shRNA沉默NSD2基因表达对OCI-Ly3细胞增殖、凋亡及Akt/mTOR信号通路的影响

Effects of Silencing NSD2 Gene by shRNA on Proliferation,Apoptosis and Akt /mTOR Signal Pathway in OCI-Ly3 Cells

目的:探讨干扰shRNA下调NSD2基因表达对弥漫大B细胞淋巴瘤OCI-Ly3细胞增殖、凋亡及Akt /mTOR信号通路的影响。方法:NSD2 shRNA慢病毒转染OCI-Ly3细胞,应用real time Q-PCR检测NSD2 mRNA的表达和Western blot法检测NSD2蛋白的表达,以鉴定NSD2 shRNA的沉默效果,应用CCK-8试验检测细胞增殖,AnnexinⅤ/PI双染流式细胞术检测细胞凋亡,Western blot测定组蛋白H3K36二甲基化水平(H3K36me2)、BAX BCL-2、caspase-3、Akt、磷酸化Akt(p-Akt)、磷酸化mTOR(p-mTOR)、磷酸化P70核糖体蛋白S6激酶(phosphorylation P70 ribosomal protein S6 kinase,p-P70S6K)的变化。结果:NSD2 shRNA慢病毒转染OCI-Ly3细胞72 h后,real time Q-PCR和Western blot检测显示NSD2的mRNA和蛋白表达均有明显下降(P<0.05),NSD2 shRNA组细胞增殖率明显低于对照和Neg shRNA组(P<0.05),NSD2 shRNA组细胞的凋亡率明显高于对照组和和Neg shRNA组(30.37±4.22)%vs(1.36±0.52)%和(2.17±1.43)%(P<0.05);促凋亡蛋白BAX表达上调,抑制凋亡蛋白BCL-2表达下调,caspase-3表达上调;H3K36me2水平较对照组明显下降,而检测Akt /mTOR通路蛋白发现,Akt总蛋白水平未明显下降,但是活性形式的磷酸化p-Akt、p-mTOR和p-P70S6K表达下调。结论:干扰沉默NSD2基因可抑制弥漫大B细胞淋巴瘤OCI-Ly3细胞增殖,诱导细胞凋亡,其机制可能是调节组蛋白H3K36me2水平变化,特异性地抑制Akt /mTOR信号通路的活性。

Objective： To investigate the effect of silencing NSD2 gene by RNA interference on the proliferation,apoptosis and the alteration of Akt /mTOR signaling pathway in diffuse large B cell lymphoma OCI-Ly3 cells.Methods：The shRNA targeting NSD2 gene was transfected into OCI-Ly3 cells by lentivirus infection.The NSD2 mRNA and protein were detected by real time Q-PCR and Western blot,respectively.The cell proliferation was detected by CCK-8 and apoptosis was measured by flow cytometry.The expressions of BCL-2,BAX,caspase-3,Akt,p-Akt,p-mTOR,pP70S6K,H3K36me2 were detected by Western blot.Results： After transfecting the OCI-Ly3 cells by NSD2-shRNA for 72 h,the expressions of NSD2 mRNA and protein both were down-regulated（ P〈0.05）,the proliferation rate of cells in NSD2 shRNA group was significantly lower than that in control and Neg shRNA groups（ P〈0.05）; the apoptosis rate of cells in NSD2 shRNA group was significantly higher than that in control and neg-shRNA group（30.37 ± 4.22） % vs 1.36± 0.52 % and 2.17 ± 1.43） %（ P〈0.05）; the expressions of BAX and caspase-3 were up-regulated,while the expression of BCL-2 was down-regulated; the H3K36me2 level significantly decreased as compared with control group,no obvious decrease of the total protein level of AKT was found,but the expressions of p-Akt,p-mTOR and p-70S6K were downregulated.Conclusion： The silencing NSD2 gene can inhibit the proliferation and induce the apoptosis of OCI-Ly3 cells,their mechanisms may relate with regulating the H3K36me2 level,specifically inhibiting the activivty of AKT/mTOR signal pathway.