重组新城疫病毒rL-IL29促进人小细胞肺癌细胞系H446内质网应激、自噬及凋亡相关蛋白的表达

Recombinant Newcastle disease virus rL-IL29 promotes the expressions of endoplasmic reticulum stress,autophagy and apoptosis related proteins in human small cell lung cancer cell line H446

目的探讨重组新城疫病毒rL-IL29对小细胞肺癌细胞系H446细胞内质网应激、自噬及凋亡相关蛋白表达的影响。方法将H446细胞随机分为对照组、新城疫病毒(NDV)组及rL-IL29组,后2组分别转染NDV和rL-IL29。CCK-8法检测细胞活性;ELISA检测细胞上清中IL-29含量;Western blot和免疫荧光检测内质网应激相关蛋白GRP78和CHOP的表达。并分别加入内质网应激抑制剂4-PBA预处理4 h,Western blot检测处理组及对照组GRP78、CHOP、自噬相关蛋白LC3Ⅰ/Ⅱ和凋亡相关蛋白cleaved-caspase-3的表达。结果感染H446细胞后,随着病毒浓度的升高,细胞活力明显降低,其中rL-IL29在10-5升高明显(P<0.05)。rL-IL29处理组细胞上清中IL-29含量明显升高(P<0.05),rL-IL29组IL-29蛋白水平较对照组明显升高(P<0.05)。NDV组、rL-IL29组中GRP78及CHOP表达明显升高(P<0.05),LC3Ⅰ/Ⅱ、cleaved-caspase-3表达明显高于对照组(P<0.01),且rL-IL29组明显高于NDV组(P<0.05)。4-PBA预处理后,4-PBA组GRP78、CHOP、cleaved-caspase-3以及LC3Ⅰ/Ⅱ蛋白表达明显降低(P<0.05)。结论 rL-IL29能促进人小细胞肺癌细胞内质网应激、自噬及凋亡相关蛋白的表达。

Objective To explore the effects of rL-IL29 （recombinant Newcastle disease virus expressing IL- 29） on endoplasmic reticulum stress, autophagy and apoptosis related protein in small cell lung cancer cell line H446. Methods H446 cells were divided into control group, Newcastle disease virus（NDV） group and rL-IL29 group randomly, the later two groups infected with NDV and rL-IL29, respectively. Cell viability was detected by CCK- 8 assay. IL- 29 content in cell supernatant was detected by ELISA assay. Western blot and immunofluorescence were used to detect the expression of GRP78 and CHOP, which related with endoplasmic reticulum stress.Then, the experimental groups were treated with 4-PBA 4 h, the expression of caspase- 3 and LC3 Ⅰ/Ⅱ protein was detected with Western blot. Results The activity of cells significantly decreased with the increasing of virus concentration, which increased significantly at 10 -5 concentration （ P 〈0.05）. The content of IL- 29 in the supernatant of rL-IL29 group increased obviously（ P 〈0.05）. The expression of GRP78, CHOP, LC3 Ⅰ/Ⅱ and caspase- 3 had a higher level in NDV and rL-IL29 group than PBS group after infected 24 h. Moreover, rL-IL29 group had high level than NDV group （ P 〈0.05）. After pre-treatment with 4-PBA 4h,the expression of GRP78, CHOP, LC3 Ⅰ/Ⅱ and cleaved-caspase- 3 decreased in NDV and rL-IL29 group（ P 〈0.05）. Conclusions rL-IL29 promotes the expression of endoplasmic reticulum stress, autophagy and apoptosis related protein in small lung cancer cells.