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miR-155 Regulates Immune Modulatory Properties of Mesenchymal Stem Cells by Targeting TAK1-binding Protein 2

Xu, Chunliang, Ren, Guangwen, Cao, Gang, Chen, Qing, Shou, Peishun, Zheng, Chunxing, Du, Liming, Han, Xiaoyan, Jiang, Menghui, Yang, Qian, Lin, Liangyu, Wang, Guan, Yu, Pengfei, Zhang, Xin, Cao, Wei, Brewer, Gary, Wang, Ying, Shi, Yufang·2013·Journal of Biological Chemistry
DOI 10.1074/jbc.m112.414862
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FIGURE1 InflammatorycytokinesinducemiR-155expression. MSCswere treated with 10 ng/ml IFN 𝛄 and 10 ng/ml TNF 𝛂 for 1 h, 6 h, 12 h and 24 h, and cells were collected in TRIzol. miR-155 ( A ), miR-27a ( B ), and miR-148a ( C ) expression were determined with the Taqman quantitative PCR kit. D , miR155 expression after treatment with different inflammatory cytokines for 24 h. miR-155 levels were determined as described in A .
FIGURE1 InflammatorycytokinesinducemiR-155expression. MSCswere treated with 10 ng/ml IFN 𝛄 and 10 ng/ml TNF 𝛂 for 1 h, 6 h, 12 h and 24 h, and cells were collected in TRIzol. miR-155 ( A ), miR-27a ( B ), and miR-148a ( C ) expression were determined with the Taqman quantitative PCR kit. D , miR155 expression after treatment with different inflammatory cytokines for 24 h. miR-155 levels were determined as described in A .CC-BY · From the PDF
FIGURE 2 miR-155 negatively regulates the immunosuppression of MSCs. MSCs were transfected with 80 nM negative control ( NC ) or miR-155 mimics ( A ) or inhibitor ( B ). After 24 h, transfected cells were harvested upon trypsinization and seeded into 96-well plates at different cell numbers per well. After 24 h, supernatant was discarded, and 4 × 10 5 splenocytes were addedperwellwith1 𝛍 g/mlanti-CD3and1 𝛍 g/mlanti-CD28.After48h,1 𝛍 Ci of [ 3 H]methylthymidine was added to each well. After another 6 h, cells were frozen, thawed, and harvested. The radioactivity of incorporated [ 3 H]thymidine was measured by scintillation.
FIGURE 2 miR-155 negatively regulates the immunosuppression of MSCs. MSCs were transfected with 80 nM negative control ( NC ) or miR-155 mimics ( A ) or inhibitor ( B ). After 24 h, transfected cells were harvested upon trypsinization and seeded into 96-well plates at different cell numbers per well. After 24 h, supernatant was discarded, and 4 × 10 5 splenocytes were addedperwellwith1 𝛍 g/mlanti-CD3and1 𝛍 g/mlanti-CD28.After48h,1 𝛍 Ci of [ 3 H]methylthymidine was added to each well. After another 6 h, cells were frozen, thawed, and harvested. The radioactivity of incorporated [ 3 H]thymidine was measured by scintillation.CC-BY · From the PDF
FIGURE 3 miR-155 negatively regulates iNOS expression in MSCs. MSCs were transfected with 80 nM negative control ( NC ) or miR-155 mimics ( A-C ) or inhibitors ( D-F ). After 24 h, medium was discarded, and fresh medium either lacking (control) or containing 10 ng/ml IFN 𝛄 and 10 ng/ml TNF 𝛂 wasadded. The level of nitrate in the supernatant ( B and E ), iNOS mRNA ( A and D ), and protein were determined 24 h later by Griess test, real-time PCR, and Western blotting analysis, respectively.
FIGURE 3 miR-155 negatively regulates iNOS expression in MSCs. MSCs were transfected with 80 nM negative control ( NC ) or miR-155 mimics ( A-C ) or inhibitors ( D-F ). After 24 h, medium was discarded, and fresh medium either lacking (control) or containing 10 ng/ml IFN 𝛄 and 10 ng/ml TNF 𝛂 wasadded. The level of nitrate in the supernatant ( B and E ), iNOS mRNA ( A and D ), and protein were determined 24 h later by Griess test, real-time PCR, and Western blotting analysis, respectively.CC-BY · From the PDF
FIGURE4 TAB2isamiR-155targetinMSCs. MSCsweretransfectedwith80nMnegativecontrol( NC ) or miR-155 mimics ( A and B ) or inhibitors ( C and D ). After 24h,mediumwasdiscarded,andfreshmediumcontaining10ng/mlIFN 𝛄 and10ng/mlTNF 𝛂 wasadded.TAB2mRNAandproteinlevelsweredetermined24h later by real-time PCR and Western blotting analysis, respectively. E , psiCheck2-TAB2-3 ′ -UTR or miR-155 target sequence mutant plasmid, psiCheck2-TAB23 ′ -UTR-mut, was transfected with internal control plasmid pRL-TK and either 0 or 50 nM miR-155 mimics into 293T cells. Cells were collected and measured 24 h later with the Dual-Luciferase assay kit.
FIGURE4 TAB2isamiR-155targetinMSCs. MSCsweretransfectedwith80nMnegativecontrol( NC ) or miR-155 mimics ( A and B ) or inhibitors ( C and D ). After 24h,mediumwasdiscarded,andfreshmediumcontaining10ng/mlIFN 𝛄 and10ng/mlTNF 𝛂 wasadded.TAB2mRNAandproteinlevelsweredetermined24h later by real-time PCR and Western blotting analysis, respectively. E , psiCheck2-TAB2-3 ′ -UTR or miR-155 target sequence mutant plasmid, psiCheck2-TAB23 ′ -UTR-mut, was transfected with internal control plasmid pRL-TK and either 0 or 50 nM miR-155 mimics into 293T cells. Cells were collected and measured 24 h later with the Dual-Luciferase assay kit.CC-BY · From the PDF
FIGURE 5 TAB2 knockdown reduces iNOS expression in MSCs. A , MSCs weretransfectedwith200nMnegativecontrol( NC ) or TAB2 siRNAs. After 48 h, TAB2 mRNA levels were determined by real-time PCR. These cells were treatedwith10ng/mlIFN 𝛄 and10ng/mlTNF 𝛂 for 24 h, and iNOS mRNAlevels ( B ) and supernatant nitrate ( C ) were determined by real-time PCR and Griess test, respectively.
FIGURE 5 TAB2 knockdown reduces iNOS expression in MSCs. A , MSCs weretransfectedwith200nMnegativecontrol( NC ) or TAB2 siRNAs. After 48 h, TAB2 mRNA levels were determined by real-time PCR. These cells were treatedwith10ng/mlIFN 𝛄 and10ng/mlTNF 𝛂 for 24 h, and iNOS mRNAlevels ( B ) and supernatant nitrate ( C ) were determined by real-time PCR and Griess test, respectively.CC-BY · From the PDF

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Citations

Cites

Research works printed but not linked: 23

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Metadata

Authors
Xu, C., Ren, G., Cao, G., Chen, Q., Shou, P., Zheng, C., Du, L., Han, X., Jiang, M., Yang, Q., Lin, L., Wang, G., Yu, P., Zhang, X., Cao, W., Brewer, G., Wang, Y., & Shi, Y.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2013-04
Volume
288
Issue
16
Pages
11074-11079
ISSN
0021-9258
DOI
10.1074/jbc.m112.414862
Cited by
84
References
2
Language
en
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