Crucial Roles Of MiRNA-558 In Regulating The Heparanase Expression In Neuroblastoma And Its Underlying Mechanisms

PART ONE mi R-558 is up-regulated and positively correlated with HPSE levels in neuroblastoma tissues and cell linesObjective:To investigate the hypothesis that mi RNA may regulate the transcription of HPSE and explore the correlation between the expression of mi RNA-558 and HPSE in neuroblastoma tissues and cell lines.Methods:To predict the enrichment of mi RNAs on the HPSE promoter regions of in NB, computational prediction was performed by the mi RNA–promoter interaction database and AGO–chromosome interaction profiling data(GSE40536); Dual-luciferase experiment was used to validate the effects of predicted micro RNAs on the promoter. The retrieval of neuroblastoma information related to the BIRC6(baculoviral IAP repeat containing 6), the host gene of mi R-558, was performed by R2 database. Real-time quantitative reverse transcriptase–polymerase chain reaction(RT–PCR) was applied to measure the mature mi R-558 levels in 30 NB specimens, normal dorsal ganglia and cultured SK-N-SH, SK-N-AS, SH-SY5 Y and SK-N-BE(2) cell lines. Western blot assay further verified the expression of HPSE level and its relationship with mi R-558 expression.Results: The analysis of mi RNA–promoter interaction database and AGO–chromosome interaction profiling data(GSE40536) revealed the enrichment of AGO1 at bases-2468/-2591 upstream the transcription start site(TSS) of HPSE. Surrounding this region, there were potential binding sites of mi R-145, mi R-558 and mi R-17, locating at bases-2330/-2345,-2314/-2332 and-2292/-2313 relative to the HPSE TSS. Dual-luciferase assay indicated that transfection of mi RNA inhibitor and mimic of mi R-558 resulted in altered promoter activities of HPSE in cultured NB cell lines, but not of mi R-145 or mi R-17. R2 revealed that the expression of BIRC6 was up-regulated in specimens from 88 well-established NB cases than that in normaldorsal ganglia(P< 0.0001). In addition, the BIRC6 levels were correlated with advanced international neuroblastoma staging system(INSS) stages(P = 0.007) and aggressiveness of neuroblastic tumors(P = 0.0079). RT–PCR showed that mature mi R-558 was up-regulated in the NB tissues and cell lines compared with normal dorsal ganglia. The western blot assay indicated the high expression of HPSE in NB tissues and cell lines. There was a positive correlation between mi R-558 expression and HPSE transcript levels in NB tissues and cell lines(R = 0.633, P < 0.0001).Conclusions: Higher mi R-558 expression was observed in NB tissues with poor differentiation(P = 0.0001) or higher HPSE protein levels(P = 0.0011). mi R-558 was up-regulated and positively correlated with HPSE levels in neuroblastoma tissues and cell lines.PART TWO The mechanisms underlying mi R-558-falilitated expression of HPSEObjective: To investigate whether mi R-558 promotes the expression of HPSE at the transcriptional or translational level.Methods: The mi RNA-558 knockdown and over-expression experiments was performed. Transfection of anti-mi R-558 inhibitor or mi R-558 precursor into NB cells, and the nascent transcript and protein levels of HPSE and VEGF were measured by Western blot and real-time quantitative RT–PCR. Computational prediction was performed by mi RNA-promoter interactions database to retrieval the binding site of mi R-558 within the 3′-UTR of HPSE.Results: Western blot and real-time quantitative RT–PCR demonstrated that transfection of the anti-mi R-558 inhibitor resulted in decreased nascent transcript and protein levels of HPSE in SH-SY5 Y and SK-N-BE(2) cells cells, accompanied by decreased expression levels of VEGF, the HPSE downstream target gene. On the otherhand, the effects of mi R-558 over-expression are opposite. Knock-down or over-expression of mi R-558 in NB cells did not affect the luciferase activities of VEGF 3′-UTR or HPSE 3’-UTR.Conclusions: mi R-558 considerably facilitates HPSE expression through transcriptional activation in NB cells,and regulates the expression of VEGF through modulating HPSE, without involvement of post-transcriptional.PART THREE mi R-558 promotes the growth, invasion, metastasis and angiogenesis of NB cells through targeting HPSE in vitroObjective: To investigate the effects of mi R-558 knockdown and over-expression on the biological features(growth, invasion, metastasis and angiogenesis) of cultured NB cells, and investigate the effects of restoration of HPSE expression.Methods: Neuroblastoma cell lines SH-SY5 Y and SK-N-BE(2) that stablely transfected with mi R-558 precursor were established. The growth, migration, invasion and angiogenesis capabilities of neuroblastoma cells were measured by MTT colorimetric, soft agar, scratch migration, Transwell migration, and tube formation assays. Rescue experiments were performed by restoration of HPSE levels through over-expression or knockdown of HPSE, respectively, in which the expression of HPSE and VEGF were detected by Western blot.Results: Western blot indicated that transfection of HPSE rescued the mi R-558 knockdown-induced down-regulation of HPSE and VEGF. In the MTT and soft agar assays, tumor cells transfected with the anti-mi R-558 inhibitor possessed the decreased viability and growth capabilities. In the scratch migration assay, mi R-558 knockdown attenuated the migration capabilities of SH-SY5 Y and SK-N-BE(2) cells. Transwell analysis showed that NB cells transfected with the anti-mi R-558 inhibitorpresented an impaired invasion capacity. The tube formation of endothelial cells was suppressed by treatment with the medium preconditioned by the transfection of NB cells with the anti-mi R-558 inhibitor. On the other hand, the effects of mi R-558 over-expression were opposite. In addition, restoration of HPSE expression via transfection of HPSE or si RNA specific for HPSE rescued the NB cells from their changes in these biological features. Meanwhile, restoration of VEGF levels only partially prevented the NB cells from mi R-558-mediated changes in the growth, migration, invasion and angiogenesis in vitro.Conclusions: mi R-558 remarkably promotes the growth, migration, invasion and angiogenesis of NB cells through targeting HPSE in vitro.PART FOUR Knockdown of mi R-558 attenuated the growth, metastasis and angiogenesis of NB cells in vivoObjective: To investigate the efficacy of mi R-558 knockdown against growth, metastasis and angiogenesis of neuroblastoma cells in vivo.Methods: The anti-mi R-558 inhibitor was injected to SH-SY5 Y cells-established subcutaneous xenograft tumors in athymic nude mice via tail vein(n=5 for each group). Administration of the anti-mi R-558 inhibitor via tail vein injection to the metastasis model(n=5 for each group); CD31-positive microvessel density and mean vessel density in nude mice were detected by immunohistochemical staining. The metastastic tumor size and quantity were detected in the lungs of nude mice by HE staining.Results: Administration of the anti-mi R-558 inhibitor via tail vein injection decreased the growth and tumor weight of SH-SY5 Y cells-established subcutaneous xenografttumors in athymic nude mice. In the experimental metastasis studies, nude mice treated with the anti-mi R-558 inhibitor established statistically fewer lung metastatic colonies. Immunohistochemical staining showed that treatment with the anti-mi R-558 inhibitor resulted in decrease in CD31-positive microvessels and mean vessel density within tumors.Conclusions: Knockdown of mi R-558 inhibited the growth, metastasis and angiogenesis of NB cells in vivo.