| Bone tumors make up about3–5%of childhood cancers and less than1%of cancers inadults. In the The first three incidence of primary malignant bone tumors are osteosarcoma35.1%, chondrosarcoma25.8%, Ewing's sarcoma16.0%[1-4]. The etiology and pathogenesisof malignant bone tumors are yet need to be further study. Despite modern treatmentprotocols that combine chemotherapy, surgery, and sometimes radiotherapy, the survivalrate for patients diagnosed with malignant bone tumors is no significantly enhanced.Therefore, scholars hope more thorough understand the pathogenesis of malignant bonetumors, and provide new target for tumor therapy.Activin A belong to the transforming growth factor beta (TGF-beta) superfamily, is amultifunctional growth and differentiation factor,play an important role in embryogenesis,cell proliferation and differentiation, and apoptosis, involved in cell cycle arrest and inductionof apoptosis. Activin A has a variety of biological activity, produced different effect in thedifferent target cell, related to the tumor occurrence and the degree of malignancy andmetastasis. Activin A can significantly increase the expression of Smad2in ovarian cancer,and further studies showed that Activin A promote the proliferation of human ovarian clearcell adenocarcinoma through autocrine role. Another study suggests that activin A promotecell proliferation and ovarian carcinogenesis through the activation of Akt and suppression ofglycogen synthase kinase (GSK). Recent studies found that activin A suppress the activity oftelomerase through down-regulated the expression of telomerase reverse transcriptase thusplay the role of suppression the tumor cell proliferation in breast cancer. Activin A inhibitedthe proliferation of hepatoma cells and induced apoptosis in liver tumors. while there is stilllack of relevant reports about the role of activin A and its specific signal transductionpathways to the bone tumors and its specific pathogenesis.Follistatin (FS), also known as the activin binding protein, is a single-chainglycoprotein,and high affinity for the activin A. Activin A and FS are usually co-expression, play an important role in the process of cell proliferation, differentiation, apoptosis andtumor formation. Anyone of the two cytokines decreased expression or overexpressionwould caused the imbalance of activin-FS system, leading to the occurrence of the disease.Activin A expression imbalance has been confirmed in the bone metastasis-prone malignanttumors such as breast cancer, lung cancer, prostate cancer, multiple myeloma. Although westill lack direct evidence of the relationship between activin A and bone primary malignanttumors, but a lot of research still prompted activin A may be related to the proliferation,migration and osteolysis of primary bone tumors. Clarify the relationship between activin Aand primary bone malignancies has very important significance for design new and moreeffective chemotherapy drugs and the establishment of a new gene therapy target.The purpose of this study is to reveal the role of the imbalance expression of activin A-FS system in primary bone malignancies, Concrete researches as follows:1the experimental route1.1Detect the mRNA levels of activin A and FS in different tissue-derived tumor celllines by the Real time PCRCultured human prostate cancer cells lines Pc-3M, DU145, human ovarian cancer cellslines Sk-ov-3, a human osteosarcoma cell lines in MG63, human lung cancer cellslinesNCI-H292, A549, human cervical carcinoma cell lines heLa, human bladder cancer celllines5637, human breastcancer cells lines MCF7, human hepatoma cell lines Hep G2,human pancreatic cancer cells lines BaxPc-1to logarithmic phase, received the cells,extracted total RNA. Detect the mRNA levels of activin A and FS by the Real time PCR.screened tumor cell lines of high expression of activin A and/or FS mRNA.1.2Verify whether osteosarcoma, lung cancer, ovarian cancer which high expressionof the FS mRNA expressed mature FS protein by immunohistochemistryThe Paraffin tissue sections were immunohistochemical staining throughconventional lost-wax and antigen repair, observed the staining results using the opticalmicroscope. verification whether the tumor tissue which high expressied FS mRNA,expressed the mature FS protein. 1.3Detect the mRNA levels of activin A and FS in osteosarcoma andchondrosarcoma by the Real time PCRIn order to analyze the relationship between tumorigenesis and the imbalanceexpression of activin A-FS system, we detected the mRNA expression levels of activin A,the FS in osteosarcoma and osteoma, chondrosarcoma and chondroma used the Real timePCR. All tumor tissue samples were taken from the tumor tissue resected in surgery.1.4Analyze the expression levels of activin A and FS in osteosarcoma,chondrosarcoma tissues by immunohistochemistry and Weastern blotAnalyzed the expression levels of activin A, FS and the cell distribution characteristicin osteosarcoma and osteoma, chondrosarcoma and chondroma tissues byimmunohistochemistry and Weastern blot.1.5Analyze the expression levels of ActR IIA in osteosarcoma, chondrosarcomatissues by immunohistochemistry and Weastern blotActivin A play its biological role through binding ActR IIA in the cell membrane, sowe detected ActR IIA levels in osteosarcoma and osteoma, chondrosarcoma and chondromatissues, to further determine the significance of activin A-FS system imbalance in theoccurrence of bone tumors.1.6Detect serum activin A and FS levelsVenous blood samples were collected from all patients in the morning of the secondday after admission, stored at80°C until analysed. Detected serum activin A and FSlevels by ELISA kit purchase from R&D Company.2Results2.1Real time PCR results showed that: osteosarcoma cells line MG63, ovariancancer cell line SKOV3, lung cancer cell line A549have high expression of the FS andactivin A mRNA.2.2Immunohistochemistry staining results showed: the bone tissues, ovariancarcinoma tissues, lung cancer tissues can be seen FS protein positive staining.2.3Real time PCR results showed that: the mRNA levels of FS and Activin A in osteosarcoma and chondrosarcoma tissues were significantly higher than its in the osteomaand chondroma tissues, and increased activin A mRNA levels were significantly higher thanthe FS mRNA levels.2.4Further compared the positive staining of FS and Activin A in osteoblast sourceof osteosarcoma with benign bone disease tissues, the results showed that the activin A andActR II A expression levels in osteosarcoma cells was significantly higher than its in theosteoma cells, matrix was also found obviously brown positive staining; while FS stainingwas no significant difference in osteosarcoma and benign bone tissue.2.5Further compared the positive staining of FS and Activin A in chondrocytessources benign cartilage disease and malignant cartilage disease the results showed thatactivin A and ActR II A expression levels in cytoplasm of chondrosarcoma cells wassignificantly higher than its in the chondroma cells; while the FS levels were nosignificantly different.2.6The Weastern blot results showed that the activin A levels were significantlyhigher in osteosarcoma tissues than the osteoma tissues, and the FS levels were nosignificant difference; the activin A levels were significantly higher in chondrosarcomatissues than the chondroma tissues, and the FS levels were no significant difference.2.7Collected peripheral blood from healthy people and patients with osteosarcoma,the results showed that peripheral blood activin A levels in patients with osteosarcoma weresignificantly higher than its in the healthy control group, FS levels were no significantdifference compared with the healthy control group.3Conclusions3.1Activin A and the FS mRNA widely present in tumor cells such as bone tumors,urinary and reproductive system tumors, liver cancer, breast cancer, pancreatic cancer, andlung cancer, High expression in osteosarcoma cells, ovarian cancer, lung cancer.3.2The results of this study reveal that the activin A-FS system imbalance may berelated to the occurrence of malignant osteogenic tumor and malignant cartilaginous tumor,Activin A is an important pathogenic factor for these diseases, Activin A may become the new target of gene therapy for malignant osteogenic tumor and malignant cartilaginoustumor.3.3Serum activin A detection may be have a certain significance for early diagnosisof osteosarcoma. |
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