Differential screening of human metastasis-associated genes of renal cell carcinoma by suppression subtractive hybridization

Objective:To construct different invasion cells' cDNA library of human renal cell carcinoma(RCC)by means of suppression subtractive hybridization(SSH),then to screen and clone metastasis-associated genes of RCC.Methods:In vitro invasion assay the Transwells coating Matrigel were performed for separation and recovery of high invasive and low invasive renal cell carcinoma cells.PolyA+ 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 adaptor1 or adaptor2.After High invasive RCC cDNA hybridized with low invasive cell cDNA twice and underwent nested PCR,the PCR products were cloned in pMD18-T Vector and transformed E.coli JM109.Some positive clones that randomly picked up were sequenced and analyzed.Results:The SSH library contained 185 clones with SSH cDNA fragments.Among 50 clones sequenced randomly,45 have insert-fragment.15 of 45 clones were sequencd and the result show they derived from 7 known genes.Conclusions:Through using SSH,we have identified seven cDNA fragments which are much higher levels in the high metastasis cancer cell clone than low metastasis variety.therefore,it is supported that they might play an important role in the metastasis of renal cell carcinoma and may very well potential application in the diagnosis and treatment of metastasis.