 on Proliferation of Saos-2 Cell Line

目的探讨c-myc启动子结合蛋白1(MBP-1)基因沉默对人骨肉瘤Saos-2细胞生长的影响。方法实验分为3组:正常对照组(未转染骨肉瘤细胞)、阴性对照组(转染错义序列组)和沉默组(转染MBP-1 sh RNA组)。设计2条MBP-1基因的RNA干扰片段及1条阴性对照si RNA,并与p SIREN-retro Q质粒连接。将重组p SIREN-retro Q质粒通过Lipofectamine 2000脂质体转染骨肉瘤Saos-2细胞。实时PCR和Western blot分别检测MBP-1表达。CCK-8法对MBP-1沉默后骨肉瘤Saos-2细胞生长进行检测。Western blot检测对照组和沉默组cyclin D1和cyclin E的表达。结果通过PCR扩增及测序,说明已成功构建MBP-1沉默及对照重组p SIREN-retro Q质粒。实时PCR结果显示,沉默组MBP-1 m RNA相对表达量与对照组相比显著下调(P<0.05)。Western blot结果显示,沉默组MBP-1蛋白表达量与对照组相比也显著下调。CCK-8法结果表明,沉默组细胞在48、72和96 h时增殖能力均比对照组显著升高(P<0.05)。沉默组cyclin D1和cyclin E的表达显著高于对照组(P<0.05)。结论 MBP-1基因沉默后骨肉瘤Saos-2细胞生长被明显促进,为寻找骨肉瘤基因治疗新靶点打下基础。

Objective To investigate the effects of c-myc promoter binding protein 1（MBP-1）gene on the proliferation of human Saos-2 osteosarcoma cells in vitro. Methods Saos-2 cells were divided into three groups：blank control group（untransfected cells）,negative group（cells transfected with missense sequence）and experimental group（cells transfected with MBP-1 sh RNA）. Two MBP-1 sh RNA sequences and one negative control sh RNA sequence were designed,synthesized and cloned into p SIREN-retro Q plasma. Then the recombinant plasmids were constructed and transfected into human Saos-2 osteosarcoma cells by Lipofectamine 2000. The expressions of MBP-1 m RNA and protein in Saos-2 cells were detected by real-time PCR and Western blot,respectively. The effects of altered expression of MBP-1 on cell proliferation were measured by CCK-8 cell proliferation assay. The expressions of cyclin D1 and cyclin E in Saos-2 were determined by Western blot. Results PCR and sequencing results indicated that the recombinant plasmids p SIREN-retro Q was constructed. The relative expression level of MBP-1 m RNA in the MBP-1si RNA transfection group was significantly decreased than that in blank control group（P〈0.05）. Compared with the blank control group,the expression levels of MBP-1 protein in the experimental group also significantly decreased. The proliferation abilities of Saos-2 cells at 48,72,and 96 hours after MBP-1 si RNA transfection were significantly increased than those in the blank control group（P〈0.05）. Compared with the blank control group,the expression levels of cyclin D1 and cyclin E protein in the experimental group also significantly increased（P〈0.05）. Conclusion Knockdown of the expression of MBP-1 gene promotes the proliferation of human Saos-2 osteosarcoma cells. MBP-1 gene may become the new target of gene therapy for osteosarcoma.