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刘世呈,李靖,黄小兵,杨彤翰,梁平,郑璐,赵弘智.BC047440-siRNA对肝癌HepG2细胞增殖抑制及其分子机制的初步探讨[J].陆军军医大学学报(原第三军医大学学报),2007,29(23):2207-2211.
LIU Shi-cheng,LI Jing,HUANG Xiao-bing,YANG Tong-han,LIANG Ping,ZHENG Lu,ZHAO Hong-zhi.siRNA-mediated silencing of BC047440 gene inhibits proliferation of human HepG2 cell[J].J Amry Med Univ (J Third Mil Med Univ),2007,29(23):2207-2211.
刘世呈,李靖,黄小兵,杨彤翰,梁平,郑璐,赵弘智.BC047440-siRNA对肝癌HepG2细胞增殖抑制及其分子机制的初步探讨[J].陆军军医大学学报(原第三军医大学学报),2007,29(23):2207-2211. DOI:
LIU Shi-cheng,LI Jing,HUANG Xiao-bing,YANG Tong-han,LIANG Ping,ZHENG Lu,ZHAO Hong-zhi.siRNA-mediated silencing of BC047440 gene inhibits proliferation of human HepG2 cell[J].J Amry Med Univ (J Third Mil Med Univ),2007,29(23):2207-2211. DOI:
目的 利用小干扰RNA(small interfering RNA,siRNA)抑制肝癌相关基因BC047440的表达,观察其受抑制后对细胞增殖能力的影响并初步分析其抑制增殖的分子机制。 方法 设计针对BC047440基因的siRNA,构建pGenSil-1-BC047440-siRNA真核表达载体。分别用脂质体法将介导siRNA的真核绿色荧光表达载体pGenSil-1-BC047440-siRNA、空载体质粒pGenSil-1转染人肝癌HepG2细胞建立稳定细胞株;随后实验分为3组:转染siRNA表达载体组(PP
2
)、转染空质粒组(PP
1
)和未处理的亲本HepG2细胞组(PP
0
)。采用荧光定量 PCR检测BC047440 mRNA表达变化;CCK-8分析细胞增殖活性;平板克隆形成实验检测单个细胞的增殖能力;最后选取NF-κB下游增殖相关基因c-myc、bcl-2、cyclin D
1
,用荧光定量PCR检测其mRNA水平的变化。 结果 成功转染并获得PP
1
、PP
2
组抗性克隆,PP
2
组与PP
0
组比较,BC047440 mRNA水平明显降低,抑制率约为75%;细胞增殖实验中PP
2
组细胞增殖能力明显降低,平板克隆形成实验结果显示PP
2
组克隆形成率为(8.42±0.82)%,明显低于PP
1
组[(39.31±5.39)%
]
和PP
0
组[(40.96±3.21)%
]
(P0.01)。 检测NF-κB下游增殖相关的几个基因时发现PP
2
组c-myc mRNA表达明显降低,约为PP
0
组的12/25;其余基因无明显变化。 结论 干扰BC047440基因表达可以抑制肝癌细胞的增殖,初步揭示BC047440基因可以对c-myc起正向调控作用,从而促进肝癌细胞的增殖,提示抑制BC047440基因可能成为控制人肝癌细胞生长的有效靶点。
Objective To observe the effect on the proliferation of HepG2 cell by inhibiting the expression of BC047440 gene. Methods Small interferin
g RNAs for BC047440 gene were designed using Ambion software and synthesized. The recombinant plasmid pGenSil-1-BC047440-siRNA was constructed with the smRNAs and pGenSil-1. The vacant vector pGenSil-1 and pGenSil-1-BC047440-siRNA vector were transfected into HepG2 cells by Lipofectamine
TM
2000 and the positive clones were screened by G418
then identified by detecting green fluorescence. BC047440 mRNA expression was detected by realtime PCR. The cell proliferation rate was analysed by CCK-. The cell colony formation rate was measured by plate colony formation assay. The proliferaion-elated genes such as c-yc
bcl-
cyclin D
1
were detected by real-ime PCR. Results We obtained stable transfectants. Endogenous BC047440 expression could be efficiently down-egulated by transfection of siRNA BC047440 gene in cultured HepG2 cell
and the cell proliferation rate of HepG2 transfected with pGenSil-1-BC047440-siRNA vector (PP
2
) was significantly inhibited as compared to that in untreated parental HepG2 cells (PP
0
) and in HepG2 transfected with vacant vector pGenSil-1 (PP
1
). The cell colony formation rate was statistically decreased in siRNA
which was respectively (8.42±0.82)% in PP
2
(39.31±5.39)% in PP
1
and (40.96±3.21)% in PP
0
(P0.01). The expressions of c-myc gene were down-regulated in PP
2
as compared to those in PP
0
and PP
1
and the expression levels of c-myc gene in PP
2
was about 12/25 of that in PP
0
. There was no significant changes in other proliferation-related genes. Conclusion BC047440 siRNA could inhibit the cell proliferation of HepG2 cell and be used as a novel method to inhibit the proliferation of hepatoma carcinoma cell. Our study reveals the relationship between BC047440 and c-myc gene.
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