The Effect And Mechanism Of MicroRNA-101-3p On The Proliferation Of Retinoblastoma Regulated By EZH2 And HDAC9

Background and Objective:Retinoblastoma(Rb)is the most common intraocular malignant tumor in infants and young children,and the survival rate is low,which poses a great threat to the vision and life of children,and has become the focus of attention of both doctors and patients.Retinoblastoma occurs before the age of 5 years old,can be monocular or binocular disease,often because of children’s pupil area pale(white pupil disease)or strabismus,there are also late visual decline,exophthalmos,glaucoma and so on to attract the attention of parents,at this time to the late,poor treatment effect.Proliferation is one of the main causes of poor therapeutic effect.Micro-RNA(micro-RNA)is a kind of non-coding single-stranded small molecular RNA with 20-24 nucleotides in length.it is widely distributed in tissues and cells and involved in the regulation of individual development,apoptosis,proliferation and differentiation.it is closely related to the occurrence and development of a variety of tumors.its action target may be anti-oncogene or oncogene.mi RNA-101-3p,in breast cancer,retinoblastoma and lymph cancer and other related diseases,confirmed that it is down-regulated in almost all tumor tissues,can inhibit the proliferation and apoptosis of tumor cells.However,its role in retinoblastoma and related mechanisms have not been reported.EZH2(Drosophila zeste gene enhancer human homologue 2)is capable of inhibiting transcription of the target gene by methylation of histones.EZH2 plays an important role in the process of malignant transformation from precancerous lesion to cancer.In addition,more and more experimental data show that the abnormal expression of EZH2 is related to the progression of malignant tumors,and the high expression of EZH2 may promote the proliferation of tumor cells and other biological characteristics.However,the mechanism by which EZH2 is regulated in retinoblastoma is unclear? Histone deacetylase HDAC9 is a member of histone deacetylase ii family,which contains deacetylase activity domain,zinc lipid domain,me F2 domain,serine binding site and localization signal.In recent years,HDAC9 has been found to be highly expressed in a variety of tumors,including retinoblastoma.the malignancy of tumor cells is often closely related to the change of cell proliferation.HDAC9 plays an important role in the regulation of tumor cell proliferation.however,the mechanism of HDAC9 regulation in retinoblastoma is unclear?In this study,the expression of miRNA-101-3p in retinoblastoma was detected by fluorescent quantitative PCR,and the relationship between miRNA-101-3p expression in retinoblastoma and clinicopathological features was clarified.The effects of mi RNA-101-3p on the proliferation of retinoblastoma were confirmed by in vitro and in vivo experiments.the effects of changing the expression of miRNA-101-3p on the proliferation of retinoblastoma cells were observed,and the effects of miRNA-101-3p expression on the proliferation of retinoblastoma cells were further confirmed by animal experiments.And whether EZH2 and HDAC9 are downstream target genes of miRNA-101-3p was verified by luciferase reporter gene analysis,and the effects of EZH2 and HDAC9 protein expression on retinoblastoma cell proliferation were observed by changing the expression of EZH2 and HDAC9 protein in retinoblastoma.Changes in proliferative capacity of retinoblastoma cells were observed by interfering with the expression of EZH2 and HDAC9 in retinoblastoma overexpressed miRNA-101-3p.The effects of mi RNA-101-3p,EZH2 and HDAC9 on the proliferation of retinoblastoma cells were investigated.Methods:1.The expression level of miRNA-101-3p in retinoblastoma tissues resected from January 2012 to October 2017 was detected by fluorescent quantitative PCR,and the experimental results were analyzed statistically.2.The expression of the overexpressed retinoblastoma cells in the tissues of retinoblastoma resected from January 2012 to October 2017 was observed by cc k8 and flow cytometry.The human retinoblastoma model of nude mice was established by stably transfected miRNA-101-3p mimics and untransformed retinoblastoma cells.3.Whether EZH2 and HDAC9 are specific downstream target genes of miRNA-101-3p is further explored through experimental methods such as luciferase reporter genes,and constructed miRNA-101-3p mimics and miRNA-101-3p negative control(NC)are transferred to retinoblastoma cells through transfection reagents,so that the expression of miRNA-101-3p is changed,and the changes of mRNA and protein expression of EZH2 and HDAC9 are detected through qpcr and western blot.4.miRNA-101-3p was expressed in the overexpressed retinoblastoma cells.EZH2 and HDAC9 overexpressed plasmids were transfected respectively.The proliferative ability of retinoblastoma was observed by CCK8 experiment.Results:1.miRNA-101-3p was lower in retinoblastoma than in normal tissues.2.miRNA-101-3p mimics were transfected into WERI-RB-1 cells and Y79 cells,the proliferation of retinoblastoma cells decreased significantly(p < 0.05).Animal experiments showed that the subcutaneous tumors of nude mice transfected with miRNA-101-3p mimics were significantly lower in volume and weight than those of blank plasmid control group(p < 0.05).3.Luciferase reporter genes confirmed EZH2 and HDAC9 as target genes downstream of miRNA-101-3p.The expression of EZH2 and HDAC9 were decreased in WERI-RB-1 cells and Y79 cells were transfected miRNA-101-3p mimics.4.Overexpression of mi RNA-101-3p in WERI-RB-1 cells and Y79 cells,transfection of EZH2 and HDAC9 overexpression plasmid,cc k8 experimental observation found that overexpression of EZH2 and HDAC9 can reverse the proliferation of miRNA-101-3p inhibited retinoblastoma.Conclusion:miRNA-101-3p is poorly expressed in retinoblastoma tissues.miRNA-101-3p inhibits the proliferation of retinoblastoma by targeting EZH2 and HDAC9.the specific mechanism is that EZH2 and HDAC9 are target genes of miRNA-101-3p.