The Study On The Role And Mechanisms Of MiR-373/SRCIN1 In Neuroblastoma Proliferation,Migration And Invasion

Objective: To explore the expression of miR-373 and SRCIN1 in neuroblastoma tissue and cell lines in order to investigate its functional role and underlying molecular mechanism of action in neuroblastoma.The results of this study will provide furthur direction and experimental basis for finding a novel therapeutic target for the treatment of neuroblastoma.Methods:1.20 high risk neuroblastoma samples and 20 low risk neuroblastoma samples were collected from Shenzhen Children's Hospital between 2013 and 2016.miR-373 expression was measured by performing q RT-PCR in tissue samples from enrolled patients with neuroblastoma.2.The expression of miR-373 in five neuroblastoma cell lines were detected by q RT-PCR.3.The q RT-PCR results demonstrated that two types of cell lines: SKNBE(2)and SH-SY5 Y expressed highest of miR-373.Hence,we chose these two cell lines to perform loss-of-function experiments.After SK-N-BE(2)and SH-SY5 Y cells were transfected with antimiR-373 or antimiR-NC lentivirus,further analyses include MTT assay,flow cytometry,transwell migration and invadion assays were used to examine cell viability,cell cycle distribution,apoptosis,migration and invadion.4.The impact of miR-373 on neuroblastoma cell growth in the subcutaneous level was determined in NOG mice.The protein levels of Ki67 in xenograft tumors were verified by immunohistochemistry.5.By using methods of miRanda,Target Scan and Pic Tar,wepredicted that SRCIN1 might be one of the potential target genes of miR-373 in neuroblastoma.Dual-luciferase reporter assay was performed to measure direct regulation of miR-373 on expression of SRCIN1.The m RNA and protein expression of SRCIN1 in SK-N-BE(2)and SH-SY5 Y cells transfected with miR-373 mimics,negative control mimics,antimiR-373 lentivirus or antimiR-NC lentivirus were all examined by q RT-PCR and western blotting,respectively.q RT-PCR was used to detect SRCIN1 m RNA expression in 20 low risk and 20 high risk neuroblastoma tissues.6.The proliferation,apoptosis,migration and invasion of antimiR-373-treated cells were then infected transiently with SRCIN1 si RNA,which were further analyzed by MTT assay,flow cytometry analysis,transwell migration and invasion assays.Results:1.The expression of miR-373 was found significantly higher in high risk neuroblastoma tissues than in low risk neuroblastoma tissues.2.Mi R-373 was highly expressed in neuroblastoma cell lines in comparison to normal tissues.3.SK-N-BE(2)and SH-SY5 Y cells that were transfected with antimiR-373 lentivirus had a reduced miR-373 expression,suggesting lower inhibited cell vability,cell cycle progression,migration and invasion and induction apoptosis.4.Inhibition of miR-373 decreased the tumorigenic ability of neuroblastoma cells,reduced the volume and weight of the transplanted tumor,and inhibited miR-373 and Ki67 expression.5.The dual-luciferase reporter assay identified that miR-373 suppresses the expression of SRCIN1 by directly targeting its 3?UTR.Overexpression of miR-373 was found to suppress SRCIN1 m RNA and protein expression,while the inhibition of miR-373 can enhance SRCIN1 m RNA and protein expression.q RT-PCR results showed that SRCIN1 expression was significantly lower in high risk neuroblastoma tissues than in low risk neuroblastoma tissues.Pearson's correlation analysis demonstrated that miR-373 was inversely correlated with SRCIN1 expression in neuroblastoma tissues.6.Knockout SRCIN1 by si RNA was found to promote cell vability,cell cycle progression,migration and invasion and induction apoptosis.Collectively,these results indicated that SRCIN1 is important for miR-373-induced malignant phenotypes of neuroblastoma cells.Conclusion:1.miR-373 expression was upregulated in neuroblastoma tissues and cell lines.2.Reducing the endogenous miR-373 levels resulted in inhibition of cell proliferation both in vitro and in vivo,also reduced the ability of neuroblastoma cells migration,invasion,and apoptosis.3.miR-373 promoted neuroblastoma cell vability,cell cycle progression,migration and invasion and reduced apoptosis through a mechanism to negatively regulate SRCIN1 expression.4.These findings suggest that miR-373 and SRCIN1 can serve as potential therapeutic targets of human NB.