shRNA干扰NOTCH1基因对套细胞淋巴瘤Akt/mTOR信号通路的影响

Effect of Silencing NOTCH1 Gene by shRNA Interference on AKT/mTOR Pathyway in Mantle Cell Lymphoma

本研究旨在探讨RNA干扰沉默NOTCH1基因对套细胞淋巴瘤Jeko-1细胞株的增殖、凋亡及Akt/m TOR信号通路的影响。针对NOTCH1基因设计短发夹RNA并将其连入p GPU6/GFP/Neo质粒中,构建NOTCH1 shRNA真核表达载体,经脂质体转染入Jeko-1细胞;应用RT-PCR及Western blot鉴定其干扰效果,M TT法绘制细胞生长曲线,流式细胞术检测细胞凋亡的变化,Western blot检测凋亡相关蛋白BCL-2、BAX、procaspase-3、procaspase-9及Akt/m TOR信号通路相关蛋白Akt、p-Akt、p-m TOR、p-P70S6K的表达。结果表明:NOTCH1 shRNA转染Jeko-1细胞后,NOTCH1基因的mRNA和蛋白表达均明显下调,有统计学差异(P<0.05);转染组增殖率明显低于NegshRNA组和空白组(P<0.05),NOTCH1 shRNA转染48 h后,凋亡率为(34.58±3.46)%,而Neg-shRNA组和空白组分别为(2.44±1.35)%、(1.72±0.64)%,有统计学差异(P<0.05);凋亡相关蛋白BCL-2、procaspase-3、procaspase-9的表达下调,而BAX表达上调;干扰NOTCH1基因后Akt总蛋白未见明显变化,而p-Akt、p-m TOR、pP70S6K的表达下降。结论:干扰沉默NOTCH1基因可抑制套细胞淋巴瘤Jeko-1细胞增殖,诱导细胞凋亡,通过去磷酸化抑制Akt/m TOR信号通路可能是其机制之一。

The study was purposed to investigate the effect of silencing NOTCH1 gene by shRNA interference on the proliferationapoptosis and the expression of AKT signaling pathway-related proteins in mantle cell lymphoma Jeko-1cell line.The hairpin-like oligonucleotide sequences targeting NOTCH1 gene were designed and transfected into Jeko-1cells by lipofectamine TM 2 000.NOTCH1 mRNA and protein were detected respectively by RT-PCR and Western blot.Cell growth was determined by MTT.Cell apoptosis was analyzed by flow cytometry.The expressions of BCL-2BAXprocaspase-3procaspase-9Aktp-Aktp-mTORp-P70S6 K were detected by Western blot.The results showed that NOTCH1 mRNA expression was markedly suppressed by the shRNA targeting NOTCH1NOTCH1 shRNA suppressed the proliferation of Jeko-1 cells and induced apoptosis of these cells.The cell apoptotic rate was（ 34.5 ± 3.4）%（2.4 ± 1.3）%（1.7 ± 0.6）% in NOTCH1 shRNANeg-shRNA and blank groupsrespectivelyand the difference between them was statistically significant（ P〈 0.01）.NOTCH1 shRNA down-regulated the expression of BCL-2procaspase-3procaspase 9p-Aktp-mTOR and p-70S6Kup-regulated the expression of BAXbut no change protein expression of Akt was observed.It is concluded that the silencing NOTCH1 gene expression by shRNA interference may inhibit Jeko-1 proliferationinduce the cell apoptosisand the mechanisms may be associated with the inhibition of Akt /mTOR signaling pathway by dephosphorylation.