Effect Of MiR-22 On Proliferation,Migration,Invasion And Apoptosis Of Retinoblastoma Y79 Cells And The Possible Mechanism

PurposeRetinoblastoma is the most common intraocular cancer in children.It has poor therapeutic effect and high mortality,which seriously threatens the vision and even life of children.Tumor initiation and development is a complex biological process involving cell proliferation,apoptosis,invasion and migration,and there are many signaling pathways involved in the process.Therefore,inhibiting the proliferation,invasion and migration of tumor cells and promoting cell apoptosis are of great significance for the control of tumor development.MicroRNA(miRNA)is a widespread class of endogenous non-coding single-stranded RNA with about 19-25 nucleotide sizes that regulates the expression of target genes by specifically inhibiting or degrading the translation of target mRNAs.miRNAs participate in the regulation of a series of cell life activities,such as cell cycle,cell differentiation,cell proliferation and apoptosis though its target genes.Studies have shown that the abnormal expression of miR-22 in a variety of tumors directly affects the occurrence and development of tumors by regulating cell proliferation or apoptosis.miR-22 has been found to be low expressed in retinoblastoma,but its specific biological function and mechanism are still unclear.In our experiment,we studied the effects of miR-22 on the proliferation,apoptosis,migration and invasion of Y79 cells,and explored its possible mechanism,providing new ideas and theoretical basis for clinical treatment of retinoblastoma.Methods 1.Transfection with miR-22 overexpression eukaryotic plasmid up-regulated the expression of miR-22.The cells were divided into normal group(N),negative control group(GV251)and miR-22 overexpression group(GV251/miR-22).CCK-8 assay was used to detect the cell proliferation in each group.Wound healing assay was used to detect the migration of cells in each group.Transwell assay was used to observe the number of invasive cells in each group.Flow cytometry was used to detect the cell apoptosis rate.2.RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of HMGB1 in the above groups.3.Transfection with miR-22 shRNA eukaryotic plasmid,and RT-qPCR was used to detect miR-22 expression.The cells were divided into normal group(N),negative control group(GV249)and miR-22 lowexpression group(GV249/miR-22-siRNA).The expression of HMGB1 mRNA and protein was detected by RT-qPCR and Western blot.4.HMGB1 shRNA eukaryotic plasmid was transfected and the expression of HMGB1 mRNA and protein was detected by RT-qPCR and Western blot.Cells were divided into normal group(N),negative control group(GV102)and HMGB1 lowexpression group(GV102/HMGB1-siRNA).CCK-8 assay was used to detect the cell proliferation in each group.Wound healing assay was used to detect the migration of cells in each group.Transwell assay was used to observe the number of invasive cells in each group.Flow cytometry was used to detect the cell apoptosis rate.5.Transfection with HMGB1 overexpression eukaryotic plasmid and the expression of HMGB1 mRNA and protein was detected by RT-qPCR and Western blot.Then,cells were co-transfected with miR-22 and HMGB1 and cells were divided into four groups:N,GV251/miR-22,GV251/miR-22+GV230,GV251/miR-22+GV230/HMGB1.Cell proliferation was detected by CCK-8 assay,cell migration was detected by wound healing assay,the number of invasive cells was observed by Transwell assay,and cell apoptosis was detected by flow cytometry.6.Y79 cells were divided into normal group(N),GV251/miR-22 group,GV102/HMGB1-siRNA group,GV230/HMGB1 group,co-transfection group(GV251/miR-22+ GV230/HMGB1).The expression of AKT and p-AKT in every group was observed by Western blot.Results 1.Transfection of miR-22 eukaryotic overexpression plasmid significantly upregulated the expression levels of miR-22(P<0.05).Compared with the negative control group,the proliferation,migration,invasion of the cells in the miR-22 overexpression group were decreased and apoptosis were elevated.It is suggested that upregulation of miR-22 inhibits cell proliferation,migration and invasion,and promotes cell apoptosis.2.Compared with the negative control group,the mRNA and protein expression levels of HMGB1 in miR-22 overexpression group were significantly lower.3.Transfection of miR-22 shRNA eukaryotic plasmid significantly reduced the expression levels of miR-22(P<0.05).Compared with control groups,the expression of HMGB1 mRNA and protein in the miR-22 lowexpression group increased significantly.4.Transfection of HMGB1 shRNA eukaryotic plasmid significantly reduced the HMGB1 mRNA and protein expression levels.Compared with control groups,in the HMGB1 lowexpression group the proliferation and migration of HMGB1 cells decreased,the number of invasive cells decreased,and the apoptosis rate increased.It is suggested that knockingdown HMGB1 expression inhibits cell proliferation,migration and invasion,and promotes cell apoptosis.5.Transfection of HMGB1 overexpression eukaryotic plasmid significantly enhanced HMGB1 mRNA and protein expression levels.Compared with GV251/miR-22+GV230 group,the proliferation,migration,invasion of cells in co-transfection group increased and apoptosis decreased.Co-transfection of miR-22 and HMGB1 reversed the effects of overexpression of miR-22 on cell proliferation,migration,invasion and apoptosis.6.Western blot results showed that the level of p-AKT in GV251/miR-22 and GV102/HMGB1-siRNA groups was significantly lower than that in the normal group(P<0.05).The phosphorylation level of AKT in GV230/HMGB1 group was significantly higher than that in normal group(P<0.05).AKT phosphorylation level in co-transfection group was significantly higher than that in GV251/miR-22 group(P<0.05).ConclusionUpregulation of miR-22 significantly inhibited cell proliferation,migration and invasion and promoted cell apoptosis.miR-22 negatively regulates the expression of HMGB1 in Y79 cells.miR-22 inhibits cell proliferation,migration,invasion and promotes cell apoptosis through HMGB1.The PI3K/AKT pathway is involved in the effects of miR-22/HMGB1 on proliferation,migration,invasion and apoptosis of Y79 cells.