| Pancreatic ductal adenocarcinoma(PDAC)is a kind of deadly tumor that account for more than 90%pancreatic cancer.The pathogenesis of PDAC is concealed,its progress is rapid.The average survival time is less than two years after surgery,and the 5-year survival rate is only about 8%.Therefore,it is so urgent to study deeply about the molecular mechanism of PDAC and develop effective therapeutic interventions or drugs.Recent studies have shown that protein methylation was the same as acetylation and phosphorylation in the progress of cancer.Protein arginine methyltransferase enzyme 5(PRMT5)is an important member of arginine methyltransferase family(PRMTs),it can symmetrically dimethylate arginine of multiple histones and non-histones.PRMT5 is highly expressed in many types of cancer and can promote the disease progression.However,the role of PRMT5 in PDAC is still unknown.Firstly,we detected the expression level of PRMT5 between normal pancreatic tissue and pancreatic cancer tissues,also detected between HPNE and PANC1,MiaPaCa2 cell lines.Subsequently,we amplified PRMT5 by PCR and made shRNA,shPRMT5;pLVX-control,pLVX-PRMT5 lentiviral virus.We used these virus to infect PANC-1 cells and got PRMT5 related stable cell lines.We had studied the different expression of PRMT5 how to impact the characteristic of PANC-1,such as NF-κB activity experimental,qRT-PCR detected the expression level of TNF-α and IL-8 genes,CCK-8 for cells proliferation,Transwell for cells invasion.In addition,because the NF-κB signal pathway palys a important role in normal cells,so it is not good idea about directly inhibiting the NF-κB activity for curing PDAC.but if we use miRNAs or drugs that inhibit the expression or methyltransferase activity of PRMT5 in order to indirectly inhibit the NF-κB activity,it should be better.miRNAs are a class of conservative,non-coding RNAs that regulate genes expression by binding with the 3’untranslated region(UTR)of target genes,result in the degradation of target genes mRNA or repression of their translation.In recent years,several studies have shown that many miRNAs were involved in the pathogenesis of PDAC.And miRNAs can inhibit the expression of PRMTs in several kinds of cancer.However,until now,there is no paper published about the interaction between miRNAs and PRMTs in PDAC.In this study,we firstly selected sereval miRNAs affected by PRMT5 expression through miRNA-seq experiment.And using bioinformatics predicting software Targetscan and published literatures,we decied to choose miR-485-5p as a candidate miRNA.After respectively transfecting miR-485-5p mimics or NC into PANC-1 cells,we detected the expression level of miR-485-5p to verify successfully transfection.We also detected the mRNA and protein expression levels of PRMT5 between miR-485-5p group and NC group.And such as NF-κB activity experimental,the expression level of TNF-α and IL-8,CCK-8 for cells proliferation,cells invasion.In addition,dual luciferase reporter assay and WB analysis were generated to validate whether miR-485-5p could inhibit PRMT5 expression.and also did mutation experiment,recovery experiment for further verification.In addition,we used AlphaLISA to high-throughput screen drugs that specifically inhibit the methyltransferase activity of PRMT5 in PANC-1 cells.we purified and identified PRMT5 protein in 293-PRMT5-Flag cells.We also detected the sensitivity of AlphaLISA method by calculating Z value.In order to accelerate the approval and clinical application of drugs in the future,the library we selected is composed of drugs that have been approved by the U.S FDA.Because some drugs that have color themselves maybe produce signal and make false positive results,we carried out the second round of screening.There was a top hit from Candesartan cilexetil as a candidate drug.We detected the expression level of PRMT5 in cells treated with or without Candesartan by WB.We measured the IC50 of Candesartan in HPNE and PANC-1 cells.And such as NF-κB activity experimental,the expression level of TNF-α and IL-8 genes,anchorage-independent growth assay,3D colony formation assay,subcutaneous xenograft model of PDAC with Candesartan.The research of this project reveal the role of PRMT5 in PDAC,and screen miRNAs that inhibit the expression level of PRMT5 and drug that inhibit the methyltransferase activity of PRMT5.It provides a new method and reference for treatment of PDAC.1.The main results were as following:1.1 High expression level of PRMT5 protein in PDAC tissue samples and cell lines.Immunohistochemical staining and WB results showed that there was higher expression level of PRMT5 protein in PDAC tissues and cells(PANC-1,MiaPaCa2)than in normal human pancreatic tissues and cells(HPNE).1.2.After establishing pLVX-PRMT5 and shPRMT5 stable cell lines,the results shown that PRMT5 promoted the progress of PDAC.Preparation of pLVX-PRMT5 and pLVX-control,as well as the shscramble and shPRMT5 lentivirus infection solution in order to get corresponding PRMT5 related stable cell lines.Compared with pLVX-control cells,the activity of NF-κB,the expression level of TNF-α and IL-8 genes were increased in pLVX-PRMT5 cells.The proliferation and invasion ability of PANC-1 cells was higher.Conversely,there was a opposite tendency in shPRMT5 cells compared with shscramble cells.1.3.After screening miRNAs that inhibits PRMT5 expression in PDAC,miR-485-5p showed anti-tumor effect in PDAC cellsBased on the results of miRNA-sequencing and TargetScan,we chose miRNA-485-5p for further study.After transfecting miR-485-5p mimics or NC into PANC-1 cells respectively,Compared with NC group,NF-κB activity level,TNF-a and IL-8 genes expression level,proliferation and invasion ability of PANC-1 cells were all decreased in miR-485-5p group.1.4 miR-485-5p inhibits the expression level of PRMT5 by targeting its 3 T-UTRCompared with NC control group,the expression level of PRMT5 mRNA and protein respectively decreased in miR-485-5p group as determined by qRT-PCR and WB;Dual luciferase reporter assay,mutation experiment and rescue assay results showed that miR-485-5p could inhibit the expression level of PRMT5 by targeting its 3 ’-UTR through seed sequence,1.5.Candesartan inhibits the methyltransferase activity of PRMT5 and the progress of PDACPRMT5 protein was purified and used for establishing AlphaLISA in order to identidy potential drugs.Finally,based on the Z value of AlphaLISA,and the results of quenching experiment.Candesartan cilexetil was selected as a new inhibitor of PRMT5 in PDAC;MTT assay showed that Candesartan had a lower toxicity in HPNE cells(IC50=70 μM)than in PANC-1 cells(IC50=18 μM).Compared with PANC-1 cells treated without Candidatan,in PANC-1 cells treated with Candesartan,the level of p65-R30me2 protein obviously decreased.the activity of NF-κB was negatively correlated with Candesartan in a dose dependent manner.TNF-a and IL-8 genes expression level were decreased.the anchorage-independent growth and 3D cloning formation ability of PANC-1 cells were negatively correlated with drug concentration in a dose dependent manner.Body weight of PDAC mice were not affected by Candesartan injection.Candesartan(10mg/kg,20mg/kg)could obviously inhibit tumor growth in PDAC mice in vivo compared to the control group(Omg/kg).PDAC mice treated with 20mg/kg candesartan showed better efficacy.2.Conclusion2.1 Compared with normal pancreatic tissues and cell lines,the expression level of PRMT5 in PDAC patient tissues and cell lines were higher.PRMT5 enhanced the activity of NF-κB pathway,resulting in elevated expression of TNF-a and IL-8 genes,increased the proliferation and invasion ability of PDAC cells.These datas indicated PRMT5 could promote PDAC progression.2.2 miR-485-5p could inhibit the expression level of PRMT5 and show an anti-tumor effect in PDAC.2.3 Candesartan could improve the progression of PDAC by inhibiting the methyltransferase activity of PRMT5. |
