| Background:Recently,WHO statistics show that the incidence of prostate cancer(PCa)is the second highest among male tumors,with 1.4 million new cases per year worldwide,and its high incidence makes it one of the most common fatal tumor diseases.The incidence of prostate cancer is closely related to geography,and is particularly high in the western countries represented by the United States and Canada,where the lifetime risk of prostate cancer is 12%,much higher than in China.With the widespread use of prostate specific antigen and other screening methods in recent years,the incidence and detection of prostate cancer in China has been increasing year by year,and some studies have found that the tumor is gradually becoming younger.The current researches suggest that age,race,genetics,diet,weight,and smoking are all associated with the development of prostate cancer,but the mechanisms responsible for the development of prostate cancer are still questionable at the molecular level.Thus,largely limitation is still existing in the treatment of prostate cancer.The current treatment including surgical,chemotherapeutic,and endocrine treatment all have their own limitations,so researchers are eager to make progress at the molecular level in order to apply the results in the clinical treatment for the better outcomes.FOXS1 is a member of the FOX protein family.This family is a class of evolutionarily conserved transcription factors named for the DNA-binding domain that contains the forkhead box structure.The FOX family plays a important role in several biological processes including metabolism,apoptosis,development,proliferation,differentiation,invasion and cell immortalization.Current studies have shown that abnormal expression of the FOX protein family is closely associated with the development of many cancers,but the effects on tumor cells vary widely and depend on the type of cell line and the member of the family.For example,FOXF2 inhibited the development of gastric cancer through the FOXF2-IRF2BPL-β-catenin signaling axis,while FOXM1 significantly promoted the invasion,metastasis and EMT of pancreatic cancer and the same is true of FOXS1.FOXS1 overexpression in medulloblastoma inhibits tumor development through the Hedgehog(Hh)pathway,while its overexpression in gastric cancer promotes tumor progression and EMT development.However,there are still no studies showing its effect on prostate cancer cell lines.Hedgehog(Hh)signal pathway is essential in embryonic development,many other studies have shown that this signal pathway is closely related to tumor progression.The HH protein family has a critical role in the Hh signal pathway and its members include SHH,DHH and IHH.SHH is closely associated with cell proliferation,and it agonizes the Hh signaling pathway by binding to the receptor called PTCH.Gli1 is located at the end of the HH cascade response and is an important factor in the Hh signal pathway,which can affect the development of several tumor cell lines.The Hh pathway has an important influence on the development of prostate cancer,and studies have shown that activation of Hh pathway can significantly enhance the malignant biological behaviors such as proliferation and invasion of prostate cancer.Objective:The role of FOXS1 on prostate cancer cell lines is still unclear,and the pathway it participating to regulate the development of prostate cancer is unknown.In this study,we combined TCGA and GEO data analysis with biological experiments to clarify the effect of FOXS1 on the ability of prostate cancer cells in proliferate,invade,migrate and apoptosis,and further investigate whether it exerts biological effects through the Hh signaling pathway.Then we observe the effects of FOXS1 on the biological behavior of prostate cancer cell lines in nude mice.The aim of our research is to identify new targets for prostate cancer therapy and provide new ideas for tumor treatment.Method:In this study,based on TCGA-PRAD and GEO databases,we analyzed the differential genes between prostate cancer and normal prostate tissue using edge R and Wilcoxon rank sum test,and analyzed the relationship between the differential expressed genes and Gleason score,tumor stage,biochemical recurrence and oncogene mutation by KS test to screen the molecules that can be used for the study.The expression of FOXS1 in 86 human prostate cancer tissues and 12 normal prostate tissues was determined by immunohistochemistry(IHC),and the RNA level and protein expression of FOXS1 in RWPE-1,C4-2,C4-2B,PC-3 and DU145 cell lines were measured by real-time quantitative polymerase chain reaction(RT-q PCR)and Western blotting(WB).According to results above,FOXS1 was identified as the target molecule in this study.Combining the results of RT-q PCR and WB,C4-2 cell line was selected to transfected by-FOXS1 plasmid to construct FOXS1 overexpression cell line called C4-2-FOXS1 OE,and DU145 cell line was selected to transfected by small interfering RNA(si RNA)to construct FOXS1 knockdown cell line called DU145-si FOXS1.The expression of FOXS1 in overexpressed and knockdown cell lines was determined by RT-q PCR,WB and Immunofluorescence(IF)to clarify whether the cell lines were successfully constructed for subsequent experiments.After successful construction of the cell lines above,the cell counting kit-8(CCK-8)and the clone formation assay were used to detect the effects on the proliferation with interference FOXS1 gene in prostate cancer cells.Transwell invasion assay was used to detect the effects on the invasion with interference FOXS1 gene in prostate cancer cells,and the wound healing assay was used to detect the effects on the migration with interference FOXS1 gene in prostate cancer cells.The effect of FOXS1 knockdown and overexpression on the apoptosis rate of prostate cancer cells was measured by flow cytometry,and the effects of FOXS1 knockdown and overexpression on EMT in prostate cancer cells were examined by measuring the expression of E-cadherin,N-cadherin,Vimentin and β-catenin by WB.To further clarify whether FOXS1 plays a molecular biological role through the Hh pathway,we measured the expression of Hh pathway star molecules including SHH,PTCH1,SMO and Gli1 by WB,and examined the effect of FOXS1 knockdown and overexpression on the Hh pathway in prostate cancer cell lines.SAG is an agonist of Hh signaling pathway.SAG 100 n M and DMSO were treated in the corresponding groups of cells for 24 h,and then the cell lines were divided into four groups which were called DU145 control(DU145-si Control)+ DMSO,DU145-si Control + SAG,DU145-si FOXS1+ DMSO and DU145-si FOXS1 + SAG.The expressions of SHH,PTCH1,SMO,Gli1,Ecadherin,N-cadherin,Vimentin and β-catenin in the above four groups were measured by WB,and the changes of proliferation,migration and invasion ability of the above four cell lines were also be detected.Thus,we can clarify whether there is a reversible effect of SAG on the knockdown effect of FOXS1 gene to determine whether FOXS1 exerts biological effects through the Hh pathway.12 BALB/c nude mice were randomly divided into 2 groups,and DU145-si Control and DU145-si FOXS1 cell lines were injected into the axillae of two groups nude mice to construct murine xenograft model.IHC was used to measure the expression of FOXS1 and Gli1 in xenograft tumors of two groups,thus verifying the effect of FOXS1 on the Hh pathway and the proliferation ability of prostate cancer cell lines in vivo.Results : In this study,TCGA-PRAD and GEO70770 were analyzed by edge R and Wilcoxon rank sum test,and the results showed that the expression of FOXS1 in prostate cancer tissues were higher than that in normal prostate tissues,and the analysis by KS test revealed that the expression of FOXS1 was positively correlated with Gleason score,tumor stage and the probability of TP53 mutation.Overexpression of FOXS1 was often accompanied by an earlier appearance of biochemical recurrence in prostate cancer.IHC assay showed that the expression level of FOXS1 in human prostate cancer was significantly higher than that in normal prostate tissues,and there was a significant positive correlation between FOXS1 expression level and Gleason score.RT-q PCR analysis showed that the expression level of FOXS1 gene in C4-2,PC-3,C4-2B and DU145 cell lines was significantly higher than that in human normal prostate cell line RWPE-1(P<0.05),and WB results were consistent with RT-q PCR.RT-q PCR,WB and IF were used to determine that C4-2-FOXS1 OE and DU145-si FOXS1 cell lines had been successfully constructed.C4-2-FOXS1 OE cell line showed a decrease in apoptosis rate,a significant increase in cell proliferation,invasion and migration ability,and a decrease in E-cadherin expression and an increase in N-cadherin,Vimentin and β-catenin expression compared with C4-2 Vector cell line.DU145-si FOXS1 cell line showed an increase in apoptosis rate,a significant decrease in cell proliferation,invasion and migration ability,and an increase in E-cadherin expression and an increase in N-cadherin,Vimentin and β-catenin expression compared with DU145-si Control.C4-2-FOXS1 OE cell line showed significantly higher expression of SHH,PTCH1,SMO and Gli1 compared to C4-2 Vector cell line.Expression of SHH,PTCH1,SMO and Gli1 were significantly decreased in DU145-si FOXS1 compared to DU145-si Control.After SAG treatment,the expressions of SHH,PTCH1,SMO,Gli1,N-cadherin,Vimentin and β-catenin were elevated in the DU145-si FOXS1+SAG and DU145-si Control +SAG groups compared with the respective control groups,while the expression of E-cadherin was decreased.The changes in the proliferation,migration and invasion ability of the four groups of cells showed that SAG could partially rescue the effects of FOXS1 knockdown in prostate cancer cell lines.In murine xenograft model,it was found that the xenograft tumor volume,wet weight of nude mice in DU145-si FOXS1 group were lower than those in DU145-si Control group.The IHC results of xenograft tumors showed that the expression of FOXS1,Gli1 in the DU145-si Control group were higher than that in the DU145-si FOXS1 group.Conclusion: In this study,the role of FOXS1 in the development of prostate cancer and its potential mechanism of action were verified for the first time.In vitro,the results showed that overexpression of FOXS1 could enhance the proliferation,migration and invasion of prostate cancer cell lines and promote the development of EMT in prostate cancer cell lines through the Hh signaling pathway.The Hh signaling pathway agonist SAG could partially reverse the inhibitory effect of FOXS1 knockdown on prostate cancer cell lines.The results in vivo were similar with that in vitro,further supporting that high expression of FOXS1 could promote the development of prostate cancer through Hh signaling pathway.Therefore,FOXS1 was expected to be a new target for diagnosis and treatment and provided a new diagnostic and therapeutic idea for prostate cancer. |
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