| Osteosarcoma is one of the most common primary bone cancers in children and adolescents, characterized by high-invasion and early pulmonary metastasis. The pathogenesis of osteosarcoma involves multiple processes and genes, but the exact mechanism has not yet been clarified. Previous studies on osteosarcoma mainly focused on genetic events, and found that the amplification, rearrangement and point mutation of oncogenes and anti-oncogenes were closely related to the development of osteosarcoma. However, recent studies suggest that cancer appears to be a process that is fuelled not only by genetic alterations, but also by epigenetic mechanism. Epigenetic abnormalities were found in almost human tumors.Genomic imprinting, as an epigenetic mechanism, plays an important role in tumorigenesis. Genomic imprinting is a genetic phenomenon by which certain genes are expressed in a parent-of-origin-specific manner. It is an inheritance process independent of the classical Mendelian inheritance. Imprinted genes are either expressed only from the allele inherited from the mother, or in other instances from the allele inherited from the father. Many imprinted genes act as oncogenes or tumor suppressor genes, and just one hit may give rise to the gene dysfunction, increasing the susceptibility of cancer. Recent data showed that DNA methylation is the molecular basis of producing and controlling genomic imprinting. The specific inactivation of parental allele is associated with the methylation of CpG island in promoter region (imprinting control region). Aberrant DNA methylation disturbs the function of imprinted genes. On the one hand, genome-wide hypomethylation appears to induce activation of oncogenes and increase genomic instability; on the other hand, hypermethylation of promoter regions plays a key role in silencing the expression of tumor suppressor genes and promoting malignant transformation. Unlike mutation, DNA methylation is a reversible epigenetic modification, which provides a new strategy for therapy of cancer.The important roles of imprinted genes and DNA methylation in cancer have attracted intensive attention of scholars. It has been found that the genes imprinted in paternal allele is consistent with a potential inhibitory effect, while those imprinted in maternal allele tend to stimulate growth. Expression of imprinted genes is in a developmental stage- and tissue-specific manner, and the abnormality of imprinted genes and methylation patterns is different in various types of cancers. In order to clarify the specific imprinted gene involved in osteosarcoma, we developed a model of osteosarcoma transformation using a known potent carcinogen MNNG and a carcinogenic promoting agent TPA to treat hFOB1.19, an immortalized human osteoblastic cell line. We also examined the global changes in expression of imprinted genes during transformation by microarray analysis, and found that ten imprinted genes were aberrantly regulated in transformed cells, of which five were located on 11p15.5 chromosome. So we speculated that chromosome 11p15.5 is closely related with the development of osteosarcoma. To determine the role and regulation mechanism of these differential imprinted genes in osteosarcoma, imprinted genes IGF-2 and TSSC3 were chosen for further analysis.IGF-2, an important potent mitogen normally expressed by the paternal allele, is involved in embryonic development, cell differentiation and other physiological processes. It contains four promoters(P1-P4), used in a development stage- and tissue-specific manner. Dysregulated expression of IGF-2 occurs in many carcinomas and sarcomas. Overexpression of IGF-2 has been associated with up-regulation of P3- and P4-driven transcripts, and loss of imprinting and hypomethylation of DMR regions and promoter regions was also observed. It was found that IGF-2 overexpression in osteosarcoma was related with significant up-regulation of the fetal transcripts driven by P3 and P4 promoters. However, the preferential promoter in osteosarcoma, and the mechanisms responsible for the promoter activation are not well understood.TSSC3, also known as IPL, is the first apoptosis-related gene found to be imprinted. It located within 11p15.5, an important tumor suppressor gene region. At present, silence of TSSC3 was only reported in wilms'tumor, complete hydatidiform mole and malignant glioma, but there are few data about the expression and the regulation mechanisms of TSSC3 in osteosarcoma. Therefore, our study contained two parts. The aim of the first part was to investigate osteosarcoma-specific expression pattern of IGF-2 transcripts and regulatory mechanism, and the aim of the second part was to clarify the regulation mechanism and the role of TSSC3 in osteosarcoma, and provide theoretical basis for developing epigenetic therapy for osteosarcoma.The main results are as follows:1. By immunohistochemical assay: positive expression rate of IGF-2 protein was 83.33% (20/24) in osteosarcoma tissue. There was no relationship between the IGF-2 expression and patient sex, age, and histological subtypes (P>0.05).2. By RT-PCR assay: adult P1 promoter was inactivated, and the fetal P3, P4 promoter were reactivated and upregulated. P3 was the most active promoter in osteosarcoma and non-tumor tissues.3. By RFLP assay:the rate of loss of imprinting of IGF-2 was low in osteosarcoma and non-tumor tissue.4. By BSP assay: the average methylation rate of DMR was no significantly different between tissue with P3 expression and those without P3 expression(P>0.05), and there was no significantly statistical relationship between DMR methylation and IGF-2 P3 expression (P>0.05).5. By BSP assay: the average methylation rate of P3 promoter was significantly different between tissue with P3 expression and those without P3 expression(P<0.05), and there was significantly statistical relationship between P3 promoter methylation and IGF-2 P3 expression (P<0.05).6. By RT-PCR assay: Loss expression of TSSC3 was detected in SaOS2 cell lines. Expression of TSSC3 in osteosarcoma tissues, with a loss rate of 54.2%, was lower than that in non-tumor tissues (P<0.05). There was a markedly significant relationship between downregulation of TSSC3 and upregulation of IGF-2(P≤0.01).7. By BSP assay: the average methylation rate of TSSC3 promoter was high in SaOS2 cells, while the methylation rate was decreased after treatment with 5-Aza-CdR (P<0.05).8. By RT-PCR, Western Blot, MTT assay, F-actin staining and flow cytometry assay: after treatment with 5-Aza-CdR, TSSC3 was re-expressed, with inhibition of cell growth and induction of cell apoptosis(P<0.05). 9. The expression vector pEGFP-C3-TSSC3 was successfully constructed, and was transfected into SaOS2 cells with liposomes. By RT-PCR and Western Blot assays: TSSC3 was recombinantly expressed in SaOS2 cells.10. By MTT, flow cytometry and Real-time PCR assays: compared with control group and blank group, inhibition of cell growth, induction of apoptosis and upregulated expression of caspase-3 was obvious in the group with pEGFP-C3-TSSC3 transfection.The main conclusions are as follows:1. The expression of imprinted gene IGF-2 was a frequent event in osteosarcoma.2. Reactivation and expression of IGF-2 P3 promoter may be an early event in osteosarcoma, which leads to upregulation of IGF-2, and participated in the development of osteosarcoma.3. IGF-2 P3 hypomethylation may contribute to the expression of P3 transcript, and may play an important role in the development of osteosarcoma, which might be a useful biomarker for early diagnosis and prognostic evaluation with osteosarcoma.4. Loss expression of TSSC3 may play an important role in osteosarcoma, and the antagonistic effect with IGF-2 may participate in the development of osteosarcoma.5. Promoter hypermethylation may be an important epigenetic mechanisms of silencing TSSC3 expression. The abnormal DNA methylation is reversible, which provide theoretical basis for developing epigenetic treatments for osteosarcoma.6. 5-Aza-CdR could reactivate the expression of TSSC3 and play an anti-tumor role in SaOS2 cells, which provide a new way for the treatment of osteosarcoma.7. TSSC3 may be a putative tumor suppressor gene in SaOS2 cells, and loss expression of TSSC3 may be responsible for the tumor cell growth and apoptotic resistance, which contribute to the tumorigenesis. TSSC3 may become a new target for treatment.In summary, our study explored the pathogenesis of osteosarcoma in epigenetic level, and the results of above study will provide a new molecular target for the early diagnosis and treatment of osteosarcoma. |
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