| Osteosarcoma,also known as osteogenesis sarcoma,is the most common primary malignant bone tumor that mainly occurs in children and adolescents.OS is usually located in the metaphysis of long bones,especially near the knee.The incidence rate is approximately four peopleper million each year.Combined surgical resection and intensive chemotherapy has improved the 5-year overall survival rate.However,the overall treatment effects and long-term free survival rate are remain unsatisfactory(50%or less).Early distant metastasis,especially the lung metastases,is one of the most important causes of death in osteosarcoma.Therefore,further study of the related genes in the occurrence and development of osteosarcoma is significant for the occurrence,evolution,prognosis and treatment of osteosarcoma.It also provides a new way and approach for the prevention,early diagnosis and treatment of osteosarcoma.Kitada and others first reported the pathogenic gene of autosomal recessivejuvenile parkinsonism(AR-JP)in 1998,and named it Parkin.The Parkin gene,also known as the PARK2 gene,has a wide range of human and rodent tissues.The abnormal expression of PARK2 is related to the occurrence and development of a variety of diseases.Previous reports have shown that mutation or deletions of PARK2 occur in 30%of human malignant tumors,including glioma,breast,liver,lung,pancreatic,and colorectal cancers,which suggested that PARK2 is an important tumor suppressor gene.The cell cycle,proliferation,invasion and migration of tumor cells,as well as cell apoptosis were regulated by PARK2 via many of signaling pathway.Now,there are no reports about the expression of PARK2 in OS,and the biological behavior and possible mechanism of PARK2 in OS remains unclear.Thus,in this study,we investigated the differential expression of PARK2 gene in osteosarcoma tissues and cell lines.Then,the OS cell lines were employed to establish a PARK2 overexpression model.The effect of PARK2 on the proliferation,cell cycle,migration and,invasion and other biological behaviors were evaluated,and its possible mechanism was also investigated.In addition,the xenograft model was used to confirm the effects of PARK2 on the growth of osteosarcoma in vivo.In order to provid more data for the diagnosis and treatment of OS.This study has been divided into three parts:(1)The differential expression of PARK2 in human OS tissue samples and cell lines;(2)The effects of PARK2 on the biological behavior of OS cells in vitro and in vivo;(3)To investigate the possible biological mechanism of PARK2 affecting the biological behavior of OS cells.Chapter ? The differential expression of PARK2 in human OS tissue samples and cell linesObjective:To evaluate the differential expression of PARK2 protein in OS and adjacent tissues,as well as in OS cell lines.Methods:Collection and screening of human OS tissues and its adjacent tissues and performed immunohistochemical staining with Parkin protein,observing the expression of Parkin protein in human osteosarcoma and its adjacent normal tissues.Assess each sample in the same method,analysis the differential expression of PARK2 in osteosarcoma and adjacent tissues.Whether the expression of PARK2 is relate to the patient's age,location and clinical stage.To investigated the level of PARK2 mRNA in SAOS2,HOS,U20S and hFOB1.19 cell lines by RT-qPCR method.The level of expression was statistically analyzed.According to the research results,the corresponding cell lines were selected for further study.Results:By immunohistochemical staining and scoring,the results showed that 76%(35/46)of the adjacent non-tumor tissues and 37%(17/46)of the OS tissues expressed the PARK2 protein(P<0.05),Moreover,the expression of PARK2 was significantly associated with higher tumor stage(P<0.05).Compare with hFOB1.19 cell,the level of PARK2 mRNA decreased significantlyin the OS cell lines.Conclusion:PARK2 gene is low expression or deletion in osteosarcoma,suggesting that the low expression of PARK2 gene may be closely related to the occurrence and development of osteosarcoma.Chapter ? The effects of PARK2 on the biological behavior of OS cells invitro and in vivoObjective:The objective of this study is to investigate the effects of PARK2 on the proliferation,cell cycle,migration,invasion and apoptosis inducing ability of OS cells.Methods:HOS and U20S cell lines were selected for PARK2 overexpression models.Transfection rates of PARK2 gene overexpression group(HOS-PARK2 and U20S-PARK2)and negative control group(HOS-NC and U20S-NC)were confirmed by western blot and immunofluorescence assay.The stably transfected cells were used to investigate biological functions of PARK2 in OS.1).The effect of PARK2 on OS cell viability was detected by CCK8,colony formation and ki67 fluorescence staining assay.The effect of PARK2 on the cell cycle was analyzed by flow cytometry assay.2).The effect of PARK2 on OS cell migration and invasion was studied by scratch test and Transwell chamber assay.3).The effect of PARK2 inducing OS cell apoptosis was detected by Hoechst nuclear fluorescence staining and the flow cytometry with annexin v-apc/7-aad double staining method.The apoptosis-related protein was evaluated by Western blot.4).20 BALB/cnu/nu nude mice were employed for in vivo experiments,the stable transfected HOS-PARK2 and HOS-NC cells were injected into the marrow cavity of the right tibiato establish a tumor formation model(n=10),and the caudal vein injection for metastases model(n = 10),for further confirm the PARK2 biology behavior on OS in vivo.Results:After HOS and U20S cell lines were stable transfected PARK2 gene and empty vector,the Parkin protein was detected by Western blot and immunofluorescence.The results demonstrate that the level of Parkin protein was significantly increased in PARK2 group relative to that in the NC group.The biological functions of the two groups were analyzed as follows:1).The growth curve of HOS and U20S cell lines analysis showed that compared with NC group,cell proliferation was significantly inhibited in PARK2 group(P<0.05).In the clone formation assay,50 cells and more than 50 cells of each clones was been analysis.The result demonstrated that the clone number and size were significantly reduced in the PARK2 group(P<0.05).Ki67 immunofluorescence staining showed that PARK2 group cell fluorescence intensity was weaker than that in NC group,and the positive cells was significantly decreased(P<0.05).In the HOS cell line,less cells were entry into the S and the G2 phase in the PARK2 group detected by flow cytometry(P<0.05).2).Compared with NC group cells,the migration and invasion ability was significantly reduced in the PARK2 group cells(P<0.05).3).The nuclear staining of Hoechst showed that the ratio of abnormal nuclear in the PARK2 group was significantly higher than that in the control group(P<0.05).Flow cytometry analysis showed that the apoptotic rate in PARK2 group was significantly higher than that in NC group(P<0.05).Compared with the NC group,the expression of protein Bcl-2 was weakened,and the protein caspase 3 and cleaved-caspase-3 was significantly up-regulated in PARK2 group.4).The result of the tibial xenograft showed that the volume of the tumor in the PARK2 group was smaller than that in the NC group at the end of 3 weeks(P<0.05).Hematoxylin and eosin(H&E)staining revealed that the PARK2 group attained fewer hemocytes and vascular structures in the Codman-triangle area than those in the NC group.In the metastasis model showed that lung metastases in the PARK2 group was significantly less than the NC group(P<0.05),and the tumoe size was also smaller than the NC group.Conclusion:PARK2 gene blocking the cell cycle,weaken the proliferation,inhibits the migration and invasion,and induces apoptosis of OS cells,which delays the growth of OS and reduces the occurrence of metastasis in vivo.It is possible that these down-regulation biological behaviors were achieved through inhibition of angiogenesis.Chapter ? To investigate the possible biological mechanism of PARK2 affecting the biological behavior of OS cells.Objective:Exploring whether PARK2 could down-regulated the vascular signaling pathway JAK2/STAT3/VEGF.Methods:The stable transfected PARK2 and NC cells were employed to detect the level of JAK2/STAT3/VEGF pathway-associated proteins by Western blot immunofluorescence assays.Moreover,interleukin 6(IL-6:25ng/ml,30 min)and stattic(10 ?M,2 h),the p-STAT3 agonist and an inhibitor,respectively,were used to activate or inhibit STAT3 phosphorylation.Then the ability of tube formation and the changs of relate protein were analysis.5 BALB/cnu/nu nude mice were used for in vivo angiogenesis model.The same number of HOS-NC cells(on the left side)and HOS-PARK2 cells(on the right side)were planted of nude mice at inguinal region,to further confirme the effect of PARK2 gene on neovascularization of OS.Results:Western blot analysis showed that the level of p-JAK2,p-STAT3 and VEGF protein in PARK2 group was significantly decreased compared with the NC group.Immunofluorescence analysis showed that the intensity of phosphorylation of STAT3 and VEGF protein of PARK2 group cells was weaker than that of the NC group,and the expression of p-STAT3 in the nucleus was significantly reduced.The tubule formation analysis showed that the cells in the PARK2 group were decreased compared to the NC group.The tubule formation ability was activated or inhibited by IL-6 and stattic,at the same time it is regulate the expression of p-JAK2,p-STAT3,and VEGF proteins too.In vivo neovascularization assays showed that there are inguinal blood vessels and osteosarcoma cells-Matrigel communication,and visible blood vessels distribution in the osteosarcoma cells-Matrigel colloidal.Compared with the NC group,less vascular distribution and reduced hemoglobin was found in the Park2 group in the body of Matrigel.Immunofluorescence analysis showed that the CD34 positive expression rate in the PARK2 group was significantly less than that in the NC group in a single field of view.Conclusion:PARK2 gene inhibits OS angiogenesis,cell proliferation,migration and invasion and more biological functions through JAK2/STAT3/VEGF signaling pathway. |
PDF Full Text Request