Analysis Of Bladder Carcinoma-Related Nuclear Matrix Proteins And Study In Their Interactions With EGFR Gene

Background and Objective: The nuclear matrix (NM) represents the insoluble structural framwork of nucleus that includes the nuclear lamina and pore complexes, residual nucleoli and internal ribonucleoprotein network. The nuclear matrix is removed nearly all the DNA and RNA from the nucleus with a series of hypertonic salt and detergent extractions and nuclearase digestion, leaves the residual structure. Nuclear matrix proteins ~NIvIPs) have been shown to play a important role in structure of chromatin, DNA replication, RNA synthesis and splicing and transport and gene expression. The NMPs have been known to be tissue and cell- type specific and associated with carcinoma. Several unique NM1Ps have been investigated in various carcinomas tissue. This study was aimed at characterizing the difference NM7Ps expression and their interaction with epidermal growth factor receptor (EGFR) gene in the human bladder transitional cell carcinoma (hBTCC). Materials and methods: The hBTCC-NMPs were prepared from fresh tissues storied at ?00C within 30 minutes of surgical removal using the Wen?s method with some modifications. Rabbit were immunized with the total hBTCC- NMPs, antiserum were collected from the immunized rabbit and the antiserum purified by sulfate amrnonium precipitation. The hBTCC-associated N7N扞Ps was analyzed by SDS-PAGE and western blot. NM in situ and paraffin tissue of hBTCC and other control tissues were detected by mi reef. i mmun ofluorescence through confo cal scanning 3 microscope. Genornic and nuclear matrix DNA(NM DNA) were extracted by phenol-chloroform method respectively, two pairs of primers were designed from 5532bp EGFR cDNA sequence, the association of EGFR gene fragment with NM and its role were detected by polymerase chain reaction (PCR). Results: The possible hBTCC-related NMPs of molecular weight 20- 4OkD and 50-2lOkD were found by western blot analysis, Indirect immunofluorescence assay showed that the proteins of the NM were largely localized in NM in situ and nuclear membrane and cytoplasm partly in liBTCC paraffin sections. Both 940 bp and 11 Obp positive EGFR fragment were amplified by PCR in genomic DNA and NM DNA with primers I - II and III-IVprirners respectively, however, when NM digested with DNase 1(1 OOIU/ml), 11 Obp fragment of amplification is still positive in NM DNA and 940bp is negative. So the binding of EGFR gene fragment at position 1361-1470 to NM is weaker than at position 3901- 40 10. Conclusions: 1 This study demonstrated that carcinoma-related NMIPs expressed in hBTCC, these NMIPs are different from total cell proteins in control tissues. It is useful to develop enzyme immunoassay in urine or serum and suggesting a potential biomarker of hBTCC. 2 The product of PCR indicates that EGFR active transcription gene fragment is tightly bound to nuclear matrix and suggesting there exist correlation between NMPs and EGFR gene, NMPs plays a important role in regulation of EGFR gene transcription or post-transcription.