| Objective:To explore the effect of PGE2 produced by the supernatant of gemcitabine on the stem transformation,cell cycle and clonal ability of pancreatic cancer cells,further explored the mechanism of PGE2 on the stem transformation and proliferation of pancreatic cancer cells.To provide new theoretical support for improving the efficacy of chemotherapy drugs,designing targeted immunotherapy and preventing recurrence of pancreatic cancer? Method:(1)Database analysis of the expression of PTGS2 in pancreatic cancer tissues and paracancer,its impact on patient prognosis.(2)The p53 wild-type pancreatic cancer cell line capan-2 was used as the experimental group,and the first-line chemotherapy drug gemcitabine was used as a chemotherapy drug.Capan-2 was seeded in 96-well plates,and different concentrations of gemcitabine were added.The cell proliferation was detected by CCK8 for 48 hours,and the IC50 of the cells was calculated to obtain the appropriate drug concentration.(3)The cells were treated with the above drug concentration and different concentrations of celecoxib for 48 hours,the medium was changed,and the supernatant was collected after 48 hours of culture.The concentration of PGE2 in the supernatant was detected by enzyme-linked immunosorbent assay(ELISA)and the supernatant was collected for further experiments.(4)Capan-2 cells were seeded in 6-well plates,and different treatment factors were given after cell attachment: medium group(control group);supernatant group;celecoxib group;DMSO group,P53 inhibitor group.After 48 hours of treatment,the cell cycle was analyzed by flow cytometry,and the cell self-renewal ability was detected by cloning experiments.(5)The expression levels of OCT-4,AKT,Sall4 and P53 mRNA were detected by real-time quantitative polymerase chain reaction(qRT-PCR).The expression levels of OCT-4,p-AKT and P53 protein were detected by Western Blot.Result:(1)Database analysis found that the expression of PTGS2 in pancreatic cancer tissues was increased,and it was unfavorable for patients to survive.(2)CCK8 results showed that different concentrations of gemcitabine can significantly inhibit the proliferation of pancreatic cancer cells.(3)ELASA results showed that PEG2 was significantly increased in the supernatant group compared with the control group,while celecoxib significantly inhibited the concentration of PEG2 in the supernatant.(4)The results of cloning experiments showed that compared with the control group,the cell cloning ability of the supernatant group was enhanced(P<0.05),while the cell cloning ability of the celecoxib group was reduced(P<0.05).(5)Compared with the control group,the proportion of G2/M+S phase of pancreatic cancer cells in the supernatant treatment group increased(P<0.05),and the cell proliferation ability increased,while the proportion of G2/M+S phase in the celecoxib treatment group Decreased,cell proliferation ability was reduced(P<0.05).(6)The expression of dry related proteins and genes was significantly increased in the supernatant of the supernatant group(P < 0.05).The expression of dry related proteins and genes in the celecoxib group was reduced(P < 0.05).(7)Further exploration of the mechanism revealed that the expression of p-AKT protein and mRNA was enhanced and the expression of p53 protein and mRNA was decreased in the supernatant treatment group;the expression of p-AKT protein and mRNA was significantly decreased in the celecoxib group,and the expression of P53 protein and mRNA was enhanced(P < 0.05).After the addition of P53 inhibitor,the expression of the stem related protein OCT-4 in the pancreatic cancer cells of the inhibitor group and the supernatant group was significantly increased;the proportion of G2/M+S phase increased(P < 0.05).Conclusions:After chemotherapy,it can cause apoptotic tumor cells to release a large amount of PGE2,thereby activating PGE2/AKT signaling pathway,leading to P53 degradation,promoting tumor cell stem transformation and cell proliferation.Celecoxib can increase the sensitivity of tumor cells to chemotherapeutic drugs. |
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