Construction Of KAI1 Eukaryotic Expression Vector And Its Function Of Inhibiting Invasion Of Ovarian Cancer Cells In Vitro

Local infiltration and distant metastasis is not only an important character ofmalignant tumours but also one of the main causes of patients' death. Ovarian canceris highly emphasized in gynecological tumors with the highest mortality and a 5-year survival rate of only 20-30%. The poor prognosis in ovarian cancer is generally due to the advanced stage of the disease at the time of diagnosis which is also one of the main causes of patients' death.KAI1 gene is a specifically metastatic suppressor gene for prostate cancer, which is located on human chromosome 11p11.2 and has been isolated from AT6.1 prostate hybrid cancer cells in 1995 by Jin-Tang Dong. It is involved in the progression of a variety of other human cancers too. We have demonstrated in our former research work that the down-regulation or loss of KAI1 protein expression might frequently occur in early stage tumors and the expression of KAI1 protein might be used as an prognostic marker which indicates the metastasis potential of ovarian epithelial cancer.To further explore its function of inhibiting invasion of ovavrian cancer cells,we divided our research work into two parts. In the first part, we extracted total RNA from human placenta tissues, then KAIl gene encoding fragment was amplified by reverse transcriptase-PCR (RT-PCR) and recombined with eukaryotic expression vector pcDNA3.1 (+). After confirmation by restrict endonuclease digestion and DNA sequencing, its eukaryotic expression vector pcDNA3.1 (+)-KAIl was constructed successfully. In the second part, the KAIl cDNA was transfected into human highly metastatic ovarian cancer cell line HO8910-PM with LipofectAMINE2000, we detected its protein steady expression by S-P immunocytochemical staning before and after transfection, observed the effect on proliferation ability by MTT assay and plate clone formation assay, compared the changes of invasion ability by cell adhesiveness assay and in vitro invasion assay. The results proved KAI1 protein steady expression in those HO8910-PM cells after having been transfeced by KAIl cDNA, showed no obviously difference of their proliferation abilities but their invasion abilities decreased sharply. We drew a conclusion that ectogenesis metastasis suppressor KAI1 gene could transfect human highly metastatic ovarian cancer cell line HO8910-PM by liposome efficiently and reduced their invasion abilities by inhibit the adhesion among cells or between cells and extracellular matrix ( ECM ). It might be a new candidate gene in gene therapy of ovarian cancer metastasis and has laid a solid foundation for further in vivo research work...