The Clinicopathological Research And Molecular Mechanisms Of Notch3 Promoting Invasion And Metastasis Of Osteosarcoma

Osteosarcoma is a common malignant bone tumor that occurs mainly in adolescents.Because of complex pathological and imaging features,pathological diagnosis of osteosarcoma is difficult in daily work.More datas about clinicopathological and prognostic factors should be summarized and analyzed.Osteosarcoma has high mortality rate.It easily metastases to the lungs.Although limb salvage and neoadjuvant chemotherapy have improved the prognosis of osteosarcoma,the survival rate has not been increased significantly in the past 30 years.It is urgent to explore new early diagnostic markers and therapeutic methods.Effective treatments for osteosarcoma depend on our understanding of mechanism of invasion and metastasis,which is still a hot and difficult issue in current studies.Notch signaling pathway is not only an important inter-cellular pathway,playing a significant role in cell proliferation,differentiation and apoptosis,but also a necessary developmental signaling pathway.Its abnormal expression can lead to abnormal tissue development and even tumorigenesis.Notch signal involves in the occurrence and development of osteosarcoma as a key factor.Notch pathway is expected to become a breakthrough in the treatment of osteosarcoma.So,investigating the effect of Notch pathway on the biological characteristics and molecular mechanism of osteosarcoma will not only help elucidate the molecular mechanism of invasion and metastasis,but also provide new targets and strategies for the diagnosis and personalized treatment.The role and mechanism of Notch 3 expression on osteosarcoma cells have not been reported.Our study is divided into the following two parts:1.Seventy cases of osteosarcoma were retrospectively studied.Histopathological observation and immunohistochemical staining with Notch1,Notch2,Notch3,Notch4,Jagged1,Jagged2,DLL4,CD133,Nanog,and SOX2 were performed in these cases.Prognosis of fifty-six cases was analyzed statistically.We also compared the Notch3 positive with negative group.The clinical and pathological characteristics of lung metastases cases were also analyzed.The aim of this study was to clarify the clinicopathological characteristics and prognostic factors of osteosarcoma,and the effect of Notch3 on the metastasis of osteosarcoma.2.After Notch3 knockdown,CCK8 assay and flow cytometry were used to detecte cell proliferation and cell cycle.Scratch assay and Boyden chamber invasion assay were used to detect migration and invasion ability.qRT-PCR and Western blot were applied to detect the expression of downstream effector Hes1 and MMP.Furtherly,we investigated the Notch3's effect on metastasis in nude mice.The main results and conclusions are as follows:Difference Expression and Prognostic Significance of Notch in 70 Cases of Osteosarcoma1.Clinical manifestations: The mean age was 25.1 years.The male to female ratio was 3:2 and 95.7% of the patient in stage?~?.2.Imaging findings: Bone destruction was shown in 87.1% of cases.Involvement of the medullary cavity and bone cortex were shown in 11.4% and 41.4% of cases respectively.Periosteal reaction was shown in 40% of cases.3.Histopathology: Ordinary osteosarcoma accounts for 88.6% of osteosarcoma.57.1% of cases showed invasion,and all 70 cases showed sarcoma cells and neoplastic osteoid tissue.4.Immunophenotype: the expression rates of Notch1,Notch2,Notch3 and Notch4 were 87.1(61/70),33.9%(23/70),68.6%(48/70)and 91.4%(64/70)respectively.The expression rates of Jagged1,Jagged2 and DLL4 were 87.1%(61/70)84.3%(59/70)and 88.6%(62/70)respectively.The expression rates of stem cell markers CD133,Nanog and SOX2 were 97.1%(68/70),10.0%(7/70)and 4.3%(3/70),respectively.5.Treatment and survival analysis: 60% of patients received chemotherapy,and 95.7% of patients underwent surgery.The mean survival time of 54 patients was 41.7 months.The 1-year,2-year and 5-year survival rates were 87%,66% and 30%,respectively.Enneking stage,preoperative chemotherapy and Notch3 expression were correlated with the prognosis and were independent prognostic factors of the tumor.Compared with the negative group,the Notch3 positive group had a higher metastasis rate.In summary,Osteosarcoma is more common in adolescents and males,with pain as the main symptom.Bone destruction is the most common imaging sign.Invasive growth,sarcoma cells and tumorous bone tissue are three important histological features.Notch3 expression is 68.6%.The 5-year survival rate of osteosarcoma is low.Enneking staging,preoperative chemotherapy and Notch3 expression are prognostic factors of osteosarcoma.Notch3 positive patients are prone to metastasis.Notch3 Knockdown inhibits the invasion and metastasis of osteosarcoma by down-regulating the expression of Hes1 and MMP71.Notch3 knockdown decreased the proliferation rate of U2 OS and MTH cells and arrested the cell cycle in G1 stage.2.Notch3 knockdown inhibited the migration and invasion of U2 OS and MTH cells.3.Notch3 knockdown decreased the expression of Hes1 and MMP7 in U2 OS and MTH cells in both mRNA and protein level.4.Hes1 knockdown decreased the expression of MMP7 in U2 OS and MTH cells.5.Notch3 knockdown decreased the lung metastasis rate and the number of metastases in nude mice in MTH group,and the expressions of Notch3,Hes1 and MMP 7 in shNotch3 group also decreased.In summary,Notch3 promotes the invasion and metastasis of osteosarcoma cells.Notch3 could regulates the invasion and metastasis of osteosarcoma by targeting the downstream target gene Hes1 and effector MMP7.