Effect of LINE1-ORF1p overexpression on the proliferation of nephroblastoma WT_CLS1 cells.
- Author:
Mei-Ling TANG
1
;
Ping XIAO
;
Ji-Zhen ZOU
;
Ding-Ding CAO
;
Yuan-Yuan LI
;
Hui-Bo CHANG
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Antibodies; analysis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Deoxyribonuclease I; analysis; genetics; metabolism; Humans; Long Interspersed Nucleotide Elements; RNA, Messenger; genetics; metabolism; Rabbits; Transfection; Wilms Tumor; genetics; metabolism; physiopathology
- From: Chinese Journal of Contemporary Pediatrics 2018;20(6):501-507
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo prepare the LINE1-ORF1p polyclonal antibody, and to study the effect of LINE1-ORF1p on the proliferation of nephroblastoma WT_CLS1 cells.
METHODSA genetic engineering method was used to achieve prokaryotic expression of LINE1-ORF1p, and rabbits were immunized with LINE1-ORF1p to prepare polyclonal antibody. Indirect ELISA was used to evaluate antibody titer, and Western blot and immunohistochemistry were used to evaluate the specific ability of antibody to recognize LINE1-ORF1p. The eukaryotic expression vector pEGFP-N1-LINE1-ORF1 was constructed and used to transfect WT_CLS1 cells. Western blot and qRT-PCR were used to measure the protein and mRNA expression of LINE1-ORF1, respectively, and cell proliferation assay and colony-forming assay were used to evaluate the effect of LINE1-ORF1p on the proliferation of WT_CLS1 cells and the formation of tumor cell clone.
RESULTSThe LINE1-ORF1p antibody prepared had a titer of >1:16 000 and could specifically recognize LINE1-ORF1p in cells and tumor tissue. WT_CLS1 cells transfected with pEGFP-N1-LINE1-ORF1 had significant increases in the mRNA and protein expression of LINE1-ORF1 and significantly enhanced cell proliferation ability and colony formation ability (P<0.05).
CONCLUSIONSLINE1-ORF1p can promote the growth of nephroblastoma cells and the formation of tumor cell clone, and may be involved in the pathogenesis of nephroblastoma.