| BackgroundOsteosarcoma is more common in adolescents and children,as a common primary malignant tumor,in all types of primary bone tumors,the incidence of osteosarcoma ranked first.Osteosarcoma is a highly malignant solid tumor.A large number of studies have shown that the occurrence of osteosarcoma is usually associated with the rapid growth of skeletal bone,but also has a high incidence,high degree of malignancy and prone to distant metastasis characteristics,the patient's long-term survival rate at a low level.The Malignant tumors have high mortality and are prone to metastasis and recurrence,so it is difficult for patients with early diagnosis and treatment.At present,the clinical treatment of osteosarcoma is a combination of surgery and individual chemotherapy combined with the combination of treatment,although the patient in the treatment of 5 years after the disease-free survival than in the past has been greatly improved.But for the occurrence of bone metastasis and recurrence of osteosarcoma patients,still failed to find effective treatment options,so the patient's short-term survival rate is still at a low level,about 3 0%.Therefore,to find a new effective detection and treatment of osteosarcoma metastasis markers is the current study of cancer and hot and difficult problems.Anti-differentiated noncoding RNA(ANCR)belongs to one of IncRNA,and it has been proved that it has differentiation and regulation function.(Enhancer of zeste homolog 2,EZH2)is one of the important factors of epigenetic regulation of EMT,and plays an important role in the process of metastasis of various malignant tumors.EZH2 can inhibit the biological activity of many genes at the gene level,and can also inhibit the tumor metastasis suppressor,so it can promote the invasion and metastasis of tumor cells.Current studies have shown that IncRNA plays an important role in the malignant process of many kinds of cells,and EZH2,which is mediated by IncRNA-ANCR in some tumors,can affect the invasion and metastasis of tumor cells.P21 and p27 belong to the family of cyclin-dependent kinase inhibitors.It was found that both of them could recognize and bind with various cyclin-cyclin dependent kinase complexes,thus inhibiting the biological function.Causing cell cycle arrest,block cell proliferation,induce apoptosis.The study found that when p21 and p27 expression abnormal abnormalities can promote tumor cell proliferation,invasion and metastasis,inhibition of apoptosis,so both have the role of tumor suppressor gene.ObjectiveIn this study,we have studied the expression of lncRNA-ANCR in osteosarcoma and its effect on the biological behavior of osteosarcoma cells.The mechanism of lncRNA-ANCR in osteosarcoma was studied.The aim of this study was to investigate the molecular mechanism of osteosarcoma As well as drug targets to lay the foundation.methods(1)Twenty patients were collected from July 2015 to March 2016 in Wuhan University Affiliated hospital of osteosarcoma surgical resection of osteosarcoma tissue specimens,while 20 patients received lumbar discectomy patients with tissue samples as a control group.The expression level of LncRNA-ANCR in the above tissue samples was detected by fluorescence quantitative PCR,and the correlation between the expression of LncRNA-ANCR and clinicopathological parameters was analyzed.(2)The expression of LncRNA-ANCR in human osteosarcoma cell lines Hos,MG-63,SW1353,MC3T3-E1,UMR-106,U20S,SaoS2,143B and human osteoblast cell line HfoB1.19 were detected by fluorescence quantitative PCR.LncRNA-ANCR has the highest expression of osteosarcoma cell lines for subsequent experimental studies.The two osteosarcoma cells were divided into WT group,Mock group,NC group and siRNA group(transfected with LncRNA-ANCR siRNA plasmid).The expression levels of LncRNA-ANCR were detected by fluorescence quantitative PCR.The proliferation of cells was detected by CCK-8 method.Apoptosis of cells was detected by flow cytometry.The invasive ability of cells was detected by Transwell chamber method.Cell migration ability was measured by cell scratches.(3)The two osteosarcoma cells were divided into WT group,Mock group,NC group and siRNA group(transfected with LncRNA-ANCR siRNA plasmid).The expression of p21 and p27 in the above-mentioned groups were detected by fluorescence quantitative PCR and western blot respectively.,EZH2 mRNA and protein expression.The expression of EZH2 mRNA was detected by fluorescence quantitative PCR in 20 cases of osteosarcoma and 20 cases of normal tissues.The correlation between LncRNA-ANCR and EZH2 was detected by RNA pull-down assay and further verified by linear correlation analysis.Results(1)? The expression of FOXM1 mRNA in tumor tissue of patients with osteosarcoma and control group was analyzed.The results showed that the expression level of LncRNA-ANCR in osteosarcoma was significantly higher than that in non-tumor tissue(P<0.01).?The expression of LncRNA-ANCR was related to the age,sex,tumor location and pathological type of osteosarcoma patients(P>0.05),but it was related to Ennecking staging and tumor metastasis in patients with osteosarcoma,that is,LncRNA-ANCR in Ennecking stage(P<0.05).The expression level of osteosarcoma in patients with osteosarcoma was significantly higher than that in non-metastatic patients(P<0.05).(2)? Compared with HfoB 1.19 cells,the expression of LncRNA-ANCR in Hos,MG-63,SW1353,UMR-106,U20S and SaoS2 cells was significantly increased(P<0.05),and MG-63 and UMR-106 cells LncRNA-ANCR expression of the highest level,so the choice of these two osteosarcoma cells for follow-up experimental study.?Compared with WT group,the expression of LncRNA-ANCR in Mock group and NC group was not significantly changed in MG-63 cells compared with WT group(P>0.05).Compared with WT group,LncRNA-ANCR(P<0.05).Compared with WT group,the expression of LncRNA-ANCR in Mock group and NC group was not significantly different from that in WT group(P>0.05).Compared with WT group,the expression of LncRNA-The expression level of ANCR was significantly decreased(P<0.05).?In MG-63 cells,the proliferation rate of each group of cells at each time point after transfection showed a gradual increase trend.Compared withWT group,the cells in Mock group and NC group were transfected at 1,2,(P>0.05).Compared with WT group,the proliferation rate of siRNA cells was significantly decreased at the 2nd,3rd and 4th days after transfection(P<0.05).In UMR-6 cells,the proliferation rate of each group of cells at each time point after transfection showed a gradual increase trend.Compared with WT group,the cells in Mock group and NC group were transfected at 1,2,(P>0.05).Compared with WT group,the proliferation rate of siRNA cells was significantly decreased at the 2nd,3rd and 4th days after transfection(P<0.05).? Compared with WT group,the apoptotic rate of Myc and NC cells was not significantly changed in MG-63 cells(P>0.05).Compared with WT group,the apoptosis rate of siRNA group was significantly higher P<0.01).Compared with WT group,the apoptotic rate of Mock and NC cells was not significantly changed in UMR-106 cells(P>0.05).Compared with WT group,the apoptotic rate of siRNA cells was significantly increased P<0.01),? In the MG-63 cells,the number of invasive cells in the Mock and NC groups did not change significantly(P>0.05)compared with the WT group.Compared with the WT group,the number of invasive cells in the siRNA group was significantly decreased P<0.01).In UMR-106 cells,the number of invasive cells in Mock and NC cells was not significantly changed(P>0.05)compared with WT group.Compared with WT group,the number of invasive cells in siRNA group was significantly decreased P<0.01).?In MG-63 cells,the migration distance of Mock and NC cells did not change significantly(P>0.05)compared with WT group,while the migration distance of siRNA group was significantly higher than that of WT group(P<0.01).In UMR-106 cells,the migration distance of Mock and NC cells did not change significantly compared with WT group(P>0.05).Compared with WT group,the migration distance of siRNA cells increased significantly(P<0.01).(3)? In the MG-63 cells,the expression level of p21 mRNA in the Mock and NC groups was not significantly different from that in the WT group(P>0.05).Compared with the WT group,the expression of p21 mRNA in the siRNA group was significantly Increased(P<0.05).In UMR-106 cells,the expression level of p21 mRNA was not significantly changed in WT group,Mock group and NC group(P>0.05).Compared with WT group,the expression level of p21 mRNA in siRNA group(P<0.05).?In the MG-63 cells,the expression level of p27 mRNA in the Mock and NC groups was not significantly different from that in the WT group(P>0.05).Compared with WT group,the expression of p27 mRNA in siRNA group was significantly Increased(P<0.05).The expression level of p27 mRNA in UMR-106 cells was not significantly different from that in WT group,Mock group and NC group(P>0.05).Compared with WT group,the expression level of p27 mRNA in siRNA group(P<0.05).? Compared with WT group,the expression levels of p21 and p27 protein in Mock group and NC group were not significantly changed(P>0.05).Compared with WT group,the expression of p21 and p27 protein in siRNA group was significantly higher than that in WT group<0.05).? Compared with normal tissues,the expression of EZH2 mRNA in osteosarcoma tissue was significantly increased(P<0-05).? There was EZH2 interaction between lncRNA and ANCR.Suggesting that IncRNA-ANCR may have an effect on the expression of EZH2.?The linear correlation coefficient analysis showed that the expression of IncRNA-ANCR was positively correlated with EZH2(r2 = 0.7541,P<0.0001).Conclusion(1)LncRNA-ANCR is highly expressed in most osteosarcoma tissues,which is associated with Ennecking surgical staging and tumor metastasis of osteosarcoma.(2)LncRNA-ANCR can promote the proliferation,invasion and metastasis of osteosarcoma MG-63 and UMR-106 cells and inhibit apoptosis.(3)LncRNA-ANCR promotes the malignant process of osteosarcoma by inhibiting the expression of p21 and p27 and promoting the expression of EZH2. |
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