Effect of LINE1-ORF1p overexpression on the proliferation of nephroblastoma WT_CLS1 cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To prepare the LINE1-ORF1p polyclonal antibody, and to study the effect of LINE1-ORF1p on the proliferation of nephroblastoma WT_CLS1 cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>

Association between Long Interspersed Nuclear Element-1 Methylation and Relative Telomere Length in Wilms Tumor / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>DNA hypomethylation of long interspersed nuclear elements-1 (LINEs-1) occurs during carcinogenesis, whereas information addressing LINE-1 methylation in Wilms tumor (WT) is limited. The main purpose of our study was to quantify LINE-1 methylation levels and evaluate their relationship with relative telomere length (TL) in WT.</p><p><b>METHODS</b>We investigated LINE-1 methylation and relative TL using bisulfite-polymerase chain reaction (PCR) pyrosequencing and quantitative PCR, respectively, in 20 WT tissues, 10 normal kidney tissues and a WT cell line. Significant changes were analyzed by t-tests.</p><p><b>RESULTS</b>LINE-1 methylation levels were significantly lower (P < 0.05) and relative TLs were significantly shorter (P < 0.05) in WT compared with normal kidney. There was a significant positive relationship between LINE-1 methylation and relative TL in WT (r = 0.671, P = 0.001). LINE-1 Methylation levels were significantly associated with global DNA methylation (r = 0.332, P < 0.01). In addition, relative TL was shortened and LINE-1 methylation was decreased in a WT cell line treated with the hypomethylating agent 5-aza-2'-deoxycytidine compared with untreated WT cell line.</p><p><b>CONCLUSION</b>These results suggest that LINE-1 hypomethylation is common and may be linked to telomere shortening in WT.</p>

Identification and analysis of galactose-1-phosphate uridyltransferase gene mutations in children with galactosemia / 中华实用儿科临床杂志

Objective To investigate the mutation of galactose-1-phosphate uridyltransferase gene (GALT gene) of galactosemia children by molecular methods.Methods Two children with galactosemia were investigated.The peripheral blood mononuclear cells were separated and total RNA was extracted.Then,whole cDNAs of GALT were amplified by reverse-transcription polymerase chain reaction;The PCR products were subcloned into T-easy vector and the positive clones were selected and sequenced;meanwhile,the PCR products were also digested by restricted enzymes and identified by restriction fragment length polymorphism.Results Two novel mutations were found in 2 children.In one child,A was changed into G in nucleotide 1006 of GALT gene,which led to amino acid residue M336V mutation.In the other child,A in nucleotide 779 of GALT gene was changed into T and led to amino acid residue H260L mutation.The 2 mutations were both missense mutation and heterozygous mutation.Conclusions Gene diagnosis is an useful method to improve the accuracy of galactosemia diagnosis and will provide valuable references for prenatal diagnosis,hematopoietic stem cell transplantation and gene therapy.