TWEAK调控Dicer促使巨噬细胞源性外泌体分选miR-7至卵巢癌细胞的机制
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摘要
目的·研究TWEAK通过何种途径促使巨噬细胞源性外泌体分选miR-7至卵巢癌细胞。方法·收集TWEAK刺激前后的巨噬细胞,real-time PCR和Western blotting检测巨噬细胞中Dicer的表达。在巨噬细胞中沉默Dicer,real-time PCR检测巨噬细胞及其外泌体中mi R-7的表达;过表达Dicer,real-time PCR检测巨噬细胞中mi R-7的表达。继而用TWEAK刺激巨噬细胞,real-time PCR检测巨噬细胞源性外泌体中mi R-7的表达,Western blotting检测TWEAK刺激前后的巨噬细胞中核因子κB(nuclear factor-κB,NF-κB)信号通路的表达;继而将NF-κB信号通路抑制,Western blotting检测TWEAK刺激前后巨噬细胞中Dicer的表达。结果·TWEAK刺激后,巨噬细胞中Dicer的表达下调。沉默巨噬细胞中Dicer的表达,巨噬细胞及其外泌体中mi R-7的表达均上调;而将巨噬细胞中的Dicer过表达,再用TWEAK刺激巨噬细胞后,与对照组相比,Dicer过表达组巨噬细胞及其外泌体中的mi R-7表达降低。TWEAK刺激巨噬细胞后,NF-κB信号通路蛋白的表达上调;而抑制NF-κB信号通路,再用TWEAK刺激巨噬细胞后,Dicer的表达无明显改变。结论·TWEAK通过活化NF-κB通路,抑制Dicer表达,从而促进巨噬细胞分选miR-7至其外泌体。
Objective·To investigate the mechanism of tumor necrosis factor like weak inducer of apoptosis(TWEAK) promoting macrophagederived exosomal miR-7 to epithelial ovarian cancer(EOC).Methods·The expression of Dicer was detected by real-time PCR and Western blotting in TWEAK-stimulated macrophages.The expression of miR-7 was detected by real-time PCR in the macrophage and macrophage-derived exosome after silencing Dicer in macrophage.While in Dicer-overexpressing macrophage,real-time PCR was used to detect the expression of miR-7.Then TWEAK was used to stimulate the macrophage,and the expression of miR-7 in the macrophage and macrophage-derived exosome was detected by real-time PCR.The expression of key proteins in the nuclear factor-κB(NF-κB) pathway was detected by Western blotting in TWEAK-stimulated macrophage.After pretreatment of NF-κB inhibitor,Western blotting was used to detect the expression of Dicer and key proteins in the NF-κB pathway in TWEAKstimulated macrophage.Results·The expression of Dicer in macrophage was down-regulated after TWEAK stimulating.The expression of miR-7 was up-regulated in Dicer-silencing macrophage and macrophage-derived exosome.While the expression of miR-7 was down-regulated in the macrophage and the macrophage-derived exosome in Dicer-overexpressing macrophage after TWEAK stimulating.The expression of key proteins in the NF-κB pathway in macrophage was also up-regulated after TWEAK stimulating.After inhibition of NF-κB signaling pathway,the expression of Dicer was not significantly changed in TWEAK-stimulated macrophage compared to the control group.Conclusion·TWEAK can active NF-κB pathway and inhibit the expression of Dicer to promote macrophage-derived exosomal miR-7 to EOC cells.
Objective·To investigate the mechanism of tumor necrosis factor like weak inducer of apoptosis(TWEAK) promoting macrophagederived exosomal miR-7 to epithelial ovarian cancer(EOC).Methods·The expression of Dicer was detected by real-time PCR and Western blotting in TWEAK-stimulated macrophages.The expression of miR-7 was detected by real-time PCR in the macrophage and macrophage-derived exosome after silencing Dicer in macrophage.While in Dicer-overexpressing macrophage,real-time PCR was used to detect the expression of miR-7.Then TWEAK was used to stimulate the macrophage,and the expression of miR-7 in the macrophage and macrophage-derived exosome was detected by real-time PCR.The expression of key proteins in the nuclear factor-κB(NF-κB) pathway was detected by Western blotting in TWEAK-stimulated macrophage.After pretreatment of NF-κB inhibitor,Western blotting was used to detect the expression of Dicer and key proteins in the NF-κB pathway in TWEAKstimulated macrophage.Results·The expression of Dicer in macrophage was down-regulated after TWEAK stimulating.The expression of miR-7 was up-regulated in Dicer-silencing macrophage and macrophage-derived exosome.While the expression of miR-7 was down-regulated in the macrophage and the macrophage-derived exosome in Dicer-overexpressing macrophage after TWEAK stimulating.The expression of key proteins in the NF-κB pathway in macrophage was also up-regulated after TWEAK stimulating.After inhibition of NF-κB signaling pathway,the expression of Dicer was not significantly changed in TWEAK-stimulated macrophage compared to the control group.Conclusion·TWEAK can active NF-κB pathway and inhibit the expression of Dicer to promote macrophage-derived exosomal miR-7 to EOC cells.
引文
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[2]Qian BZ,Pollard JW.Macrophage diversity enhances tumor progression and metastasis[J].Cell,2010,141(1):39-51.
[3]Hu Y,Li D,Wu A,et al.TWEAK-stimulated macrophages inhibit metastasis of epithelial ovarian cancer via exosomal shuttling of micro RNA[J].Cancer Lett,2017,393:60-67.
[4]Gu L,Dai L,Cao C,et al.Functional expression of TWEAK and the receptor Fn14 in human malignant ovarian tumors:possible implication for ovarian tumor intervention[J].PLo S One,2013,8(3):e57436.
[5]Wang X,Ivan M,Hawkins SM.The role of microrna molecules and micrornaregulating machinery in the pathogenesis and progression of epithelial ovarian cancer[J].Gynecol Oncol,2017,147(2):481-487.
[6]Haga CL,Velagapudi SP,Childs-Disney JL,et al.Rapid generation of mi RNAinhibitor leads by bioinformatics and efficient high-throughput screening methods[J].Methods Mol Biol,2017,1517(2):179-198.
[7]Fareh M,Yeom KH,Haagsma AC,et al.TRBP ensures efficient Dicer processing of precursor micro RNA in RNA-crowded environments[J].Nat Commun,2016,7(2):13694-13705.
[8]Song MS,Rossi JJ.Molecular mechanisms of Dicer:endonuclease and enzymatic activity[J].Biochem J,2017,474(10):1603-1618.
[9]Devasthanam AS,Tomasi TB.Dicer in immune cell development and function[J].Immunol Invest,2014,43(2):182-195.
[10]Xu S,Guo K,Zeng Q,et al.The RNaseⅢenzyme Dicer is essential for germinal center B-cell formation[J].Blood,2012,119(3):767-776.
[11]Kuipers H,Schnorfeil FM,Fehling HJ,et al.Dicer-dependent micro RNAs control maturation,function,and maintenance of Langerhans cells in vivo[J].JImmunol,2010,185(1):400-409.
[12]Baer C,Squadrito ML,Laoui D,et al.Suppression of micro RNA activity amplifies IFN-γ-induced macrophage activation and promotes anti-tumour immunity[J].Nat Cell Biol,2016,18(7):790-802.
[13]Lee Y,Biswas SK.Rewiring macrophages for anti-tumour immunity[J].Nat Cell Biol,2016,18(7):718-720.
[14]Melo SA,Sugimoto H,O'connell JT,et al.Cancer exosomes perform cellindependent micro RNA biogenesis and promote tumorigenesis[J].Cancer Cell,2014,26(5):707-721.
[15]Park MH,Hong JT.Roles of NF-κB in cancer and inflammatory diseases and their therapeutic approaches[J].Cells,2016,5(2):15-21.
[16]Wang Y,Sun L,Song Z,et al.Maspin inhibits macrophage phagocytosis and enhances inflammatory cytokine production via activation of NF-κBsignaling[J].Mol Immunol,2017,82:94-103.
[17]Richmond A,Yang J.The role of NF-κB in modulating antitumor immunity[J].Oncoimmunology,2016,5(1):e1005522.