| BackgroundOsteosarcoma is the most common primary malignant bone tumors mostly in children and teenagers. It often occurs in the rich blood long bone shaft metaphysic , with some characteristics just like malignant degree high, transfer rate early, progress quickly and mortality high and so on. And it often leads to clinical treatment failures and patients die. Currently some researchers think that cell proliferation and death is closely related with cancers occurrence and development. And make cell tolerance radiation and chemotherapy stimulation. Therefore, inducing tumor cell apoptosis became a kind of new killer tumor cells treatment strategies.Cell Fas-associated death domain-like IL-1 beta converting enzyme inhibitory protein, c-FLIP is a kind of new discovery of apoptosis inhibiting protein, it belongs to the new members of the family of antiapoptotic. Specific to express in malignant melanoma, Kaposi tumor, colorectal cancer and non-hodgkin's lymphoma, It has control of apoptosis and apoptosis of the dual role of cell division. Due to its specific expression and promote the growth of tumors function, that makes it become the new targets cancer treatment.ObjectiveTo investigate the expression of c-FLIP protein and unravelits clinical significance in Osteosarcoma.And to investigate the inhibitory effect of RNA interference(RNAi) on c-FLIP expression and protein level of MG-63,as well as its effect on cell proliferative ability,apoptosis level.Methods1. 47 cases of osteosarcoma, 25 of osteochondroma, and 8 of normal bone tissue which have a complete clinical information were collected. The expression of c-FLIP was detected with Immunohistochemistry method (SP) to analyze the relation between c-FLIP and osteosarcoma differentiation degree, tissue types, lymphatic metastasis.2. Design small interference clips for preparation of many c-FLIP genes, then be transfected into osteosarcoma cell MG-63 by positive ion LipofectamineTM2000 liposome. Transfect effiency was assessed by fluorescence microscope.Use RT - PCR, the western blot test respectively the c-FLIP mRNA and protein level. And screening the siRNA of the most effective silence.3. The most efficiency of siRNA was transfected into MG-63 cell.The cell cycles were measured by Flow cytometry.The cell apoptosis were measured by flow cytometry and TUNEL. Cultured MG-63 cells were also divided into two groups: control group and interference group. Results1. The positive expression rate of c-FLIP in 47 patients with osteosarcoma was 83 %(39/ 47) , which was significantly higher than that in the patients with osteochondroma (16%, P<0.05) .Analysis of the clinical date, a negative correlation was found between the expression of c-FLIP and sex, age, time, tumor size, pathology types, as well as the metastasis of osteosarcoma(P>0.05), but there was a positive correlation with clinical Ennecking stages(P<0.05).2. The silence efficiency of siRNA-3 on the c-FLIP mRNA and protein expression reduce the most significant.3. Then transfect siRNA-3 into osteosarcoma cell line MG-63, cell proliferation activity is obviously decreased. Flow cytometric analysis display, The apoptosis ratio of experimental group was obviously higher than the apoptosis ratio of control group (P<0.05). Compared with control group, interference group increased the cell ratio of G1 phase (P<0.05). TUNEL method to detect shows,apoptosis cells in interference groups (30%) were significantly more than those in control groups (6%) (P<0.05).Conclusion1. The expression of c-FLIP is potentialized in osteosarcoma, and it is positively correlated with the Ennecking stages,but have nothing to do with tissue type and lymphatic metastasis.2. The c-FLIP siRNA is successfully transfected to MG-63 cells. c-FLIP siRNA can effectively suppress c-FLIP mRNA and protein expression in osteosarcoma cell line MG-63,and at the same time,there are marked difference on proliferation ability and apoptosis rate of MG-63 cell. |
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