Effect Of Mdm2 Gene Silenced By Rnai On Cell Proliferation In Human Osteosarcoma U-20s Cell Line

Background and objectivesOsteosarcoma, aslo known as osteogenic sarcoma, consists of sarcomatous osteoblasts and its osteoid tissues, newly formed bones. Osteosarcoma is the most common and malignant in bone neoplasms. It occurs mostly in teenagers, and about 70% incidences located in distal femur, proximal tibia and metaphysis of proximal humerus. The bone neoplasms has the characteristic of fast progression, early metastasis and poor prognosis. In the past, no more than 20% of the patients can live for five years after operation. The pathogenesis of osteosarcoma is still unkown, but it is found that the osteosarcoma's occurrence and development is closely relevant with P53, MDM2 genes mutation and expression level.About half of human cancers due to TP53 mutation or missing. P53 protein is a most important inhibitor in cancers. And murine double minute 2 (MDM2) has the regulating function of feedback inhibition on the activity of p53. MDM2 was overexpression in most tumor cells, it can combine with p53, and regulates the activity and stability of p53 transcripton in order to inhibit p53's function. Now, the methods of inhibiting the combination of p53 and MDM2 to activate p53 have been becomed the focus ways on antitumor drugs research. But it is a great challenge to discover the nonpeptidic pharmaceutical inhibitor aiming to MDM2. Although some MDM2 inhibitors were been found, their effects were still unsatisfactory on antitumor. Therefore this study aim to do some initial research by RNAi.In this study, we plan to construct siRNA expression vector targeting directly MDM2 gene and transfect it into osteosarcoma cell line (U-20S), then detect the silencing effect of the expression of MDM2. And meanwhile, observe the effect of inhibiting MDM2 expression on the growth and apoptosis of osteosarcoma cell line (U-20S) in vitro.MethodsAccording to the principle of designing siRNA sequence, we used the Ambion and promega siRNA sequence designing and analysis software to scan human MDM2 cDNA exon (NM₀02392). siRNA sequences targeting MDM2 were selected and definited by BLAST homology analysis. Afterwards three pairs of DNA oligonucleotides targeting directly MDM2 annealed into double strands DNA. The annealed products were purifed and cloned into siRNA expression vector (pSilence 2.1) that has been cut by BamHI and HindIII restriction enzyme. The positive ones (pSilencer2.1-MDM2si373,pSilencer2.1-MDM2si646 and pSilencer2.1-MDM2si946) were identified and confirmed by restrictive endonuclease digestion and DNA sequencing. Eventually, the positive recombinants (pSilencer2.1-MDM2si373,pSilencer2.1-MDM2si646 and pSilencer2.1-MDM2si946) were transfected into human osteosarcoma cell line (U-20S), while empty vector group and untransfected group cells were taken as control groups. The mRNA and protein expression level of MDM2 in each group cells was detected by RT-PCR and Western-blot; Cell cycle, cell proliferation, and apoptosis were detected by FCM, and MTT assay respectively.Results1. 58 target siRNA oligonucleotides were obtained by preliminary screening. After retrieveing and optimizing homologization sequence, 3 target siRNA sequences were located at 373-391 (CAAGAGACCCUGGUUAGAC) , 646-664 UCAUCGGACUC AGGUACAU) and 946-964 (UCUACAGGGACGCCAUCGA) .2. The pSilencer2.1-MDM2si373, pSilencer2.1-MDM2si646 and pSilencer2.1 - MDM2si946 were constructed and comfirmed by BamHI and HindIII restrictive endonuclease digestion. After sequenced, the target sequences were coincident completely with the design.3. After U-20S cells transfected by the recombinant plasmids pSilencer2.1-MDM2si373 (group1), pSilencer2.1-MDM2si646(group 2), pSilencer2.1 -MDM2 si946 (group 3) respectively, the expression of MDM2 mRNA were significantly inhibited compared to those in empty vector control group and untransfected group (P<0.05). Each group of the ralative expression of MDM2 mRNA is 0.12±0.02(group 1), 0.21±0.03(group 2), 0.27±0.03(group 3), 0.78±0.11 (empty vector control group), and 0.82±0.13(untransfected group).4. Western blot shows that the expression of MDM2 protein(90KD) were much lower in experimental groups (group 1-3) than in empty vector control group and untransfected group, and that in group 1 and group2 was lowest among all groups.5. The cell proliferation in the 3 experimental groups were inhibited significantly. The proliferation levels were significant different among the experimental groups and control groups (P<0.05).6. The apoptosis ratio in group1-3 was 14.6%±2.1%,13.9%±2.6%,10.3%±1.9% respectively, and that in empty vector control group and untransfected group was 5.7%±0.8% and 4.6%±0.9%, which were dramatically higher than that of empty vector control group and untransfected group. There were significant difference between them (P<0.05).Conclusions1. siRNA vectors pSilencer2.1-MDM2si373 and pSilencer2.1-MDM2si646 aiming to MDM2 gene 373-391 (CAAGAGACCCUGGUUAGAC) and 646-664 ( UCA UCGGACUCAGGUACAU) can obviously decrease the MDM2 expression after they was transfected into osteosarcoma cell line (U-20S). And these two sequences were comfirmed to be desirable target sequences.2. The MDM2 gene silenced by RNAi contributes to reduce the proliferation and promote apoptosis in human osteosarcoma cell line (U-20S).