The Role Of KLF7 In Promoting The Development Of Prostate Cancer By FFAs And Its Molecular Mechanism

Object: Our previous study found that compared with non cancer individuals,the serum octanoic acid(FFA C8:0)content in patients with prostate cancer(PCa)was significantly higher.In this study,we collected the tissue and the data of benign prostatic hyperplasia(BPH)and PCa patient and on the basis of the establishment of obese prostate tumor bearing mice model induced by high fat diet,detect the expression of KLF7,IL-6 and tumor related factors p21,Ki67 in tumor tissue.Analyze the relationship between these factors and the occurrence of PCa.On the basis of human prostate cancer cell lines PC3 and 22RV1 cultured in vitro.Objective to observe whether high level of FFA C8:0 can promote the proliferation,invasion and migration of PCa cells by up regulating the expression of KLF7,and to explore the possible mechanism.Through these studies,we try to clarify the molecular mechanism of obesity induced PCa,and provide theoretical basis for the prevention of obesity induced PCa and the screening of clinical therapeutic molecular targets.Methods: 1.From September 2018 to September 2019,the general data of 60 cases of fresh prostate tissues from patients with BPH(n=10)and PCa(n =13),and 60 cases of paraffin embedded prostate tissues from patients with BPH and PCa were collected in Shihezi people’s hospital.The protein expression levels of KLF7,IL-6,p21,Ki67 and MMP2 were detected by Western Blot and immunohistochemical technique,analyze the correlation among the factors.2.To establish a high-fat diet induced obese mice model with prostate tumor in situ(n=10),with normal diet mice(n=5)as the control,monitor the weight and Lee’s index of mice,to detect the content of blood lipids and Glu;to compare the tumor formation rate of PCA cell PC3-luc in the prostate of two groups of mice by in vivo imaging technology;and to compare the tumor formation rate of PCa cell PC3 in the prostate of two groups of mice by Western blot The expression of KLF7,IL-6,p21 and Ki67 in tumor bearing mice were detected by blot and immunohistochemistry.3.The expression of KLF7 was up / down regulated in PC3 cells,and detectethe expressions of IL-6 and p21.detect The biological behavior of PC3 cells.4.200 μM FFA C8:0 was used to treat PCa cell line PC3 in vitro,and HY-112562 was added to specifically block GPR84 when cell was treated with FFA C8:0.Western blot was used to detect the protein expression levels of KLF7,IL-6 and tumor related factor p21;CCK8,clone formation assay,transwell method and scratch test detect the effects on the proliferation,invasion and migration of PC3 cells.5.Use the weighted gene function enrichment analysis software(http://www.cuilab.cn/weat)and Knock TF(http://www.cuilab.cn/weat)database to analyze and predict the possible downstream target genes of KLF7.6.SPSS 25.0 software was used for data analysis.T-test was used for normal distribution data,rank sum test was used for non normal distribution data,and one-way ANOVA was used for comparison among multiple groups.The difference was statistically significant(P< 0.05).Results: 1.The expression of KLF7,IL-6,Ki67 and MMP2 was high,while p21 was low in PCa patients 1.1 Compared benign prostatic hyperplasia with prostate cancer,the serum prostate-specific antigen(PSA)level of PCa patients was significantly higher(P<0.05).The body weight,BMI,TC,TG,LDL and Glu levels of PCa patients were higher than those of BPH patients.1.2 Using the weighted gene function enrichment analysis tool wet software,we found that KLF7,p21,NF-κ B and other genes belong to SMAD2 target gene set(P<0.001)and SMAD3 target gene set(P<0.001).According to the gene ontology annotation of Knock TF database,KLF7,IL-6 and p21 are involved in the adipose synthesis pathway.These results suggest that IL-6 and p21 may be downstream target genes of KLF7.1.3 Immunohistochemical results showed that the expression of KLF7,IL-6,Ki67 and MMP2 in PCa was significantly higher than BPH(P<0.05),the expression of p21 was significantly lower than BPH(P<0.05).The results of correlation analysis showed that the expression of KLF7 was positively correlated with serum PSA(r= 0.220,P=0.004)and the expression of IL-6(r=0.219,P=0.047)and Ki67(r= 0.312,P<0.001)in prostate tissue,and negatively correlated with p21(r =-0.211,P=0.014).1.4 Compared with BPH patients,the expression of KLF7 m RNA and protein in PCA patients was significantly higher(P < 0.05).2.The tumorigenicity of PC3 cells was enhanced in obese mice induced by high-fat diet.KLF7,IL-6 and Ki67 were highly expressed in tumor tissues,while p21 was low expressed 2.1 After feeding male BALB/C nude mice with high fat diet(HFD,n=10)for 4 weeks,the body weight of HFD group mice(21.24±1.136 g)was significantly higher than that of normal chow diet(NCD,n=5)(17.48±0.335 g),and more than 20% of the body weight of NCD group mice;the Lee’s index of HFD group mice(14.25±0.315)was significantly higher than that of NCD mice(13.63±0.244).It is suggested that the obesity mouse model induced by high fat diet is successfully constructed.2.2 After the successful establishment of diet induced obese mice model,PC3-Luc cells with fluorescence were injected into the prostate of NCD group and HFD group mice respectively.After 9 weeks of continuous feeding,the body weight and Lee’s index of HFD group mice were still significantly higher than the group of NCD,the contents of TG,TC and FFA in serum were significantly higher than NCD group mice.In vivo imaging results showed that the tumor formation rate of PC3-Luc cells in HFD group(9/10,90%)was significantly higher than that in NCD group(3/5,60%),The weight and volume of tumor tissue in HFD group(0.33±0.3g)were higher than those in NCD group(0.15±0.06 g;160.2±67.7 mm3).2.3 The results of immunohistochemistry showed that the expression of KLF7 and Ki67 in HFD group increased,while the expression of p21 decreased significantly.Western blot results showed that the expression of IL-6 in HFD group was higher than that in NCD group.3.High concentration of FFA c8:0 can promote the proliferation,invasion and migration of prostate cancer cells by up regulating KLF7 and downstream target genes 3.1 After PC3 cells were stimulated with 50 μM,100 μM,200 μM,500 μM,1000 μM and 2000 μM FFA C8:0 respectively,it was found that 200 μM FFA C8:0 treatment for 48 h was the best concentration and time.3.2 After treated with 200 μM FFA C8:0 for 48 h,the expressions of KLF7,IL-6 and MMP2 in PC3 cells were significantly higher than control group(P=0.05),the expression of p21 were significantly lower than control group(P<0.05);200 μM FFA C8:0 could significantly promote the effect on the biological behavior of PC3 cells(P<0.05).3.3 After up regulating the expression of KLF7 in PC3 cells,compared with the control group,the expressions of IL-6 and MMP2 were significantly increased(P<0.05),the expression of p21 were significantly decreased(P<0.05);overexpression of KLF7 could significantly promote the the effect on the biological behavior of PC3 cells(P<0.05).3.4 Compared with the control group,the expressions of IL-6 and MMP2 in PC3 cells were significantly decreased(P<0.05),the expression of p21 were significantly increased(P<0.05);inhibition of KLF7 expression could significantly inhibit the the effect on the biological behavior of PC3 cells(P<0.05).3.5 Compared with the control group,the expressions of IL-6 and MMP2 in PC3 cells were significantly decreased,the expression of p21 were significantly increased after downregulation of KLF7 expression by si RNA.Inhibition of KLF7 expression could significantly inhibit the effect on the biological behavior of PC3 cells.The above differences were statistically significant(P<0.05).4.High concentration FFA c8:0 can promote the proliferation,invasion and migration of prostate cancer cells by activating gpr84 and up regulating KLF7 and downstream target genes When 200 μM FFA C8:0 was added to PC3 cells and HY-112562 specifically blocked GPR84,compared with FFA C8:0 alone group,the expressions of KLF7 and IL-6 in PC3 cells were decreased,while the expression of p21 was increased;GPR84 blocker could significantly reverse the effect of FFA C8:0 on the effect on the biological behavior of PC3 cells.(P<0.05).Conclusions: 1.High expression of KLF7,IL-6 and low expression of p21 are closely related to the occurrence and development of obesity related PCa.2.High level FFA C8:0 up regulates the expression of KLF7 through GPR84,which promotes the biological behavior of PCa cells.3.IL-6 and p21 may be downstream target genes of KLF7;KLF7 can promote the biological behavior of PCa cells by up regulating the expression of IL-6 and down regulating the expression of p21.