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Expression and Clinical Relevance of MiR-302 b in OosteosarcomaObjective: To explore the differential expression of miRNA-302 b in osteosarcoma tissues and osteosarcoma cells,and the correlation between miRNA-302 b and clinical characteristics of osteosarcoma patients.Methods: A total number of 31 osteosarcoma patients were collected,including tumor tissues and paratumoral tissues.The expression of miRNA-302 b was detected by RT-PCR in the two groups.RT-PCR was used to detect the relative expression of miRNA-302 b in osteosarcoma cell lines(MG63,U2 OS,143B,Sao2)and osteoblasts(human hFOG1.19,mouse MC3T3-E1).We collected clinical information of osteosarcoma patients,including age,sex,tumor location,metastasis or not,and pathological grade of osteosarcoma.Based on the median expression of miR-302 b in osteosarcoma tissues(0.81),osteosarcoma patients were divided into two groups: the high expression group(>0.81)and the low expression group(<0.81).Chi-square test was used to analyze the correlation between the expression of miRNA-302 b and the clinical characteristics of osteosarcoma patients.Results: The expression level of miRNA-302 b in osteosarcoma tissues was significantly lower than that in adjacent tissues(n=31,t=10.29,P <0.001).The relative expression level of miRNA-302 b in MG63,U2 OS,U2OS,143 B,Sao2 cells were 0.973±0.071,1.239± 0.092,0.620 ± 0.086,1.746 ± 0.092,respectively.And the relative expression level of miRNA-302 b in hFOG1.19 and MC3T3-E1 cells were 8.172±0.424,6.053±0.441,respectively.The relative expression of miRNA-302 b in MG63,U2 OS,143B and Sao2 cells was lower than that in human hFOG1.19 cells and mouse MC3T3-E1 cells,respectively(P<0.05).There was no significant differences in age,sex and location of osteosarcoma between the high expression group and the low expression group.86.7%(13/15)of osteosarcoma patients had metastasis in the low expression group of miRNA-302 b,which was higher than 56.3%(9/16)in the high expression group of miRNA-302b(P=0.035).The high pathological grade of osteosarcoma patients in the low expression group of miRNA-302 b accounted for 93.3%(14/15),which was significantly higher than that in the high expression group of miRNA-302b(50.0%(8/16),(P=0.02).Conclusion: MiRNA-302 b was down-regulated in osteosarcoma and miRNA-302 b was closely related to the pathological grade and metastasis of osteosarcoma.Part ? The Effect of miR-302 b on Regulating Osteosarcom Cell Proliferation,Apoptosis,Invasion and Metastasis In Vitro and the Possible Mechanism.Objective: To explore the regulatory effect of miR-302 b on proliferation,invasion and metastasis of osteosarcoma cells and the possible molecular mechanism.Methods: Transient transfection of miR-302 b mimic up-regulated the expression of miR-302 b in osteosarcoma 143 B and MG63 cells respectively.MTT was used to detect the proliferation of osteosarcoma cells at 24,48,72 and 96 hours after transfection.After transfection of miR-302 b mimic for 48 hours,the invasive ability of osteosarcoma cells was detected by Transwell assay,and the migration ability was detected by scratch assay.Potential target genes were detected,and the interaction between miR-302 b and target gene 3'-UTR was validated by double luciferase reporting system,and the regulation of Runx2 on downstream genes was explored.The expression of Runx2 in OS cells(MG-63,U2 OS,143B,Saos2)was detected by qRT-PCR and was compared with osteoblasts(hFOB1.19,MC3T3-E1).The relative expression of Runx2 mRNA was detected in 31 pairs of osteosarcoma patients and adjacent tissues.Rescue experiment was designed.The experiment was divided into four groups: mimic + Runx 2-ctrl,mimic + Runx 2 up-regulation,Runx 2 up-regulation,mimic-NC + Runx 2 up-regulation,control group(mimic-NC + Runx 2-ctrl),Transwell assay and scratch assay were performed to evaluated invasive ability the migration ability,respectively.Results: In osteosarcoma 143 B and MG63 cells,OD values at 24,48,72 and 96 h in the miR-302 b mimic transfection group were significantly lower than those in the control group(P < 0.05).The total apoptotic rate of osteosarcoma cells in the miR-302 b mimic transfection group was significantly higher than that in the control group(P < 0.05).In osteosarcoma 143 B and MG63 cells,the number of osteosarcoma cell transmembrane cells and cell migration rate in the miR-302 b mimic transfection group were lower than those in the control group.The expression of Runx2 mRNA and its protein in 143 B cells in the miR-302 b mimic transfection group was significantly lower than that in the control group.The expression of Runx2 gene in the miR-302 b inhibitor transfection group was significantly higher than that in the control group,with statistical significance.The results of double luciferase reporter gene experiment showed that the luciferase activity of pmirGLO-Runx2-3'-UTR-wt was significantly inhibited by miR-302 b transfection,but the luciferase activity of pmirGLO reporter gene carrying mutant Runx2-3'-UTR was not affected.The expression level of Runx2 in osteosarcoma cell lines was significantly higher than thosed in two osteoblast cells.The expression level of Runx2 in osteosarcoma tissues was significantly higher than that in paratumoral tissues.Upregulation of miR-302 b inhibited the expression of RUNX2,OPN,MMP-2,MMP-9,MMP-13,MMP-14 and VEGF.Downregulation of miR-302 b promoted the expression of RUNX2,OPN,MMP-2,MMP-9,MMP-13,MMP-14 and VEGF.The protein and mRNA expression levels of Runx2,OPN,MMP2 and MMP9 genes in the co-up-regulation group of miR-302 b and Runx2 were significantly higher than those in the up-regulation group of miR-302 b.Conclusion: Up-regulation of miR-302 b inhibits proliferation,invasion and migration of osteosarcoma cells and promotes apoptosis.miR-302 b may regulate the invasion and metastasis of osteosarcoma by targeting Runx2.Part ? Study on the Effect of MiR-302 b on Regulation of Osteosarcoma Proliferation and Lung Metastasis in vivoObjective: To investigate the effect of microRNA-302 b on osteosarcoma proliferation and lung metastasis in vivo.Methods: A nude mice model of tibial osteosarcoma in situ was established.After the success of tumor-bearing,the mice in the treatment group(n=5)were injected with microRNA-302 b agomir which can up-regulated the expression of miR-302 b in vivo.The mice in the control group(n=5)were injected with a negative control substance(microRNA-302 b agomir-NC).The changes of tumor volume in both group was detected.After the treatment,the nude mice were executed and X-ray was performed to examine the tumors in situ.The destruction of bone tissue was observed by HE staining,and the number of metastatic nodules in lung tissue was detected.Results: The volume of tibial tumors of nude mice treated with miRNA-302 b agomir was statistically lower than that of control group on 7,14,21 and 28 days after administration.HE staining showed that the rate of tumor necrosis in the treatment group was higher than that in the control group.The X-ray results of mice showed that the severity of tibial destruction in the treatment group was lower than that in the control group.The nude mice were sacrificed 28 days after administration and the lung tissues were dissected.The number of HE-stained lung tissue metastasis tumors was observed under microscope.The results showed that the number of lung tissue metastasis tumors in miR-302 b agomir administration group was significantly lower than that in the control group.Conclusion: Up-regulation of miR-302 b inhibits tumor growth and lung metastasis in osteosarcoma of nude mice in vivo. |
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