| Background and objectiveOsteosarcoma is the most common malignant tumor of bone in young people, in which pulmonary metastasis is the first reason for death. Once the pulmonary metastasis is developed, the prognosis will turn to be poor with nearly 100% mortality rate for the patients with unresectable pulmonary metastasis. It's still a focus and difficulty in clinical research on raising the cure rate of pulmonary metastasis in clinic. Since nearly 50 percent pulmonary metastasis were developed during the treatment, the cure rate of osteosarcoma will be raised markedly if certain measures are taken at the beginning to prevent pulmonary metastasis from developing. This will be the best methods to decrease the mortality rate of osteosarcoma.Tumor metastasis is a multi-step process in which many molecules are involved. Ezrin is regarded as the main regulatory factor in it. It has been proved that ezrin is correlated with prognosis and metastasis in many malignant tumors. Khanna discovered that there was a high expression of ezrin in high metastatic osteosarcoma cell strain than that in low metastatic stain in 2001. Later researches demonstrated that suppression of expression of ezrin caused a down regulation of the survival rate for osteosarcoma cells in lung distinctively. All theses raise a cue that ezrin may be used as a gene therapy target for preventing osteosarcoma metastasis.RNA interference (RNAi) is an effective technology for gene silence developed in recent years, and it has many advantages such as high specificity, high efficiency, easy manipulation, controllable silence level, short experiment cycle, wide applicability and cheapness. RNAi developed rapidly and has been widely used in research on gene function, tumor and antiviral therapy.Research on ezrin in tumor metastasis in vivo was taken on experimental pulmonary metastasis model recently. Such experiments can not reflect the process of osteosarcoma metastasis truly due to the limitation of model and can not display the effect of ezrin on primary tumor. Our research has the following purposes:⑴constructing the recombined small hairpin RNAs (shRNA) plasmid targeted to ezrin gene;⑵knocking down the ezrin gene of UMR106 cells (rat osteosarcoma cell strain);⑶exploring the changes of abilities of proliferation, mobility, adhesion and invasion after knocking down ezrin in the cells;⑷researching the effect of ezrin on primary osteosarcoma growth and metastasis in orthotopic transplantation osteosarcoma model of rats.Methods⑴Small hairpin RNAs (shRNA) targeted to ezrin coding gene (villin2) were designed and synthesized according to the principle mentioned by Elbashir and Reynolds. It was cloned into pGenesil-1 which worked as a transcription vector to construct recombined plasmid named pshRNA1-villin2, pshRNA2-villin2 and pshRNAHK- villin2 (negative control plasmid). The recombined plasmid was extracted in middle quantity and transferred into UMR106 cells by LipofectamineTM 2000. RT-PCT and western blot were used to detect mRNA and protein of ezrin respectively.⑵The ezrin of UMR106 cells was suppressed by transfection of optimal recombined plasmid, and the changes of abilities of cell proliferation, adhesion, motility and invasion were evaluated by MTT, adhesion test and transwell chamber.⑶The rat osteosarcoma model with high pulmonary metastasis was established by orthotopic transplantation of UMR106 cells. The UMR106 cells were resuspended by serum-free media or collagen gel and injected into medullary cavity of proximal tibia of 3-week-age SD rats. The rats were given immunosuppressive agents and antibiotics after operation. Tibial primary tumor and pulmonary metastasis were observed in gross. Alteration of bone was checked by X-ray and tumor characters were detected by HE stain.⑷The UMR106 cells transfected by optimal recombined plasmid were selected by culturing in G418 and injected into the proximal tibia of SD rats according to method of⑶. Nude cells and cells transfected by pshRNAHK-villin2 were used as control. Time of tumor developing, rat survival and pulmonary metastasis were recorded. Distribution of transfected cells in body was detected by fluorescence microscope and RT-PCR.Results⑴Three recombined plasmids, pshRNA1-villin2, pshRNA2- villin2 and pshRNAHK-villin2, were constructed successfully. The transfection rate is 90% by LipofetamineTM 2000. The mRNA of ezrin was suppressed to 23.12% and 30.25% contrast to normal cells detected by RT-PCR, and ezrin protein was knocked down to 12.32% and 25.56% measured by western blot in cells transfected by pshRNA1-villin2 and pshRNA2-villin plasmid respectively. The pshRNA1-villin2 is better than pshRNA2-villin2.⑵Biology behaviors of UMR106 were changed dramaticly after ezrin was inhibited by pshRNA1-villin2:①ability of proliferation was suppressed by 26.63%±20.65%;②ability of adhesion decreased to 52.9% of that of normal cells;③ability of mobility and invasion were also suppressed, the suppression rate were 45.34±5.91% and 50.69±5.96% respectively. ⑶The osteosarcoma model with high pulmonary metastasis was established successfully by orthotopic transplantation of UMR106 cells into medullary cavity of proximal tibia of SD rats, and the rate of tumor formation and pulmonary metastasis were both 100%. The destruction of bone and the neoplastic bone were seen in the X-ray, osteoid tissue induced by tumor was detected in microcopy and all these are consistent with human osteosarcoma. The time of tumor formation in serum-free media group was shorter than that of collagen gel group (10.0±1.65 vs 17.8±0.87 p<0.0001). The pulmonary metastasis foci in serum-free media group were more than that of collagen gel group (155.25±36.63 vs 91.5±29.56, p<0.0001). The rats of serum-free media group died earlier than collagen gel group (21.4±6.67 days vs 40.6±9.52 days, p<0.0001).⑷After selective culture for 2 months in G418, UMR106 cells stably transfected by pshRNA1-villin2 were more than 50% of total cells. The ezrin mRNA was 60.25% of normal cells detected by RT-PCR, and ezrin protein were knocked down to 50.56% of normal cells measured by western blot. The osteosarcoma model established by cells after selective culture has obvious difference with the two control groups, the results are demonstrated as the following:①ezrin had no effect on tumor formation and growth of UMR106 cells since no difference could be found in tumor formation time among pshRNA1-villin2 group, normal cells group and pshRNAHK-villin2 group (10.17±0.90 days vs 10.17±0.95 days and 10.08±0.88 days,p>0.05);②the survival time of pshRNA1-villin2 group was obviously longer than normal cells group and pshRNAHK-villin2 group (42.8±17.28 days vs 21.3±6.72 days and 22.4±7.42 days,p<0.0001);③the pulmonary metastasis foci of pshRNA1-villin2 group was much lesser than normal cells group and pshRNAHK-villin2 group (60.9±57.78 vs 120.4±41.92 and 117.6±36.48 , p<0.0001);④it was proved by fluorescence microscope and RT-PCR that cells whose ezrin was knocked down could grow normally in primary tumor, but could not be found in the pulmonary metastasis focus.Conclusions⑴The ezrin protein in UMR106 cells can be knocked down effectively by recombined plasmid pshRNA1-villin2 and pshRNA2-villin2. The effect of pshRNA1-villin2 is better than pshRNA2-villin2.⑵Ezrin is very important for cell proliferation, adhesion, mobility and invasion. These functions can be weakened by knocking down ezrin expression in UMR106 cell, at also the transfer capability of cell is attenuated.⑶The osteosarcoma model established by orthotopic transplantation of UMR106 resuspension cells into medullary cavity of proximal tibia of SD rat has high pulmonary metastasis rate, which is a perfect model for studying the pulmonary metastasis of osteosarcoma. The model established by cells resuspended in serum-free media, has shorter tumor formation and survival time, more pulmonary metastatic foci, and is fit for the studying the mechanisms of pulmonary metastasis. The model established by cells resuspended in collagen gel, has longer tumor formation and survival time, lesser pulmonary metastic foci, and is fit for studying the treatment of pulmonary metastasis.⑷Ezrin is a necessary factor for pulmonary metastasis of osteosarcoma, but has no effect on the growth of primary tumor. Since the suppression of ezrin can prevent the pulmonary metastasis of osteosarcoma in rat model, ezrin can be served as a target in gene therapy for preventing pulmonary metastasis in clinic.
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