KIF1A过表达对氧糖剥夺-再灌注损伤PC12细胞的作用机制研究

The effects of KIF1A overexpression on oxygen-glucose deprivation and reperfusion induced PC12 cell injury

目的:研究Kinesin-3家族成员蛋白(Kinesin-3 family member1A,KIF1A)对氧糖剥夺-再灌注诱导的PC12细胞活力、自噬和凋亡的影响,为进一步研究KIF1A在脊髓缺血再灌注损伤治疗方面提供理论依据。方法:PC12细胞(美国ATCC公司)分为四组:A组,对照组,无处理;B组,氧糖剥夺再灌注(oxygen glucose deprivation/reperfusion,OGD/R)组,PC12细胞用无糖DMEM培养基,于含混合气体(95%N2和5%CO2)的37℃恒温箱内密闭缺氧培养4h;C组,pcDNA3.1空质粒组,PC12细胞转染pcDNA3.1空质粒48h,进行OGD/R处理;D组,pcDNA3.1-KIF1A质粒组,PC12细胞转染pcDNA3.1-KIF1A质粒48h,进行OGD/R处理。B、C、D组经OGD/R处理后,更换常规培养基,正常孵育24h,收集细胞总RNA及蛋白质,进行实时定量PCR(quantitative real-time PCR,qRT PCR)和Western blot检测KIF1A mRNA和蛋白的表达情况;CCK8检测细胞存活率变化;凋亡ELISA及Caspase-3活性检测试剂盒检测细胞凋亡及Caspase-3活性;Western blot检测各组自噬相关基因LC3-Ⅰ、LC3-Ⅱ、P62以及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路的蛋白表达变化。结果:与A组(1.00±0.00)相比,B组细胞KIF1A mRNA(0.41±0.05)和蛋白表达水平(0.52±0.07,P<0.05)显著下调;细胞活力[(51.60±7.35)%,P<0.05]显著降低。与B组(1.00±0.00)相比,C组空质粒对KIF1A mRNA(0.91±0.13)及蛋白质(1.08±0.08)表达,细胞活力[(51.60±7.35)%vs(47.30±4.16)%],细胞凋亡(1.95±0.18 vs 2.08±0.16,P>0.05)等无显著影响。而D组KIF1A过表达后能显著上调KIF1A mRNA(2.63±0.16)以及蛋白表达(2.51±0.18,P<0.05),显著缓解OGD/R引起的细胞存活率下降[(51.60±7.35)%vs(86.40±9.03)%]及凋亡[1.95±0.18 vs 1.36±0.12,P<0.05];与B组相比,D组自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ的比值(1.68±0.14 vs 1.19±0.09,P<0.05)及pmTOR的表达(1.00±0.00 vs 1.26±0.02,P<0.05)显著受抑制,P62表达显著升高(0.53±0.05 vs 0.89±0.09,P<0.05)。结论:KIF1A过表达可促进缺血再灌注损伤诱导的PC12细胞存活、抑制细胞自噬与凋亡,其机制可能与抑制mTOR通路相关。

Objectives:To investigate the effects of the Kinesin-3 family member,Kinesin-3 family member 1A(KIF1A)on cell survival,autophagy and cell apoptosis capacity of PC12 cells after oxygen-glucose deprivation and reperfusion,and to provide theoretical basis for further study of KIF1A in the treatment of spinal cord ischemia reperfusion injury.Methods:PC12 cells were divided into four groups as follows:group A,Control,no treatment;group B:oxygen glucose deprivation/reperfusion(OGD/R),PC12 cells were cultured in a glucose-free DMEM medium in a 37℃incubator containing mixed gases(95%N2 and 5%CO2)for 4h;group C:cells were transfected with pcDNA3.1 plasmid for 48h and then underwent OGD/R;group D:cells were transfected with pcDNA3.1-KIF1A plasmid for 48h and then underwent OGD/R.For group B,C,D,after OGD/R,normal complete medium were changed for 24h incubation.Total RNA and proteins were isolated for quantitative real-time PCR(QRT-PCR)and Western blot analysis.CCK8 was used to observe cell viability.Cell death ELISA kit and Caspase-3 activity detection kit were used to detect apoptosis and caspase-3 activity.The expression levels of autophagy related proteins and mammalian target of rapamycin(mTOR)signaling were investigated by Western blot.Results:Compared with group A(1.00±0.00),OGD/R treatment(group B)could significantly decrease mRNA(0.41±0.05)and protein expression levels(0.52±0.07)of KIF1A in PC12 cells(P<0.05)and inhibit cell viability[(1.00±0.00)%vs(51.60±7.35)%;P<0.05].Compared with group B,pcDNA3.1transfection in group C had no significant influence on KIF1A mRNA(0.91±0.13)and protein expression(1.08±0.08),cell viability[(51.60±7.35)%vs(47.30±4.16)%]and apoptosis[1.95±0.18 vs 2.08±0.16,P>0.05].However,compared with group B,KIF1A overexpression in group D significantly up-regulated KIF1A mRNA(2.63±0.16)and protein(2.51±0.18,P<0.05)expression levels,and promoted the survival rate[(51.60±7.35)%vs(86.40±9.03)%,P<0.05]and apoptosis(1.95±0.18 vs 1.36±0.12,P<0.05).KIF1A overexpression inhibited the ratio of autophagy-related protein LC3-Ⅱ/LC3-Ⅰ(1.68±0.14 vs 1.19±0.09,P<0.05)and expression of pmTOR(1.00±0.00 vs 1.26±0.02,P<0.05),and promoted P62 expression(0.53±0.05 vs 0.89±0.09,P<0.05).Conclusions:Overexpression of KIF1A in PC12 cells could significantly promote OGD/R induced cell survival and inhibited OGD/R induced autophagy and apoptosis.mTOR pathway may be involved in the protective mechanism of KIF1A.