Studies On The Role And Related Mechanisms Of MircoRNA In Osteosarcoma

Osteosarcoma is the most common malignant bone tumor, and accounting for17%ofprimary bone tumors,42%of primary malignant bone tumors. It characterized by highincidence of adolescence, early lung targeted metastasis and poor prognosis. The currenttreatment of osteosarcoma is still based on surgery and chemotherapy. With thedevelopment of therapeutic method, the survival rate of patients suffer from osteosarcomahad been improved, However, a lot of characteristics deteriorate the prognosis ofosteosarcoma,such as local tumor recurrence postoperation, early lung targeted metastasis,resistance of chemotherapeutics. In recent years, although a lot of research in themechanism of osteosarcoma development and metastasis had been done, themolecular mechanisms are still elusive, especially in certification and fuctional research ofmolecule which are specifically expressed in osteosarcoma. Thus, a fundamental quest inthe mechanism of osteosarcoma development and metastasis is imperative. Looking for thenovel molecular markers in osteosarcoma.about early diagnosis, prognosticevaluation of and biotherapeutics, may has significant effect in improving the currentsituation of diagnosis and treatment. As we know, early diagnosis in post-operativemetastasis of osteosarcoma relies in the screening of specifical molecular markers and theestablishment of an effective, convenient, non-invasive diagnostic system. The treatment ofmetastatic osteosarcoma patients mainly depends on screening of novel molecular markersrelated with metastasis. Recently, both aspects described above are lacking of favourableclinical value, but researching on mircoRNA genomics maybe can offer a promisingresearch strategy in these two areas.In this study, we use osteosarcoma cell lines and clinical samples as models to explore thesignificance of abnormally espressed mircoRNA in the development and metastasis ofosteosarcoma. The whole study was divided into three parts. Part1, Using genechiptechnique to screen the abnormally expressed mircoRNA in osteosarcoma clinical samples.Part2, Studies on the role and related mechanisms of mircoRNA-143in osteosarcoma. Part3, Studies on the role and related mechanisms of mircoRNA-148a in osteosarcoma.Part1Using miRNA genechip technique to screenthe deregulated mircoRNA in osteosarcoma clinical samplesObjective: Using genechip technique to screen the abnormally expressed mircoRNA in osteosarcoma clinical samples, and using statistical softwares to analysis the experimentaldata. Methods: Using osteosarcoma clinical samples as objects and adjacent tissues ascontrol groups, and using miRNA2.0genechip which purchased from Affymetrix company,to detect human species miRNA expression profiling. In order to obtain the differentialmiRNAs expression between osteosarcoma samples and adjacent tissues, we apply theselecting standard as ratio≥2.0or≤0.5. Results: The miRNA2.0genechip contains1205human spices related miRNAs and1105pre-miRNAs. Between osteosarcoma samples andadjacent tissues there have272up-regulated miRNAs and96down-regulated miRNAs insample ca-1,406up-regulated miRNAs and218down-regulated miRNAs in sample ca-2,and108up-regulated miRNAs and40down-regulated miRNAs in both samples.Conclusion：Differentially expressed miRNAs may play an important regulatory rolein the development and metastasis of osteosarcoma.Part2Studies on the role and related echanisms ofmicroRNA-143in osteosarcomaObjective: Studies on the role and related echanisms of miR-143in the development andmetastasis process of osteosarcoma which was shown down-regulated from miRNA2.0genechip. Methods: In this study, we use osteosarcoma cell lines MG-63, U2OS,osteoblasts hFOB1.19and clinical samples as objects. Using cell culture, RNA extractionand qRT-PCR, transfection, analysis of cell viability in vitro, detection of apoptosis,tumorigenicity assay in nude mice,3’UTR luciferase reporter assay, western blot analysisand statistical analysis as experimental methods to explore the value in the cellproliferation, apoptosis and tumorigenicity of osteosarcoma. For statistical analysis, wechoose Student’s t test to compare the difference among experimental groups and controlgrops, and P<0.01was taken to indicate significance. Results:1. miR-143isdown-regulated in osteosarcoma cell lines and primary tumor samples.2. miR-143reducescell viability, promotes cell apoptosis and suppresses tumorigenicity.3. AntiapoptoticBcl-2is a direct target of miR-143.4.Proapoptotic function of miR-143is mainly throughthe targeting of Bcl-2. Conclusion：Human important antiapoptotic Bcl-2is identified to bea novel direct target of miR-143in osteosarcoma, and miR-143may exert its proapoptoticfunction via inhibiting Bcl-2expression. Part3Studies on the role and related echanisms ofmicroRNA-148a in osteosarcomaObjective: Studies on the role and related echanisms of miR-148a in the development andmetastasis process of osteosarcoma which was shown up-regulated from miRNA2.0genechip. Methods: In this study, we use osteosarcoma cell lines MG-63, U2OS,osteoblasts hFOB1.19and92clinical samples as objects. Using cell culture, RNAextraction and qRT-PCR, transfection, analysis of cell viability in vitro, detection ofapoptosis, tumorigenicity assay in nude mice,3’UTR luciferase reporter assay, western blotanalysis and statistical analysis as experimental methods to explore the value in the cellproliferation, apoptosis and tumorigenicity of osteosarcoma. For statistical analysis, wechoose Student’s t test to compare the difference among experimental groups and controlgrops, and P<0.01was taken to indicate significance. Results:1.Increased miR-148aexpression in osteosarcoma.2. miR-148a increase is significantly correlated with theprogression and prognosis of osteosarcoma.3. miR-148a promotes osteosarcoma growthboth in vitro and in vivo.4. miR-148a promotes PI3K pathway activation by targetingPTEN expression. Conclusion：Increasing of miR-148a expression may inhibit PTENexpression, which in turn promotes PI3K pathway activation in osteosarcoma. miR-148aplays important role in osteosarcoma progression and prognosis, and determination ofmiR-148a expression may be used in pathology identification and prognosis prediction inosteosarcoma.