神经纤毛蛋白质1、存活素双基因联合沉默抑制鼻咽癌细胞生长增殖的研究  

Inhibitory effect of dual short hairpin RNAs targeting neuropilin-1 and Survivin genes on growth and proliferation of nasopharyngeal cancer cells in vitro and in vivo

作　　者：孙瑾[1] 宋英[1] 曹华[1] 桑建中[1] Sun Jin;Song Ying;Cao Ying;Sang Jianzhong(Department of Throat-Head-Neck Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院咽喉头颈外科,郑州450052

出　　处：《中华实验外科杂志》2023年第2期330-333,共4页Chinese Journal of Experimental Surgery

摘　　要：目的构建同时表达神经纤毛蛋白质1(NRP-1)和存活素(Survivin)双基因的短发夹RNA(shRNA)真核表达质粒,通过体内外实验,观察其对鼻咽癌细胞CNE-2Z生长增殖的影响。方法构建同时表达NRP-1和Survivin双基因的质粒(Plasmid-1),并构建单独表达NRP-1和Survivin基因质粒(分别为Plasmid-2、Plasmid-3)。转染入鼻咽癌CNE-2Z细胞株,于荧光显微镜观察质粒转染及表达情况;以噻唑蓝(MTT)法检测细胞增殖能力;定量聚合酶链反应(PCR)及免疫印迹法(Western blot)在mRNA水平和蛋白水平上检测目的基因抑制作用。建立稳定转染裸鼠移植瘤模型,体内裸鼠成瘤实验观察质粒对肿瘤的抑制效果。原位缺口末端标记法(TUNEL)法检测移植瘤细胞凋亡率。多组间比较采用单因素方差分析。结果通过测序证实重组shRNA真核表达载体构建成功。各重组质粒表达载体成功转染CNE-2Z细胞,可见大量的癌细胞表达绿色荧光。MTT显示细胞增殖受到抑制,且双基因干扰质粒对细胞增殖的抑制作用明显强于单基因干扰质粒,差异有统计学意义(0.209±0.021比0.392±0.013、0.384±0.014,F=354.121,P<0.05)。反转录-聚合酶链反应(RT-PCR)证实双基因联合沉默组和单基因沉默组均能有效抑制目的基因mRNA表达,双基因组NRP-1 mRNA(0.20±0.06)表达明显低于NRP-1单基因组(0.27±0.05),差异有统计学意义(F=198.437,P<0.05);双基因组Survivin mRNA表达(0.13±0.02)明显低于Survivin单基因组(0.17±0.01),差异有统计学意义(F=245.647,P<0.05)。Western blot检测结果双基因组对NRP-1和Survivin蛋白的表达抑制率分别为67.41%和79.24%,与单基因组比较均有统计学意义(F=129.354、216.411,P<0.05)。体内抑瘤实验证实,与NRP-1单基因干扰组[(0.628±0.013)cm3]、Survivin单基因干扰组[(0.601±0.301)cm3]比较,双基因干扰组[(0.205±0.002)cm3]瘤体积生长明显受到抑制,差异有统计学意义(F=49.214,P<0.05),抑瘤率为81.71%。TUNEL实验表明�Objective To construct dual short hairpin RNAs(shRNAs)targeting neuropilin-1(NRP-1)and Survivin genes and explore the effects of RNA interference(RNAi)targeting two different genes on the growth and proliferation of nasopharyngeal carcinoma(NPC)CNE-2Z cells in vitro and in vivo.Methods ShRNA plasmids targeting NRP-1 and Survivin were constructed and were respectively transfected into human NPC CNE-2Z cells.The expression of fluorescein-labeled plasmids in NPC CNE-2Z cells was observed by fluorescence microscopy.Cell proliferation was detected by methyl thiazolyl tetrazolium(MTT)assay.The inhibitory effects on target genes were evaluated with reverse transcriptase-polymerase chain reaction(RT-PCR)and Western blotting at the levels of mRNA and protein,respectively.The inhibitory effect of plasmids on xenograft tumors was observed in nude mice.Apoptosis was determined with terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL).The comparison between multiple groups was made by one-way analysis of variance ANOVA.Results ShRNA recombinant plasmid expression vectors were successfully constructed.The MTT results showed that the growth of cells treated with dual shRNA was apparently inhibited(0.209±0.021 vs.0.392±0.013,0.384±0.014,F=354.121,P<0.05).The expression level of NRP-1 mRNA in Plasmid-1(0.20±0.06)was significantly lower than that in Plasmid-2(0.27±0.050,F=198.437,P<0.05).The expression level of Survivin mRNA in Plasmid-1(0.13±0.02)was significantly lower than that in Plasmid-2(0.17±0.01,F=245.647,P<0.05).In Plasmid-1 group,the protein inhibition rate of NRP-1 and Survivin was 67.41%and 79.24%respectively,which were more effective than those of single shRNA groups(F=129.354,216.411,P<0.05).The tumor volumes in Plasmid-1 group,Plasmid-2 group and Plasmid-3 group were(0.205±0.002)cm3,(0.628±0.013)cm3 and(0.601±0.301)cm3,respectively.A values of xenograft tumors in nude mice were significantly lower in dual shRNAs group than single shRNA groups with P<0.05,and the tumor inhibitory rate was

关 键 词：神经纤毛蛋白质1 存活素 短发夹RNA 基因沉默 

分 类 号：R739.63[医药卫生—肿瘤]