ABSTRACT
OBJECTIVE@#To screen the effective target sequences of laryngeal carcinoma related gene LCRG1 using RNAi.@*METHODS@#PCR site mutation method was used to reconstruct pSuper vector. Five pairs of siRNA sequences designed by siRNA software were annealed and inserted into the reconstructed pSuper vector. The reconstructed pSuper 362,398,432,789,903,and pSuper vectors were transfected into Hela cell lines and selected with the appropriate drugs to get resistant and pool cells, respectively. The colonies were identified by RT-PCR or real-time RT-PCR analysis. The silence effects were observed by cloning formation analysis.@*RESULTS@#pSuper vector was reconstructed to restore Bgl II restriction enzyme sites using PCR mutation. The RT-PCR or real-time RT-PCR Results of pool clones showed 362, 398, and 432 pool clones all had better effects of LCRG1 gene-silence, especially 362 pool clones. The expression level of LCRG1 mRNA of selected 362 group anti-puromycin clones A2 and A5 was decreased. The Results of clone forming efficiency revealed that the cellular proliferation in A2 of 362 group was significantly higher than that of the vector and control Hela cells (P<0.05).@*CONCLUSION@#The reconstructed pSuper vector is successfully constructed. The 362 group has better gene silence and has 2 effective 362 group anti-clones, suggesting that methodology has important values in studYing the function and molecular mechanism of LCRG1.
Subject(s)
Humans , Base Sequence , Cloning, Molecular , Gene Silencing , HeLa Cells , Laryngeal Neoplasms , Genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA Interference , RNA, Small Interfering , Genetics , Transfection , Tumor Cells, Cultured , Tumor Suppressor Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>This study was designed to establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous cell carcinoma tissue and paired tumor-adjacent normal bronchial epithelial tissue, and to identify differential expression of tumor-associated proteins by using proteome analysis.</p><p><b>METHODS</b>Comparative proteome analysis of human lung squamous carcinoma and paired normal bronchial mucosa adjacent to tumors from 20 cases were carried out. Total proteins of the carcinoma tissue and normal bronchial mucosa were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).</p><p><b>RESULTS</b>(1) Seventy-six differentially expressed proteins were screened by analyzing the electrophoretic maps of the 20 carcinoma and control mucosa tissues. (2) Sixty-eight differential proteins were identified by peptide mass fingerprinting (PMF). Some proteins were products of oncogenes and others were involved in the regulation of cell cycle and signal transduction. (3) The expression of three proteins mdm2, c-Jun and EGFR, correlated with lung squamous carcinoma, were detected by immunohistochemical staining and Western blot analysis. The results showed that the expression of mdm2, c-Jun and EGFR were up-regulated in lung squamous carcinomas, whereas down-regulated in control normal mucosa. It was consistent with our proteome analysis results. Those results suggested that those proteins may play roles in the carcinogenesis of lung squamous carcinoma.</p><p><b>CONCLUSION</b>sixty-eight differentially expressed proteins were successfully characterized by comparative proteome analysis. Those results may provide scientific foundation for screening the molecular biomarkers which can be used in diagnosis and treatment of lung squamous carcinoma, as well as to improve patients' prognosis and provide a new clue for carcinogenesis research of lung squamous cell carcinoma.</p>