The Role Of LncRAN E2F3SR And MiR-181b In Retinoblastoma Progression

THE ROLE OF LONG NON-CODING RNA E2F3 SR IN RERINOBLASTOMA PROGRESSION OBJECTIVE:Retinoblastoma is a kind of intraocular malignant tumor which reaches The highest incidence among children and 4% of malignant tumor happened in children are retinoblastoma.There are 5000 primary patients of retinoblastoma in China every year and they are 20% of primary patients throughout the world.Patients with retinoblastoma often suffer from blind,loss of eyeball and even death.To investigate the underly mechanism of retinoblastoma progression is significant for the living quality of patients and it can efficiently improve the survival rate of retinoblastoma patients.long non-coding RNA regulates the progession of malignant tumor at different levels,such as epigenetic mechanism,transcription regulation and Post-transcriptional regulation,it has significant biological functions and increasing evidence suggests that aberrant long non-coding RNAs(lnc RNAs)are significantly correlated with the pathogenesis,development and metastasis of cancers.Preliminary study has show that E2F3 SR was such a kind of Lnc RNA which originate from the intron region of E2F3 gene locus.E2F3 SR expression from this region at a high level which indicates that it may play an important role in the initiation and progression of retinoblastoma.However the underly molecular mechanism of E2F3 SR in retinoblastoma is still unkown.In this study,we aimed to explore the potential mechanism by which E2F3 SR promotes RB progression,provide new clinical strategies for retinoblastoma therapy.METHODS:1.The Cancer Genome Atlas data set was interrogated to explore whether E2F3 SR expression levels are associated with the clinicopathological factors and prognosis of patients with retinoblastoma;2.Realtime PCR was uesd to determine the expression level of E2F3 SR in retinoblastoma tissues and cell lines Y79、WERI-Rb-1,normal Retinal pigment epithelium and its cell line ARPR-19 respectively;3.RNA fluorescence in situ hybridization(FISH)analyses and nuclear/cytoplasmic RNA fractionation were performed to determine the subcellular localization of E2F3 SR,the rapid amplification of c DNA ends was performed to determine the real full lenghth of E2F3SR;4.E2F3 SR shRNA was designed and lentiviral vector-mediated shRNA knockdown cell lines sh E2F3SR-Y79 、 sh E2F3SR-WERI-Rb-1 and the corresponding mock cell lines were generated;5.CCK8 assay,colony formation assay,softagar assay and flow cytometry cell cycle were performed to examine the impact of E2F3 SR on cell survival and proliferation of retinoblastoma;the effect of E2F3 SR on tumor growth was also assessed in vivo by a tumor xenograft model in nude mice;6.we used Chromatin Isolation by RNA Purification assay combine with Mass Spectrometer to investigate the proteins or peptides which binding to E2F3 SR.RNA Binding Protein Immunoprecipitation assay was performed to examine the binding vadality between E2F3 SR and the candidate proteins or peptide.7.Microarray analysis was used to elucidate the mechanisms by which E2F3 SR contributes to the progression of retinoblastoma and to find its potential target gene.Chromatin Immunoprecipitation Assay was performed to vadidate the binding region of target gene’s promoter and E2F3 SR and to understand the underly mechanism of how does E2F3 SR promote the progression of retinoblastoma.RESULTS:1.E2F3 SR expression is up-regulated in retinoblastoma;2.High-level of E2F3 SR is correlated with poor prognosis of patients with RB;3.E2F3 SR is located in the nucleus and its full lenghth was 451 bp which was a little bit differ from the data from UCSC database;4.E2F3 SR promotes retinoblastoma cell proliferation and colony formation in vitro and in vivo,knockdown of retinoblastoma promotes cell cycle arrest;5.E2F3 SR was bind to hnRNLP in retinoblastoma.6.E2F3 SR recruits hnRNLP to the promoter region of E2F3 which play a key role in retinoblastoma cell cycle.7.The abundant binding hnRNLP on E2F3 gene’s promoter region promote the activation of E2F3 transcription.CONCLUSIONS:In this study,we verified that the new finding long non-coding RNA E2F3 SR was up-regulated in retinoblastoma and it’s cell lines and plays a vital role in retinoblastoma cell proliferation and survival.Knockdown of E2F3 SR inhibited the growth of retinoblastoma in vitro and in vivo.We showed that this might be partly attributed to E2F3 SR binding to hnRNLP and thus facilitating cell survival.These results suggest that E2F3 SR may be an attractive prognostic factor and therapeutic target in retinoblastoma.THE MECHANISM STUDY OF MIR-181 B IN RERINOBLASTOMA PROGRESSION OBJECTIVE: MiR-181 b have been found to be closely related to tumor formation.Our previous research results identify that MiR-181 b promote UM progression and we also find that MiR-181 b was highly expressed in Rb cell.In this study,we aimed to explore the potential mechanism by which MiR-181 b promotes RB progression.METHODS: Detection of miR-181 expression both in Rb celllines HOX-RB44 and Y79.Overexpression and knockdown of miR-181 in Rb cells by transfecting lentiviral virus to evaluate the growth of Rb.Bioinformatics forecast miR-181’target regulative position on m RNA of NLK.luciferase reporter assay identified that miR-181 negatively regulate the m RNA of NLK by targating it’s 3’UTR.the expression of NLK and it’s downstream Wnt/β-catenin signaling pathway were quantified using real-time PCR and Western blot to identify the role of miR-181 b on Rb growth.RESULTS: miR-181 was significantly upregulated in Rb celllines HOX-RB44、Y79.Ablation of miR-181 inhibited the proliferation of Rb cells and overexpression of miR-181 promote the proliferation of Rb cells.NLK was the direct target of miR-181.High level of NLK inhibited the Wnt/β-catenin signaling pathway and it’s downstream gene β-catenin、cyclin D1、c-myc.CONCLUSIONS: Our analyses demonstrated that miR-181 regulated the progression of retinoblastoma by directly targeting NLK and affecting Wnt/β-catenin signaling pathway.