摘要
目的构建并筛选Smoothened(SMO)基因的RNAi表达质粒,了解Hedgehog信号通路影响乳腺癌细胞增殖的作用机制。方法以脂质体LipofectamineTM2000介导转染乳腺癌MCF-7细胞。转染后48 h,采用实时定量RT-PCR技术检测转染细胞中SMO基因mRNA的转录水平,筛选有效的SMO RNAi质粒,Western blot检测SMO蛋白在乳腺癌MCF-7细胞中的表达,CCK-8法比较转染前后乳腺癌细胞增殖变化,实时定量RT-PCR检测SMO短发夹状RNA(shRNA)对cyclinD1 mRNA表达的影响。结果 4个SMO shRNA表达载体SMO shRNA-1、SMO shRNA-2、SMO shRNA-3和SMO shRNA-4经测序鉴定证明插入正确。转染48 h后,与MOCK组相比,SMO shRNA-1、SMOshRNA-3、SMO shRNA-4组SMO mRNA表达量均下降(P=0.015、0.002、0.000),其中转染SMO shRNA-4的MCF-7细胞中SMO mRNA表达水平低于SMO shRNA-1、SMO shRNA-2、SMO shRNA-3组(P=0.007、0.000、0.046)。SMOshRNA-4干扰抑制效率为87%。Western blot可检测到SMO在MCF-7中的表达,干扰片段SMO shRNA-4的抑制效果较好。转染SMO shRNA-4的MCF-7细胞增殖受到明显抑制(P<0.01),转染SMO shRNA后cyclinD1 mRNA表达下降(P=0.000)。结论已成功构建并筛选出SMO基因的RNAi表达质粒,SMO shRNA对乳腺癌细胞增殖有明显的抑制作用,Hedgehog信号通路可通过活化cyclinD1诱导细胞恶性增殖。
Objective To construct and screen the RNAi expression vector for Smoothened（SMO） gene and explore the effects of Hedgehog signaling on the proliferation of breast cancer cells.Methods Breast cancer MCF-7 cells were transfected for 48 hours by the mediation of LipofectamineTM 2000.The transcription level of SMO mRNA and expression of SMO protein in transfected cells detected by realtime RT-PCR and Western blot methods.CCK-8 kit and real time RT-PCR were employed to study the effects of SMO mRNA transfection on the proliferation of breast cancer cells and cyclinD1 mRNA.Results It was verified by partial nucleotide sequencing that the constructed eukaryotic vectors expressing shRNA of SMO were correct.Contrast to MOCK group,the SMO mRNA expression in SMO shRNA-1 group,SMO shRNA-3 group,SMO shRNA-4 group declined（P=0.015,0.002,0.000）,moreover the SMO mRNA expression in SMO shRNA-4 group obviously lower than other 3 groups（P=0.007,0.000,0.046）.The inhibitor rate of SMO shRNA-4 was 87%,meanwhile the expression of SMO protein decreased.MCF-7 cell proliferation and cyclinD1 mRNA significantly inhibited under the condition of transfected by SMO shRNA-4.Conclusions The RNAi expression vector for SMO gene is successfully constructed and screened.SMO shRNA visibly inhibite the proliferation of breast cancer cells.Hedgehog signaling can induce cell proliferation by actived cyclinD1.
出处
《山东医药》
CAS
北大核心
2011年第8期13-16,共4页
Shandong Medical Journal
基金
国家自然科学基金资助项目(30870968)
教育部高等学校博士学科点专项科研基金(200801610001)
作者简介
通讯作者，E—mail：lilianhong@dlmedu．edu．cn
