| Objective: Nrf2,a key factor in cell redox reaction,the role of which played in the prevention and treatment of cancer is a focus of research.However,due to the special dual role of Nrf2,the mechanism of its role in the progression of prostate cancer is still unclear.Glogi phosphoprotein-3(GOLPH3),which is located in Golgi body,is highly correlated with the evolution of prostate cancer,promoting the transformation of prostate cancer from hormone dependence to castration-resistance.The increased expression of GOLPH3 indicates the enhancement of prostate cancer.This thesis intends to explore the effect of Nrf2 on the proliferation of prostate cancer cells and its mechanism,through the perspective of redox reaction and GOLPH3.Methods:(1)Hormone-dependent human prostate cancer cell LNCa P,castration-resistant human prostate cancer cell DU-145 were cultured,observe the condition of their growth under microscope.(2)The expression of Nrf2 protein in LNCa P and DU145 was detected by Western blot.(3)Flow cytometry was used to detect the contents of reactive oxygen species(ROS)in LNCa P and DU145 cells.(4)The recombinant plasmid p EGFP-Nrf2 and the no-load plasmid p EGFP-N1 were transiently transfected into LNCa P and DU145 cells,forming LNCa P-Nrf2-OE,LNCa P-Con,and DU145-Nrf2-OE,DU145-Con respectively.(5)Cell wax blocks were prepared from LNCa P and DU145 with heavy plasmid and no plasmid,and the expression of GOLPH3 protein in the two groups was detected by immunohistochemistry(IHC).(6)The expression of GOLPH3 protein in LNCa P-Nrf2-OE,Nrf2-Con,and DU145-Nrf2-OE,DU145-Con was detected by Western Blot.(7)Real-time fluorescence quantification(RT-PCR)was used to detect the effects of Nrf2 overexpression on the expression of GOLPH3 m RNA in LNCa P-Nrf2-OE,Nrf2-Con,and DU145-Nrf2-OE,DU145-Con.(8)Keap1 wereoverexpressed in LNCa P-Nrf2-OE and DU145-Nrf2-OE and LNCa P-Nrf2-OE-Keap1 and DU145-Nrf2-OE-Keap1 were bulit,and the inhibition of GOLPH3 expression were used to establish LNCa P-Nrf2-OE-GOLPH3 L and DU145-Nrf2-OE-GOLPH3 L,and the growth curves of LNCa P and DU145 with heavy load plasmid,no-load plasmid,Keap1 overexpression and GOLPH3 inhibition were plotted by MTT method.(9)Cell colony formation experiments by Soft AGAR were performed to detect the differences in the cloning and formation rates of LNCa P group and DU145 group with heavy load plasmid,no-load plasmid,Keap1 overexpression and GOLPH3 L LNCa P group and DU145 group,respectively.Results:(1)DU145 cells grew faster than LNCa P cells.(2)Western blot results showed that the expression of Nrf2 protein in LNCa P cells was higher than that of DU145.(3)The content of ROS in LNCa P cells was higher than that in DU145.(4)The results of IHC and Western blot showed that the overexpression of Nrf2 could reduce the expression of GOLPH3 protein in LNCa P-Nrf2-OE and DU145-Nrf2-OE.(5)RT-PCR results showed that Nrf2 overexpression could reduce the expression of GOLPH3 m RNA in LNCa P-Nrf2-OE and DU145-Nrf2-OE.(6)The results of MTT assay showed that the overexpression of Nrf2 significantly slowed the growth rate of prostate cancer cells,and the overexpression of Keap1 significantly increased the cell growth rate.The effect of Keap1 on LNCa P-Nrf2-OE was more obvious than that of DU145-Nrf2-OE,and GOLPH3 L significantly increased the cell growth rate.(7)The results of soft AGAR cell colony formation showed that overexpression of Nrf2 could inhibit cell colony formation,Keap1 could inhibit the action of Nrf2 to promote cell colony formation,Keap1 had a more obvious effect on LNCa P-Nrf2-OE than DU145-Nrf2-OE,and GOLPH3 L significantly promoted the cell colony formation of LNCa P-Nrf2-OE and DU145-Nrf2-OE in vitro.Conclusion: Nrf2 inhibits the proliferation and colony formation of prostate cancer cells by maintaining intracellular redox homeostasis and reducing the expression of GOLPH3. |
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