摘要
目的探讨西格列汀(Sit)调节CC趋化因子配体2(CCL2)-CC趋化因子受体2(CCR2)信号轴对前列腺癌细胞迁移、侵袭和免疫逃逸的影响。方法将前列腺癌PC3细胞分为CK组、西格列汀低剂量(Sit-L)组、西格列汀高剂量(Sit-H)组、Sit-H+pcDNA-NC组、Sit-H+pcDNA-CCL2组。克隆形成实验检测细胞增殖;Transwell实验检测细胞迁移和侵袭;流式细胞术检测细胞凋亡率;酶联免疫吸附法(ELISA)检测细胞上清中趋化因子配体(CXCL-2、CXCL-8)水平;实时荧光定量聚合酶链反应(qRT-PCR)法检测细胞中CCL2 mRNA、CCR2 mRNA表达水平;蛋白免疫印迹法(Western blot)检测细胞增殖细胞核抗原(PCNA)、基质金属肽酶9(MMP-9)、半胱氨酸蛋白酶3(cleaved caspase-3)、CCL2、CCR2蛋白表达;将上述各组细胞与NK-92MI细胞共培养,细胞计数(CCK-8)法检测NK-92MI细胞免疫杀伤率;ELISA检测共培养细胞上清液中肿瘤坏死因子-α(TNF-α)、γ-干扰素(IFN-γ)水平。结果与CK组相比,Sit-L组、Sit-H组和Sit-H+pcDNA-NC组PC3细胞克隆形成率、细胞迁移和侵袭个数、CXCL-2、CXCL-8水平、CCR2 mRNA和CCL2 mRNA的表达水平、PCNA、MMP-9、CCL2、CCR2蛋白表达水平显著降低(P<0.05),细胞凋亡率、cleaved caspase-3蛋白表达水平显著升高(P<0.05);过表达CCL2可减弱Sit对PC3细胞的抑制作用(P<0.05);与CK-NK组共培养组比较,Sit-L-NK组、Sit-H-NK组和Sit-H+pcDNA-NC-NK组共培养组NK-92MI细胞免疫杀伤率、细胞上清中TNF-α、IFN-γ水平显著升高(P<0.05);与Sit-H+pcDNA-NC-NK共培养组比较,Sit-H+pcDNA-CCL2-NK共培养组NK-92MI细胞免疫杀伤率,细胞上清中TNF-α、IFN-γ水平显著降低(P<0.05)。结论Sit通过抑制CCL2-CCR2信号轴可抑制前列腺癌细胞增殖、迁移、侵袭和免疫逃逸,并促进其凋亡。
Objective To investigate the effects of sitagliptin(Sit)on the migration,invasion and immune escape of prostate cancer cells by regulating the CC chemokine ligand 2(CCL2)-CC chemokine receptor 2(CCR2)signaling axis.Methods PC3 cells were divided into CK group,sitagliptin low dose(Sit-L)group,Sitagliptin high dose(Sit-H)group,Sit-H+pcDNA-NC group and Sit-H+pcDNA-CCL2 group.Cell proliferation was detected by clonal formation assay.Cell migration and invasion were detected by Transwell assay.The apoptosis rate was detected by flow cytometry.The chemokine ligands(CXCL-2,CXCL-8)in the supernatant were detected by enzyme-linked immunosorbent assay(ELISA).Re‐al-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression levels of CCL2 mRNA and CCR2 mRNA in cells.Western blot was used to detect the expression of proliferating cell nuclear antigen(PCNA),matrix metallopeptidase 9(MMP-9),cysteine protease 3(cleaved caspase-3),CCL2,and CCR2.The above groups of cells were co-cultured with NK-92MI cells,and CCK-8 method was used to detect the immune killing rate of NK-92MI cells.ELISA was applied to detect the levels of tumor bad factor-α(TNF-α)andγ-interferon(IFN-γ)in the supernatant of co-cultured cells.Results Compared with CK group,PC3 cell clonal formation rate,number of cell migration and invasion,CXCL-2,CXCL-8 levels,CCR2 mRNA and CCL2 in Sit-L,Sit-H and Sit-H+pcDNA-NC groups mRNA expression levels,protein expression levels of PCNA,MMP-9,CCL2,and CCR2 were significantly de‐creased(P<0.05),and apoptosis rate and cleaved caspase-3 protein expression levels were significantly increased(P<0.05).Overex‐pression of CCL2 could weaken the inhibitory effect of Sit on PC3 cells(P<0.05).Compared with the CK-NK group,the immune kill‐ing rate of NK-92MI cells and the levels of TNF-αand IFN-γin the cell supernatance of the Sit-L-NK group,SIT-H-NK group and Sit-H+pcDNA-NC-NK group were significantly increased(P<0.05).Compared with the Sit-H+pcDNA-NC-NK co-culture group,the im‐mune killing rate of NK-92MI cells and the levels of TNF-αand IFN-γin the cell supernatant of the Sit-H+pcDNA-CCL2-NK co-cul‐ture group were significantly decreased(P<0.05).Conclusion Sit can inhibit the proliferation,migration,invasion and immune es‐cape of prostate cancer cells and promote their apoptosis by inhibiting the CCL2-CCR2 signal axis.
作者
陈莉洁
陈晨
CHEN Lijie;CHEN Chen(The First Outpatient Department of the 960th Hospital of the Joint Logistics Support Force,PLA,Jinan 250000,Shandong,China)
出处
《医学研究与战创伤救治》
北大核心
2025年第1期22-27,共6页
Journal of Medical Research & Combat Trauma Care
