Knokdown Of Survivin Expression By Small Interfering RNA Induce Apoptosis In Human Osteoscoma Cell Line U-2OS

IntroductionThe inhibitor of apoptosis protein (IAP) family consists of a group of structurally related proteins with anti - apoptotic properties. A novel member of this family is the survivin protein, which is absent from most adult tissues, but is expressed in melanoma, as well as in most major types of human cancer. Survivin expression in tumors has been associated with increased aggressiveness and decreased patient survival. Survivin is a member of the inhibitor of apoptosis protein (IAP) family, that has been implicated in both control of cell division and inhibition of apoptosis. Specifically, its anti - apoptotic function seems to be related to the ability to directly or indirectly inhibit caspases. Survivin is selectively expressed in the most common human neoplasms and appears to be involved in tumor cell resistance to some anticancer agents and ionizing radiation. On the basis of these findings survivin has been proposed as an attractive target for new anticancer interventions. Several preclinical studies have demonstrated that down - regulation of survivin expression/function, increased the apoptotic rate, reduced tumor - growth potential and sensitized tumor cells to chemotherapeutic drugs in vitro and in vivo models of different human tumor types.Osteosarcoma is a highly aggressive disease with a poor prognosis. It was suggest that genetic charges associated with the development of osteosarcoma involve over expression of survivin. The discovery that synthetic 21 - 23 nucleo-tide RNA duplexes (siRNAs) can trigger an RNA interference response in mammalian cells and induce strong inhibition of specific gene expression has opened the door to the therapeutic use of siRNAs. In order to get more look for new gene therapy for osteosarcoma, we used RNA interference (RNAi) technology to ex-amine the effects of small - interfering RNA ( siRNA) on cell apoptosis and biological behaviors of U -2OS cells. The planning of targeted sequence is the key step of RNAi experiment. There are five methods in the execution of RNAi in current research. The chemically synthesized small interference RNA (siRNA) can effectively decrease the expression of targeted gene, but the expensive cost restricts its widely application. Vector mediated RNAi is suitable for the research after specific sequences are screened and identified, while the proce -dure of screening is comparatively complicated. Cock - tail and siRNA expression frame methods cant be applied in the screening of targeted sequence. Considering above factors, we conducted T7 transcription in vitro to acquire siRNA, avoiding shortcomings of other methods. Our results in consistent with the concept that survivin is involved in the regulation of apoptosis.ObjectiveThe first part: To investigate the efficency of construct the small interferen-ing RNA ( siRNA ) of survivin suppress the expression of survivin in human os-teosarcoma cell line U - 2OS after knocked down survivin gene by siRNA synthesized in vitro transcription. The second part: Investigate cell proliferation and apoptosis statue of human osteoscoma cell line U -20S and the change of chem-sensitivity to cisplatin after Silencing of survivin in U - 2OS cell line after knockdown of survivin. The third part;Study the relationship between matrix metallo proteinase 9 ( MMP - 9) and survivin in Osteosarcoma and its clinical significance.MethodsThe first part: Survivin - siRNA transcription in vitro system was firstly sta-blished in our work. Small interfering RNA was transfected into U - 20S cells to inhibit expression of survivin. Lipofectamine 2000 mediated transient transfec-tion was conducted to transmit the siRNA into U -20S cells. Three pairs of specific targeted sequence were selected in the coding region of U - 2OS mRNA.Transfection of U - 2 OS siRNA was conducted with Lipofectamine 2000 in Os-teosarcoma carcinoma cell line U - 2OS. mRNA and protein levels of survivin were detected by reverse transcription - polymerase chain reaction ( RT - PCR) and Westen blot, proliferation inhibition rate of U - 2OS cells was analyzed by MTT assay. The second part;The antiapoptosis effects of sequence specific siR-NA were assessed with flowcytometry and TUNEL. MTT experiment showed the proliferation ability of osteoscoma cells was inhibited. By use of transwell -room, the difference in invasion and metastasis ability between the transfected and non - transfected cell lines was tested. Cell apoptosis was assayed by and TUNEL. The chemosensitivity of transfected cells to cisplatin was measured by MTT. The apoptosis percent of surviving - siRNA was tested by Annexin - V. The third part: The expression, correlation and their clinical significance of survivin and MMP - 9 in 32 patients were detected by immunohistochemistry method. The relationship between matrix metallo proteinase 9 (MMP - 9) and survivin in Osteosarcoma and its clinical significance were analysised by the statistics software spss.ResultsThe first part: Sequence specific siRNAs targeting survivin down - regulated mRNA expression significantly. The survivin protein positive expression rates in specific siRNA group, nonspecific group and control group was a significant difference between survivin siRNA group and other groups ( P < 0. 001 ). Specific group showed slow proliferation and there was a significant difference compared with other groups (P < 0.001). The expression of surviving in siRNA - transfected group decreased by 72% in comparison to untransfected group. After treatment of siRNA, proliferation inhibition rate and apoptosis rate of U -2OS cells were increased. The second part: The apoptosis percentage of interference group was higher than that of nonspecific and control groups (P <0.001). No significant difference in apoptosis rate was found between the control group and nonspecific group. There was a significant difference between interference group and other groups, TUNEL assay also show that the apoptosis body were in-creased after transfected with survivin siRNA. The invasion and metastasis ability of U - 2 OS cells are decreased after trancfect survivin siRNA into the cell line. The third part:The positive expression rates of MMP -9 and survivin were 63. 6% and 84. 6% ,respectively in osteosarcoma, which were higher than that of normal bone tissues (P <0. 05). The survivin expression had positive correlation with MP - 9 (P <0.05).ConclusionsiRNA can efficiently suppress survivin gene expression, inhibit proliferation in osteosarcoma cell line U -2OS.siRNA can efficiently increase cell apoptosis statue of human osteoscoma cell line U -2OS and the change of chemsensitivity to cisplatin after Silencing of survivin in U -2OS cell line.The expression of survivn in osteosarcoma was markedly correlation with the expression of MMP -9.