MiR-4516 Promotes The Growth Of Retinoblastoma By Targeting PTEN Through The AKT Signaling Pathway

BcakgroudRetinoblastoma(RB)is the most common intraocular malignant tumor in children,which usually occurs in infants under 5 years old.The neonatal morbidity rate is 1/16000-18000.In China,there are about 1100 newly diagnosed children every year.The most common clinical symptoms of children with RB are white pupil and strabismus.Delayed diagnose due to lack of self-conscious symptoms in the early stage.Most of the patients in China have developed to stage D,stage E or even extraocular stage,which losts the best change for treatment,causing great harm to the children and their families,and a huge economic burden on the society.At present,it is believed that the development of retinoblastoma is related to the double mutation of Rb1 allele,however,it has been found that the inactivation of Rb1 gene is not only one reason leading to retinoblastoma.More and more evidence shows that other genes(such as MYCN)and epigenetic changes---non-coding RNA,are necessary for the development of RB.MicroRNAs(miRNAs)is a group of endogenous,non-coding short-stranded RNAs,that inhibits the translation of target genes by interacting with the 3'UTR of target gene m RNA,similars to proto-oncogenes or tumor suppressor genes in human tumors.It has been found that miRNA has high sensitivity and specificity in the diagnosis of many kinds of tumors.The abnormal expression of miRNAs and its target genes in RB will provide a new direction for the study of molecular mechanism of retinoblastoma,diagnosis and treatment.We use gene chip screening technique to dynamically observe the changes of serum miRNA in patients with RB before treatmrnt with intravenous chemotherapy of VEC regimen,after one course of chemotherapy and after 3 courses of chemotherapy.It was suggested that the expression of mi R-4516 was down-regulated after treatment,and miR-4516 may be related to the development of RB.It has been reported that miR-4516 is abnormal expressed in thyroid papillary carcinoma,small cell lung cancer,liver cancer,prostate cancer and acute myeloid leukemia.miR-4516 represents a negative prognostic factor in patients with malignant gliomas and acts as oncogenes through regulating PTPN14/Hippo pathway.Therefore miR-4516 may be a new potential therapeutic target for malignant gliomas.As studies have shown that there is a close relationship between miR-4516 and the development of tumors,however,the role of miR-4516 in RB has not been reported.PurposeThe purpose of this study is to explore the effect and mechanism of miR-4516 on the growth of retinoblastoma through basic experiments of tumor tissues and cells.In the first part,the expression of miR-4516 in retinoblastoma and Y79 cells was studied,and the effect of miR-4516 on proliferation and apoptosis of Y79 cells was observed.The second part predicts the target gene of miR-4516 by bioinformatics software and verifies its binding with the target gene.The third part focuses on the regulation that miR-4516 affects target genes and downstream signaling pathway.Methods1.qRT-PCR was used to detect the expression of miR-4516 in RB tumor tissues and RB cells(Y79).2.CCK-8 cell proliferation assay,flow cytometry and Hoechst cell staining were used to detect the effect of miR-4516 on the proliferation and apoptosis of Y79 cells.Western blot was used to detect the effect of miR-4516 on the expression of apoptosis-related protein p53 and Bcl2 in Y79 cells.3.The potential downstream target genes of miR-4516 were predicted by Targetscan,miRWalk and miRanda bioinformatics methods,and the binding between miR-4516 and downstream target genes was verified by dual luciferase reporter gene experiment.4.qRT-PCR and Western blot were used to detect the effect of miR-4516 on the mRNA and protein expression of PTEN gene in Y79 cells.5.CCK-8 cell proliferation assay,flow cytometry and Hoechst cell staining were used to detect the effect of miR-4516 on the proliferation and apoptosis of Y79 cells through target gene PTEN.6.Western blot was used to detect the effect of miR-4516 on the expression of key proteins in PTEN/AKT signal pathway in Y79 cells: PTEN,AKT,pAKT,MDM2,P53,Bcl2.Result1.The expression level of miR-4516 in RB tumor tissue was higher than that in normal retinal tissue(P<0.05),and the expression level of miR-4516 in Y79 cells was higher than that in normal control(P<0.01).2.Inhibition of miR-4516,the proliferation of Y79 cells was inhibited,the apoptosis was increased,the expression of P53 protein was increased,and the expression of Bcl2 protein was decreased.Overexpression of miR-4516 promoted the proliferation of Y79 cells,the apoptosis was decreased,the expression of P53 protein was decreased and the expression of Bcl2 protein was increased.3.Bioinformatics predicted that PTEN is a potential downstream target gene of miR-4516.Dual luciferase reporter gene experiments have confirmed the binding of miR-4516 and 3'UTR of PTEN mRNA(908-915bp).4.After miR-4516 mimics transfection into Y79 cells,the expression of PTEN mRNA and protein was inhibited.5.Co-transfection miR-4516 inhibitor and si-PTEN can reverse the inhibitory effect of miR-4516 inhibitor on the proliferation of Y79 cells,and co-transfection miR-4516 mimics and pcDNA-PTEN can reverse the promoting effect of miR-4516 mimics on the proliferation of Y79 cells.6.Co-transfection miR-4516 inhibitor and si-PTEN can reverse the promoting effect of miR-4516 inhibitor on apoptosis of Y79 cells,and co-transfection miR-4516 mimics and pcDNA-PTEN can reverse the inhibitory effect of miR-4516 mimics on apoptosis.7.After miR-4516 mimics transfected into Y79 cells,the expression of PTEN and p53 decreased,while the expression of AKT,pAKT,MDM2 and Bcl2 increased.Co-transfection miR-4516 mimics and pcDNA-PTEN can reverse the expression of PTEN/AKT signal pathway related proteins induced by miR-4516 mimics in Y79 cells.8.After miR-4516 inhibitor transfected into Y79 cells,the expression of PTEN and P53 increased,while the expression of AKT,pAKT,MDM2 and Bcl2 decreased.Co-transfection miR-4516 inhibitor and si-PTEN can reverse the expression of PTEN/AKT signal pathway related proteins induced by miR-4516 inhibitor in Y79 cells.Conclusions1.miR-4516 is overexpressed in retinoblastoma tissues and Y79 cells.2.miR-4516 promotes the proliferation and inhibits the apoptosis of Y79 cells.3.The target gene of miR-4516 is PTEN.4.miR-4516 acts as oncogenes by regulating PTEN/AKT signaling pathway,inhibits the apoptosis of Y79 cells and promots the proliferation of retinoblastoma.