shRNA靶向沉默P2X7R表达对鼻咽癌细胞增殖、凋亡及PI3K／Akt和Wnt／β-catenin通路的影响∗

摘要

Objective To explore the effect of short hairpin RNA ( shRNA) mediated purinergic receptor P2X ligand⁃gated ion channel 7 ( P2X7R) silencing on cell proliferation, apoptosis and signal pathway in nasopharyngeal carcinoma. Methods After optimization of the transfection conditions of shRNA, two shRNA vector fragments ( shRNA⁃1, shRNA⁃2) of P2X7R gene were trans⁃fected into HONE1 cells, respectively. The HONE1 cells transfected with nonsense sequence was assigned as empty transfection group and cells without any treatment was used as control group. Quantitative real⁃time PCR ( QPCR) was used to detect the expression of P2X7R at 24, 48, 72 and 96 h after transfection and the infection vector with high inhibition rate was screened for subsequent experi⁃ment. The proliferation inhibition rate was detected at 24, 48, 72 and 96 h after transfection by thiazolyl blue ( MTT) . The cell apopto⁃sis was detected by flow cytometry at 48 and 96 h after transfection. The mRNA levels of apoptosis related genes were detected by QPCR method. The protein changes of phosphatidylcholine 3⁃kinase/protein kinase B ( PI3K/Akt ) pathway and Wnt/β⁃catenin pathway were detected at 96 h after transfection by Western blotting. Results The level mRNA of P2X7R in transfected group was lower than those in the blank group and the control group ( P<0�05) . The shRNA⁃1 carrier with high interference efficiency was select⁃ed for the functional study. Compared to empty transfection group and control group, there were increased inhibition rate, apoptosis rate and mRNA levels of pro⁃apoptosis protein bid and Bax but decreased mRNA levels of anti⁃apoptosis protein Bcl⁃2 and Mcl⁃1 in shRNA⁃1 transfected group with statistical difference ( P<0�05) . Compared with the control group, the protein level of PTEN was elevated and protein level of p⁃Akt in PI3K/Akt pathway were decreased in shRNA⁃1 transfection group ( P<0�05) . As for the Wnt/β⁃catenin path⁃way, there were decreased expression of p⁃β⁃catenin, Cyclin D1 and C⁃myc in transfection group (P<0�05). Conclusion The inhibi⁃tion of P2X7R gene expression by shRNA can inhibit the proliferation and induce apoptosis, which may be related to the inhibition of PI3K/Akt and Wnt/β⁃catenin pathway, and it has a certain value for the prevention and treatment of nasopharyngeal carcinoma.%目的：探讨靶向P2X7受体（ P2X7R）的小发夹RNA（ shRNA）对人鼻咽癌HONE1细胞增殖、凋亡及信号通路的影响。方法根据预实验结果优化shRNA转染条件，将2条靶向抑制P2X7R基因的shRNA载体片段（ shRNA⁃1、shRNA⁃2）分别高效转染HONE1细胞，同时设转染无义序列（ Blank）的空转染组及不进行任何处理的对照组。分别于转染24、48、72、96 h后采用实时荧光定量PCR（ QPCR）检测P2X7R表达水平以筛选抑制率高的转染载体用于后续试验。四甲基偶氮唑盐（MTT）法检测各组转染24、48、72、96 h后的增殖抑制率，流式细胞术检测各组转染48、96 h后的细胞凋亡率，QPCR法检测各组凋亡相关基因的mRNA水平，Western blotting检测各组转染96 h后磷脂酰肌醇3⁃激酶／蛋白激酶B（ PI3K／Akt）通路和Wnt／β⁃连接素（β⁃catenin）通路中的蛋白变化。结果转染组的P2X7R mRNA水平均低于空转染组和对照组（ P＜0�05），且选择干扰效率高的shRNA⁃1载体进行功能学研究；与空转染组和对照组比较，shRNA⁃1转染组的HONE1细胞增殖抑制率、凋亡率及促凋亡基因Bid、Bax的mRNA水平均升高，而抗凋亡基因Bcl⁃2、Mcl⁃1的mRNA水平均降低，差异有统计学意义（ P＜0�05）；与对照组比较，shRNA⁃1转染组处理后PI3K／Akt通路中PTEN蛋白水平均升高，而p⁃Akt水平均降低（ P＜0�05），Wnt／β⁃catenin通路中p⁃β⁃catenin、Cyclin D1和C⁃myc水平均降低（ P＜0�05）。结论通过shRNA抑制P2X7R基因表达可抑制鼻咽癌细胞增殖并诱导凋亡，可能与抑制PI3K／Akt、Wnt／β⁃catenin通路有关，对鼻咽癌的防治有一定价值。