Lactate dehydrogenase A promotes the growth of human melanoma cell line MUM-2B
10.13389/j.cnki.rao.2018.0121
- VernacularTitle:乳酸脱氢酶A促进人脉络膜黑色素瘤细胞的生长
- Author:
Yuan-Yuan LIU
1
;
Xiao-Yu ZHU
;
Ya-Geng WU
;
Ying LIU
;
Xiao-Jie XU
;
Dan LIU
Author Information
1. 锦州医科大学附属第一医院
- Keywords:
lactate dehydrogenase A;
eukaryotic expression;
human choroidal melanoma MUM-2B cells;
clone
- From:
Recent Advances in Ophthalmology
2018;38(6):519-522
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the eukaryotic expression vector of LDHA with Flag label and detect its effects on the growth of human choroidal melanoma (CM) MUM-2B cells.Methods CM cells line MUM-2B subcultured by the Military Academy of Sciences were divided into two groups:experimental group and control group.The experimental group was transiently transfected with Flag-LDHA plasmid,and the control group was transiently transfected with Flag plasmid.Using the Flag-LDHA with GST label as a template,the LDHA gene was amplified by polymerase chain reaction (PCR),which then was inserted into eukaryotic expression vector of Flag,and the recombinant plasmid Flag-LDHA was identified by bacterial liquid PCR,double enzyme digestion and sequencing,both which were transiently transfected into human CM MUM-2B cells after successful identification,and finally,its expression was determined by Western blot.The biology behaviors of melanoma cell line MUM-2B transfected with Flag-LDHA and Flag plasmid were analyzed by counting Kit-8 (CCK8) assays.Results The coding region sequence of LDHA gene of approximately 1000 bp was harvested from PCR amplification,which was successfully cloned into the Flag vector.Compared with the control group,the PCR result of the bacterial liquid in the experimental group was positive.The double enzyme digestion results showed that eukaryotic expression vector of Flag with a length of about 4000 bp Flag vector and a 1000 bp LDHA gene band.And the sequencing results indicated that the inserted sequence was completely in consonance with the coding sequence of the LDHA gene.Western blot results showed the successful expression of recombinant plasmid Flag-LDHA in MUM-2B melanoma cells.CCK8 assays demonstrated that Flag-LDHA recombinant plasmid could promote the growth of melanoma cell line MUM-2B.Conclusion The eukaryotic expression vector of Flag-LDHA was successfully constructed,which can promote the growth of melanoma cell line MUM-2B.This will lay the foundation for studying the function of LDHA in the initiation and progression of human CM.
