| Objective: Osteosarcoma is the most frequent type of malignant bone tumor in children and adolescents and is associated with a high propensity for lung metastasis. The 5-year survival of patients with metastatic osteosarcoma is only 20 %. Therefore, it is very important to further understand the mechanism of osteosarcoma metastasis. PLA2G16 is classified as a Group XVI phospholipase A2(PLA2G16) and is expressed in most normal tissues. Previous studies suggested that overexpression of PLA2G16 has been related with poor clinical prognosis in osteosarcoma patients with lung metastasis, but PLA2G16 is how to promote the transfer of the specific mechanism has not been elucidated. The aim of this study was to investigate the association and mechanism of PLA2G16 with pulmonary metastasis in osteosarcoma. Methods: 1. Quantitative real-time PCR and Western blot were used to examine PLA2G16 m RNA and protein in osteosarcoma cell lines. HOS, Saos2 and MG63 cell lines were selected for cell experiment in vitro. 2. Lentivirus were used to construct overexpression of PLA2G16 in osteosarcoma cell lines, then MTT assay, wound-healing assay and Matrigel Transwells assay were conducted to explored the cell proliferation, migration and invasion function. 3. Overexpression and knocking down PLA2G16 expression by lentivirus and si RNA, then western blot was used to detect the expression level of p-Erk1/2, Erk1/2, p-m TOR, m TOR, p-Akt, Akt and other classic downstream signaling pathways 4. Quantitative real-time PCR was used to examine PLA2G16 m RNA in primary osteosarcoma patients(18 patients without metastases and 17 patients with metastases). 5. According to the above experimental results, immunohistochemistry(IHC) staining of PLA2G16 and p-ERK1/2 were performed on tissue microarrays from 88 osteosarcoma patients. Evaluation of PLA2G16 and p-ERK1/2 expression and clinical pathological characteristics and prognosis of patients Results: 1. According to the q RT-PCR analysis and western blot results, the relative level of PLA2G16 expression in HOS cell was significantly higher than its expression in Saos2 and MG63 cells. As compared to the control, the cell proliferation, migration and invasion ability of Saos2 and MG63 cells which overexpressed PLA2G16 were significantly increased. 2. Both the conventional cell culture and fetal bovine serum overnight starvation stimulation conditions were able to obviously increase the phosphorylation level of ERK1/2 in Saos2 and MG63 cells which overexpressed PLA2G16. And the phosphorylation level of ERK1/2 in knockout PLA2G16 HOS cell lines which was significantly decreased. The results indicated that MAPK is a downstream signal pathway of PLA2G16. 3. According to the q RT-PCR analysis, the relative level of PLA2G16 m RNA expression in metastatic primary osteosarcoma tissues(mean ± SD: 3.65 ± 0.60) was clearly higher than that in non-metastatic tissues(mean ± SD: 2.49± 0.69; P < 0.05). The expression levels of PLA2G16 m RNA were found to be significantly increased by 1.47-fold, on average, in the metastatic group compared with the non-metastatic group. According to the IHC analysis, there was a significant correlation between the expression of PLA2G16 and p-ERK1/2(P=0.002). PLA2G16 and p-ERK1/2 were associated with lung metastasis(both P < 0.05), but not with gender, age at diagnosis, tumor location or histological classification(P>0.05). 4. PLA2G16 and p-ERK1/2 positive rates(75.8% vs 41.8%, P=0.002; 81.8% vs 54.5%, P=0.010; respectively) in patients with lung metastasis were significantly higher than those non-metastatic ones. PLA2G16 and p-ERK1/2 yielded an area under the ROC curve(AUC) of 0.670 and 0.636, respectively. Combined ROC analyses revealed an AUC of 0.706. 5. PLA2G16 and p-ERK1/2 are both the prognosis factors of OS and MFS in osteosarcoma. and when PLA2G16 and p-ERK1/2 expression were taken into consideration together, the 5-year OS(51.1%) and MFS(44.7%) rates in the group positive for PLA2G16 and p-ERK1/2 were the worst, and the prognosis of PLA2G16- and p-ERK1/2- group were the best(85.4% and 85.7%, respectively; P=0.020, P=0.016). The 5-year OS(61.5%) and MFS(54.1%) rates for the PLA2G16-p-ERK1/2+/PLA2G16+p-ERK1/2- group were in between the group with the worst and the group with best rates(P<0.05). Conclusion: PLA2G16 could enhance the proliferation, invasion and metastasis ability by activating MAPK signaling pathway in osteosarcoma which means that PLA2G16 may be a potential therapeutic target and prognostic factor of osteosarcoma. PLA2G16 combined with p-ERK1/2 could be more useful prognostic factor for osteosarcoma patients than either one. |
PDF Full Text Request