LncRNA ANRIL Co-operated With PRC-mediated DNA Damage Repair Enhance Gemcitabine Chemoresistance In Pancreatic Cancer

PurposePancreatic cancer is one of the most deadly malignancies,The 5-year overall survival rate was 8.Because of the lack of diagnostic symptoms early in the disease,When diagnosed with pancreatic cancer,between 80%and 85%of patients lost surgical opportunities^[1-2].For these unresectable pancreatic cancer patients,Chemotherapy（mainly based on the combination of gemcitabine and gemcitabine）is essential^[3-4].Gemcitabine is a pyrimidine metabolite,the main metabolism in vivo is active diphosphate（d Fd CDP）and triphosphate nucleoside（d Fd CDP）,First,it causes DNA damage inside the cell,thus causing pancreatic cancer cell death through anti-proliferation and pro-apoptosis.However,most of them have a very poor prognosis,the overall survival of chemical resistance is less than 1 year^[5-6].Therefore,to elucidate the molecular mechanism of chemical resistance is an important way to improve the prognosis of pancreatic cancer patients.The causes of chemotherapeutic resistance in pancreatic cancer are very complex.Current studies indicate that it may be to apoptosis,^[7]related to EMT^[8]、epigenetic changes and pancreatic cancer stem cell（PCSCs）formation^[9],Abnormal activation and enhancement of DDR ability is an important basis for tumor resistance to chemotherapy.Previous research has found that LncRNA ANRIL plays an important role in the development of pancreatic cancer by promoting EMT behavior,PRC family is involved in DDR^[10],in some tumors EZH2 and Ring1B can aggregate to the site of dna damage to promote repair.LncRNA plays an important role in tumorigenesis and development,^[11].LncRNA It can form a scaffold that organizes protein complexes to form functions.This study deeply explored the role and mechanism of ANRIL combination PRC promoting gemcitabine resistance in pancreatic cancer cells,as well as the interaction mechanism of the three in the process of chemotherapy-induced dna injury repair,and provided new targets and strategies for the treatment of pancreatic cancer.Methods1 ANRIL is overexpressed in pancreatic cancer cells with resistant gemcitabineGemcitabine-resistant pancreatic cancer cells Panc-1-GR and Bxpc-3-GR were induced by low concentration increment combined with high dose intermittent shock in vitro;The expression of E-cadherin and Vimentin in Panc-1-GR/Panc-1 and Bxpc-3-GR/Bxpc-3 were detected by Western blots;incubate cells with different concentrations of gemcitabine for 48 h,Growth inhibition assay using Cell Counting Kit-8 reagents according to the manufacturer’s protocol,Panc-1-GR/Panc-1,received IC50 value of Bxpc-3-GR/Bxpc-3 cells;The expression of ANRIL in Panc1-GR/Panc-1cells was detected using a partially complementary locking nucleic acid（LNA）probe;Real-time fluorescence quantitative PCR detection of ANRIL expression in Panc-1-GR/Panc-1 cells.2 ANRIL enhancing gemcitabine resistance in pancreatic cancerThe stable cell line with ANRIL knockdown was constructed by gene knockdown technique;the expression of ANRIL was RT-PCR detected with the increase of gemcitabine drug concentration;the sensitivity of pancreatic cancer cells to gemcitabine after ANRIL knockdown was detected by growth inhibition test;the effect of clone formation test on the proliferation of pancreatic cancer cells after knockout;Western blot detection ANRIL effect of gemcitabine on dna injury of pancreatic cancer cells after knockout,comet assay,laser confocal detection ANRIL injury and repair of pancreatic cancer cells after knockout.3 ANRIL combined EZH2、Ring1B promotes chemoresistance by enhancing DDR of pancreatic cancer cellsRIP and protein immunoprecipitation experiments confirmed that ANRIL can bind specifically to Ring1B、EZH2;cloning and growth inhibition experiments detected the sensitivity and proliferation ability of Panc-1 cells to gemcitabine after knockout;expression of Ring1B、EZH2 and the effect of gemcitabine on pancreatic cancer cell dna injury after knockout;comet assay,laser confocal detection and damage and repair of pancreatic cancer cells after knockout.4 ANRIL combined with PRC can enhance DDR of pancreatic cancer cells by regulating RAD51 and BRCART-PCR was used to detect the effects of Panc-1-GR/Panc-1,Bxpc-3-GR/Bxpc-3and when ANRIL,Ring1B and EZH2 were knocked out,respectively,on BRCA,RAD50,RAD51,MRN,XRCC2,KU70,KU80 in HR and NHEJ repair pathways.The expression of BRCA,RAD50 and RAD51 after ANRIL,Ring1B and EZH2 elimination were detected by Western blotting.Results:1 The pancreatic cancer cell line Panc-1-GR/Bxpc-3-GR with resistant gemcitabine was constructedThe gemcitabine IC50 value of Panc-1-GR was 10 times that of Panc-1,and the gemcitabine IC50 value of Bxpc-3-GR was 8 times that of Bxpc-3.Vimentin was up-regulated and E-cadherin was down-regulated in both Panc-1-GR and Bxpc-3-GR.2 ANRIL enhances gemcitabine resistance in pancreatic cancerCompared with Panc C-1 and Bxpc-3,the expression of ANRIL in Panc-1 GR and Bxpc-3-GR was significantly increased.With the increase of gemcitabine concentration,the expression level of ANRIL also increased gradually.After ANRIL knockout,pancreatic cancer cells were more sensitive to gemcitabine.After the elimination of ANRIL,gemcitabine caused more serious DNA damage and showed weaker DNA repair ability in pancreatic cancer cells.3 Anril combined with EZH2 and Ring1B enhances DDR of pancreatic cancer cells to promote chemotherapy resistanceANRIL can specifically bind to Ring1B.After EZH2 and Ring1B deletion,pancreatic cancer cells were more sensitive to gemcitabine and caused stronger DNA damage,with significantly reduced repair ability and proliferation ability.4 ANRIL combined with EZH2 and Ring1B can enhance DDR of pancreatic cancer cells by regulating RAD51 and BRCAAfter the elimination of ANRIL,EZH2 and Ring1B,the expression levels of BRCA1,BRCA2 and RAD51 were significantly decreased.Conclusions:LncRNA ANRIL can joint EZH2,Ring1B jointly promote pancreatic cancer chemotherapy of gemcitabine resistance,through the three HR pathway of BRCA1 and BRCA2,RAD51,when pancreatic cancer cells treated with gemcitabine,collaborative ability of pancreatic cancer cell’s DNA repair,inhibits cell apoptosis,subsequent to pancreatic cancer cells to gemcitabine resistance.This study provides a new target for the treatment of PDAC,a new biomarker for the prognosis of PDAC treated with gemcitabine,and a new theoretical basis for chemotherapy resistance of PDAC patients with gemcitabine.