Effect Of FGF21 On Activation Of Stellate Cells In Inflammatory Environment Of Pancreatic Cancer Cells And Its Mechanism

Background: Pancreatic ductal adenocarcinoma(PDAC)is a highly lethal malignant tumor of the digestive system.Its 5-year survival rate has not exceeded 9% in decades.Therefore,finding specific targets for the effective treatment of PDAC has been the direction of clinical and scientific researchers.In our previous study,we found that the expression of fibroblast growth factor 21(FGF21)in pancreatic ductal adenocarcinoma was significantly lower than that in normal pancreatic tissue.After exogenous administration of FGF21,the growth of pancreatic cancer in mice was inhibited,the liver metastasis was significantly reduced and the survival time was prolonged,which suggested that FGF21 could inhibit the growth and metastasis of pancreatic cancer,but the mechanism is not clear.By consulting the relevant literature,we found as follows:(1)FGF21 is an atypical member of the FGF family and has no obvious mitogenic effect.FGF21 plays a role in inhibiting inflammation,pancreatic fibrosis and stellate cell activation in acute and chronic pancreatitis;(2)Inflammation is a common pathological phenomenon of pancreatic cancer and plays an important role in the occurrence and development of pancreatic cancer;(3)Inflammation enhances the activation of pancreatic stellate cells(PSC)by inflammatory mediators such as(IL-6)and growth factors(PDGF,TGF-? and FGF)produced by pancreatic cancer cells(PCC).PCC promotes the proliferation,migration and production of extracellular matrix of PSC;(4)Activated PSC promotes the progression and resistance to chemotherapy and radiotherapy of pancreatic cancer further by promoting the proliferation,migration,metastasis and epithelial interstitial degeneration of PCC and inhibiting its apoptosis.Therefore,we speculate that FGF21 may prevent the growth and metastasis of pancreatic cancer cells by inhibiting the activation of PSC in the inflammatory environment of pancreatic cancer cells.Objective: In order to determine whether FGF21 can suppress the activation of PSC to achieve its prevention effect on pancreatic cancer,we observed the effect of FGF21 on the proliferation and activation of PSC in the inflammatory environment of pancreatic cancer cells and explored its related mechanism.It is hoped that it will provide a new direction for the treatment of pancreatic cancer.Methods: This study is divided into two parts: 1 Inhibitory effect of FGF21 on PSC proliferation and activation in human PCC inflammatory environment(1)Establishment of inflammatory environment in human pancreatic cancer cells Capan-1 and MIA Pa Ca-2 RT-q PCR was used to detect the relative expressions of IL-6 or IL-8 m RNA in Capan-1 and MIA Pa Ca-2 respectively after inducing by CCK for 0,30 min,6h,12 h and 24 h and the optimal effect time of CCK was determined.(2)Effect of FGF21 on the proliferation of human pancreatic stellate cells.The immortalized human pancreatic stellate cells were divided into three groups: control group,Capan-1 or MIA Pa Ca-2 CCK supernatant group and Capan-1 or MIA Pa Ca-2 CCK+FGF21 supernatant group.CCK-8 assay was used to measure the viability of stellate cells in each group.(3)Effect of FGF21 on the activation of human pancreatic stellate cells The immortalized human pancreatic stellate cells were divided into three groups: control group,Capan-1 or MIA Pa Ca-2 CCK supernatant group and Capan-1 or MIA Pa Ca-2 CCK+FGF21 supernatant group.The expression of ?-SMA,an activation index of stellate cells,was detected by Western blot.2 Molecular mechanism of FGF21 inhibiting PSC activation in the inflammatory environment of human pancreatic cancer cells(1)Effects of FGF21 on the expression of EGR1 and PDGFB in human pancreatic cancer cells Capan-1 and MIA Pa Ca-2 in the inflammatory environment 1)The expression of EGR1 and PDGFB m RNA in Capan-1 and MIA Pa Ca-2 CCK group and CCK+FGF21 group were measured by RT-q PCR.2)The expression of EGR1 and PDGFB protein in Capan-1 and MIA Pa Ca-2 CCK group and CCK+FGF21 group were determined by Western blot.(2)Effect of FGF21 on AMPK/EGR1 signal pathway of human pancreatic cancer cells Capan-1 and MIA Pa Ca-2 in the inflammatory environment after adding AMPK inhibitor(Compound C)The expressions of p-AMPK and EGR1 proteins were detected by Western blot in Capan-1 and MIA Pa Ca-2 CCK group,CCK +FGF21 group and CCK +FGF21+Compound C group.Results: 1 FGF21 inhibits the proliferation and activation of PSC in human PCC inflammatory environment(1)Establishment of inflammatory environment in human pancreatic cancer cells Capan-1 and MIA Pa Ca-2 RT-q PCR results showed that the relative expression of IL-6 m RNA in Capan-1 6h group and IL-8 m RNA in MIA Pa Ca-2 24 h group induced by CCK was significantly higher than that in other groups(P<0.05),the difference was statistically significant.(2)Effect of FGF21 on the proliferation of human pancreatic stellate cells CCK-8 assay showed that the proliferation of PSC in Capan-1 and MIA Pa Ca-2 CCK supernatant group was stronger than that in the control group,while Capan-1 and MIA Pa Ca-2 CCK+FGF21 supernatant group could inhibit the promotive effect of CCK supernatant group on the proliferation of PSC(P < 0.05),the difference was statistically significant.(3)Effect of FGF21 on the activation of human pancreatic stellate cells The results of Western blot showed that compared with the control group,the expression of ?-SMA was increased in Capan-1 and MIA Pa Ca-2 CCK supernatant group(P<0.05),while Capan-1 and MIA Pa Ca-2 CCK+FGF21 supernatant group inhibited the promotive effect of CCK supernatant group on the expression of ?-SMA significantly(P <0.05),the difference was statistically significant.2 Molecular mechanism of FGF21 inhibiting PSC activation in the inflammatory environment of human pancreatic cancer cells(1)Effects of FGF21 on the expression of EGR1 and PDGFB in human pancreatic cancer cells Capan-1 and MIA Pa Ca-2 in the inflammatory environment 1)RT-q PCR results showed that the relative expression levels of EGR1 and PDGFB m RNA in Capan-1 and MIA Pa Ca-2 CCK+FGF21 group were significantly lower than those in CCK group(P<0.05),the differences were statistically significant.2)Western blot results showed that the relative expression levels of EGR1 and PDGFB protein in Capan-1 and MIA Pa Ca-2 CCK+FGF21 group were significantly lower than those in CCK group(P<0.05),the differences were statistically significant.(2)Effect of FGF21 on AMPK/EGR1 signal pathway in human pancreatic cancer cells Capan-1 and MIA Pa Ca-2 in the inflammatory environment after adding AMPK inhibitor(Compound C)The results of Western blot showed that compared with Capan-1 and MIA Pa Ca-2 CCK group,the expression of p-AMPK was increased and the expression of EGR1 was decreased(P<0.05)in CCK+FGF21 group,while Compared with CCK+FGF21 group,the expression of p-AMPK was inhibited and the expression of EGR1 was promoted in CCK+FGF21+ Compound C group(P <0.05),the differences were statistically significant.Conclusion: FGF21 may prevent tumor by attenuating the proliferation and activation of stellate cells in the inflammatory environment of pancreatic cancer cells and the mechanism may be that FGF21 inhibits the expression of EGR1 through activating the AMPK pathway of pancreatic cancer cells in inflammatory environment and results the expression of PDGFB decreased.The specific mechanism needs to be further studied.