稳定靶向干扰候选抗药基因UGT1A9鼻咽癌细胞株建立

目的构建针对UGT1A9基因的shRNA慢病毒干扰表达载体;建立稳定UGT1A9干扰鼻咽癌细胞株,测定干扰效率。方法应用荧光定量PCR法检测UGT1A9 mRNA在鼻咽癌成瘤高转移细胞株5-8F和成瘤非转移细胞株6-10B之间的差异表达水平。构建重组UGT1A9 shRNA表达质粒pLentiU6/UGT1A9,经293FT细胞包装后,产生的慢病毒感染5-8F细胞,荧光定量PCR检测干扰效率。结果 UGT1A9在鼻咽癌细胞株中明显高表达。PCR、测序验证pLentiU6/UGT1A9-shRNA重组质粒构建成功;经293FT细胞病毒包装,感染5-8F细胞后,与阴性对照组和未干扰组相比,可明显抑制UGT1A9 mRNA水平。结论成功构建了稳定靶向干扰候选抗药基因UGT1A9的鼻咽癌细胞株,为研究UGT1A9可能参与介导鼻咽癌抗药的分子机理奠定了基础。

Establishment of Nasopharyngeal Carcinoma Cells With Stably Targeting Interfering Anti-drug UGT1A9 Gene

Objective To construct lentivirus interfering expression vector targeting to UGT1A9.To establish the nasopharyngeal carcinoma cells with stably targeting interfering UGT1A9 gene and examine the interference efficiency.Methods UGT1A9 mRNA expression level was detected in NPC 5-8F and 6-10B cells.Recombinant UGT1A9 shRNA-expression plasmid pLentiU6/UGT1A9-shRNA was constructed,and the 5-8F cell was infected by the lentivirus containing the shRNA expression vector produced in 293FT cells,the mRNA level of UGT1A9＇s interfere efficiency was detected by qRT-PCR.Results The mRNA level of UGT1A9 in NPC cell lines 5-8F was significantly upregulated compared to 6-10B cells.Successfully constructing pLentiU6/ UGT1A9-shRNA recombinant plasmid was identified by PCR and sequencing analysis.The mRNA level of 5-8F cell infected with UGT1A9 shRNA lentivirus was significantly lower than the negative control group and the blank control 5-8F cell.Conclusions UGT1A9 is highly expressed in NPC 5-8F cells compared to 6-10B cells.Successfully constructed pLentiU6/UGT1A9-shRNA recombinant plasmid can stably downregulate the expression of UGT1A9 in NPC 5-8F cell line after the expression vector was intergrated into cells.