| Part Ⅰ Study on the expression profile of microRNAs in chordomaObjective:To investigate the differential expression profile of microRNAs(miRNAs)between chordoma and fetal nucleus pulposus by using microRNA microarray.Methods:Twenty-eight freshly frozen chordoma tissues and ten fetal(12-28 weeks old)nucleus pulposus tissues were collected and their total RNAs were isolated routinely.Expressions of miRNAs in 3 chordoma tissues and 3 fetal nucleus pulposus tissues were detected by using microRNA microarray.Furthermore,some of the differentially expressed miRNAs were identified through real-time RT-PCR analysis.Results:From the microarray results,we found that a set of aberrant miRNAs could help us to clearly differentiate chordoma tissues from fetal nucleus pulposus tissues.Specifically,42 miRNAs were significantly dysregulated in chordoma compared with fetal nucleus pulposus tissues(P<0.001),which consisted of 19 upregulated miRNAs(miR-150,miR-497,miR-21,miR-29b,miR-223,miR-143,miR-222,miR-221,miR-4261,miR-374b,miR-185,miR-93,miR-770,miR-1260a,miR-4485,miR-1233-1,miR-365a,miR-211,miR-193a)and 23 downregulated ones(miR-5196,miR-4433,miR-4754,miR-4707,miR-3156,miR-3652,miR-1290,miR-3137,miR-623,miR-3917,miR-4656,miR-4710,miR-33b,miR-4685,miR-4690,miR-9,miR-1471,miR-1273f,miR-1229,miR-1246,miR-6127,miR-320e,miR-320d).The subsequent quantitative RT-PCR analysis results indicatedthat miR-21and miR-150 were significantly upregulated and miR-1290 and miR-623 were significantly downregulated in chordoma tissues compared with fetal nucleus pulposus tissues(P<0.05),which was in accordance with the microarray results.Conclusions:Our results revealed that 42 differentially expressed miRNAs were related to chordomas.This profile may provide potential diagnosis biomarkers and therapeutic targetsfor chordoma patients.Part Ⅱ Expression of miR-1290 in sacral chordoma and its association with invasion and recurrenceObjective:To investigate the expression level of miR-1290 in chordoma and its association with the clinical significance,so as to provide evidence for chordoma prognosis assessment and target for molecular therapy.Methods:We detected the expression of miR-1290 in 28 frozen chordoma tissue samples and 10 fetal nucleus pulposus tissue samples by qRT-PCR,and furtherly analyzed its association with the clinicopathological features which includes age,gender,tumor size,tumor location,invasion and recurrence.Results:qRT-PCR results showed that the expression of miR-1290 in chordoma tissues was significantly lower than that in fetal nucleus pulposus tissues(P<0.05).The results of Chi-Square analysis showed miR-1290 expression was significantly related to the surrounding tissue invasion and tumor recurrence(P<0.05),while the statistical result indicate that the miR-1290 expression had not any association with age,gender,tumor size and tumor location(P>0.05).The results of Kaplan-Meier survival analysis showed the recurrence-free survival time was significantly shorter in miR-1290 low expression group(46.1±7.20 m)than that in miR-1290 high expression group(115.9±14.61 m)(P<0.05).Conclusions:The expression of miR-1290 was significantly down-regulated in chordoma,it was closely related to tumor invasion and recurrence,which may serve as a potential biomarker for recurrence and prognosis assessment.Part Ⅲ Effect of miR-1290 on proliferation and invasion function of chordoma cellObjective:To explore the effect of miR-1290 on the proliferation and invasion of chordoma cell.Methods:The expression level of miR-1290 cells in chordoma cells were regulated by transfection of miR-1290 mimic and inhibitor.The experiment subjects were divided into miR-1290 mimic,mimic NC,miR-1290 inhibitor and inhibitor NC groups according to different transfection.The transfection efficiency was detected by qRT-PCR method.The changes of cell proliferation were assessed by cell clone formation assay,and the invasion of chordoma cells were assessed by transwell invasion test.Results:qRT-PCR results showed that miR-1290 was upregulated in miR-1290 mimics transfection group,and miR-1290 was downregulated in miR-1290 inhibitors transfection group(P<0.05).Cell clone formation assay showed that the number of cell clone formation in miR-1290 mimics group was significantly lower than that in miR-1290 mimic NC group(P<0.05),while the number in miR-1290 inhibitor group was significantly higher than that in miR-1290 inhibitor NC group(P<0.05).Transwell invasion test showed that the number of cells that pass through the membrane in miR-1290 inhibitor group was significantly higher than that in miR-1290 inhibitor NC group(P<0.05),while the number of cells in miR-1290 mimic group was significantly lower than that in miR-1290 mimic NC group(P<0.05).Conclusions:The upregulation of miR-1290 can inhibit chordoma cell proliferation and invasion of chordoma,and the downregulation of miR-1290 expression can promote chordoma cell proliferation and invasion of chordoma.Part Ⅳ Predicting and verifying the target gene of miR-1290 in chordomaObjective:To predict the target gene of miR-1290 which wasassociated with proliferation and invasion in chordoma,and verify it at the proteinand gene level.Methods:The target genes of miRNA-1290 were predicted by using TargetScan,microRNAorg and PITA databases,then get the intersection of prediction results.Expression of Robol was detected in chordoma tissues by using immunohistochemical method.Expression of Robol was detected in miR-1290 inhibitor NC group,inhibitor group,miR-1290 mimic group and miR-1290 mimic group NC by Western blot in chordoma cell.The relationship between the expression of miRNA-1290 and 3’UTR Robol was identified by dual luciferase reporter assay.Results:The intersection of the prediction results for TargetScan,microRNAorg and PITA database included 762 target genes,Robol is an important potential target gene of miR-1290.Immunohistochemistry results showed that the expression of Robol in sacral chordoma was significantly higher than that in fetal nucleus pulposus(82.1%vs 20%,P<0.05).The expression of miR-1290 in chordoma tissues was negatively correlated with the expression level of Robol protein(P<0.05).The results of Western blot showed that the expression of Robol was significantly increased in miR-1290 inhibitor transfection group,while the expression of Robol decreased significantly in miR-1290 mimic transfection group.Dual luciferase reporter assay showed that upregulation of miR-1290 significantly reduced the firefly luciferase activity of Robol-3’UTR(P<0.05).Conclusions:Robol is a downstream target gene of miR-1290 in chordoma,miR-1290 can negatively regulate Robol gene.Part V miR-1290 promotes chordoma cell’s proliferation and invasion through regulating the expression of Robo1 geneObjective:To explore the mechanism of miR-1290 regulation of Robol gene to promote chordoma cell proliferation and invasion.Methods:The expression of Robol in chordoma cells was downregulated by siRobol.The expression of Robol proteins after siRobol transfection was detected by Western blot.The changes of cell proliferation were detected by cell clone formation assay and the invasion of chordoma cells were detected by transwell invasion test.U-CH1 cells were co-transfected by siRobol and miR-1290 inhibitors or miR-1290 mimics.The changes of proliferation and invasive ability of chordoma cells after co-transfection were detected by cell clone formation assay and transwell invasion test.Results:Western blot results showed that the transfection of siRobol can significantly reduce the expression of Robol protein in chordoma cells.Cell cloning formation assay and Transwell invasion experiment results showed that downregulation of Robol can inhibit the proliferation and invasion ability of chordoma cells.The results of cell cloning formation assay showed that the proliferation ability of Robol siRNA+miR-1290 inhibitor group was significantly higher than that of Robol siRNA+inhibitor NC group(P<0.05),while the proliferation ability of Robol siRNA +miR-1290 mimics group was significantly lower than that of Robol siRNA+mimics NC co-transfection group(P<0.05).Transwell invasion assay results showed that the invasion ability of Robol siRNA+miR-1290 inhibitor group was significantly higher than that of Robol siRNA+inhibitor NC co-transfection group(P<0.05),while the invasion ability of Robol siRNA+miR-1290 mimics group was significantly lower than that of Robol siRNA+mimics NC co-transfection group(P<0.05).Conclusions:miR-1290 promotes the chordoma cell’s proliferation and invasion through negatively regulating the expression of Robol gene. |
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