Abstract：Objective: By constructing β-catenin siRNA lentiviral vectors, the correlation of Wnt pathway with cisplatin resistance of non-small cell lung cancer was explored. Methods: According to the β-catenin gene mRNA sequence, we designed and synthesized three pairs for β-catenin gene shRNA1, shRNA2, shRNA3. Forming double stranded DNA and pLentiLox3.7 lentiviral vector were connected and transformed. The positive clones were selected and the shRNA constructor was identified by DNA sequencing.The recombinant lentivirus was obtained by packaging cells 293T. The titer of virus was tested according to the expression level of GFP. A549 cells were infected to screen out the effective inhibition of the target. After cisplatin-resistant cell line A549/DDP were infected by lentivirus in 24 h, different concentrations of cisplatin were used to interfere with the cells 48 h, MTT assay was used to detect the cells proliferation. According to the cells were infected by lentivirus or not, divided into A549 group, A549/DDP group, A549/DDP+ siRNA group, A549/DDP+siNC group. After 8μg/mL cisplatin was used to interfere with the cells in each group, the expression of LC3 Ⅱ and β-catenin was tested by real-time PCR and western blotting. Results: DNA sequencing showed that the β-catenin shRNA lentiviral vector was constructed successfully and screen out the effective inhibition of the target. At the same concentration, the inhibitory effect of cisplatin on A549 cells was significantly higher than that of A549/DDP cells(P<0.05). After siRNA lentivirus infecting A549/DDP cells, the inhibitory effect of cisplatin on A549/DDP cells was increased. Compared with A549 group, treated with 8 μg/mL cisplatin for 48 h, the expression levels of β-catenin mRNA and protein in A549/DDP group were increased, meanwhile the expression of LC3Ⅱ mRNA and protein were also increased(P<0.05). Compared with A549/DDP group, after treating with cisplatin, the expression levels of β-catenin mRNA and protein in A549/DDP and siRNA cells group were decreased, and the expression of LC3 Ⅱ mRNA and protein were also decreased. Conclusion: The Wnt pathway played an important role in the formation of the cisplatin resistence in non-small cell lung cancer, which was associated with autophagy.
［Key words］lung cancer; Wnt pathway; drug resistence; autophagy
宋萍, 连小怡, 郑金旭, 葛莉萍, 刘超, 蒋婷, 朱黎容, 丁明. β-catenin siRNA慢病毒载体的构建及其对A549耐药细胞生长及自噬的影响[J]. 江苏大学学报:医学版, 2017, 27(03): 213-.
SONG Ping, Lian-Xiao-Yi, Zheng-Jin-Xu, Ge-Li-Ping, Liu-Chao, Jiang-Ting, Zhu-Li-Rong, Ding-Ming. Construction of lentiviral vector of RNA interference of β-catenin and its effect on the growth and autophagy in A549 resistant cell lines. Journal of Jiangsu University(Medicine Edition), 2017, 27(03): 213-.
2017, Vol. 27 
