一个编码4个不同基因RNAi的shRNA质粒的构建及其对CNE-2Z细胞增殖、凋亡和小鼠鼻咽癌移植瘤的影响

Construction and identification of shRNA eukaryotic vector system expressing four different genes and effects of shRAN vector on proliferation and apoptosis of CNE-2Z cells

目的:构建同时表达VEGF、c-myc、survivin和hTERT基因的shRNA真核表达质粒,研究其对人鼻咽癌细胞系CNE-2Z的干扰作用。方法:根据VEGF、c-myc、survivin和hTERT基因序列,各设计2条寡核苷酸序列,合成互补DNA链,插入真核表达质粒pGenesil-1中,同时构建单基因(VEGF)质粒pGenesil-2。分别转染CNE-2Z细胞,分为多基因质粒(P-1)组和单基因质粒(P-2)组,并设空白对照(BC)组和阴性对照(NC)组。采用MTT法检测细胞增殖活性。采用实时定量PCR和Western-blot检测4种基因在人鼻咽癌细胞系CNE-2Z中的表达。体内裸鼠成瘤实验观察单基因和多基因质粒干预对肿瘤的抑制作用。结果:通过酶切和测序证实重组真核表达载体构建正确。MTT显示细胞增殖受到抑制,且多基因质粒对细胞增殖的抑制作用明显强于单基因质粒。目的基因在mRNA和蛋白水平上表达均下降。裸鼠实验显示P-1组抑制肿瘤细胞增长的作用要强于P-2组。结论:成功构建一个载体编码4个不同基因的shRNA质粒载体系统。转染鼻咽癌细胞后,与单独沉默VEGF相比,同时干扰VEGF、c-myc、survivin和hTERT基因可以更好地抑制细胞增殖。

Aim：To construct and identify a VEGF,c-myc,survivin,hTERT shRNA multigenes eukaryotic expression vector system and explore the effects of RNAi targeting four different genes on the apoptosis and proliferation of nasopharyngeal carcinoma （NPC） CNE-2Z cells.Methods：According to VEGF,c-myc,survivin, hTERT gene sequence, two oligonucleotide sequences were designed and synthesized complementary DNA chain. Then inserted into pGenesil vector and constructed single VEGF plasmid and multiple plasmids.These plasmids were respectively transfected into human NPC CNE-2Z cells and xenograft tumors in nude mice. Cell proloferation was detected with MTT assay. The inhibitory effects on target genes were evaluated with Real-time PCR and Western blot at the levels of mRNA and protein, respectively. The inhibitory effect of plasmids on xenograft tumors was observed in nude mice.Results： Restriction endonuclease and sequence analysis of inserted fragment confirmed sequencing results of recombinant eukaryotic expression vector were correct. MTT showed that CNE-2Z cell proliferation in multi-gene plasmid group was more significantly inhibited than a single gene. The expression of mRNA and protein of four different genes were all decreased in p-1 group. and the interference effect of multiple genes was better than the single-gene.Nude mice experiments showed that multi-gene plasmid was more effective than single-gene plasmid in inhibiting the growth of tumor cells.Conclusion： shRNA vector system expressing four different genes has been successfully constructed, which could more effectively inhibit NPC proliferation and induce apoptosis than single plasmid.