Effects of PKM1 gene on the migration of ocular melanoma cells
10.13389/j.cnki.rao.2018.0005
- VernacularTitle:丙酮酸激酶M1(PKM1)对眼黑色素瘤细胞迁移的影响
- Author:
Yuan-Yuan LIU
1
;
Xiao-Yu ZHU
;
Ying LIU
;
Xiao-Jie XU
;
Ling LI
;
Qi-Nong YE
;
Dan LIU
Author Information
1. 锦州医科大学附属第一医院
- Keywords:
ocular B16 melanoma cells;
pyruvate kinase M1 gene;
cloning;
eukaryotic expression
- From:
Recent Advances in Ophthalmology
2018;38(1):27-30
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the eukaryotic expression vector of pyruvate kinase M1 (PKM1) gene labeled with pXJ-40-myc and detect its biological activity in ocular B16 melanoma cells.Methods Ocular B16 melanoma ceils were randomly divided into experimental and control group,and the experimental group was transfected with pXJ-40-myc-PKM1 plasmid and the control group was transfected with pXJ-40-myc plasmid.Then PKM1 gene was amplified by PCR with human liver cDNA library as the template.The recombinant plasmid pXJ-40-myc-PKM1 was identified by bacteria PCR and double enzyme digestion,followed by transfection of pXJ40-myc-PKM1 and pXJ-40-myc plasmid into B16 melanoma cells,and finally,the expression of PKM1 protein was verified by the Western blot,while wound healing assay was used to detect the effects of PKM1 on the migration of ocular melanoma ceils.Results The length of PKM1 gene was 1800bp,which was consistent with the expected size.Compared with the control group,the result of bacteria PCR was positive.The length of double enzyme digestion was 4000 bp and 1800 bp respectively.Western blot results showed that recombinant plasmld pXJ-40-myc-PKM1 was successfully expressed in ocular B16 melanoma cells.Compared with the control group,wound healing assay showed that recombinant plasmid could inhibit the migration of ocular B16 melanoma cells.Conclusion The eukaryotic expression vector of pXJ-40-myc-PKM1 is successfully constructed,which can suppress the migration of ocular B16 melanoma cells.
