The Effect And Mechanism Of MicroRNA-129-5p And UHRF1 On Retinoblastoma

Retinoblastoma(RB)is the most common primary intraocular malignancy in children.Accumulating evidences have clarified that microRNAs(miRNAs)modulated signaling molecules by acting as oncogenes or tumor-suppressor genes in RB.Thus,in our study,we aimed to investigate the function of miR-129-5p in RB cells through PI3K/AKT signaling pathway by targeting PAX6.Two RB cell lines,Y79 and WERI-Rb-1,were selected in our study,followed by transfection of miR-129-5p inhibitor or si-PAX6 to explore the regulatory role of miR-129-5p in RB cell proliferation,invasion and migration.Dual-luciferase assay was used for the detection of targeting relationship between miR-129-5p and PAX6.Besides,western blot analysis was applied to detect expression of cell cycle-related factors(CDK2 and Cyclin E)and PI3K/AKT signaling pathway-related factors(p-AKT and AKT).Nude mice tumorigenesis experiment was used to evaluate the effect of miR-129a-5p on RB growth in vivo.miR-129-5p was down-regulated in RB cell lines.miR-129-5p directly targeted the 3′-untranslated region of PAX6.Artificial down-regulation of miR-129-5p promoted cell proliferation,migration and invasion in RB cell lines Y79 and WERI-Rb-1,and promoted RB growth in vivo via PI3K/AKT signaling pathway,which could be reversed by transfection with silencing PAX6.This study provides evidences that RB progression was suppressed by overexpressed miR-129-5p via direct targeting of PAX6 through PI3K/AKT signaling pathway,which may provide a molecular basis for better treatment for RB.This study aimed to investigate the effect of silencing ubiquitin-like with PHD and RING finger domains 1(UHRF1)on the proliferation and apoptosis of retinoblastoma(RB)cells and to clarify the molecular mechanism of the UHRF1 gene in the development of RB.Human RB WERI-Rb-1 cells were selected and assigned into a blank group(WERI-Rb-1 cells with no transfection),NC-shRNA group(WERI-Rb-1 cells infected with NC-shRNA virus)and UHRF1-shRNA group(WERI-Rb-1 cells infected with pGC-UHRF1-shRNA-LV-GFP#(39–1)virus).The mRNA and protein expression of UHRF1 was detected by RT-qPCR and Western blot analysis.The effect of silencing UHRF1 on the proliferation and apoptosis of WERI-Rb-1 cells was assessed by MTT assay,EdU assay,flow cytometry,and Hoechst staining.Furthermore,the expression of cell cycle-related factor(cyclin D1),apoptosis-related factors(caspase-9,Bcl-2 and Bax),and PI3K/Akt signalling pathway-related factors(p-PI3 K,PI3K,p-Akt)were measured via Western blot analysis.The RNA interference plasmid UHRF1-shRNA was successfully constructed.After WERI-Rb-1 cells were infected with UHRF1-shRNA,decreased mRNA and protein expression of UHRF1 was found.WERI-Rb-1 cells infected with UHRF1-shRNA showed inhibited proliferative ability and increased apoptosis.In the UHRF1-shRNA group,more cells arrested at the G0/G1 phase and less cells at the S and G2/M phases.WERI-Rb-1 cells infected with UHRF1-shRNA had increased expression of caspase-9 and Bax and decreased expression of Bcl-2 expression and decreased levels of p-PI3 K and p-Akt.In conclusion,our study demonstrated that silencing UHRF1 could inhibit the proliferation of RB cells and promote apoptosis.The mechanism may be caused by the downregulation of the proportion of Bcl-2/Bax expression and the promotion of the expression of caspase-9 through the PI3K/Akt signalling pathway.