α-Galactosidase A gene mutation induced autophagic dysfunction and its mechanism
10.3760/cma.j.issn.1006-7876.2015.07.003
- VernacularTitle:转染突变型α-半乳糖苷酶A基因诱导细胞自噬异常及其作用机制探讨
- Author:
Chunxiao XU
;
Jing LIU
;
Mande KANG
;
Yawen ZHAO
;
Wei ZHANG
;
Zhaoxia WANG
;
Yun YUAN
- Publication Type:Journal Article
- Keywords:
Fabry disease;
alpha-Galactosidase;
Microtubule-associated proteins;
Autophagy;
Apoptosis regulatory proteins
- From:
Chinese Journal of Neurology
2015;48(7):551-555
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of α-galactosidase A (GLA) gene mutation on cell autophagy and to elucidate its mechanism preliminarily.Methods Two families were diagnosed by ultrastructural pathological examination,GLA gene activity test and GLA gene mutation screening.Mutant type recombinant expression plasmid of two pedigrees (pcDNA3.1-GFP-ex1 (EX1 group),pcDNA3.1-GFP-ex3 (EX3 group)) and wild type recombinant expression plasmid of GLA (pcDNA3.1-GFP-GLA,GLA group) were constructed.Hela cell line (control group) was transiently transfected with recombinant expression plasmid according to lipofectin transfection.The relative gene expression of Beclin-1 was measured with real-time PCR,and protein expression level of LC3-Ⅱ/LC3-Ⅰ,Beclin-1 and P62/SQSTM1 was examined by Western blotting.Results The LC3 protein values of groups EX1,EX3,GLA and control were 1.495 ± 0.064,1.490 ± 0.020,1.285 ± 0.021,1.260 ± 0.042,respectively;P62/ SQSTM1 values were 0.555 ± 0.086,0.480 ± 0.084,0.785 ± 0.439,0.980 ± 0.278,respectively;Beclin-1 mRNA 2-△Ct values were 0.011 ±0.003,0.008 ±0.002,0.005 ±0.001,0.003 ±0.001,respectively;Beclin-1 protein values were 1.178 ±0.098,1.209 ±0.092,0.931 ±0.100,0.796 ±0.184,respectively.Compared with the wide type group,the level of LC3-Ⅱ/LC3-Ⅰ protein was significantly higher in the mutant type groups(t =5.118,4.984;P =0.007,0.008),though no statistically significant difference was found in the expression levels of P62/SQSTM1 (t =1.052,1.400;P =0.323,0.199).Besides,the expression levels of Beclin-1 mRNA (t =3.800,2.445;P =0.005,0.040) and protein (t =2.424,2.729;P =0.042,0.026) were significantly higher in the mutant type groups.Conclusions GLA gene mutation can induce cell autophagic dysfunction,and signaling pathway of autophagic activation may be Beclin-1 dependent.
