Study On STK15 Gene Overexpression And P53 Gene Mutation In Centrosomal Abnormality Of Laryngeal Carcinoma

IntroductionThe centrosome is the major microtubule organizing center ( MTOC) of mammalian cells. It maintains genomic stability by establishing the bipolar spindles during cell division and executes accurate segregation of chromosomes during mitosis. Aberrations in the centrosome duplication cycle result in the formation of monopolar or multipolar spindles, and it has been long proposed that such aberrations may cause aneuploidy and contribute to cancer development. Many types of tumors feature a high proportion of cells with centrosomal abnormalities and centrosome anomalies have been reported to arise at early stages of tumor formation and to expand concomitant with tumor progression. Centrosome amplification in tumors and cell lines has been linked to numerous genetic aberrations, including mutation of p53 and overexpression of STK15.Mutations of the p53 gene are widely detected in all kinds of human cancers. Point mutations in exons 5-8 occur frequently in many human tumors including human laryngeal squamous cell carcinoma ( LSCC) and are extensively studied. However, intronic point mutations are rare and totally unknown for human laryngeal squamous cell carcinoma. Intronic mutations may affect gene regulation through aberrant splicing or disruption of critical DNA protein interactions. Any mutation in the porypyrimidine (py) tract and the branch point consensus sequence of introns that affected splicing did so by interfering with efficient spliceosome assembly and splicing of pre - mRNAs. Impairment of p53 function could result in centrosome abnormalities that might be mediated by overexpression of STK15 through transactivation - independent manner.STK15 (also known as BTAK, Aurora -A) is a human serine/threonine kinase and essential for chromosome segregation and centrosome functions. TheSTK15 gene is amplified, and its transcript is also highly expressed in various cancers. Overexpression of STK15 induces increased numbers of centrosome, aneuploidy, and transformation of the cells.In order to explore p53 gene intronic mutation, it's influence to p53 protein expression, and the relationship of p53 gene mutation to STK15 abnormal expression in the centrosomal abnormalities of human LSCC, we detected both la-ryngeal squamous cell carcinoma tissues and paired normal tissues from 55 patients. PCR - SSCP combined with sequencing analysis and Western blot were used to test the p53 gene intronic mutations and p53 protein expression; Meanwhile RT-PCR was used to detect the STK15 gene expression; Combined with tissue culture, centrosomal abnormalities were detected by immunofluorescence on the frost slides of above samples and tumor tissue cells; Chromosomal instability was examined by routine and high - resolution G - banding. This study will provide the evidence to probe the occurrence mechanism and the scientific basis for early diagnosis and therapy of LSCC.Materials and methods1. Materials1. 1 Human laryngeal squamous cell carcinoma tissue1. 2Reagents for RT - PCR, PCR - SSCP and PCR fragments purification1. 3 Reagents for Western Blot1. 4 Monoclonal anti -