| Osteosarcoma(OS)is a malignant tumor originating from mesenchymal tissue,which is mostly seen in the adolescent population,resulting in poor prognosis due to its invasive growth and early pulmonary metastasis.Previous treatment measures including limb amputation,postoperative irradiation and chemotherapy,but the 5-year survival rate is only 36%.In recent years,the use of neoadjuvant chemotherapy combined with limb salvage surgery has ameliorated the prognosis of patients significantly,the patient's quality of life is also improved,However,in case of drug-resistant and those who do not afford the side-effects of chemotherapy,the prognosis is still not optimistic.To this end,the research of the characteristics and genetic specificity of osteosarcoma gave the birth of some targeted drugs and disclose related gene targets,some drugs such as Endostar have been put into clinical use,and plays as an important role in tumor treatment,these findings push the treatment of osteosarcoma into a new phase of precision treatment.By combining these new targeted drugs and gene loci,irradiation which was thought to be less sensitive to osteosarcoma,has become a new therapeutic tool.Some studies confirmed that multiple genes were associated with irradiation sensitivity of osteosarcoma.In the study of C Blattmann,after the inhibition of histone decarboxylase inhibitors(Histone deacetylase inhibitors,HDACIs)two osteosarcoma cell lines(Khos-24os,SAOS2)and two kinds of striated muscle sarcoma cell lines(A-204,RD)showed significant sensitivity to irradiation,compared with the control group,there were also some significant differences in the biological characteristics of cellular proliferation and apoptosis in the experimental group.N Chen confirmed that through knocking out the autophagy protein-5 to induce the silence of NRF2 could make the disappearance of irradiation resistance of the osteosarcoma cell line U20S,and the expression level of related gene and the protein such as P65,Bcl-2 decreased,while the p53 and the Bax elevated,and they finally confirmed that its corresponding cell pathway is ERK 1/2 pathways.FB Mantovani etc,also gave research of epidermal growth factor receptor(epidermal growth factor receptor,EGFR)and confirmed its role in the enhancement of osteosarcoma cells' sensitivity to irradiation,and confirmed the pkb/akt signaling pathway both in-vivo and in-vitro.In this background,we studied the relationship between ubiquitin-conjugating enzyme 2T(ubiquitin-conjugating enzyme T,UBE2T)gene and irradiation sensitivity of osteosarcoma.The UBE2T gene belongs as a member of the ubiquitin-binding enzyme family.Ubiquitin proteasome pathway is the main pathway of protein degradation,which is also an important step in the invasion and metastasis of malignant tumors,in which ubiquitin is identified and decomposed by ubiquitin-activating enzyme(El),ubiquitin-conjugating enzyme(E2)and ubiquitin ligas(E3)to the substrate protein,which then make the protein decomposing to polypeptide.Previous studies have confirmed that ubiquitin-conjugating enzyme plays an important role in the sensitization of tumor irradiation.A study of irradiation therapy for cervical cancer confirmed that by silencing the ubiquitin-conjugating enzyme C(UBE2C),the Siha and Hela's sensitivity to irradiation was enhanced,and the siha cells which was significantly resistant to irradiation became highly sensitive to irradiation.Comparing with the control group,the proliferation and invasiveness of the cells also decreased significantly.Another study of breast cancer confirmed that the expression of UBE2C gene in breast cancer tissue was significantly higher than that of para-cancer tissue,silencing ube2c increased the sensitivity of breast cancer cells HBL100 and hek293t to irradiation,tamoxifen,and Trazodone.Another study of breast cancer has concluded that silencing ube2c could promote the sensitization of breast cancer to irradiation.UBE2T is an important member of the family of ubiquitin-conjugating enzymes,it plays an important role in epithelial mesenchymal transformation(EMT)and mesenchymal epithelial transformation(MET),and it was also found to be an important gene target in the growth,invasion and metastasis of various tumors,such as lung cancer,breast cancer,gastric cancer,etc.Recent studies has confirmed it also played as an important role in the growth and invasion of osteosarcoma.Combining with recent findings,we believe that UBE2T may also play an important role in the irradiation sensitization of some tumors,so we chose UBE2T as the target to study its enhancement of radio-sensitization in osteosarcoma cells and clarify the function and mechanism of this combination therapy on pulmonary metastasis.Through the combination of clinical and basic methods,we found the expression of UBE2T in patients with pulmonary metastasis was higher than those who don not have pulmonary metastasis.Through in-vitro experimental study,we found the growth,cloning and migration of tumor cells was inhibited by SiRNA-UBE2T when combined with irradiation,so were the tumor growth and pulmonary metastasis in mice.Through these research,we found the enhancement of SiRNA-UBE2T on the radio-sensitivity of osteosarcoma cells,which may provide a new potential target for the diagnosis and treatment of metastatic osteosarcoma.Part?the research of UBE2T expression in the human osteosarcoma specimens and cell linesObjective:To study the expression of UBE2T in human osteosarcoma specimens including the tumor tissue and para-tumor tissue,and to compare the expression of UBE2T in in-situ tumor of the osteosarcoma patients with the pulmonary metastasis and non-pulmonary metastasis.At the same time,UBE2T expression was studied in four osteosarcoma cell lines to determine its expression abundance in osteosarcoma cell lines.Methods:1.Immunohistochemical method was used to study the specimens of clinically collected osteosarcoma patients,analyze the expression of UBE2T in tumor tissue and para-tumor tissue,and compare the expression of UBE2T in tumor of the osteosarcoma patients with the pulmonary metastasis and non-pulmonary tmetastasis.To investigate the expression of UBE2T in tumor tissues whether has the association with osteosarcoma invasiveness.2.q-PCR was used to detect the expression abundance in osteosarcoma cell lines U20S,Saos-2,MG-63 and HOS.Results:1.UBE2T was positive in tumor tissues of osteosarcoma patients and negative in their para-tumor tissue;2.The expression level of UBE2T is higher in patients with pulmonary metastasis than those with non-pulmonary metastasis;3.UBE2T was highly expressed in U20S,Saos-2,MG-63 and HOS osteosarcoma cell lines,and the expression abundance was highest in U20S.Conclusions:By comparing tumor with para-tumor tissues and the patients with pulmonary metastasis and non-pulmonary metastasis,we believe that UBE2T is positively correlated with the invasiveness and pulmonary metastasis of osteosarcoma.Part?Radiosensitizing effect of SiRNA-UBE2T on growth and migration of osteosarcoma cells line U20SObjective:To construct UBE2T interfering RNA vector(SiRNA-UBE2T)and evaluate its enhancement with irradiation in the growth,cloning and migration of osteosarcoma cell line U20S.Methods:1.UBE2T interfering RNA vector(SiRNA-UBE2T)was constructed through molecular biology and cell biology.The expression of UBE2T was determined by q-PCR and Western Blot.2.Osteosarcoma cell U20S was divided into groups(SiRNA-Ctrl,SiRNA-UBE2T,SiRNA-UBE2Tcombined with irradiation group(IR+SiRNA-Ctrl)and SiRNA-UBE2T combined with irradiation group(IR+SiRNA-UBE2T)).The proliferation of U20S cells was evaluated by Celigo cell counting method and CCK-8,and the clone of U20S was evaluated by plate colony formation assay.The migration was verified by wound healing method using U20S cells after combined therapy.We also used flow cytometry to test its appotosis rate.Results:1.The UBE2T interfering RNA vector(SiRNA-UBE2T)was successfully constructed,which can significantly reduce 83.22%expression of UBE2T in U20S.2.In the SiRNA-UBE2T combined with irradiation group,the proliferation,cloning and migration rate of U20S cells were significantly lower than those of the other three groups,and its appotosis rate is the highest.Conclusion:SiRNA-UBE2T combined with irradiation can significantly reduce the proliferation,cloning and migration rate of U20S cells,while raise its appotosis rate,the SiRNA-UBE2T had radio-sensitization effect on U20S cells.Part ? SiRNA-UBE2T combined with irradiation on tumor growth and pulmonary metastasis of tumor-bearing miceObjective:To construct a U20S tumor-bearing mouse model and evaluate the effect of SiRNA-UBE2T combined with irradiation on tumor growth and pulmonary metastasis,and try to explore its mechanism and target gene.Methods:1.Severe combined immunodeficient mice(SCID)were purchased and subcutaneously inoculated with osteosarcoma cells U20S in the back to construct a SCID tumor-bearing model.2.The mice were divided into 4 groups(SiRNA-Ctrl,SiRNA-UBE2T,IR+SiRNA-Ctrl group and IR+SiRNA-UBE2T group)to evaluate the tumor growth in situ,and observe the pulmonary metastasis,lung wet weight after sacrifice..3.Immunohistochemistry was used to evaluate the expression of vimentin and apoptosis-related proteins(Bax,Caspase-3)in each group of tumors.Results:1.The tumor in the IR+SiRNA-UBE2T group was significantly smaller than that in the other three groups,and the tumor size was reduced at the 6th week,and pulmonary metastasis occurred in none of mice in this group;2.The expression level of Vimentin was the lowest in IR+SiRNA-UBE2T group,while the expression level of Bax and Caspase-3 was the highest.Conclusion:SiRNA-UBE2T combined with irradiation can significantly attenuate the growth of osteosarcoma in mice and reduce the probability of pulmonary metastasis,that is due to the radio-sensitization effect of SiRNA-UBE2T on tumor cells.The mechanism may be achieved by Vimentin and Bax,Caspase-3. |
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