内质网应激时重组腺病毒Ad-PERK siRNA对人软骨肉瘤SW1353细胞增殖和凋亡的影响

Effects of recombinant adenovirus Ad-PERK si RNA on the proliferation and apoptosis of human chondrosarcoma SW1353 cells in endoplasmic reticulum stress

目的 :探讨靶向蛋白激酶样内质网激酶(protein kinase-like endoplasmic reticulum kinase,PERK]基因的小干扰RNA(small interfering RNA,si RNA)重组腺病毒在内质网应激(endoplasmic reticulum stress,ERS)时对人软骨肉瘤SW1353细胞增殖和凋亡的影响。方法:构建靶向PERK基因的si RNA重组腺病毒Ad-PERK si RNA,并将其感染SW1353细胞后,RT-PCR和蛋白质印迹法检测SW1353细胞中PERK m RNA和蛋白的表达。应用衣霉素(tunicamycin,TM)建立ERS模型;在ERS状态下,MTT法检测Ad-PERK si RNA感染后SW1353细胞的增殖情况,FCM法检测感染后SW1353细胞的细胞周期和细胞凋亡率,透射电子显微镜下观察感染后SW1353细胞超微结构的变化,蛋白质印迹法检测感染后SW1353细胞中cleaved caspase 3、caspase 12、CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)和磷酸化c-Jun氨基端激酶(phospho-c-Jun-N-terminal kinase,p-JNK)蛋白的表达。结果 :成功构建获得重组腺病毒AdPERK si RNA。Ad-PERK si RNA感染后,SW1353细胞中PERK m RNA和蛋白的表达水平下调(P<0.05)。在ERS状态下,Ad-PERK si RNA可促进SW1353细胞的增殖(P<0.05),Ad-PERK si RNA感染组S期细胞所占比例高于感染空载体腺病毒Ad-RFP的阴性对照组和未感染的空白对照组(P<0.05),Ad-PERK si RNA感染组SW1353细胞的凋亡率低于阴性对照组和空白对照组(P<0.05),透射电子显微镜下观察发现阴性对照组和空白对照组SW1353细胞存在明显的凋亡,Ad-PERK si RNA感染组SW1353细胞中cleaved caspase 3、caspase 12、CHOP和p-JNK蛋白的表达水平均明显低于阴性对照组和空白对照组(P<0.05)。结论 :重组腺病毒Ad-PERK si RNA可以促进ERS状态下SW1353细胞的增殖,并抑制其凋亡。

Objective：To investigate the effects of small interfering RNA（si RNA） targeting protein kinase-like endoplasmic reticulum kinase（PERK） gene recombinant adenovirus on the proliferation and apoptosis of human chondrosarcoma SW1353 cells in endoplasmic reticulum stress（ERS）.Methods：Recombinant adenovirus Ad-PERK si RNA targeting PERK gene was constructed,and then it was infected into SW1353 cells.The expression levels of PERK m RNA and protein in SW1353 cells after infection with Ad-PERK si RNA were detected by reverse transcription-PCR（RT-PCR） and Western blotting,respectively.ERS model was induced by tunicamycin（TM）.Under the ERS condition,the proliferation of SW1353 cells after infection with Ad-PERK si RNA was determined by MTT assay,the cell cycle distribution and apoptosis were measured by fl ow cytometry（FCM）,the change of the microscopic structure was observed under a transmission electron microscope,and the expression levels of cleaved caspase 3,caspase 12,CCAAT/enhancer-binding protein homologous protein（CHOP） and phospho-c-Jun-N-terminal kinase（p-JNK） proteins were detected by Western blotting.Results：The recombinant adenovirus Ad-PERK si RNA was successfully constructed.The expression levels of PERK m RNA and protein in SW1353 cells after infection with Ad-PERK si RNA were decreased（P 〈 0.05）.Under the ERS condition,the proliferation of SW1353 cells after infection with Ad-PERK si RNA was promoted（P 〈 0.05）.The ratio of S phase in AdPERK si RNA infection group was higher than those in negative control adenovirus Ad-RFP infection group（as a negative control） and non-infection group（as a blank control）（both P 〈 0.05）.The apoptotic rate of SW1353 cells after infection with Ad-PERK si RNA was lower than those of the negative control and the blank control groups（P 〈 0.05）.The apoptosis structures of SW1353 cells in the negative control and the blank control groups were clearly observed under a transmission electron microscope.The expression levels of cleaved caspase 3,caspase 12,CHOP and p-JNK proteins in SW1353 cells of Ad-PERK si RNA infection group were lower than those of the negative control and the blank control groups（P 〈 0.05）.Conclusion：The recombinant adenovirus Ad-PERK si RNA can promote the proliferation of SW1353 cells and inhibit the apoptosis under the ERS condition.