AKT2-siRNA对肺癌细胞NCI-H446沉默作用的研究

背景与目的 肺癌是目前发病率较高的恶性肿瘤之一,其病死率居恶性肿瘤的首位,随着分子生物学研究的不断进展,人们越来越清楚地认识到肺癌的发生和发展是一个多基因参与的多步骤的过程,RNAi作为基因治疗中一种新的效率高、特异性强的治疗手段愈来愈多地受到人们的重视.本研究应用RNA干扰(RNAi)技术,以AKT2为靶基因,设计并构建重组真核表达载体,转染肺癌细胞NCI-H446,观察其对AKT2基因的沉默作用.方法利用已知AKT2的mRNA基因序列设计并合成具有短发夹结构的两条寡核甘酸序列及一条无关对照序列,与pGEM-TEasy载体连接后以T7和SP6为引物进行PCR鉴定,对表达载体pRNAT-U6.2及阳性克隆产物进行双酶切,将得到的基因及线性化的表达载体再次连接,利用PCR的方法进行重组体的筛选鉴定及测序分析,利用脂质体转染肺癌细胞NCI-H446,RT-PCR检测AKT2-mRNA沉默效果.结果 将合成的DNA序列克隆到表达载体上,经PCR扩增筛选鉴定和测序鉴定证实为所需序列,转染肺癌细胞NCI-H446后,AKT2-mRNA表达明显降低.结论 成功构建的靶向AKT2-RNA的干扰重组表达载体可有效抑制肺癌细胞NCI-H446 mRNA的表达.

Silencing effect study of Akt2-siRNA aimed at lung cancer cell NCI-H446

Background and objective Lung cancer is one of the most frequent malignant tumors and at the first place of mortality in today's world, It becomes more and more clear that the initiation and development of lung cancer is multistep processes involved in many genes. RNAi gets more and more attention in gene therapy because of its high efficiency and specificity. With RNAi technology, eukaryotic expression vectors aimed at AKT2 siRNA was designed and constructed, which is transfected into human small lung cancer cell line NCI-H446. The silencing effect was observed. Methods Using the known sequence of AKT2's mRNA (M95936), we designed two oligonucleotides that have short hairpin configuration and one oligonucleotide as control. DNA double strands was annealed, and cloned into vector- pGEM-T. Then, blue-white selection and T7/SP6 PCR was used to screen the positive clones, the target DNA and the express vector pRNAT-U6.2 were cut down by BamHI and XhoI restriction enzyme, and linked with each other; use PCR test to filtrate the positive clones and sequenced the target DNA, then transfected it into human small lung cancer cell line (NCI-H446), cultured and screened by G418, RT-PCR was used to detect the expression level of AKT2 mRNA in NCI-H446. Results validate the positive sequence by PCR and, transfect them to lung cancer cell line (NCI-H446), AKT2-mRNA's expression was evidently declined. Conclusion The expression vector pRNAT-U6.2-AKT2 was constructed successfully and the expression of Akt2 mRNA in small lung cancer cell line (NCI-H446) was obviously silenced after transfection.