Construction and function detection of EMMPRIN glycosylation mutantion plasimid
10.13481/j.1671-587x.20140324
- VernacularTitle:EMMPRIN糖基化突变质粒的构建及功能检测
- Author:
Chaoyue ZHAO
;
Runmin SONG
;
Hui QIN
;
Na WANG
;
Liu YANG
;
Jiang LI
- Publication Type:Journal Article
- Keywords:
extracellular matrix metalloproteinase inducer;
point mutation;
glycosylation;
cell proliferation
- From:
Journal of Jilin University(Medicine Edition)
2014;(3):583-587
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct in the extracellular matrix metalloproteinase inducer (EMMPRIN)glycosylation single point mutation plasmid,and to explore its relationship with tumor cell proliferation.Methods PCR point mutantion technology was used to construct the mutantion plasimid of EMMPRIN glycosylation single point.After successful mutation, the function of mutantion plasmids were detected. Western blotting was used to detect the expression of EMMPRIN protein,immunofluorescence method was used to detemine the morphological changes of the cells,and MTT assay was performed to detect the relationship between mutantion pasmid and tumor cell proliferation.Results Confirmed by restriction enzyme digestion and sequencing,the 44th,the 152th,and the 186th Asn were successfully mutated to Gln in the sequence of EMMPRIN;EMMPRIN/GFP (N44Q), EMMPRIN/GFP(N152Q),and EMMPRIN/ GFP (N186Q)glycosylation single point mutation plasmids were constructed.Compared with wild-type,thel morphology of the cells was significantly changed,the core division of mutant-type cells was significantly reduced, the number of filopodia was reduced. The results of MTT assay showed that the survival rate of the cells in wild-type group were significantly increased compared with control group (P<0.05 );the survival rates of the cells in EMMPRIN (N44Q) group, EMMPRIN (N152Q) group and EMMPRIN(N186Q)were significantly decreased compared with wild-type group(P<0.05).Conclusion Mutant-type EMMPRIN can inhibit the proliferation of tumor cells;with the duration increasing, the inhibitory effect is weakened.There is a correlation between EMMPRIN glycosylation and proliferation of tumor cells.
