Gene Silence Of CD147 By RNAi Influence The Biological Behavior Of Ovarian Cancer Cells HO-8910pm

Ovarian cancer is one of the three most common malignant tumours in female reproductive system, Early disease causes minimal, nonspecific, or no symptoms and develops quickly. Therefore, almost 80% patients are diagnosed in an advanced stage,and the overall 5-year survival rate is only 20-30% .The investigation of the molecular mechanism of tumour invasion and migration has been the hot point.The tumour cell microenviroment is important in the proliferation and invasion of the tumour,MMPs play a important role in tumour cell microenviroment.Molecular CD147,namely EMMPRIN(Excellular Matrix Metalloproteinase Inducer), as the major stimulator of MMPs, is found in all kinds of human tumor cells. It indicats that CD 147 is closely related with malignant tumour.Lots of researches have proved that the expression of CD 147 is obviously improved in human ovarian cancer,which demonstrate that CD 147 plays a important role in ovarian cancer invasion and migration. Former researches in Our library have shown that CD 147 expression in malignant and borderline ovrian tumor are obviously higher than benign one and that the MMPs secretion ability of fibroblast cells can be inhibited by antisense RNA ,at the same time the invasion ability of ovarian tumor cell can be weaken. Therefor We want to construct shRNAexpression vector of CD147 to silence the expression of CD147 then to explore itsfunction of inhibiting invasion of ovarian tumor.AIM:1. To construct CD 147 shRNA eukaryotic expression vector;2. To Observe the effect of CD 147 gene silence in ovarian cancer cells HO-8910pm on biological behavior;3. To prove that silenceing gene CD 147 by RNAi can inhibit invasion and metastasis ability of ovarian cancer cell METHODS:1. According to the human CD 147 cDNA on Genebank,we designed there different shRNA targeting the coding sequence of CD 147, the pGE-CD147 shRNA was constructed by inserting the designed shRNA to the eukaryotic expression vector pGE-l.The vector was identified by PCR restriction endonuclease digestion and partial nucleotide sequencing;2. The ovarian cancer cell strain HO-8910pm was transfected by pGE-CD147 shRNA 1,2,3. After selected by G418 ,the CD 147 expression in the positive clones was detected by RT-PCR and Westernblot.Choose the most efficient one,then observe the silence effect of CD 147 by immuno-histochemical S-P method, CLSM;3. MTT assay was carried out to observe the effect on proliferation ability, in vitro invasion assay was carried out to assess changes of invasion ability;4. Transfected cells were transplanted subcutaneously in nude mice to observe the tumorigenesis.RESULTS:1. It was verified by PCR restriction endonuclease digestion and partial nucleotide sequencing that the constructed eukaryotic vector expressing shRNA of CD 147 was correct;...