Screening The Metastasis Functional Genes By Combining Suppression Subtractive Hybridization With CDNA Microarray

Tumor metastasis is a biochemical process involving multiple factor, multiple procedures,the procure of tumor metastasis phenotype is associated with diverse metastasis-related genes expressions, thus, seeking novel metastasis functional genes is of vital importance for lucubrating gene tumor metastasis molecular biology and searching for new treatment targets. Currently, various kinds of gene triage techniques are widely applied in searching for metastasis-related genes, among which suppression subtractive hybridization is a range of comparatively mature screening technique introduced by Diatchenko et al in 1996 and it is featured by simple procedure, high sensitivity, low sham-positive rate, and efficiently isolating low abundance differential expressive gene and etc. Nevertheless, there is still a few affinitive expressive. genes were retained without pruning at the same time of obtaining relatedly full-scale differential products via SSH, furthermore, SSH can only screen out differential expressive gene,but cannot quantify its expressive difference, this has caused certain obstacles for anaphase analysis. Genetic chip can detect ponderous genes'expressions at the same pace, thus it was used in the anaphase analysis for decreasing Lib to facilitate anaphase analysis work. In this experiment, the prophase constructed prostatic carcinoma cell line PC3M-1E8 and PC3M-2B4 decreasing hybridizationLib sequences were made into genetic chips to hybridize with PC3M - 1E8 andPC3M-2B4 cDNA for further screening out larger difference expressive genes and performing sequencing and function probation. Annexin II were selected to identify their mRNA expressions in 35 fresh clinic specimens and three matched-pairs of tumor cell lines with high and low metastasis potentials for eating the correlation between Annexin II expression and tumor metastasis.Methods1.The protophase constructed prostatic carcinoma cell line PC3M-1E8 and PC3M-2B4 subtractive hybridization Lib was constructed into genetic chips,and hybridized with PC3M-1E8 and PC3M-2B4 cDNA,then the hybridizational signals were analysed.2. The significantly differential expressive sequences in genetic chips were selected and performed sequencing as well as homology analysis in GeneBank.3. The expressing level of annexin II in the mating tumor cells of different metastatic potential was verified utilizing RT-PCR; annexin II expressing level in clinic prostatic carcinoma specimens was detected using real-time quantitative PCR and its relationship with tumor metastasis was approached.4. CXCL14 eukaryotic expressive carrier was constructed by genetic engineering to transcribe SKOV3 cell, so as to screen stably expressing CXCL14 clone SKOV3 /CXCL14. The impact of extrinsic over-expressive CXCL14 on ovarian carcinoma cell SKOV3 proliferation, metastasis and invasion was studied applying cfse, unilayer intention test and etc.Results1.Over all 56 sequences had more than two folds of expressive difference in PC3M-1E8 and PC3M-2B4 via genetic chip test,17 known genes and 2 unknown EST were included in sequencing and homology analysis.2.annexin II was highly expressed in lowly metastatic tumor cell line and was lowly expressed in highly metastatic tumor cell line. Its expressive level in prostatic carcinoma was correlated with Gleason score.4 . Eukaryotic expressive carrier pcDNA3.1/CXCL14 was successfully constructed, stable-expressing clones were screened out. cfse, unilayer intention test confirmed that extrinsic expressive CXCL14 SKOV3 cell migration and proliferative ability were weakened.ConclusionPC3M - 1E8 and PC3M-2B4 two-way subtracted hybridization Lib was successfully constructed with suppressive decreasing hybridization technique, the Lib sequences were further validated with DNA array technique, part of the relatedly larger differential expressive sequences were selected to perform sequencing, 17 known genes and 2 unknown EST were discovered via Genebank homology analysis .To verify annexin II expressive activity in tumor cell lines of different metastatic potential and clinical prostatic carcinoma specimens,annexin II abnormal expression is closely related to tumor metastasis,in prostatic carcinoma,its expressive level is negatively correlated with prostatic carcinoma metastasis.SKOV3 extrinsic over-expressing CXCL14's ability of inhibiting its proliferative and metastatic potential was found in further approaching the mechanisms of CXCL14.