Screening Of Osteosarcoma Pulmonary Metastasis-asscoiated Molecules

Osteosarcoma (OSA) is the most common malignant tumor of bone. Even with amputation, it had once been reported to have a poor prognosis. Over the past 30 years, surgical intervention and neoadjuvant chemotherapy have improved limb salvage rate and considerably raised the survival of affected patients to 60-70%. However, approximately one-thirds of all the OSA patients develop lung metastasis with much lower survival rate. Like other malignant tumors, the improvements of therapy on OSA's metastasis depends on the understanding of correspond mechanisms. Through several decades work, especially the improvements of high-density research techniques such as micro-array, proteomics, mass spectra, bioinformatics have made the systematic studies possible on tumor metastasis. The framework of metastasis has been elucidated and many key molecules have been identified too. As a result, new diagnosis methods and therapies have been carried out including molecular imaging, gene therapy and molecular targeting therapy. On the other hand, due to the lower incidence and absence of characteristic and genome-wide research works of OSA, the mechanism of osteosarcoma pulmonary metastasis needs to be disclosed. [Purpose]To screen the human osteosarcoma pulmonary metastasis associated molecues and validate them preliminarily.[Methods]1. To establish the cell sublines with differential metastatic potential by the orthotopic transplantation and in vivo serial screening.2. To assess the metastasis related biological characters through cell invasion assay, cell heterogenetic adherence assay, clone formation assay, calcium nodules formation assay, immunohistochemestry , apoptosis detecting assay, cytoskeleton staining.3. To screen the differential expressed genes by Affymetrix U133plus 2.0 gene chip.4. To screen the differential expressied MicroRNAs by LC MicroRNA chipμParaflo TM miRHuman₁1.0₀80411.5. To screen the differential ecpressed protein spots by DIGE 2-DE combining the MALDI-TOF-MS.6. To analyse the chips and proteomics results and identify the candidate molecules.7. To confirm the chip assay and proteomic results in differential OSA cell lines and OSA patients'tissues through qRT-PCR , Western Blot ,immuohistochemistry staining.[Results]1. We established high-metastatic OSA cell sublines named SOSP-9607F5M2 (abbreviated as F5M2, which corespond spontaneous metastasis reached 100%) and low-metastatic sublines named as SOSP-9607F4 (abbreviated as F4,which corespond spontaneous metastasis remained at approximately 30%). Additionally, we screened another high-metastatic OSA cell sublines named Saos-2M2(which spontaneous metastasis kept at 90%) .These two paired of sublines (F5M2 vs F4; Saos-2 vs Saos-2M2) also originated from same parent line and had the distinguished high-metastatic potential.Thus they could be regarded as great tools in the future OSA metastatic research works.2. Cell sublines with different metastatic potential had different biology characters.We found these two cell sublines had distinguished difference in such tumor metastasis related biological characters as proliferation, anti-apoptosis, invasion while there was no clear difference between them including heterotypic adherence, motion, cytoskeleton, angiogenesis and bone formation potential.3. Gene expression profile of cell sublines with differential metastatic potential There are totally 484 over 1.7-fold changed gene (783 probes) with 277 up-regulated and 207 down-regulated genes between F5M2 and F4.4. MicroRNA expression profile of cell sublines with differential metastatic potential Different MicroRNA expression between F5M2 and F4 were examined by the MicroRNA microarray, there are totally 76 MicroRNAs differential expressed. Among them, 43 MicroRNA up expressed, 33 MicroRNAs down expressed.5. Protein expression expression profile of cell sublines with differential metastatic potentialWe totally screened 83 differential expressed protein spots by using 2-D DIGE and combining mass spectrum techniques. Among the 37 interested differential spots, 24 were identified up -regulated and 13 were identified down--regulated. 6. Bioinformatics analysisBy using bioinformatics softwares, we finally indentified four molecules (EREG,CHST2 ,ALDOA ,SULT1A3) as candidate pulmonary metastasis associated molecules.7. ValidationWe validated 9 differential expressed genes by qRT-PCR, all showed similar trends with the result of gene expression profile. Five of six MicroRNAs were examined coordinated trends with MicroRNA chip. The results of Western Blot confirmed ALDOA and SULT1A3 differential expressed between F5M2 and F4.8. Functional study of pulmonary metastasis associated candidate moleculesFirstly, we confirmed four proteins (EREG,CHST2,ALDOA,SULT1A3) expressed in 6 OSA cell lines, then we validated the distinguished differential expression of such molecules between OSA survival group and metastasis group patiens by using immuonohistichemistry staining.[Conclusion]We successfully established the cell subclones with differential metastasis potentials and screen the four candidate OSA pulmanary metastasis associated molecues combining such high-density techniques as gene microarray, MicroRNA microarray and differential proteomics. Through bioinformics analysis, four OSA pulmonary metastasis associated molecues, EREG, CHST2, ALDOA and SULT1A3, were identified and then validated preliminarily in OSA cell lines and OSA tissues.