Differential expression of Epstein-Barr virus-encoded RNA and several tumor-related genes in various types of nasopharyngeal epithelial lesions and nasopharyngeal carcinoma using tissue microarray analysis.

Studies have revealed that Epstein-Barr virus (EBV) infection, genetic aberration, and environmental factors are of importance in the development of nasopharyngeal carcinoma (NPC), although the definite mechanism remains to be fully elucidated. The aim of our study is to investigate using tissue microarray analysis whether differential expression of EBV-encoded small RNA-1 (EBER-1) and several tumor-related genes were associated with NPC carcinogenesis. Immunohistochemistry and in situ hybridization were performed on tissue microarrays containing 148 NPCs and 164 noncancerous nasopharyngeal epithelia (NPE) with different morphologic features. We found that overexpressions of EBER-1 hybridization signals, p53, p21ras, and bcl-2 proteins and loss expressions of p16 and p27 proteins were significantly increased in NPC tissues compared with normal NPE and hyperplastic NPE (P

[Function of a novel brain-specific gene LRRC4].

OBJECTIVE: To study the suppressive effect of LRRC4 gene on human glioma U251 cells and further investigate its biological functions. METHODS: H&E, DNA and AgNORs stainings were performed on LRRC4-transfected U251 cells, mock-transfected U251 cells and non-transfected U251 cells, respectively. Quantitative analysis including cell morphometry, DNA content, DNA ploidy, silver stained argyrophilic nucleolar organizer regions (AgNORs) were investigated by image analysis. Flow cytometry was employed to determine the difference of cell cycle distribution and MTT staining was used to elucidate the activity of the LRRC4-transfected U251 cells. RESULTS: The morphological cell parameters such as area, perimeter and diameter, DNA content, chromosomal aneupoloidy, mean area of AgNORs particles and mean nucleus area of the LRRC4-transfected U251 cells were remarkably decreased compared to those of the mock-transfected and non-transfected U251 cells (P < 0.05, P < 0.01). Meanwhile, significant accumulation of cells in G(0)/G(1) phase but decrease of cells in S and G(2)/M phase, was observed in transfected U251 cells compared to those of the mock-transfected and non-transfected U251 cells (P < 0.05, P < 0.01). MTT staining showed that proliferation activity of both the mock- and non-trasfected U251 cells was significantly higher than that of the U251 cells transfected with LRRC4 gene (P < 0.01). CONCLUSION: LRRC4 gene might be involved in tumor suppression by restraining DNA synthesis and the nucleoli organizer regions-associated proteins, keeping the cell cycles in phase G(0)/G(1) and reducing proliferation activity of the glioma cells. Morphometry combined with other techniques such as flow cytometry and MTT staining can well elucidate the biological function of novel genes.

A susceptibility locus at chromosome 3p21 linked to familial nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) poses one of the serious health problems in southern Chinese, with an incidence rate ranging from 15 to 50/100,000. Chromosome translocation t(1;3) and frequent loss of heterogeneity on short arms of chromosome 3 and 9 have been reported to be associated with NPC, and a genome-wide scan identified an NPC susceptibility locus on chromosome 4p15.1-q12 recently. In our study, we collected samples from 18 families at high risk of NPC from the Hunan province in southern China, genotyped with a panel of polymorphic markers on short arms of chromosomes 3, 9, and 4p15.1-q12. A locus on 3p21 was identified to link to NPC with a maximum logarithm of odds for linkage score of 4.18. Fine mapping located the locus to a 13.6-cM region on 3p21.31-21.2, where a tumor suppressor gene cluster resided. Our findings identified a novel locus for NPC and provided a map location for susceptibility genes candidates. In contrast to a recent study, no significant evidence for NPC linkage to chromosomes 4 and 9 was observed.

[Analysis of splicing variants in NASG 3'UTR, down-regulated in nasopharyngeal carcinoma, and its expression in multiple cancer tissues].

BACKGROUND & OBJECTIVE: NASG gene, a tissue-specific gene of human nasopharyngeal epithelium was isolated by suppression subtractive hybridization. This study was designed to analyze splicing variants in NASG 3'untranslated region (UTR) and its expression profiling in multiple cancer tissues. METHODS: The PCR primers were designed in NASG 3'UTR around the splicing variants and reverse transcription-polymerase chain reaction (RT-PCR) was performed. The PCR products were separated and sequenced. The expression patterns of NASG were detected by RT-PCR among nasopharyngeal carcinoma (NPC) cell line HNE1, primary human embryo nasopharyngeal epithelial cells, NPC biopsies, and normal adult nasopharyngeal epithelial tissues. Its expression profiling in multiple cancer tissues were tested by cancer profiling array hybridization. RESULTS: There were three splicing variants in NASG 3'UTR. NASG was identified to be down-regulated in NPC cell line HNE1 and 71% of the NPC biopsies, but up-regulated in 25% lung of the cancer biopsies, and not express in other cancer tissues and normal tissues. CONCLUSION: There were three splicing variants in NASG 3'UTR. Its abnormal expression may be an important molecular event in NPC and lung cancer.

[Expression of a new cloned nitroreductase gene NOR1 and purification of expressed product].

BACKGROUND & OBJECTIVE: NOR(1)is a good candidate of tumor suppressor/susceptibility gene associated with nasopharyngeal carcinoma. This study was designed to construct the prokaryotic expression vector and to investigate the expression of nitroreductase gene NOR(1)in Escherichia coli and to purify expressed product. METHODS: Total RNA was subtracted from normal nasopharyngeal carcinoma tissue. The full length of NOR(1)gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHIand XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI,then the NOR(1)gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR(1)was identified by sequencing and digested with restriction enzymes. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express GST fusion protein. The result was confirmed by Western blot analysis and the purified targeted protein was obtained by affinity chromatography. RESULTS: The 1.25kb NOR(1)gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). It existed not only in supernatant but also in precipitation of broken bacteria. The result was confirmed by Western blot analysis,and the purified targeted protein was obtained by affinity chromatography. CONCLUSION: The successes in construction of expression vector of NOR(1), expression and purification of GST/NOR(1)fusion protein make it possible to prepare for the polyantibodies for NOR(1).

[Information behavior of 7 STR loci on chromosome 9p in gene scanning].

To get genotype and allele frequency distributions of seven short tandem repeat (STR) loci of chromosome 9p,D9S288,D9S157,D9S1748,D9S171,D9S161,D9S1817 and D9S1805 in Chinese Han population in Hunan area,blood samples were collected from the random Han individual in Hunan and the whole genomic DNA was extracted.STR loci were amplified by multiplex-PCR technique and genotyped by ABI 377 sequencer.Seventy-five alleles were detected,with frequencies ranging from 0.002 to 0.800,and constituted 243 genotypes. All the seven loci met Hardy-Weinberg equilibrium. The statistical analysis of seven STR loci showed H(heterozygosity) ranging from 0.347 to 0.844,DP(discrimination power) ranging from 0.346 to 0.841,PPE(probabilities of paternity exclusion) ranging from 0.308 to 0.738 and PIC(polymorphic information content) ranging from 0.328 to 0.822. The result indicated that there was a significant difference between Han ethnic group and the white and the black.