| Background and Objective:Retinoblastoma(RB)is a kind of primary intraocular malignant tumor,which occurs most frequently in children.Recently,with the development of surgical procedures such as enucleation and various chemotherapy methods,the survival rate of retinoblastoma patients has improved.However,the mortality of RB remains high because of its limited sensitivity to chemotherapy.Therefore,understanding the molecular events of chemoresistance in retinoblastoma and formulating molecular targeted treatment strategies are very important for the treatment of this malignant disease.Long non coding RNAs(lnc RNAs)are a group of non-coding RNAs subsets with no obvious protein coding function,and their lengths exceed 200 nucleotides.lnc RNAs involves in multilevel gene expression regulation,containing transcriptional regulation by recruiting complexes modified by chromatin,and post-transcriptional adjustment through interacting with protein,m RNA or mi RNA..New evidence indicates the viewpoint that lnc RNAs regulate many characteristics of cancer,containing chemoresistance,apoptosis and proliferation.lnc RNA UCA1 is located on chromosome 19p13.12,it was first discovered in the bladder cancer,which facilities the progression of cancer in various tumors through the regulation of proliferation,migration,apoptosis,as well as invasion of cell.More importantly,in addition to its carcinogenic function,lnc RNA UCA1 also regulates drug resistance in various cancers.For instance,lnc RNA UCA1 facilitates the proliferation of cell and leads to 5-fluorouracil resistance in patients with colon cancer.Reduced expression of lnc RNA UCA1 can increase chemosensitivity and apoptosis of TSCC cells induced by CDDP.However,the role of lnc RNA UCA1 in ca RBoplatin resistance is unclear.In the present paper,we investigated the effect of lnc RNA UCA1 on the ca RBoplatin resistance and simultaneously explored the therapeutic importance of RB patients with ca RBoplatin resistant.Methods:1.Retinoblastoma cell lines WERI-RB-1 and Y79 were used to construct subcutaneous tumor-bearing models,and ca RBoplatin treatment was used to construct ca RBoplatin resistant retinoblastoma WCR3 rd and YCR3 rd.The expression of lnc RNA UCA1 in retinoblastoma cell lines Weri-RB-1Y,Y79 and ca RBoplatin resistant retinoblastoma WCR3 rd and YCR3 rd was detected by QRT-PCR.2.Interference technique,plate cloning and flow cytometry were used to observe the changes of cell sensitivity to ca RBoplatin after changing the expression of lnc RNA UCA1 in ca RBoplatin resistant retinoblastoma wcr3 rd and ycr3rd;The effect of changing the expression of lnc RNA UCA1 in ca RBoplatin resistant retinoblastoma on ca RBoplatin sensitivity was observed by subcutaneous nude mouse tumor bearing model;3.The expression of AXL and c-Met in ca RBoplatin resistant retinoblastoma was observed by interference technology,Western blot and QRT PCR.Interference technology,plate cloning and flow cytometry were used to determine whether lnc RNA UCA1 affected the sensitivity of ca RBoplatin resistant retinoblastoma to ca RBoplatin by regulating the expression of AXL and c-Met.4.The distribution of lnc RNA UCA1 in retinoblastoma was detected by QRT PCR.The relationship between lnc RNA UCA1 and mi R-206 was clarified by RNA immunocoprecipitation assay(RIP)and Luciferase Report.To detect the relationship between lnc RNA UCA1 and mi R-206,and to determine whether lnc RNA UCA1 regulates the expression of AXL and c-Met through mi R-206,so as to affect the sensitivity of ca RBoplatin resistant retinoblastoma to ca RBoplatin.Results:1.UCA1 is highly expressed in ca RBoplatin-resistant RB cellsCompared with parental cells,the sensitivity of the third generation retinoblastoma ycr3 rd and wcr3 rd cells to ca RBoplatin decreased significantly.After ca RBoplatin treatment,the apoptosis of ca RBoplatin resistant retinoblastoma cells decreased and the growth ability increased.In order to identify the expression of lnc RNA in ca RBoplatin resistant retinoblastoma cells,I found that the expression of lnc RNA UCA1 in the third generation retinoblastoma ycr3 rd and wcr3 rd cells was significantly increased compared with the parent cells.2.AXL and c-Met are responsible for UCA1-mediated ca RBoplatin resistance in RBWe further sought to identify the mediators of UCA1-driven ca RBoplatin resistance.Growing evidence supports activation of alternative RTKs as playing key roles in drug resistance.Thus,we screened multiple RTKs in resistant cells and found that UCA1 knockdown reproducibly decreased AXL and c-Met at their phosphorylation levels as well as m RNA and protein expression.However,other RTKs,such as the epidermal growth factor receptor(EGFR),was not changed after downregulation of UCA1.Consistently,high expression of AXL and c-Met was observed in the ca RBoplatin-resistant xenografts.Next,we determined whether AXL and c-Met were functionally involved in UCA1-mediated ca RBoplatin resistance.Restoration of AXL and c-Met recapitulated the ca RBoplatin-resistant phenotype in UCA1-knockdown-resistant cells.Conversely,concurrent knockdown of AXL and cMet abolished UCA1 mediated ca RBoplatin resistance with regard to cell viability,while knockdown of each alone had a limited effect.In addition,BMS777607,a smallmolecule inhibitor of both AXL and c-Met,resensitized UCA1-overexpressing cells to ca RBoplatin.Taken together,the genetic and pharmacological data indicate that AXL and c-Met are responsible for UCA1-mediated ca RBoplatin resistance.3.UCA1 functions as a ce RNA for mi R-206 to facilitate AXL and c-Met expression in RB cellsPrevious study has shown that UCA1 functions as ce RNAs for specific mi RNAs,leading to the liberation of corresponding mi RNA-targeted transcripts.To test this hypothesis,we first transfected Y79 cells with Dicer-specific si RNAs to efficiently block mi RNA biogenesis.Intriguingly,UCA1-mediated upregulation of AXL and cMet was abolished when Dicer was knocked down.To confirm the pivotal roles of mi RNAs in this effect we performed an RIP assay on Ago2,which is the core component of the RNA-induced silencing complex.Overexpression of UCA1 in Y79 cells led to the increased enrichment of Ago2 on UCA1 but substantially decreased enrichment on AXL and c-Met transcripts.In parallel,UCA1 knockdown in YCR3 rd cells elicited a significant increase in the recruitment of Ago2 to AXL and c-Met transcripts compared with control cells.These results suggested that UCA1 could compete with AXL and c-Met transcripts for the Ago2-based mi RNA-induced repression complex.Next,we further analyzed the distribution of UCA1 in RB cells.The results demonstrated that UCA1 was existed in both cytoplasm and nucleus in Y79 cells,but the ratio of UCA1 in cytoplasm is higher than that of in nucleus.Morover,we performed a search for mi RNAs that have complementary base pairing with UCA1 by using Sta RBase v2.0.The bioinformatics analysis data revealed that the mi RNA response elements(MREs)of mi R-206 shared by UCA1 and the 3’ UTR of AXL and c-Met.Furthermore,comparable copy numbers of UCA1 and mi R-206 were detected per ca RBoplatin-resistant cell,which is important for ce RNA-mi RNA interaction.Moreover,transfection of mi R-206 mimics significantly inhibited the luciferase activity of p GL3-UCA1/wt,which contained UCA1 at the 3’ UTR of Rluc,while p GL3-UCA1/mut showed no response to mi R-206 mimics.These results demonstrated that UCA1 contained functional mi R-206 binding sites.In addition,inhibition of mi R-206 with the mi R-206 inhibitor was sufficient to restore ca RBoplatin resistance following UCA1 knockdown.Conversely,the introduction of mi R-206 mimics abolished UCA1-mediated ca RBoplatin resistance.Next,we investigated whether UCA1-mediated sequestration of mi R-206 was responsible for the upregulation of AXL and c-Met.p GL3 reporters containing AXL or c-Met 3’ UTR with wild-type or mutant(mut)mi R-206 MREs were constructed.The luciferase activity of AXL and cMet reporters was decreased upon UCA1 knockdown and was rescued by mi R-206 inhibitor,while the luciferase activity of mut reporters was unchanged.Conversely,the luciferase activity of AXL and c-Met wild-type reporters but not the mut ones was elevated upon pc DNA3.1-UCA1 transfection in a dose-dependent manner,whereas mi R-206 overexpression abolished this effect.These results were further confirmed at both the RNA and protein levels of AXL and c-Met.Collectively,these data demonstrated that UCA1 functions as a molecular sponge for mi R-206 to facilitate expression of AXL and c-Met Conclusion:In conclusion,the expression of lnc RNA UCA1 increased significantly in ca RBoplatin resistant retinoblastoma.It was further confirmed that lnc RNA UCA1 promoted the expression of AXL and c-Met in vivo and in vitro,which led to ca RBoplatin resistance of retinoblastoma cells.Mechanism studies confirmed that lnc RNA UCA1 regulates the expression of AXL and c-Met through mi R-206,resulting in resistance of retinoblastoma cells to ca RBoplatin,and lnc RNA UCA1 acts as the ce RNA of mi R-206,thereby regulating the expression of mi R-206 target genes AXL and c-Met. |
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