| Osteosarcoma,which has high aggressiveness,is a common primary malignant bone tumor in children and adolescents.In the past 30 years,although the 5-year survival rate for patients with non-metastatic osteosarcoma has increased to 60%-70%with a combination of surgery and multiple adjuvant therapies,the clinical prognosis remains poor for patients with local metastasis and recurrence.Therefore,to reduce the recurrence rate and improve the survival rate,it is still an urgent need to find new therapeutic targets and treatment methods for osteosarcoma patients.In recent years,more and more studies have found that long non-coding RNA that with almost no protein-coding capacity plays an important role in various cellular regulatory processes,including gene expression,chromatin remodeling,post-transcriptional processing and transcription.Long non-coding RNA has also been found to has closely relationship with the the occurrence and development of a variety of human cancers,and plays an important role in carcinogenesis or cancer inhibition.Currently,it has been found that a variety of long non-coding RNA,such as RP5-919F19,RP11-739N20,CRNDE,PCBP1-AS1 and TP73-AS1 are closely related to the occurrence and development of a variety of cancers.Among them,TP73-AS1 has been proved to be abnormally expressed in a variety of tumor tissues and cells,such as breast cancer,hepatocellular carcinoma,brain glioma and esophageal squamous,etc.The abnormal expression of TP73-AS1 can generally promotes the proliferation,invasion and metastasis of tumor cells,reduces its sensitivity to chemotherapy drugs,and inhibits cell apoptosis.Although numerous studies have shown that TP73-AS1 is involved in the progression and prognosis of human cancers,the role and underlying mechanisms of TP73-AS1 in human osteosarcoma remain unclear.In this study,the expression,functional role and underlying mechanisms of TP73-AS1 in osteosarcoma were investigated.It was found that TP73-AS1 was over expressed osteosarcoma tissues and cells,and it promoted the proliferation,migration and invasion of osteosarcoma cells.At the same time,the underlying molecular mechanism of TP73-AS1 in the progression of osteosarcoma was explored,and it was found that TP73-AS1 may act as a sponge for miR-142 in osteosarcoma.TP73-AS1 positively regulates Racl expression by inhibiting miR-142.This study provides new insights into the molecular function of the TP73-AS1/miR-142/Racl signaling pathway in osteosarcoma,which may provide a new target for the treatment of osteosarcoma.Experimental aim:1.To understand the expression of long non-coding RNA TP73-AS1 in osteosarcoma tissues and cell lines,as well as the relationship between the TP73-AS1 expression and the overall survival rate of osteosarcoma patients.2.TP73-AS1 gene knockdown inhibited the proliferation and invasion of osteosarcoma cell,and tumor growth in vitro.3.miR-142 can be used as a direct target of TP73-AS 1,and the inhibition of miR-142 reverses the inhibitory effect of TP73-AS1 gene knockdown on the proliferation and invasion of osteosarcoma cells.4.To investigate the role of long non-coding RNA TP73-AS1 as a sponge for miR-142 in the positive regulation of Racl in osteosarcoma cells and the role of the TP73-AS1/miR-142/Rac1 pathway in osteosarcoma.Main Contents:The first charpter is the expression and role of long non-coding RNA TP73-AS1 in clinical osteosarcoma samples and different osteosarcoma cell linesMethods:1.qRT-PCR was used to detect the expression of osteosarcoma cells in tissues.2.CCK-8 assay and evaluation of cell proliferation;Transwell migration and invasion analysis Cell invasion assay was performed in the suspended cell culture compartment.Results:1.The expression of TP73-AS1 in patients with osteosarcoma was up-regulated.2.The high expression of TP73-AS1 in osteosarcoma patients was generally associated with the stage of the tumor and distant metastasis of tumor of the patients.3.The expression of TP73-AS1 in osteosarcoma cell lines was up-regulated.4.In vitro experiments verified that TP73-AS1 knockdown could reduce the proliferation and invasion of osteosarcoma cells.Part Ⅱ.The underlying mechanism of TP73-AS1 in osteosarcoma was investigatedMethods:1.Tumor model of in vivo transplantation was built.2.The correlation of the TP73-AS1/miR-142/Racl signaling pathway in osteosarcoma was determined by immunoblotting luciferase gene,qRT-PCR detection,CCK-8 and Transwell migration and invasion analysis.Results:1.TP73-AS1 interacts directly with miR-142 and inhibits its expression.2.Inhibition of miR-142 can reverse the effect of TP73-AS1 knockdown on osteosarcoma cells.3.TP73-AS1 positively regulates the expression of Racl protein by inhibiting miR-142.4.Animal experiments verified that knockdown of TP73-AS1 could inhibit the growth of osteosarcoma.The full text summary1.It was found that the expression of long non-coding RNA TP73-AS1 was up-regulated in osteosarcoma patients.2.The relative expression level of long non-coding RNA TP73-AS1 in osteosarcoma tissues has not significantly correlated with the age,sex,or tumor size of osteosarcoma patients,but was generally associated with the stage of the tumor and distant metastasis of tumor of the patients.3.Knockdown of TP73-AS1 significantly upregulated the expression of miR-142 in osteosarcoma cells of U2OS and MG-63.4.TP73-AS1 can directly interact with miR-142 and inhibit its expression.The expression of miR-142 in osteosarcoma tissues and cells was lower than that in normal tissues and cells,and there was a negative correlation between the expression of TP73-AS1 and miR-142.Tp73-as1 knockdown reduced the proliferation of osteosarcoma cells,but inhibition of miR-142 significantly reversed the inhibition of TP73-AS1 knockdown on the proliferation,migration and invasion of osteosarcoma cells,suggesting that TP73-AS1 promoted the proliferation,migration and invasion of osteosarcoma cells by acting as miR-142 sponge.5.TP73-AS1 was found to positively regulate Racl expression by inhibiting MiR-142 in osteosarcoma.6.TP73-AS1 knockdown significantly inhibited the growth rate of tumor volume in mice.The expression of TP73-AS1 and Racl was down-regulated,but the expression of miR-142 was up-regulated,showing a negative correlation.The dissertation includes three chapters:In charpter 1,the long non-coding RNA TP73-AS1 in clinical osteosarcoma samples and different osteosarcoma cell lines were investigated.By investigating the osteosarcoma tissues of 46 patients using qRT-PCR,it was found that the TP73-AS 1 was remarkably up-regulated in osteosarcoma tissues.Then,the correlation between the expression of TP73-AS1 in tumor tissue and the clinicopathological characteristics of osteosarcoma patients was investigated.The results reveal that the high expression of TP73-AS1 in osteosarcoma patients was generally associated with the stage of the tumor and distant metastasis of tumor of the patients.In addition,in vitro cell experiments showed that TP73-AS1 was over-expressed in various different kinds of osteosarcoma cells.TP73-AS1 knockdown can significantly inhibit the proliferation,migration and invasion of osteosarcoma cells.These results indicated that the overexpression of TP73-AS1 may promot the occurrence and development of osteosarcoma.In charpter 2,the underlying mechanism of TP73-AS1 in osteosarcoma was investigated.By conducting online prediction algorithm and luciferase reporter assay,it was found that miR-142 contained putative binding sites with TP73-AS1.The expression of TP73-AS1 and miR-142 in osteosarcoma tissues and cells was negatively correlated,and TP73-AS1 knockdown significantly up-regulated the expression of miR-142 in U2OS and MG-63 cells.In addition,miR-142 inhibition reverses the effects of proliferation,migration,and invasion for TP73-AS1 knockdown in osteosarcoma.Using a series of in vitro cell experiments,the TP73-AS1 that as a sponge of miR-142,can positively regulates Racl protein expression in osteosarcoma cells was demonstrated.In vivo experiments showed that TP73-AS1 knockdown could significantly inhibit the tumor growth rate on mice.Moreover,tissue analysis results showed that the expressions of TP73-AS1 and Racl were down-regulated,while the expressions of miR-142 were up-regulated after TP73-AS1 knockdown.These results provide important references for the study of the TP73-AS1/miR-142/Racl signaling pathway in osteosarcoma and may provide a new targets and biomarkers for the diagnosis,treatment and prognosis of osteosarcoma.In charpter 3,an introductce about the osteosarcoma and its cruuently main diagnostic techniques and treatment methods is given,with focus on the main recently research on the role of long non-coding RNA in osteosarcoma and the effect and underlying mechanisms of long non-coding RNA TP73-AS1 in various different tumor. |
