Construction and identification of a eukaryotic expression vector for the small interfering RNA targeting nucleostemin gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a eukaryotic expression vector for the small interfering RNA (siRNA) targeting nucleostemin (NS) gene.</p><p><b>METHODS</b>The siRNA targeting NS gene was designed according to the sequence of NS mRNA available in GenBank. Three siRNA sequences were obtained, and the corresponding cDNAs were synthesized and inserted into plasmid pRNAT-U6.1 for constructing the recombinant plasmids, which were transformed into E.coli DH5alpha strain. The plasmids, after identification by PCR and DNA sequencing, were transfected into EC9706 cell line via liposome, and the mRNA and protein expressions of NS gene in the cells were determined by RT-PCR and Western blotting, respectively.</p><p><b>RESULTS</b>Three recombinant plasmids were identified by PCR and sequence analysis, the results of which showed correct insertion of the designed sequences in the plasmids. RT-PCR and Western blotting showed substantially decreased mRNA and protein expressions of NS gene in the transfected cells.</p><p><b>CONCLUSION</b>The recombinant plasmid expressing the siRNA targeting NS gene has been successfully constructed, which provides the basis for studying RNA interference of the NS gene.</p>

Expression of nucleostemin mRNA and protein in the esophageal squamous cell carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the mRNA and protein expression of nucleostemin (NS) in human esophageal squamous cell carcinoma.</p><p><b>METHODS</b>The mRNA and protein expression of NS were detected in 31 mucosal atypical hyperplasia specimens, 62 esophageal squamous cell carcinoma specimens and the matched normal esophageal mucosa samples by RT-PCR and immunohistochemistry method, respectively.</p><p><b>RESULTS</b>The positive expression rate of NS protein in normal esophageal mucosa, atypical hyperplasia and esophageal squamous cell carcinoma was 17.7% (11/62), 41.9% (13/31) and 69.4% (43/62), respectively. There was a significant difference among the above three groups (chi2 = 33.676, P < 0.01). The expression levels of NS mRNA in esophageal squamous cell carcinoma (0.971 +/- 0.121) was significantly higher than that in the atypical hyperplasia (0.913 +/- 0.085) and also in the normal esophageal mucosa (0.866 +/- 0.103; F = 14.829, P < 0.01). The expression level of both NS protein and mRNA was positively correlated with histological grade, infiltration depth, and lymph node metastasis (P < 0.05), but not with age, gender or pathological type (P > 0.05).</p><p><b>CONCLUSION</b>Our results indicate that nucleostemin mRNA and protein are over-expressed in human esophageal squamous cell carcinoma, and it may be related with its oncogenesis.</p>

Detection of methylation of hMSH2 gene promoter region of esophageal cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect methylation in promoter region of hMSH2 gene in esophageal cancer.</p><p><b>METHODS</b>Specimens of cancer and normal tissues freshly removed from 32 cases of esophageal cancer patients without previous radiotherapy, chemotherapy or other treatment were preserved at -80 degrees C within 30 min. Methylation specific PCR (MSP) was used to detect methylation of mismatch repair gene (MMR) hMSH2 in promoter region in esophageal cancer and normal esophageal tissues.</p><p><b>RESULTS</b>The frequencies of methylation of hMSH2 gene in promoter region of cancer and normal esophageal tissues were 32.4% (11/32) and 0/30 (0%), respectively, and significant difference was found between the two groups (P < 0.01). The frequency of methylation in elder patients (> or = 70 years old) was significantly higher than that in younger patients (< 70 years old) (P < 0.05). Methylation was less frequently found in grade I-II (18.2%) than in grade III-IV (70.0%) (P < 0.05).</p><p><b>CONCLUSION</b>Methylation of hMSH2 gene in promoter region is related to patients' age and histopathological grade of the esophageal cancer.</p>