| Osteosarcoma is the most common primary bone cancer occurring in young adultsand the third highest cause of cancer-related death after leukemia and nervous-systemcancers in children and adolescents under20years of age. Consistent with its highincidence in adolescents, Osteosarcoma preferentially develops in the metaphyseal regionof the most rapidly growing bones such as the distal femur, with approximately60%ofcases originating around knee area. Osteosarcoma is often highly agressive with20%patients presenting overt distal organ metastases (most are lung) at initial diagnosis. Overthe past third decades, the5-year survival for patients with localized disease has dramaticincreased from less than20%to70%due to the combination using of surgery,neoadjuvant, and adjuvant chemotherapy. Unfortunately, for patients with metastatic orrecurrent disease remain have a poor prognosis, with only30%surviving at5years.Respiratory failure resulted from pulmonary metastasis is the main cause of death inpatients with osteosarcoma. At present, our knowledge of the mechanisms underlyingosteosarcoma metastasis is still limited. Along with extensive research in osteosarcoma, anumber of biomarkers have been identified in osteosarcoma, such as aberrant modification of tumor suppressor-gene Rb, P53genes and oncogene c-MYC, c-FOS genes.Notch is evolutionarily conserved signaling pathways in the development ofmulti-cellular organisms. Its temporal-spatial expression effects can specify diversecellular events, including proliferation, differentiation, apoptosis, stem cell maintenance,and binary cell-fate specification. Emerging evidence indicate that Notch signalingpathway is aberrantly activated in a variety of human cancers, including T-cell acutelymphoblastic leukemia, lung, colorectal, prostate, and breast carcinomas. Jagged1is oneof important ligand for Notch and it is also associated with cancer progression because it’soverexpression predicting poor prognosis for patients with prostate cancer and breastcancer. Experimental studies have demonstrated that the expression of Jagged1wassufficient to promote cancer invasion and metastasis. The role of Notch signaling inosteoblast differentiation, proliferation and lineage commitment was recently identified.Given that osteosarcoma is believed to arise from mesenchymal stem cells (MSCs) orosteoprogenitor cells due to a disruption in the osteoblast differentiation pathway, wespeculate Notch signaling may function as an oncogene in osteosarcoma development. Infact, sevaral groups have observed the activation of Notch in osteosacoma, but wetherNotch ligand Jagged1involved in osteosarcoma remain a question. To determine the roleof Jagged1in osteosarcoma progression, we carried out the following research work.Objective:1. To investigate the expression and significance of Jagged1inosteosarcoma patients.2. To investigate the effect of silence Jagged1by siRNA onosteosarcoma cell proliferation, cell cycle, migration and invasion.Methods:1.68formalin-fixed paraffin-embedded osteosarcoma tissue samples were obtainedbetween January2007and January2009from our laboratory. The expression of Jagged1in osteosarcoma tissues were examined by immunohistochemical stain and evaluated bytwo pathologist. The data was analylized with clinical characteristic of patients.2. The expression of Jagged1and Notch receptors were determined in twoosteosarcoma cell lines, the lower metastic F4and the higher metastic F5M2, bysemi-quantative PCR, Real-time PCR and Western-blot. 3. Chemically synthesized Jagged1-siRNA and control-siRNA were transfeted intoF5M2cell lines, and the effect of Jagged1silence mediated by siRNA were analylized bysemi-quantative PCR, Real-time PCR and Western-blot. The GFP-labelledJagged1-shRNA plasmid was also constructed and introduced into F5M2cells bylipofectmine2000.4. MTT, plate colon formation assay were conducted to determine the influence ofJagged1silence in F5M2cells proliferation and colony formation capability, cell cyclewere detected by Flow Cytometry and data were analyzed using the Cellquest Prosoftware.5. Transwell assays were performed to determine the influence of Jagged1silence inF5M2cells migration and invasion.Results:1. Immunohistochemical results indicated that Jagged1expression was stronglypositive in69%osteosarcoma samples (47/68), it was more noteworthy that in24metastatic sampples, the positive stain is87.5%including17strong positive cases(+++),4moderate positive cases(++), whereas among non-metastatic samples, the positive stain is59%（26/44）, including6strong positive cases(+++),20moderate positive cases(++). Theresults indicate that Jagged1expression was positive correlated with tumor metastasis(P=0.0154).2. Semi-quantative PCR and Western-blot results showed that Jagged1expressionlevel was higher in F5M2cells than its expression in F4cells. Real-time PCR illustratedthat Jagged1expression is2.8times higer in F5M2cells compare to F4cells. Among4Notch receptors, only Notch1expression was elevated in F5M2cells.3. After transfecting Jagged1-siRNA into F5M2cells, the expression of Jagged1waseffectively silenced by80%compared to control-siRNA transfected cells. However, wefailed to establish the shRNA mediated long-term Jagged1silencing cells.4. Silence Jagged1expression significantly suppressed F5M2cells prolifrarion asdetermind by MTT assay, and the number of cell colones in Jagged1-siRNA group wasextremely less than control group (67±6vs33±5), the difference was significantly. Cell cycle assay illustrated that after48h Jagged1-siRNA transfection, the population of G1phase was significantly increased and the population of S phase was obviously decreasedin F5M2cells compared to control cells.5. In transwell assay, we demonstrated that blocked Jagged1expression by siRNAsignificantly inhibiting F5M2cell migration and invasion.Conculusion: in our work, we showed that Jagged1were positively expressed inmajority of osteosarcoma patients and strong Jagged1expression may positively relatedwith osteosarcoma metastasis. Blocking Jagged1expression inhibited osteosarcoma cellproliferation and impaired tumor cell migration and invasion capability. |
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