[Study on mitochondrial function of ND1 gene with 3316 G-->A mutation in human diabetes].
OBJECTIVE
To explore the effects of ND1 gene with 3316 G-->A mutation upon mitochondrial function and elucidate its role in the development of human diabetes.


METHODS
The eukaryotic expression vector pcDNA3.1B and E. coli DH5alpha were used to construct the recombinant plasmid (pcDNA3.1B-ND1) of wild-type and 3316 G-->A mutant type ND1 gene. And the recombinant plasmids were analyzed by restriction enzyme digestion and DNA sequencing. Two siRNAs (mtND11 and mtND12) specific for human mtNDA ND1 gene were designed, synthesized and then transfected into Hela cells for silencing endogenous mtDNA ND1 gene. The gene-silencing effects were analyzed by RT-PCR, SDS-PAGE and MitoCapture mitochondrial apoptosis detection kit. Later the two types recombinant plasmids were transfected into Hela cells in which endogenous mtDNA ND1 gene was silenced. After 48 h culture, the Hela cells were collected for determination of mitochondrial proteins by SDS-PAGE.


RESULTS
Both mtND11 and mtND12 could decrease mtDNA ND1 expression and mtND11 caused a smaller decrease. The expression of mitochondrial protein in 3316 G-->A mutant type recombinant decreased.


CONCLUSIONS
The normal expression of mitochondrial ND1 gene maintain the function of mitochondrial respiratory chain and cell proliferation. The 3316 G-->A mutation in mitochondrial ND1 gene might be related to the down-regulated expression of mitochondrial protein and the diabetes mellitus pathogenesis.
Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan, China.
Guan-hua
GH
Deng
Xin
X
Zhou
Zhi-yu
ZY
Pang
Song-mei
SM
Liu
Yan
Y
Xie
D049109:Cell Proliferation; D004272:DNA, Mitochondrial / Q000235:genetics*; D003920:Diabetes Mellitus / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009245:NADH Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D009245:NADH Dehydrogenase; D034741:RNA, Small Interfering; C515075:NADH dehydrogenase subunit 1, human