| Objective:Pancreatic cancer(PC)is known as a fatal malignant tumor accompanied by high mortality and poor prognosis,which has increasingly become a heavy health burden worldwide.Although surgery,chemotherapy,and radiation therapy can mildly alleviate symptoms of patients with PC,PC still faces unsatisfactory clinical outcomes due to the lack of effective diagnosis biomarkers to predict the prognosis and monitor PC at the initial stage.Therefore,it is urgent to seek out other potential therapeutic targets for PC treatment.Overexpression of PD-L1 is reported in PC patients with poor clinical outcomes.PD-L1 acts as a negative regulatory molecule for T cell activation to mediate tumor progression through alteration of immune surveillance status.Recently,programmed death-ligand 1(PD-L1),an immune checkpoint,has been applied for oncotherapy as a therapeutic target for multiple tumors.It is generally known that miRs,can control the expression of multiple target genes,by which modulating various biological processes and immune surveillance.Strikingly,several microRNAs(miRs)have been demonstrated to downregulate PD-L1 thus improving the treatment efficacy of targeted therapy.miR-195-5p has been revealed to be implicated in various human cancers.The tumor suppressive role of miR-195 in PC has been proposed in a previous study.Competing endogenous RNAs(ceRNAs)are regarded as RNA transcripts that proceed mutual communication through the reduction of targeting concentration of miRs.Long noncoding RNAs(lncRNAs)serve as ceRNAs to modulate cancer progression.Currently,a series of lnc RNAs have been proved to be vital in tumor formation in PC.LINC00473 overexpression has been found in tumor tissues that antagonizes the inhibitory role of miR-195 in the pathogenesis of Wilms tumor.There were binding sites between miR‐195‐5p and LINC00473,also miR‐195‐5p and PD‐L1 via the online target prediction.Based upon the above,a hypothesis is presented that LINC00473 sponges miR-195-5p to affect PC development through regulating PD-L1.Therefore,the study was designed to testify this hypothesis to offer a better understanding for the underlying molecular mechanisms in PC progression.Methods:1.The PC tissues were obtained from 134 patients who had received pancreatectomy in the Department of pancreatic oncology of Shengjing Hospital of China Medical University from December,2008 to January,2012.Patients experienced36-month follow-up by the end of December,2014.Afterward,the Kaplan-Meier method was utilized to analyze the survival period.In addition,the normal pancreatic tissues were obtained from 20 patients with benign pancreatic cysts.The resected tissues were preserved in-80°C liquid nitrogen for subsequent analysis.The study was permitted by the Ethics Committee of Shengjing Hospital of China Medical University.2.Immunohistochemistry was used to detect the expression of PD-L1 in pancreatic cancer and normal pancreatic tissues.3.The expression of PD-L1,LINC00473 and miR-195-5p in PC tissues were tested by the reverse transcription quantitative polymerase chain reaction(RT-qPCR).4.The expression of LINC00473 in five PC cell lines was tested by the RT-qPCR,and the cell line with the highest expression of LINC00473 was obtained for the subsequent experiments.5.The targeting relationship between miR-195-5p and PD-L1,miR-195-5p and LINC00473 was detected by the dual luciferase reporter genes.6.Fluorescence in situ hybridization(FISH)technology was applied for visualization of LNC00473 subcellular localization.7.The interaction between LINC00473 and miR-195-5p was assessed by the RNA pull down and RNA immunoprecipitation(RIP)assay.8.The cell treatment was conducted with following mimics,inhibitors or siRNAs:miR-195-5p mimic or its NC,miR-195-5p inhibitor or its NC,PD-L1 inhibitor(Atezolizumab),si-LINC00473,si-LINC00473 NC,LINC00473 NC,LINC00473overexpression vector.9.EdU staining,TUNEL,scratch test and Transwell assay were conducted to detect PC cell proliferation,apoptosis,migration and invasion following the cell treatment.10.The expression of Bcl-2,Bax,MMP-2 and MMP-9 in PC cells were tested by RT-qPCR and Western blot.11.The expression of IL-10,IFN-γand IL-4 in PC cells cocultured with CD8~+T cells were tested by ELISA.12.Annexin V-fluorescein isothiocynate(FITC)/propidium iodide(PI)staining was conducted to detect apoptosis of CD8~+T cells.13.The expression of PD-L1,PD-1 in PC cells were tested by RT-qPCR and Western blot following the cell treatment.Results:1.Immunohistochemical staining results showed that PD-L1 mainly exhibited on the membrane.The positive rate of PD-L1 in the PC tissues(57.69%)was higher than that in the normal pancreatic tissues(23.53%).Besides,the clinicopathological data analysis showed that positive PD-L1 expression was closely related with tumor size,tumor stage,LNM,and distant metastasis(P<0.05).The overall survivals of patients were assessed by Kaplan-Meier analysis,which indicated that patients with PC with positive PD-L1 had shorter overall survival than those patients with negative PD-L1.2.Relative to normal pancreatic tissues,LINC00473 and PD-L1 were elevated,while miR-195-5p was declined in PC tissues(P<0.05).In contrast to the normal pancreatic cell line H6C7,elevated LINC00473 was detected in PC cell lines SW-1990,Panc-1,Bx PC-3,AsPC-1,and CAPAN-2,with the highest LINC00473 expression found in the SW-1990cell line.Hence,SW-1990 cell line was selected for the following cell experiments.3.The luciferase activity detection manifested that miR-195-5p mimic reduced the luciferase activity of PD-L1-WT(P<0.05),implying that miR-195-5p could target PD-L1.The association of mi R-195-5p and PD-L1 was further explored in cells with treatment of miR-195-5p mimic or its NC,Atezolizumab(PD-L1 inhibitor),and miR-195-5p inhibitor or its NC to interfere the expression of miR-195-5p and PD-L1.The expression of miR-195-5p was remarkably increased in the cells introduced with miR-195-5p mimic and obviously depleted in the cells treated with both miR-195-5p inhibitor and Atezolizumab(P<0.05).Thus,miR-195-5p could target and downregulate PD-L1expression.4.FISH was conducted for LINC00473 subcellular localization and LINC00473 was mainly found in the cytoplasm.5.miR-159-5p was speculated to bind to LINC00473 and their interaction was subsequently verified by luciferase activity assay.miR-195-5p mimic declined the luciferase activity of LINC00473-WT(P<0.05),while the luciferase activity of LINC00473-MUT,had no significant change.The results of RNA pull-down showed that LINC00473 expression co-precipitated with WT-miR-195-5p was remarkably increased than that with MUT-miR-195-5p and Bio-NC.RIP further proved that LINC00473 could bind to miR-195-5p.Next,the cells were transfected with or without si-LINC00473.The findings indicated that,the transfection of si-LINC00473 caused an elevated miR-195-5p expression and a decreased PD-L1 level,whereas the transfection of miR-195-5p inhibitor reduced the mi R-195-5p expression and increased the PD-L1 level(P<0.05).All in all,LINC00473 could function as ceRNA to increase the expression of PD-L1 by competitively binding to miR-195-5p.6.EdU staining indicated that the cell viability was significantly reduced by the transfection of miR-195-5p mimic or the treatment of Atezolizumab.Transfection of miR-195-5p mimic and the treatment of Atezolizumab led to an elevated apoptosis and Bax expression whereas reduced Bcl-2 expression(P<0.05).The results on cell migration and invasion showed that cell migration and invasion and the expression of MMP-2 and MMP-9 showed a significant reduction by the treatment of miR-195-5p mimic or Atezolizumab(P<0.05).Therefore,overexpressed miR-195-5p or downregulated PD-L1could facilitate the apoptosis and repress the proliferation,migration and invasion abilities of PC cells.7.To analyze the function of LINC00473 in PC,the loss-of-function and gain-of-function experiments were conducted.The result of EdU detection on cell proliferation showed that the cell viability was remarkably attenuated by the transfection of si-LINC00473 yet obviously increased by the transfection of LINC00473 overexpression vector(P<0.05).The transfection of si-LINC00473 resulted in elevated apoptosis and Bax expression yet reduced Bcl-2 expression,whereas the LINC00473 overexpression caused reduced apoptosis and Bax expression yet increased Bcl-2 expression(P<0.05).Results of the scratch test and Transwell assay on cell migration and invasion showed that the cell migration and invasion and the expression of MMP-2 and MMP-9 were significantly reduced when cells treated with si-LINC00473,but obviously increased in cells with restored LINC00473 expression(P<0.05).Thereby,LINC00473 depletion rescued the function of miR-195-5p on PC,accelerated PC cell apoptosis whereas inhibited its proliferation,invasion,and migration.8.The si-LINC00473 NC,si-LINC00473,LINC00473 NC,LINC00473 overexpression vector,miR-195-5p inhibitor NC,and miR-195-5p inhibitor were,respectively,delivered into the SW-1990 cells which were then cocultured with CD8~+T cells.The levels of cytokines were determined by ELISA.The delivery of si-LINC00473 exhibited a reduced level of pro-tumor cytokine IL-10 yet increased levels of antitumor cytokines IFN-γand IL-4,whereas the delivery of LINC00473,resulted in a higher IL-10 level but lower levels of IFN-γand IL-4(P<0.05).Flow cytometry results revealed that the apoptosis of CD8~+T cells was dramatically attenuated by transfection of si-LINC00473yet elevated by LINC00473 overexpression(P<0.05).The expression of PD-L1 and PD-1was significantly reduced by the transfection of si-LINC00473 whereas markedly increased by LINC00473 overexpression(P<0.05).All in all,it indicated that CD8~+T cell activation could be suppressed by LINC00473 through upregulating the expression of PD-L1 via sponging miR-195-5p.Conclusion:1.Enhanced PD-L1 was suggested to be correlated with a poor prognosis.Relative to normal pancreatic tissues,LINC00473 and PD-L1 were elevated,while miR-195-5p was declined in PC tissues.2.LINC00473 could function as ceRNA to increase the expression of PD-L1 by competitively binding to miR-195-5p.3.Enforced miR-195-5p expression suppresses PC progression through inhibiting PD-L1.4.LINC00473 competitively binds to miR-195-5p to upregulate PD-L1,thereby facilitating the proliferation,migration and invasion and repressing the apoptosis of PC cells.LINC00473 could function as ceRNA to drive the progression of pancreatic cancer.5.LINC00473 competitively binds to miR-195-5p to upregulate PD-L1,thereby inhibiting the activation of CD8~+T cells. |
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