RNA干扰下调人胆管癌细胞系Hucct-1乳酸脱氢酶-A表达的体外研究
DOI:
作者:
作者单位:

作者简介:

通讯作者:

中图分类号:

基金项目:

江苏省兴卫工程重点人才专项基金(H201116)


Knock-down expression of lactate dehydrogenase-A (LDHA) by RNA interference in cholangiocarcinoma cell line Hucct1 in vitro
Author:
Affiliation:

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    目的:体外实验观察乳酸脱氢酶-A(lactate dehydrogenase-A,LDHA)短发夹RNA对胆管癌细胞系Hucct1 LDHA表达的抑制效果,筛选出针对LDHA基因有效的干扰靶点,并检测LDHA表达下调后,细胞增殖活力的改变情况-方法:设计合成干扰LDHA表达的寡核苷酸片段,经退火-连接等步骤克隆到线性化pGPU6/GFP/Neo真核表达载体,转化入DH-5α大肠杆菌中扩增,经质粒抽提纯化后测序鉴定-重组质粒用脂质体法转染胆管癌细胞系,荧光显微镜观察转染效率;并分别利用RT-PCR及Western blot技术检测LDHA mRNA及蛋白表达,验证短发夹RNA的干扰效果-将干扰效率最高的质粒转染Hucct-1,MTT法检测干扰组较对照组细胞增殖的变化-结果:成功构建了靶向LDHA基因的3个shRNA质粒表达载体,脂质体法平均转染效率为(80 ± 5)%-荧光定量RT-PCR和Western blot显示瞬时转染si-LDHA-1-si-LDHA-3的Hucct1细胞LDHA mRNA和蛋白表达均较阴性对照组有不同程度的下降,其中si-LDHA-3干扰效果尤为明显-MTT法检测显示转染组Hucct1细胞较阴性对照组增殖减慢,转染后96 h两组差异具有统计学差异(P < 0.05)-结论:重组质粒能有效干扰胆管癌细胞Hucct1中LDHA基因的表达,经瞬时转染筛选出了有效干扰靶位,下调LDHA的表达,明显抑制胆管癌细胞系Hucct-1的增殖活力-

    Abstract:

    Objective:To investigate inhibitory effect of lactate dehydrogenase-A (LDHA) short hairpin RNA (shRNA) on the expression of LDHA in human cholangiocarcinoma cell line Hucct-1 and observe the effect of LDHA down-expression on the proliferation of Hucct-1 cells. Methods: Three pairs of oligonucleotides targeting LDHA were cloned into linearized pGPU6/GFP/Neo eukaryotic expression vector after annealing. Hucct-1 cells were transfected with the recombinant plasmids that were identified by sequencing. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to evaluate the effect of RNA interference on the transcription of LDHA gene and the expression of LDHA protein respectively. Results: DNA sequencing confirmed that three recombinant plasmids were successfully constructed. The si-LDHA-1 and si-LDHA-3 showed higher interfering efficiency. Compared with that of negative control,the protein expression inhibition rates of si-LDHA-1 and si-LDHA-3 were (50 ± 3)% (P < 0.05) and(70 ± 2)%(P < 0.05). MTT assay showed that the proliferation of LDHA knocked-down Hucct1 cell (si-LDHA-3) was slower than negative control Hucct1 cell(si-NC),and there was significant difference in the absorbance value between transfected cells and negative control cells at 96 h after transfection(P < 0.05). Conclusion: The recombinant plasmid can effectively knockdown the expression of LDHA in Hucct1 cells,and two efficient interfering targets in LDHA gene are screened out. Inhibition of LDHA expression evidently results in inhibition of Hucct-1 proliferation.

    参考文献
    相似文献
    引证文献
引用本文

于亚平,傅 赞,韩 涛,廖敏琪. RNA干扰下调人胆管癌细胞系Hucct-1乳酸脱氢酶-A表达的体外研究[J].南京医科大学学报(自然科学版),2012,(7):913-917

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2011-12-08
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期:
  • 出版日期:
通知关闭
郑重声明