摘要
目的构建5号染色体开放阅读框13(chromosome 5 open reading frame 13,C5orf13)基因siRNA慢病毒载体,探讨其对胃癌SGC-7901细胞增殖、迁移和侵袭的影响。方法针对人C5orf13基因设计3个不同的siRNA靶点,构建重组质粒,包装GV115慢病毒。取对数生长期SGC-7901细胞,分为KD1组、KD2组、KD3组和阴性对照组,前3组分别转染3个不同siRNA靶点的GV115慢病毒,阴性对照组转染阴性对照慢病毒。转染72 h,用嘌呤霉素筛选稳转细胞株,4组转染效率均>90%时,采用实时荧光定量PCR法检测4组细胞C5orf13 mRNA相对表达量,筛选出表达最低的KD2组细胞为C5orf13敲低组。取SGC-7901细胞为空白对照组。采用MTT实验检测空白对照组、阴性对照组、C5orf13敲低组转染后培养0、24、48、72、96 h时细胞增殖吸光度(optical density,OD)值,采用细胞划痕实验检测细胞迁移能力,采用Transwell小室实验检测细胞侵袭能力。结果KD1组(0.45±0.04)、KD2组(0.25±0.02)、KD3组(0.49±0.04)细胞C5orf13 mRNA相对表达量均低于阴性对照组(1.00±0.04)(P<0.05),KD1组、KD3组均高于KD2组(P<0.05),KD1组与KD3组比较差异无统计学意义(P>0.05)。空白对照组、阴性对照组、C5orf13敲低组转染后培养0 h时细胞增殖OD值比较差异无统计学意义(P>0.05);C5orf13敲低组转染后培养24、48、72、96 h时细胞增殖OD值(0.30±0.01、0.51±0.03、0.62±0.03、0.93±0.13)均低于空白对照组(0.36±0.03、0.64±0.03、0.81±0.04、1.39±0.10)和阴性对照组(0.36±0.02、0.63±0.02、0.79±0.05、1.30±0.07)(P<0.05),空白对照组与阴性对照组比较差异均无统计学意义(P>0.05)。C5orf13敲低组细胞迁移率[(0.24±0.01)%]及侵袭率[(3.07±0.13)%]均低于空白对照组[(0.41±0.05)%、(4.91±0.66)%]和阴性对照组[(0.38±0.03)%、(4.70±0.23)%](P<0.05),空白对照组与阴性对照组比较差异均无统计学意义(P>0.05)。结论构建的C5orf13基因siRNA慢病毒载体可明显下调SGC-7901细胞C5orf13表达,抑制SGC-7901细胞增殖、迁移及侵袭能力。
Objective To construct a lentiviral siRNA expression vector for chromosome 5 open reading frame 13(C5orf13)gene,and to investigate its influence on the proliferation,migration and invasion of SGC-7901 cells.Methods Three different siRNA targets were designed for human C5orf13 gene,recombinant plasmids were constructed,and GV115 lentivirus was packaged.The SGC-7901 cells in logarithmic growth phase were divided into KD1,KD2,KD3,and negative control groups,and the first three groups were transfected with GV115 lentivirus with different siRNA targets,and negative control group was transfected with negative control lentivirus.After 72 h of transfection,the positive cells were screened with puromycin.When the transfection efficiency of four groups was over 90%,the relative expression of C5orf13 mRNA in four groups was detected by real-time fluorescence quantitative PCR,and the cells with the lowest expression in KD2 group were screened out as C5orf13 knockdown group.The SGC-7901 cells were taken as blank control group,the cell proliferative optical density(OD)values were detected by MTT assay in blank control group,negative control group and C5orf13 knockdown group at 0,24,48,72 and 96 h after transfection,the cell migration ability was detected by scratching assay,and the cell invasion ability was detected by Transwell assay.Results The relative expression of C5orf13 mRNA was lower in KD1 group(0.45±0.04),KD2 group(0.25±0.02)and KD3 group(0.49±0.05)than that in negative control group(1.00±0.04)(P<0.05),was higher in KD1 group and KD3 group than that in KD2 group(P<0.05),and showed no significant difference between KD1 group and KD3 group(P>0.05).The OD value showed no significant difference at 0 h among blank control group,negative control group and C5 orf±3 knockdown group(P>0.05).The OD values at 24,48,72 and 96 h were lower in C5orf13 knockdown group(0.30±0.01,0.51±0.03,0.62±0.03,0.93±0.13)than those in blank control group(0.36±0.03,0.6±0.03,0.81±0.04,1.39±0.10)and negative control group(0.36±0.02,0.63±0.02,0.79±0.05,1.30±0.07)(P<0.05),and showed no significant differences between blank control group and negative control group(P>0.05).The migration rate and the invasion rate were lower in C5orf13 knockdown group[(0.24+0.01)%,(3.07±0.13)%]than those in blank control group[(0.41±0.05)%,(4.91±0.66)%]and negative control group[(0.38±0.03)%,(4.70±0.23)%](P<0.05),and showed no significant differences between blank control group and negative control group(P>0.05).Conclusion The constructed C5orf13 gene siRNA lentiviral vector can significantly down-regulate the expression of C5orf13 in SGC-7901 cells,and inhibit the proliferation,migration and invasion ability of SGC-7901 cells.
作者
张高丽
刘立业
ZHANG Gao-li;LIU Li-ye(Clinical Laboratory,Henan Provincial People’s Hospital,Fuwai Central China Cardiovascular Hospital,Zhengzhou University People’s Hospital,Zhengzhou,Henan 451464,China;Center for General Surgery,General Hospital of Western Theater Command,Chengdu,Sichuan 610083,China)
出处
《中华实用诊断与治疗杂志》
2022年第7期662-666,共5页
Journal of Chinese Practical Diagnosis and Therapy
基金
河南省医学科技攻关计划联合共建项目(LHGJ20210116)
四川省卫生健康委科研课题(16PJ026)。
