| Object: In this study,we collected serum samples from non-cancer control individuals and patients with prostate cancer(PCa),constructed a PCa tumor-bearing mouse model in bone marrow cavity under obesity,and analyzed the correlation between serum free fatty acids(FFA)and the occurrence and development of PCa.Based on co-culture of bone marrow adipocytes(BMA)and PCa cells in vitro,observe whether high levels of FFA can promote CCL2 secretion in BMA via up-regulating GPRs/KLF7 to promote the proliferation,invasion and migration ability of PCa cells,and explore possible molecular mechanisms.Through the above research,we try to elucidate the possible molecular mechanism of bone metastasis in PCa caused by obesity,and provide basic theoretical basis for finding molecular targets for clinical treatment.Methods:(1)General information and serum of 10 PCa patients and 10 non-cancer individuals were collected from September 2017 to September 2018 in the First Affiliated Hospital of Shihezi University School of Medicine,and the levels of FFA,TG,TC,HDL,LDL and GLU and the expression of CCL2 were measured in serum.(2)Constructed a PCa tumor-bearing mouse model in the bone marrow cavity under obesity by a high-fat diet(n= 8),and a PCa tumor-bearing mouse in the bone marrow cavity by a normal diet(n= 4)was used as a control to dynamically monitor mice body weight and Lee's index,serum total FFA,TG,TC,HDL,LDL and GLU contents in serum of mice were measured.The vivo imaging was used to compare the tumorigenic rate of PCa cells PC-3 in bone marrow cavity between two groups of mice;HE staining was used to compare the distribution of BMA in the bone marrow cavity between two groups of mice;the expression levels of KLF7/CCL2 in the bone marrow cavity of mice was detected by q RT-PCR and immunohistochemical techniques.(3)Cultured adult human bone marrow mesenchyme stem cells in vitro and induce differentiation into mature BMA,use 0.3 m M palmic acid(PA)stimulates BMA;transfected over-expressed plasmids or si RNAs to up/down-regulate KLF7 expression,and antagonized GPRs or down-regulated KLF7 with PA stimulates BMA;BMA supernatants treated with the above groups were co-cultured with PCa cells PC-3/22RV1,respectively.(4)q RT-PCR and Western Blot technology were used to detect GPRs/KLF7/CCL2 expression levels in BMA,and ELISA was used to detect CCL2 secretion levels in supernatants of BMA;CCK8 and Transwell methods were used to detect proliferation,invasion and migration ability of PC-3/22RV1 cells.(5)Use SPSS 17.0 software for data analysis.The t-test was used for the normal distribution data,the rank sum test was used for the data not conforming to a normal distribution,and the one-way ANOVA was used to compare the data between multiple groups.P <0.05 was defined as the difference statistically significant.Results: 1.Serum levels of total FFA and CCL2 in PCa patients are significantly higher than those in non-cancer individuals.(1)The levels of total FFA in the serum of PCa patients are significantly higher than those of non-cancer individuals(P <0.05).Comparing the FFA profiles of 25 kinds different carbon chain lengths,it has been found that the 21 kinds of FFA levels in the serum of PCa patients are higher than those of non-cancer individuals.Among them,the pro-inflammatory and cancer-promoting saturated fatty acid PA(16: 0)accounts for a relatively large proportion;(2)The expression level of CCL2 protein in the serum of PCa patients was significantly higher than that of non-cancer individuals(P <0.05).2.Obesity promotes the adaptive growth of PCa cells in mice bone.(1)After male BALB/c Nude mice were fed 60% high-fat diet for 3 weeks,the weight and Lee's index of the mice were significantly higher than those in the normal diet group(P <0.05);(2)After 7 weeks of high-fat feeding,the levels of total FFA,TG,HDL and GLU in the serum of mice were significantly higher than those in the normal diet group(P <0.05);(3)Four weeks after the injection of human PCa cells PC-3-Luc into the left leg femur of mice under high-fat diet,the tumorigenic rate(100%)of bone marrow PCa cells in mice was higher than that in the normal diet group(25%).3.Under the state of obesity,BMA distribution and KLF7/CCL2 expression levels in mice bone marrow were significantly increased.(1)Under obesity,the number of BMA,the area of single BMA,and the proportion of BMA in the bone marrow cavity of mice were significantly higher than those in the normal diet group(P <0.05);(2)Under obesity,the m RNA and protein expression levels of KLF7/CCL2 in the bone marrow cavity of mice were significantly higher than those in the normal diet group(P <0.05).4.High concentration of PA promotes PCa cell proliferation,invasion and migration by stimulating BMA secrete CCL2.(1)BMA were stimulated with 0.1 m M,0.2 m M,and 0.3 m M PA,respectively.It was found that the m RNA and protein secretion levels of CCL2 significantly increased in BMA under the action of 0.3 m M PA(P <0.05),and after high concentration(0.3m M)PA stimulated BMA,the m RNA and protein expression levels of GPR40/ GPR120/KLF7 were significantly increased(P <0.05);(2)After co-culture of high concentration(0.3m M)PA stimulated BMA supernatant and PCa cells,the proliferation,invasion and migration ability of PC-3 cells were significantly facilitated(P <0.05),and the proliferation ability of 22RV1 cells were significantly facilitated(P <0.05);(3)Add the CCR2 receptor antagonist RS102895 to the supernatant of BMA stimulated with high concentration(0.3m M)PA and co-culture with PCa cells.Compared with the group of PA-stimulated BMA supernatants,add CCR2 receptors antagonists can significantly inhibit the proliferation,invasion and migration ability of PC-3 cells and 22RV1 cells(P <0.05).5.High concentration(0.3m M)PA can promote the secretion of CCL2 by BMA via up-regulating KLF7 and enhance the biological behavior of PCa cells.(1)After successfully up-regulated KLF7 in BMA,the m RNA and protein expression levels of CCL2 in BMA were significantly increased(P <0.05),and the CCL2 protein secretion levels in the supernatant of BMA were significantly increased(P <0.05);After up-regulation KLF7,BMA supernatant was co-cultured with PCa cells,the proliferation,invasion and migration ability of PC-3 cells were significantly facilitated(P <0.05),and the proliferation and migration ability of 22RV1 cells were significantly facilitated(P <0.05);(2)After successfully down-regulated KLF7 in BMA,CCL2 m RNA and protein expression levels in BMA were significantly reduced(P <0.05),and CCL2 protein secretion levels in BMA supernatants were significantly reduced(P <0.05);After down-regulation KLF7,BMA supernatant was co-cultured with PCa cells,the proliferation,invasion and migration ability of PC-3 cells were significantly reduced(P <0.05),and the proliferation ability of 22RV1 cells was significantly reduced(P <0.05);(3)After high concentration(0.3m M)PA stimulated BMA and down-regulated KLF7,the CCL2 m RNA and protein expression levels in BMA was significantly reduced(P <0.05),and the protein secretion level of CCL2 in BMA was significantly reduced compared with the PA stimulated group(P <0.05);After down-regulation KLF7 with PA stimulated,BMA supernatant was co-cultured with PCa cells.Compared with PA stimulated group,the BMA supernatants after down-regulation of KLF7 significantly inhibited proliferation,invasion and migration ability of PC-3 cells(P <0.05),and significantly inhibited the proliferation and migration ability of 22RV1 cells(P <0.05).6.High concentration(0.3m M)PA up-regulates KLF7/CCL2 in BMA through GPR40/ GPR120,thereby promoting proliferation,invasion and migration ability of PCa cells.(1)When high concentration(0.3m M)PA stimulates BMA while using the GPR40 receptor antagonist GW1100,compared with PA stimulated group,the expression level of KLF7/CCL2 m RNA in BMA after antagonizing GPR40 were significantly reduced(P <0.05),and CCL2 protein secretion level in BMA was significantly reduced(P <0.05);(2)After antagonizing GPR40 as the same time as high concentration(0.3m M)PA stimulate,BMA supernatant was co-cultured with PCa cells.Compared with BMA supernatant after PA stimulated,BMA supernatant with antagonizing GPR40 can significantly inhibit proliferation,invasion and migration ability of PC-3 cells(P <0.05),and significantly inhibited the proliferation ability of 22RV1 cells(P <0.05);(3)When high concentration(0.3m M)PA stimulate BMA while using the GPR120 receptor antagonist AH7614,compared with PA stimulated group,the expression level of KLF7/CCL2 m RNA in BMA after antagonizing GPR120 were significantly reduced(P <0.05),and CCL2 protein secretion level in BMA was significantly reduced(P <0.05);(4)After antagonizing GPR120 as the same time as high concentration(0.3m M)PA stimulate,BMA supernatant was co-cultured with PCa cells.Compared with BMA supernatant after PA stimulated,BMA supernatant with antagonizing GPR120 can significantly inhibit proliferation,invasion and migration ability of PC-3 cells(P <0.05),and significantly inhibited the proliferation and migration ability of 22RV1 cells(P <0.05).7.CCL2 promotes the proliferation,invasion and migration ability of PCa cells through CCR2.(1)Under the stimulation of 100ng/m L CCL2 recombinant protein,the proliferation,invasion and migration ability of PC-3 cells were significantly facilitated(P <0.05),and Ki67/MMP2 m RNA expression levels were significantly increased(P <0.05);(2)Use the CCR2 antagonist RS102895 while stimulated with 100ng/m L CCL2 recombinant protein,the proliferation,invasion and migration ability of PC-3 cells were significantly inhibited(P <0.05),and the Ki67/MMP2 m RNA expression levels were significantly inhibited(P <0.05);(3)Under the stimulation of 100ng/m L CCL2 recombinant protein,the proliferation ability of 22RV1 cells was significantly increased(P <0.05),and the expression level of Ki67 m RNA cells was significantly increased(P <0.05);(4)Use the CCR2 antagonist RS102895 while stimulated with 100ng/m L CCL2 recombinant protein,the proliferation,invasion and migration ability of 22RV1 cells were significantly inhibited(P <0.05),and the Ki67 m RNA expression levels were significantly inhibited(P <0.05).Conclusions: Under the state of obesity,high concentration of palmitic acid(PA)may promote the bone metastasis of PCa cells by up-regulating the expression of GPR40/GPR120/KLF7/CCL2 in bone marrow adipocytes. |
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