| Background:Pancreatic ductal adenocarcinoma(PDAC)is a highly lethal malignant tumor in the world.Its clinical manifestations are concealed,its disease progresses rapidly,and its resistance to treatment is severe,resulting in high mortality.Pancreatic cancer is a highly invasive tumor,and almost 90% of patients with pancreatic cancer have metastasis,and the presence of pancreatic inflammation provides a favorable environment for metastasis and infiltration of pancreatic cancer.The inflammatory microenvironment formed by inflammatory cells and inflammatory mediators in pancreatitis promotes the growth,invasion and distant metastasis of pancreatic cancer by activating inflammatory signals NF-k B,STAT3,and COX-2.Fibroblast growth factor 21(FGF21)belongs to the atypical member of the FGF family.Previous studies in our group found that FGF21 was highly expressed in normal pancreatic acinar cells,but was significantly inhibited in pancreatic ductal adenocarcinoma;We speculate that FGF21 may be involved in the development and progression of pancreatic cancer.Subsequently,we reviewed the literature and found that when a variety of damage factors act on mouse acinar cells,FGF21 in pancreatic tissue can rapidly increase and inhibit the production of inflammatory factors and the activation of inflammatory signals in pancreatic tissue to reduce inflammation damage of acinar cells.In other words,FGF21 with high expression levels in normal pancreatic tissue can be further elevated in the inflammatory environment and inhibit pancreatic inflammation,while the expression of FGF21 is low in pancreatic cancer,the specific mechanism is not clear,but we suspect the low expression of FGF21 in pancreatic cancer may weaken the resistance to inflammatory damage,thereby promoting the malignant biological behaviors such as growth,migration and invasion of pancreatic cancer cells.Then,if exogenously increases the level of FGF21 in pancreatic cancer,whether it would inhibit the progression of pancreatic cancer by inhibiting the inflammatory microenvironment of pancreatic cancer.Objective:In order to verify our hypothesis,we firstly investigated whether it can inhibit the migration and invasion behavior of pancreatic cancer cells in inflammatory environment by exogenous administration of FGF21.Secondly,we further explored the molecular mechanism of FGF21 inhibiting the migration and invasion of pancreatic cancer cells.We hope to provide a new idea for the treatment of pancreatic cancer.Methods:The study is divided into two parts:1.FGF21 inhibits the migration and invasion of human pancreatic cancer cells Capan-1 and As PC-1 in an inflammatory environment.(1)Construction of inflammatory environment of human pancreatic cancer cells Capan-1 and As PC-1.The transcription of IL-6 mRNA under different concentrations of lipopolysaccharide(LPS)in Capan-1 and As PC-1 cells was detected by q RT-PCR to screen the optimal concentration of LPS.(2)Effects of FGF21 on migration and invasion of human pancreatic cancer cells Capan-1 and As PC-1.1)The migration of FGF21 to human pancreatic cancer cells Capan-1 and As PC-1 in inflammatory environment was observed by scratch test,and the optimal concentration of FGF21 was screened.Capan-1 and As PC-1 cells were treated with lipopolysaccharide and different concentrations of FGF21.The experimental groups were as follows: Control group,FGF21(1 ug/ml)group,LPS group,FGF21(1 ug/ml)+ LPS group,FGF21(2 ug/ml)group + LPS group,FGF21(4 ug/ml)group + LPS group.The concentration of FGF21 was screened according to the results of the scratch test.2)The effect of FGF21 on the migration of human pancreatic cancer cells Capan-1 and As PC-1 in inflammatory environment was detected by Transwell assay.The experimental groups were Control group,FGF21 group,LPS group,FGF21+LPS group.3)The effect of FGF21 on the invasion of human pancreatic cancer cells Capan-1 and As PC-1 in inflammatory environment was detected by Transwell assay.The experimental groups were Control group,FGF21 group,LPS group,FGF21+LPS group.2.Molecular mechanism of FGF21 inhibiting the migration and invasion of human pancreatic cancer cells Capan-1 and As PC-1 in inflammatory environment.(1)Activation of AMPK/NF-k B signaling pathway in human pancreatic cancer cells Capan-1 and As PC-1 by FGF21.(Experimental group: Control group,FGF21 group,LPS group,FGF21+LPS group)1)The protein expression of P-AMPK,AMPK,P-P65 and P65 in each group was detected by Western Blot.2)The situation of NF-k B P65 transferring into the nuclear in each group was detected by immunofluorescence.3)The transcription of MMP9 and IL-6 m RNA in each group was detected by q RT-PCR.4)Western blot was used to detect the protein expression of MMP9 in each group of cells.(2)Activation of AMPK/NF-k B signaling pathway in pancreatic cancer cells Capan-1 and As PC-1 in inflammatory environment after addition of AMPK inhibitor(Compound C).(Experimental group: LPS group,FGF21+LPS group,FGF21+LPS+Compound C group)1)The expression of P-AMPK,AMPK,P-P65 and P65 protein in each group was detected by Western blot.2)The situation of NF-k B P65 transferring into the nuclear in each group was detected by immunofluorescence.3)The transcription of MMP9 and IL-6 m RNA in each group was detected by q RT-PCR.4)Western blot was used to detect the protein expression of MMP9 in each group of cells.Results:1.FGF21 inhibits the migration and invasion of human pancreatic cancer cells Capan-1 and As PC-1 in an inflammatory environment(1)Construction of inflammatory environment of human pancreatic cancer cells Capan-1 and As PC-1The results of qRT-PCR showed that the expression of IL-6 mRNA in Capan-1 and As PC-1 cells increased with the increase of LPS concentration(P<0.05).(2)Effects of FGF21 on migration and invasion of human pancreatic cancer cells Capan-1 and As PC-11)The results of scratch test showed that the wound closure rate of Capan-1 and As PC-1 cell lines in LPS group was significantly higher than that in Control group and FGF21 group,and the wound closure rate afterFGF21 treatment was significantly reduced,and with FGF21 The concentration of the stains increased and the effect of wound closure was gradually increased(P<0.05).There was no significant difference in the wound closure rate between the Control group and the FGF21 group(P>0.05).2)Transwell migration results showed that the cell migration rates of Capan-1 and As PC-1 cells in the LPS group were significantly higher than those in the Control group and the FGF21 group,while the FGF21+LPS group migrated compared with the LPS group.The rate was significantly lower(P<0.05),and the cell migration rate between the Control group and the FGF21 group was not statistically significant(P>0.05).3)The results of Transwell invasion experiment were consistent with the trend of Transwell migration experiment.The cell invasion rate of Capan-1 and As PC-1 cell lines in LPS group was significantly higher than that in Control group and FGF21 group,while the invasion rate of FGF21+LPS group was significantly higher than that of LPS group(P<0.05).There was no statistically significant difference in cell migration between Control group and FGF21 group(P>0.05).2.Mechanism of FGF21 inhibiting the migration and invasion of human pancreatic cancer cells Capan-1 and As PC-1 in inflammatory environment(1)Activation of AMPK/NF-k B signaling pathway in human pancreatic cancer cells Capan-1 and As PC-1 by FGF211)Western blot results showed that the protein expression of P-AMPK was lower in LPS group and Control group,while P-AMPK was significantly increased in FGF21 group and FGF21+LPS group.There was no significant difference in the expression of P-P65 between Control group and FGF21 group.The protein expression of P-P65 in LPS group was significantly higher than that in Control group,while the protein expression of P-P65 in FGF21+LPS group was significantly lower,The difference was statistically significant(P<0.05).And there was no significant difference in the expression of AMPK and P65 in each group(P>0.05).2)Immunofluorescence results showed that NF-k B P65 transferring into the nuclear was significantly enhanced in the LPS group,and the P65 nuclear transduction after FGF21 treatment was significantly inhibited.3)q RT-PCR results showed that MMP9 and IL-6 m RNA were increased in LPS group,and MMP9 and IL-6 m RNA transcription was decreased in FGF21+LPS group(P<0.05).There was no significant difference between the FGF21 group and the Control group(P>0.05).4)Western blot results showed that compared with Control group and FGF21 group,MMP9 protein expression was increased in LPS group,MMP9 protein expression was inhibited after FGF21 treatment,the difference was statistically significant(P<0.05).There was no significant difference between the FGF21 group and the Control group(P>0.05).(2)Activation of AMPK/NF-k B signaling pathway in human pancreatic cancer cells Capan-1 and As PC-1 by FGF21 after addition of AMPK inhibitor1)Western blot results showed that compared with FGF21+LPS group,the protein expression of P-AMPK was decreased in FGF21+LPS+Compound C group,while the protein expression of P-P65 was increased(P<0.05),were in each.There was no significant difference between AMPK and P65 protein expression in each group(P>0.05).2)Immunofluorescence results showed that NF-k B P65 transferring into the nuclear in the LPS group increased,and decreased after FGF21 treatment,while increased again in the FGF21+LPS+Compound C group.3)q RT-PCR results showed that compared with FGF21+LPS group,MMP9 and IL-6 m RNA transcription increased in FGF21+LPS+Compound C group,the difference was statistically significant(P<0.05).4)Western blot results showed that compared with FGF21+LPS group,the expression of MMP9 protein in FGF21+LPS+Compound C group increased,the difference was statistically significant(P<0.05).Conclusions:FGF21 can inhibit the migration and invasion of human pancreatic cancer cell lines Capan-1 and As PC-1 in inflammatory environment.The possible molecular mechanism is to inhibit NF-k B by activating AMPK,thereby inhibiting the transcription and expression of MMP9 and IL-6 in pancreatic cancer cells.FGF21 plays an important role in the progression of pancreatic cancer in an inflammatory environment,and the detailed mechanism remains to be further studied. |
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