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

English Keywords:Kinesin-3 family member 1A  PC12 cell  Oxygen-glucose deprivation and reperfusion  Apoptosis  Mammalian target of rapamycin

English Abstract:

  【Abstract】 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.