Study On The Effect Of Gemcitabine On Immune Regulation Of Pancreatic Carcinoma And Its Molecular Mechanism

[Objective]1.Study the immunoregulatory effect of gemcitabine in the treatment of pancreatic cancer and its molecular mechanism;2.Investigate the expression of soluble ULBP2 in serum of patients with pancreatic cancer,and explore the correlation between soluble ULBP2 expression and clinicopathological parameters and prognosis,and to explore the diagnostic value of serum ULBP2 level in pancreatic cancer.[Methods]1.ELISA was used to detect the level of soluble ULBP2 protein in pancreatic cancer cell culture supernatant with or without gemcitabine added,and the level of membrane type ULBP2 protein was detected by flow cytometry.2.Establish of co-culture system with human pancreatic cancer cells and human NK cells,and ananlyze NK celly cytotoxicity against pancreatic cancer with or without gemcitabine via CCK8 assay.3.The effect of gemcitabine on the expression of ADAM10 gene and protein in pancreatic cancer cells was detected by quantitative PCR and western blot.The AD AMI 0 siRNA was further transfected into pancreatic cancer cells.After validation of the down-expression of ADAM10 in pancreatic cancer,the expression of soluble ULBP2 protein from pancreatic cancer cells culture supernatant was analyzed by ELISA,while cell proliferation was detected by CCK8 assay.4.The expression of ADAM10 was determined using immunohistochemical analysis,and the expression of sULBP2 in serum of pancreatic cancer patients was detected by ELISA.Then statistical analysis was used to analyze the correlation between ULBP2 and ADAM10 expression and clinicopathological characteristics5.The expression of cyclic RNA,lncRNA and mRNA in pancreatic cancer cells with or without gemcitabine were analyzed by whole transcriptome sequencing.Further biological analysis was used to classiby the differential gene according to its function and pathway classification,to find differential genes that may be involved in immune regulation.[Results]1.We cultured 2 pancreatic cancer cell lines,PANC-1 and MIA PACA-2 cells and analyzed culture supernatants from the two cell lines.The level of sULBP2 decreased after gemcitabine was added to the culture medium of PANC-1 and MIA PACA-2 cells.FACS analysis showed ULBP2 was expressed on the cell surface on PANC-1 and MIA PACA-2 cells in the membrane form,and gemcitabine upregulated ULBP2 surface expression.Treatment with gemcitabine was observed to have markedly augmented membrane-bound ULBP2 expression and significantly decreased sULBP2 in PANC-1 cells and MIA PACA-2 cells.2.We cultured NK92 cell lines and evaluated the cytotoxicity of NK92 cells to PANC-1 or MIA PACA-2 cells using the CCK-8 assay.We co-cultured NK92 cells and PANC-1 or MIA PACA-2 cells,with or without gemcitabine.Treatment with gemcitabine was shown to enhance NK cytotoxicity to PANC-1 and MIA PACA-2 cells,whereas sULBP2 protein decreased NK cytotocity to PANC-1 cells or MIA PACA-2 cells remarkably.3.We cultured PANC-1 cells and MIA PACA-2 cells with or without gemcitabine.Realtime PCR and western blot results showed that gemcitabine treatment downregulate ADAM10 expression in PANC-1 cells and MIA PACA-2 cells.Then PANC-1 cells or MIA PACA-2 cells were transfected with siRNA against human ADAM 10 or control siRNA.The downregulation of ADAM 10 transcripts and AD AM 10 protein was monitored by real-time RT-PCR and by western blotting,respectively.Knockdown of ADAM10 for PANC-1 cells and MIA PACA-1 cells resulted in 40.95%and 42.7%reduction of sULBP2 levels in the culture medium,respectively4.We next investigated serum levels of ULBP2 by ELISA assay in 45 PD AC patients and 45 healthy individuals,and the sULBP2 levels of PD AC patients were significantly higher(p<0.001)than in healthy controls.Based on ROC analysis of PD AC patients and healthy controls,the cut-off value of 16.11 pg/ml was used to divide the serum sample into groups that were negative or positive for sULBP2.The expression of ADAM10 was determined using immunohistochemical analysis,which showed that ADAM10 staining was mainly located in the cytoplasm of tumor cells with varying staining intensity.A significant difference was observed in the serum ULBP2 levels with regard to the CA199 levels(p=0 013),lymph node metastasis(p=0 009)and overall survival(p=0.045).There was no significant correlation between the ADAM10 expression with age,gender,tumor size,perineural invasion,or lymph node metastasis(P>0.05,respectively).Serum ULBP2 was found to be positively correlate with ADAM10 expression.5.The whole transcriptase sequencing analysis showed that 72 circular RNA were up-regulated and 56 circular RNA were down-regulated;and 1025 mRNA expression was up-regulated,656 mRNA expression was down-regulated.Through the biological analysis,the differential genes were classified according to their function and pathways,and the differential RNA-miRNA interaction was predicted by the popular miRNA target gene prediction software to infer the cyclic RNA that could be used as "miRNA sponge".[Conclusions]1.Gemcitabine inhibits the extracellular shedding of ULBP2 from pancreatic cancer cells.Solube ULBP2 bind 1 activation receptor NKG2D on NK cel through competing with membrane form ULBP2 protein,thus promoting NK cell killing of pancreatic cancer cells.2.Gemcitabine reduce ADAM10 protease expression from pancreatic cancer cells,thereby inhibiting its protein cleavage of ULBP2 protein.ADAM10 siRNA can down-regulate the expression of ADAM10 protein in pancreatic cancer cells and down-regulate the expression of soluble ULBP2 protein in pancreatic cancer cell culture supernatant.3.The expression level of soluble ULBP2 in serum of patients with pancreatic cancer was significantly higher than that of normal healthy volunteers,and correlated with lymph node metastasis and overall survival of pancreatic cancer.The level of ULBP2 in serum was positively correlated with the expression of ADAM10 in tumor tissues.4.The expression of cyclic RNA,lncRNA and mRNA in pancreatic cancer cells with or without gemcitabine treatment was analyzed by the whole transcriptome sequencing.The results showed that 72 circular RNA were up-regulated and 56 circular RNA were down-regulated,and 1025 mRNA expression was up-regulated,656 mRNA expression was down-regulated.Some of them are related to immune tolerance induction and immune cell differentiation regulation,which will be further investigated in the next step.