Quantitative Proteomics Analysis In Glioblastoma Cell Lines After LncRNA HULC Silencing

Background and purpose:Glioblastoma multiforme（GBM）,a life-threatening brain tumor,is the most common central nervous system（CNS）tumor in adults.Since it is highly destructive,aggressive and invasive,the patient’s survival is extremely terrible^[1].In the past few decades,we have made a lot of efforts in the treatment of GBM^[6,7],but disappointingly,the cure rate and prognosis are still not satisfactory.Moreover,the efficacy of some new therapies needs to be further evaluated^[4].The treatment of GBM is particularly difficult,not only because the brain tissue for surgical resection is quite limited,but also because most remaining tissue is resistant to radiotherapy or chemotherapy.And it is more worrying that GBM is prone to recurrence in situ.Targeted therapy to tumors is referred to those drugs designed for some specific carcinogenic sites.Since there is no or few effects to other surrounding normal cells,targeted therapy has received a lot of attention.Long noncoding RNAs（lncRNAs）has become one of the emerging hotspot of related research.Highly up-regulated in liver cancer（HULC）,a member of lncRNAs,was first discovered as a result of its high expression in hepatocellular carcinoma^[11,12].It has been proved that the level of HULC in glioma cells is significantly higher than that in normal cells.And Experiments in vitro have further verified that HULC can promote the proliferation of glioma cells^[16].However,there are only a few studies that have focused on HULC’s mechanistic role in GBM.Recently,the vigorous development of genomics and proteomics has brought a breakthrough for seeking biomarkers or drug targets.In the present study,we analyzed the differentially expressed proteins of GBM cells after lncRNA HULC knockdown compared to negative control（NC）in an attempt to find out some targeted proteins or key signaling pathways involved in the pathology of GBM.We hope it might be helpful for the study of the mechanisms and targeted therapy to GBM.Methods:1.Two cell lines of U87 were constructed by vector construction,lentiviral packaging and infection:HULC-si RNA and NC.q RT-PCR was performed to validate the transfection efficiency of HULC silencing vector.2.Mass spectrometry（MS）based on tandem mass tags（TMT）labeling was used to integrate quantitative data and generate proteomic profiles for the two cell lines.GO and KEGG pathway enrichment analyses were used to identify differentially expressed proteins.3.Western blot was performed to verify the difference in the expression level of key target proteins between the two cell lines.4.Colony formation,Transwell,and wound-healing assays were used to investigate the functional effects of HULC knockdown on U87 cells.Results:1.The relative level of lncRNA HULC in HULC-si RNA was significantly lower than that in NC detected by q RT-PCR,suggesting that the two stably-transfected cell lines were successfully constructed.2.We identified 112 up-regulated proteins and 24 down-regulated proteins from a total of4,360 quantified proteins.GO enrichment illustrated that these proteins were mainly involved in organelle structure,catalysis,cell movement,and material metabolism.KEGG pathway analysis indicated that some of these proteins were significantly enriched in tight junction,metabolic pathways,and arachidonic acid metabolism.By searching for shared proteins in significantly enriched pathways,it is further found that PLA2G4A might be a target of HULC.3.PLA2G4A was a shared protein in several enriched pathways.HULC silencing significantly down-regulated the expression of PLA2G4A using Western blot.4.Compared with NC,the colony formation rate,invasive cells and the wound healing rate in HULC-si RNA were significantly decreased by colony formation,Transwell,and wound-healing assays.They demonstrated that HULC knockdown inhibited the proliferation,invasion,and migration of GBM cells probably by targeting PLA2G4A.Conclusion:Lnc RNA HULC changed the proteomic characteristics of U87 cells.Specifically,HULC knockdown might target PLA2G4A to alter the behaviors of GBM cells.This study provides a new perspective on the mechanisms and potential drug targets of GBM treatment.