摘要
目的:探讨鞘胺醇激酶1(Sphingosine kinascs-1,SPHK1)对M2巨噬细胞极化作用的调控以及对膀胱癌细胞上皮间质转化(Epithelial-mesenchymal transition,EMT)的影响机制。方法:选取巨噬细胞系Raw264.7进行原代培养,构建Control-NC(SPHK1空白质粒转染Raw264.7细胞系)、LV-SPHK1组(过表达SPHK1质粒转染Raw264.7细胞系)、si-SPHK1组(敲除SPHK1质粒转染Raw264.7细胞系);Western-blot检测各组细胞内SPHK1及M2巨噬细胞标志物(CD206、ArgI)蛋白表达;免疫荧光检测M2巨噬细胞标志物(CD206、ArgI)蛋白荧光强度;在上述各组巨噬细胞与膀胱癌T24细胞共培养24h后,采用MTT法检测各组T24细胞的增殖活性;划痕、Transwell实验检测各组T24细胞迁移、侵袭能力;Western-blot检测各组T24细胞增殖、侵袭相关蛋白(Survivin、MMP-2、MMP-9)及EMT相关蛋白(E-Cadherin、Vimentin、N-Cadherin)浓度表达。结果:与Control-NC组相比,LC-SPHK1组SPHK1蛋白表达明显提升,si-SPHK1组SPHK1蛋白表达明显下调(P<0.05);SPHK1过表达质粒转染可以上调M2巨噬细胞标志物CD206、ArgI蛋白表达及荧光强度(P<0.05);SPHK1沉默质粒转染可以下调M2巨噬细胞标志物CD206、ArgI蛋白表达及荧光强度(P<0.05)。随着细胞培养时间的延长,各组膀胱癌T24细胞均体现出增殖活性提升趋势(P<0.05);与Control-NC组相比,SPHK1过表达会提升T24细胞增殖活性、迁移及侵袭能力,SPHK1沉默则会降低T24细胞增殖活性、迁移及侵袭能力(P<0.05);SPHK1过表达会提升T24细胞内Vimentin、N-Cadherin蛋白表达,降低E-Cadherin蛋白表达(P<0.05);SPHK1沉默则会降低T24细胞内Vimentin、N-Cadherin蛋白表达,提升E-Cadherin蛋白表达(P<0.05)。结论:SPHK1过表达可以提升M2型巨噬细胞极化程度,促进膀胱癌肿瘤细胞增殖、迁移、侵袭能力及EMT进程,最终提升膀胱癌的远处扩散能力,值得临床进一步研究。
Objective:To investigate the regulation of sphingosine kinascs-1(SPHK1)on M2 macrophage polarization and its mechanism on epithelial mesenchymal transition(EMT)in bladder cancer cells.Methods:Raw264.7 macrophages were cultured using the primary culture method.The cells were divided into the following groups:Control-NC(empty SPHK1 plasmid-transfected Raw264.7 cells),LV-SPHK1(SPHK1 overexpression plasmid-transfected Raw264.7 cells),and si-SPHK1(SPHK1 knockdown plasmid-transfected Raw264.7 cells).Western blot was used to detect the expression of SPHK1 and M2 macrophage markers(CD206 and ArgI)proteins in each group.Immunofluorescence was used to detect the fluorescence intensity of M2 macrophage markers(CD206 and ArgI)proteins.After co-culturing the macrophages of each group with bladder cancer T24 cells for 24 hours,the MTT method was used to detect the proliferation activity of T24 cells in each group.The scratch and Transwell assays were used to detect the migration and invasion abilities of T24 cells in each group.Western blot was used to detect the expression levels of proliferation-and invasion-related proteins(Survivin,MMP-2,and MMP-9)and EMT-related proteins(E-Cadherin,Vimentin,and N-Cadherin)in each group.Results:Compared with the Control-NC group,the SPHK1 protein expression was significantly increased in the LV-SPHK1 group and significantly decreased in the si-SPHK1 group(P<0.05).The overexpression of SPHK1 upregulated the expression and fluorescence intensity of M2 macrophage markers CD206 and ArgI proteins(P<0.05).The knockdown of SPHK1 downregulated the expression and fluorescence intensity of M2 macrophage markers CD206 and ArgI proteins(P<0.05).With the extension of cell culture time,the proliferation activity of T24 cells in each group showed an increasing trend(P<0.05).Compared with the Control-NC group,SPHK1 overexpression increased the proliferation,migration,and invasion abilities of T24 cells,while SPHK1 knockdown decreased the proliferation,migration,and invasion abilities of T24 cells(P<0.05).SPHK1 overexpression increased the expression of Vimentin and N-Cadherin proteins and decreased the expression of E-Cadherin protein in T24 cells(P<0.05).SPHK1 knockdown decreased the expression of Vimentin and N-Cadherin proteins and increased the expression of E-Cadherin protein in T24 cells(P<0.05).Conclusion:Overexpression of SPHK1 can promote the polarization of M2 macrophages,promote the proliferation,migration,invasion,and EMT of bladder cancer cells,and ultimately increase the distant metastasis ability of bladder cancer.This finding is worthy of further clinical research.
作者
王晨静
郭晓丹
马振禹
黄秀英
张伟
WANG Chenjing;GUO Xiaodan;MA Zhenyu(The Second Hospital of Baoding City,Hebei Baoding 071000,China)
出处
《河北医学》
CAS
2024年第3期381-386,共6页
Hebei Medicine
基金
河北省保定市科技计划项目,(编号:2041ZF021)。
