人环氧合酶-2(hCOX-2)编码基因的克隆及其反义核酸转染胃癌细胞的初步研究

Cloning of human cyclooxygenase-2(COX-2)encoding gene and study of gastric cancer cell transfected with its antisense vector

目的环氧合酶-2与肿瘤发生的关系已成为肿瘤研究的新热点之一,本研究旨在获得 hCOX-2编码基因序列,构建其正、反义真核表达载体,并用反义重组载体转染 COX-2高表达的胃癌细胞,以进一步研究其生物学作用及其在肿瘤发生中的作用机制。方法按文献报道的hCOX-2核苷酸序列设计合成 PCR 引物,利用总 RNA 抽取试剂盒从 COX-2高表达的培养的人胃癌细胞系 SHC7901中提取细胞总 RNA,反转录合成 cDNA,再用PCR 方法扩增目的基因序列,PCR 产物经 DNA 序列测定证实后,利用引物5端引入的 EcoRⅠ,Xba Ⅰ酶切位点,通过粘端连接,将所获目的基因分别按正、反两个方向定向克隆入真核表达载体 pcDNA3.1中,对两种重组质粒分别进行相应的酶切鉴定,采用脂质体介导的方法用 COX-2反义核酸转染 SGC7901细胞,经 G418筛选后,随机挑选细胞克隆,通过 RT-PCR 检测COX-2 mRNA 水平的变化。结果应用 RT-PCR 反应扩增获得大小约1.9kb 的特异性片段,经 DNA 序列分析,证实与文献报道的 hCOX-2编码区序列一致,重组正义表达载体 pcDNA3.1/hCOX2(+)用 EcoRⅠ,Xba Ⅰ双酶切后,产生大小约5.4kb,1.9kb 的片段;重组反义表达载体 pcDNA3.1/hCOX2(-)通过 PstⅠ酶切,产生大小约4.5kb,1.5kb 与1.3kb 的片段,与预期结果相符,转染细胞经筛选后,随机挑选、扩增了3个细胞克隆,其中1个克隆稳定表达 COX-2反义核酸,RT-PCR 提示其 COX-2 mRNA 水平显著下降.结论成功克隆了 hCOX-2编码基因序列,并构建了其正、反义真核表达载体.反转录 PCR 是克隆基因的简便、有效方法.为扩增高 GC 含量的 cDNA 模板,可在 PCR 反应体系中加入适量的 DMSO,并采用较高退火温度.在 PCR 引物中加入限制性内切酶位点,可以方便地实现基因的定向克隆.COX-2反义核酸在胃癌细胞系 SGC7901中得到稳定转染,转染细胞 COX-2mRNA 表达显著受抑制.

AIM To obtain human COX-2 encoding gene,construct its sense,antisense eukaryotic vectors and transfect its antisense vector into COX-2 highly expressing gastric cancer cell line in order to further study its biological activities end role in tumorigenesis. METHODS We amplified hCOX-2 cDNA with RT-PCR. Primes were designed according to the reported nuclear acid sequence of the gene.Using total RNA isolation kit, total RNA was isolated from cultured human gastric cancer cell line SGC7901 which had high expression of COX-2, and then reverse transcripted to cDNA.PCR product was confirmed by DNA sequencing and then directionelly cloned into eukaryotic expression vector pcDNA3.1 in two different directions using endonuclease sites introduced in primers.The two recombinant vectors were identified by endonuclease digestion.Antisense recombinant vector was transfected into SGC7901 cell line using lipofectamine. Positive clones were selected by G418 and COX-2 mRNA level was detected by RT-PCR RESULTS We obtained a 1.9kb fragment by RT-PCR whose sequence was consistent with hCOX-2 cDNA reported in literature.The recombinant sense vector pcDNA3.1/hCOX2(+)generated two fragments of 5.4kb and 1.9kb by EcoR Ⅰ,Xba Ⅰ digestion,while the recombinant antisense vector pcDNA3.1/hCOX2(-) generated 4.5kb,1.5kb and 1.3kb fragments by Pst Ⅰ digestion.The results accord with expected.After G418 selection,three clones of transfected cell were cultured. One of them steadily expressed COX-2 antisense RNA.RT- PCR showed a low level of COX-2 mRNA. CONCLUSION hCOX-2 encoding gene was successfully amplified with RT-PCR and its sense,antissense eukaryotic vectors were successfully constructed.RT- PCR is a simply and effective strategy for gene cloning. DMSO as well as two-step PCR is helpful for amplification of GC rich templates.Endonuclease sites introduced by primes simplify the construction of recombinant vectors. The COX-2 antisense RNA can be expressed steadily in human gastric cancer cell line SGC7901 and lower COX-2 mRNA level.