[Effect of LINE1-ORF1p overexpression on the proliferation of nephroblastoma WT_CLS1 cells].

OBJECTIVE: To prepare the LINE1-ORF1p polyclonal antibody, and to study the effect of LINE1-ORF1p on the proliferation of nephroblastoma WT_CLS1 cells. METHODS: A 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. RESULTS: The 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). CONCLUSIONS: LINE1-ORF1p can promote the growth of nephroblastoma cells and the formation of tumor cell clone, and may be involved in the pathogenesis of nephroblastoma.

Hypermethylation of AKT2 gene is associated with neural-tube defects in fetus.

INTRODUCTION: Neural-tube defects (NTDs) are common birth defects of complex etiology. Although many studies have confirmed a genetic component, the exact mechanism between DNA methylation and NTDs remains unclear. METHODS: In this work, we investigated the alteration of methylation from placental tissues obtained from 152 normal infants or with NTDs in 130 children with neural-tube defects. Genome-wide changes in DNA methylation were measured using the NimbleGen microarray. The expression levels of 12 genes were also determined, and two genes (AKT2 and CDC25C) showed low expression in NTDs by quantitative real-time PCR analysis. Then, the methyhlated region of AKT2 promoter sequences were confirmed by massARRAY. RESULTS: A total of 150 differentially methylated regions (81 low methylated regions and 69 high methylated regions) were selected by microarray. The expression levels of AKT2 and CDC25C showed lower expression in NTDs. And the percentage of methyhlated region of AKT2 promoter were increased in NTDs. CONCLUSIONS: DNA mythelation was one of the possible epigenetic variations correlated with the occurrence of NTDs, and AKT2 may be a candidate gene for NTDs.