The Effect And Mechanism Of MiR-140-5p And PRC1 On Retinoblastoma

Background and purpose:Retinoblastoma is an ocular malignancy occurring in childhood.RB affects the eyes of children at a very young age and accounts for 5% of blindness in children.miRNAs have been identified to be involved in many physiological and pathological processes,including proliferation,death,differentiation,and metabolism.Mi R-140-5p is low expression and acts as a tumor suppressor in various types of human cancers.However,the potential role of miR-140-5p in retinoblastoma(RB)remains unknown.The protein regulator of cytokinesis 1(PRC1)acts on the cytokinesis phase and is the key protein molecule of cytokinesis.It has been discovered recently that the Wnt/ ? ? catenin pathway functions importantly in retinoblastoma,suggesting its prognostic and molecular therapeutic values.However,the current information regarding PRC1 gene silencing interacting with the Wnt/??catenin pathway in retinoblastoma cells remains to be minute,and warrants further research.The aim of this study was to investigate the potential role of miR-140-5p in retinoblastoma,and to prove that PRC1 can inhibit the proliferation,invasion and angiogenesis of retinoblastoma cells by inhibiting Wnt / ?-Catenin pathway.This study explored new potential biomarkers of Rb and provided new ideas for the diagnosis and treatment of Rb.Method:1.We performed the miRNA microarray analysis to identify the miRNA expression profiles in RB tissues and normal retinas,the q RT-PCR was used to measure the miR-140-5p level in RB tissues(n=50)and normal retinas(n=8).We also investigated the correlation between mir-140-5p level and clinicopathological features in Rb.The Y79 and Weri-Rb1 cells were transfected with miR-140-5p mimics or mimics NC,and the q RT-PCR was used to assess the overexpression efficiency of miR-140-5p.We performed targetscan and Miranda to predict the target gene of miR-140-5p,and further confirmed that the target gene expression is regulated by miR-140-5p.WB assay was used to detect the expression of target genes in Y79 cells and Weri-Rb1 cells.The q RT-PCR assay was used to determine the m RNA level of target genes in Rb tissues(n = 50).2.The c-Met was identified as a potential target gene of miR-140-5p.To further explore the role of c-Met in RB cell,c-Met was inhibited by si-c-Met in Y79 or Weri-Rb1 cells,and then cell viability,apoptosis,and cell cycle were assessed using CCK-8 assay and flow cytometry,respectively.To investigate whether c-Met is a functional target of miR-140-5p,the Y79 or Weri-Rb1 cells were transfected with miR-140-5p mimics/inhibitor or were co-transfectedwithmiR-140-5p mimics and pc DNA-c-Met plasmid.Then,we performed the CCK-8 assay and flow cytometry analysis to evaluate cell proliferation,apoptosis,and cell cycle in each group.To investigate whether miR-140-5p inhibit RB cell growth and cycle through modulating c-Met/AKT/mTOR signaling pathway,the Y79 and Weri-Rb1 cells were transfected with miR-140-5p mimics or mimics NC and the p-c-Met,c-Met,p-AKT,AKT,p-mTOR,mTOR,p-S6,and S6 were analyzed by Western blot analysis.3.Immunohistochemistry was used to detect the expression of PRC1,VEGF and GSK-3 ? phosphorylation in retinoblastoma tissues of normal control group,Rb differentiation group and Rb undifferentiated group.Immunohistochemistry was used to further determine the relationship between PRC1 expression and primary/invasive retinoblastoma tissues.RT q PCR was used to detect the expression of PRC1 in human RB cell lines Y79(HTB-18),Weri-Rb1(HTB-169),SO-Rb50,HXO-RB44 and human retinal pigment epithelium(RPE)cells.With the purpose of determining the effect of silencing PRC1 on related pathway,the expression patterns of PRC1 and Wnt/??catenin pathway ?related genes were evaluated.RB cells were divided into blank group,NC group,si RNA-PRC1 group,Li Cl(activator of Wnt / ?-catenin signaling pathway)group and si RNA-PRC1 + Li Cl group.MTT assay was used to detect the effect of PRC1 gene silencing on the proliferation of retinoblastoma cells.Flow cytometry was used to investigate whether rpc1 affected the cell cycle distribution of retinoblastoma,and Transwell assay was used to detect the invasiveness of retinoblastoma cells.Finally,the effect of PRC1 gene silencing on angiogenesis of HUVECs was detected in vitro.Result:1.The miR-140-5p was down regulated in Rb tissues and cells,and was associated with low survival rate.Consistent with miRNA microarray data,the expression level of miR-140-5p in RB tissue was significantly lower than that in normal retina.Low expression of miR-140-5p indicates poor prognosis in Rb patients.Overexpression of miR-140-5p inhibited the growth of Rb cells.The miR-140-5p can inhibit RB cells by inhibiting cell proliferation,inducing apoptosis and cell cycle arrest.2.c-Met is the target gene of miR-140-5p in RB cells.The expression of miR-140-5p and c-Met is negatively correlated.The c-Met silencing can inhibit the proliferation of RB cells and induce apoptosis and cell cycle arrest.Overexpression of miR-140-5p reduced the protein expression level of c-Met,and pc DNA-c-Met could prevent the overexpression of miR-140-5p from inhibiting c-Met expression.The overexpression of c-Met in Y79 and Weri-Rb1 significantly eliminated the inhibitory effect of miR-140-5p on RB cell growth and cell cycle arrest.Overexpression of miR-140-5p significantly reduced the expression of pc-Met,p-AKT,p-mTOR and p-S6 in Y79 cells and Weri-Rb1 cells.3.The expression of PRC1,VEGF and the phosphorylation of GSK-3? in the retinoblastoma differentiated group and the retinoblastoma undifferentiated group were higher than those in the control group.Compared with the retinoblastoma undifferentiated group and the retinoblastoma differentiated group,the expression of PRC1 and VEGF And the phosphorylation degree of GSK-3? increased significantly.We found that the expression of PRC1 in retinoblastoma in situ and aggressive retinoblastoma groups was higher than that in the control group.Compared with the primary retinoblastoma group,the expression of PRC1 was significantly higher in the aggressive retinoblastoma group.PRC1 m RNA expression increased in HXORB44 and WERI-Rb-1 cell lines.The silencing of PRC1 gene inactivates the Wnt/?-catenin pathway.PRC1 gene silencing inhibited the proliferation,cell cycle,invasion and angiogenesis of retinoblastoma cells.Conclusion:1.The expression of miR-140-5p is down-regulated in RB tissues,and the low expression level of miR-140-5p indicates a poor prognosis for RB patients.This suggests that miR-140-5p may be a new type of biomarker in the clinical diagnosis of RB.The miR-140-5p can inhibit RB cells by inhibiting cell proliferation,inducing apoptosis and cell cycle arrest.2.c-Met is the functional target of miR-140-5p in RB cells.The expression of miR-140-5p and c-Met is negatively correlated.Down-regulation of c-Met expression can inhibit the proliferation of RB cells,promote apoptosis and decrease the ratio of cells in G0/G1 phase.The miR-140-5p inhibits RB cells by regulating the expression of c-Met.Importantly,miR-140-5p may have a tumor suppressor effect in RB by inhibiting the c-Met/AKT/mTOR signaling pathway.The results of the study indicate that miR-140-5p can be used as a potential biomarker and therapeutic target for the prognosis of RB patients.3.The expression of PRC1 protein in retinoblastoma tissue is positively correlated with angiogenesis and aggressiveness.The silencing of PRC1 gene can inactivate the Wnt/?-catenin signaling pathway.PRC1 gene silencing can block retinoblastoma cells in the G0/G1 phase,thereby promoting cell apoptosis.PRC1 gene silencing inhibited the proliferation and invasion of retinoblastoma cells,and inhibited the angiogenesis of retinoblastoma cells.