Effect Of FGF21 On The Proliferation,Autophagy Of Prostate Cancer Cells Under High Glucose Environment And Its Mechanism

PART Ⅰ EXPRESSION LEVELAND CLINICAL SIGNIFICANCE OF FGF21 IN PROSTATE CANCERObjective: To investigate the expression level of FGF21 in prostate cancer and its relationship with the clinicopathological characteristics of prostate cancer patients.Methods: Immunohistochemistry was used to detect the expression of FGF21 in 42 cases of prostate cancer patient tissues and 24 cases of benign prostatic hyperplasia patient tissues.The correlation between the expression level of FGF21 in prostate cancer patient tissues and the clinicopathological features of prostate cancer was analyzed by statistical test.The m RNA and protein expression levels of FGF21 in normal prostate epithelial cells(RWPE-1)and prostate cancer cell lines(LNCaP,PC3,DU145,and 22RV1)were detected by real-time quantitative PCR and Western blotting,respectively.Results: Immunohistochemical staining results showed that the positive rate of FGF21 in PCa and BPH specimens was 38.1%(16/42 cases)and 70.8%(17/24 cases),respectively,and the expression difference was statistically significant(p<0.05).The expression of FGF21 protein and the clinicopathological characteristics of prostate cancer patients showed that the clinicopathological stage and Gleason score were significantly correlated with the expression of FGF21(p<0.05).But Age,PSA and lymph node metastasis were not significantly correlated with FGF21expression(p>0.05).The results of real-time fluorescence quantitative PCR and Western blotting showed that compared with normal prostate epithelial cells RWPE-1,the expression level of FGF21 in prostate cancer cells LNCaP,PC3,DU145 and decreased significantly(p<0.01).Conclusion: FGF21 is low expressed in prostate cancer patients and is closely related to the clinicopathological characteristics of prostate cancer patients.PART Ⅱ EFFECTS OF FGF21 ON THE PROLIFERATION,APOPTOSIS,MIGRATION AND INVASION OF PROSTATE CANCER CELLS UNDER HIGH GLUCOSE ENVIRONMENTObjective: To investigate the effect of FGF21 on the proliferation,apoptosis,migration and invasion of prostate cancer cells under high glucose environment.Methods: The pFGF21 and the control plasmid pN1 were extracted to transfect LNCaP cells of prostate cancer.The normal glucose environment of cell growth was simulated with 5m M glucose concentration as Control group and the high glucose environment of cell growth was simulated with 25 m M glucose concentration as HG group.The experiment was divided into the following four groups(Control-pN1,Control-pFGF21,HG-pN1,HG-pFGF21).CCK-8 and colony formation assay was used to detect the effect of FGF21 on proliferation of LNCaP cells in prostate cancer.The effect of FGF21 on apoptosis of LNCaP cells in prostate cancer was analyzed by flow cytometry.The effect of FGF21 on prostate cancer LNCaP cell migration and invasion were determined by the scratch assay and transwell matrigel invasion assay,respectively.The effect of FGF21 on tumorigenic ability of prostate cancer LNCaP cells was detected by subcutaneous tumorigenicity test in nude mice,and the effect on body weight,tumor volume and tumor weight of nude mice was analyzed by statistical test.The m RNA and protein expression levels of FGF21,Ki67 and BCL2 were detected by real-time quantitative PCR and Western blotting.Results: pFGF21 and the control plasmids pN1 were successfully extracted,and LNCaP cells of prostate cancer were successfully transfectd.CCK-8 and colony formation experiments showed that high glucose promoted the proliferation of prostate cancer cells,while FGF21 could improve the effect of high glucose on the proliferation of prostate cancer cells and inhibit the proliferation of prostate cancer cells.The results of flow cytometry showed that high glucose inhibited the apoptosis of prostate cancer cells,and FGF21 could improve the effect of high glucose on the apoptosis of prostate cancer cells and promote the apoptosis of prostate cancer cells.The results of the scratch experiment and transwell matrigel experiment showed that high glucose promoted the migration and invasion of prostate cancer cells,and FGF21 could improve the effect of high glucose on the migration and invasion of prostate cancer cells.Subcutaneous tumorigenicity test in nude mice results showed that the weight of the nude mice did not significantly change in four groups,pFGF21 is not toxic to nude mice;compared with the HG-pN1 group,the tumor volume and tumor weight of the HG-pFGF21 group were significantly reduced(p<0.01).Real-time quantitative PCR and Western blotting results showed that when FGF21 was overexpressed,compared with the HG-pN1 group,Ki67 and BCL2 expression were decreased in the HG-pFGF21 group(p<0.05).Conclusion: FGF21 inhibited the proliferation,migration and invasion of LNCaP cells in prostate cancer and promoted the apoptosis of LNCaP cells.PART Ⅲ EFFECT OF FGF21 ON THE AUTOPHAGY OF PROSTATE CANCER CELLS AND ITS MECHANISM UNDER HIGH GLUCOSE ENVIRONMENTObjective: To investigate the effect of FGF21 on the autophagy of prostate cancer cells and its mechanism under high glucose environment.Methods: The effect of FGF21 on autophagosome formation of prostate cancer LNCaP was detected by immunofluorescence and transmission electron microscopy.m TOR activator MHY1485(2u M,6h)was used to treat LNCaP cells of prostate cancer to explore the mechanism pathway of FGF21 affecting autophagy of prostate cancer cells under high glucose environment.The m RNA and protein expression levels of FGF21,LC3 B,P62 were detected by real-time quantitative PCR and Western blotting.Results: The results of immunofluorescence experiment and transmission electron microscopy showed that high glucose inhibited the formation of autophagosomes,and FGF21 could improve the effect of high glucose on the formation of autophagosomes in prostate cancer LNCaP cells and promote the formation of autophagosomes.Real-time quantitative PCR and Western blotting results showed that when FGF21 was overexpressed,compared with the HG-pN1 group,Ki67 and BCL2 expression were decreased in the HG-pFGF21 group(p<0.05).Western blotting results showed that Akt-m TOR pathway related proteins were low expressed in in the HG-pFGF21 group compared with the HG-pN1 group.When MHY1485 was added in the high glucose environment,the expression level of Akt-m TOR pathway related proteins was significantly increased(P<0.01),and the effect of FGF21 on LNCaP cells was weakened in the high glucose environment with significant significance(P<0.01).Conclusion: FGF21 promotes the autophagy of prostate cancer cells by inhibiting Akt-m TOR pathway under high glucose environment.