Cloning Of Metastasis-related Genes Expressed In Renal Cell Carcinoma By Suppression Subtractive Hybridization

Renal Cell Carcinoma(RCC) is an urologic familiar malignancy.The incidence of RCC is about 3%in full-length cancer.The incidence and death toll increased by degrees every year.The major treatment of RCC is surgical operation.There had a thick skin such as Chemotherapy,Actinotheraphy and Internal secretion therapy, especially in terminal RCC.Therefore,how to achieve diagnosis and therapy early about RCC is an exigent unfathomed problem in clinical working.Suppression Subtractive Hybridization(SSH) is a reliable strategy for screening and cloning differentially expressed genes which based on subtractive hybridization and combined with suppressive polymerase chain reaction.Appling of Suppression Subtraetive Hybridization,we construct the library which contains the differently expressing subtracted cDNAs between renal cell carcinoma cell lines ACHN-1 and ACHN-2 with different metastasis potential.A serias metastasis-related biology markers and therapy targets,may be construct by using the metastasis-related genes expressed in renal cell carcinoma in the future.Objective:To construct the library which contains the differently expressing subtracted cDNAs between renal cell carcinoma cell lines ACHN-1 and ACHN-2 with different metastasis potential,screen and clone metastasis-related genes expressed in renal cell carcinoma; metastasis potential were used to analysis with suppression subtractive hybridization (SSH):Total RNA was isolated from high invasive cells and matched low invasive cell ,respectively.Then double-strand cDNA were synthesized and restricted by RsaI.High .invasive renal cell carcinoma cell cDNA were divided into two groups and ligated with either adaptorl or adaptor2.After High invasive RCC CDNA hybridized with low invasive cell cDNA twice and underwent nested PCR.The hybridization production were directly inserted into T/A cloning vector and transformed E.coli JM109 to set up the subtractive library.Partial positive bacteria clones picked randomly were identified by colony PCR method and sequenced,then analyze the sequences with the BLAST software,the different expression of metastasis-related genes in renal cell carcinoma between primary and metastatic focus were determined by emi-quantitative RT-PCR analysis.;Results:Construct the library which contains the differently expressing subtracted cDNAs between renal cell carcinoma cell lines ACHN-1 and ACHN-2 with different metastasis potential.The subtracted library contains 185 positive bacteria clones. Random analysis of 50 clones with colony PCR method showed that 90%clones contained 500-1200 bp inserts,7 gene fragments were found by sequence and BLAST analysis software;Conclusion:Suppression Subtractive Hybridization(SSH) is a reliable strategy for screening and cloning differentially expressed genes,The metastasis-related genes expressed in renal cell carcinoma can be used to constructing a serias metastasis-related biology markers and therapy targets for further study;...