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MicroRNA-155 Targets SMAD2 and Modulates the Response of Macrophages to Transforming Growth Factor-β

Louafi, Fethi, Martinez-Nunez, Rocio T., Sanchez-Elsner, Tilman·2010·Journal of Biological Chemistry
DOI 10.1074/jbc.m110.146852
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Image 1
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FIGURE 2 SMAD2proteinlevelsaredown-regulatedwhenTHP1-155cells overexpress miR-155. A , THP1-155 were treated or not with doxycycline ( Dox ) to de-repress or not the expression of miR-155 transgene, respectively. Cells were collected at different time point and subjected to RNA extraction; cell lysate of the same samples were also collected. RNA from these samples wasusedtoquantify miR-155 and the housekeeping RNU44 levels with a specific microRNA RT-qPCR. All values are expressed as -fold induction of the nontreated THP1-155 cells (time = 0 h). B , protein extract from the same sample wassubjected to Western blotting, and SMAD2 levels were determined. As a normalizing control, 𝛃 -actin was also detected in the same blot. Values represent percentages of SMAD2 normalized against 𝛃 -actin and compared with control. C , the same RNA was extracts were also used to perform a standard RT-qPCR to determine SMAD2 mRNA levels. Values were normalized against GAPDH.
FIGURE 2 SMAD2proteinlevelsaredown-regulatedwhenTHP1-155cells overexpress miR-155. A , THP1-155 were treated or not with doxycycline ( Dox ) to de-repress or not the expression of miR-155 transgene, respectively. Cells were collected at different time point and subjected to RNA extraction; cell lysate of the same samples were also collected. RNA from these samples wasusedtoquantify miR-155 and the housekeeping RNU44 levels with a specific microRNA RT-qPCR. All values are expressed as -fold induction of the nontreated THP1-155 cells (time = 0 h). B , protein extract from the same sample wassubjected to Western blotting, and SMAD2 levels were determined. As a normalizing control, 𝛃 -actin was also detected in the same blot. Values represent percentages of SMAD2 normalized against 𝛃 -actin and compared with control. C , the same RNA was extracts were also used to perform a standard RT-qPCR to determine SMAD2 mRNA levels. Values were normalized against GAPDH.CC-BY · From the PDF
FIGURE 3 In THP1-155 cells TGF 𝛃 1-induced increases of phosphoSMAD2, protein levels are reduced when miR-155 is overexpressed. THP1-155 cells were cultured with or without doxycycline for 96 h, then cells were starved for 12 h before the addition of TGF 𝛃 1 (10 ng/ml). Lysates of these cells were taken after 1, 2, and 3 h to quantify the levels of phospho-SMAD2/SMAD2 and phospho-SMAD3/SMAD3 protein by Western immunoblotting. As a normalizing control, 𝛃 -actin was also detected in the same blot. Densitometry values across the top represent the percentage of phospho-SMAD2 normalized against 𝛃 -actin and compared with control.
FIGURE 3 In THP1-155 cells TGF 𝛃 1-induced increases of phosphoSMAD2, protein levels are reduced when miR-155 is overexpressed. THP1-155 cells were cultured with or without doxycycline for 96 h, then cells were starved for 12 h before the addition of TGF 𝛃 1 (10 ng/ml). Lysates of these cells were taken after 1, 2, and 3 h to quantify the levels of phospho-SMAD2/SMAD2 and phospho-SMAD3/SMAD3 protein by Western immunoblotting. As a normalizing control, 𝛃 -actin was also detected in the same blot. Densitometry values across the top represent the percentage of phospho-SMAD2 normalized against 𝛃 -actin and compared with control.CC-BY · From the PDF
FIGURE 4 A , overexpression of miR-155 reduces the TGF𝛃 dependent transcriptional activity. THP1-155 cells were treated with or without doxycycline for 96 h to de-repress or not the expression of the miR-155 transgene. Cells were then treated or not with TGF𝛃 1 (10 ng/ml) for 48 and assayed in RT-qPCR to determine the levels of SERPINE1 mRNA. B , THP-1 cells were transfected with pre-miR-155, pre-miR-221, or pre-miR control oligonucleotides. 48 h after transfection, cells were treated or not with TGF𝛃 1 for an additional 48 h period and then assayed by RT-qPCR to determine the levels of SERPINE1 mRNA. C , THP-1 cells were transfected with different siRNAs: anti-SMAD2#1, anti-SMAD2#2, and siRNA-Control#1. 48 h after transfection cells were treated or not with TGF𝛃 1 for an additional 48 h period and then assayed by RT-qPCR to determine the levels of SERPINE1 mRNA. D , THP1155 cells were treated with doxycycline as in A . Cells were then transfected with the CAGA12 LUC vector and pRLTK vector as a normalizing vector. Cells were then starved for 12 h before the addition or not of TGF𝛃 1 (10 ng/ml). 24 h later cell lysates were collected and subjected to a dual luciferase activity assay. Values in D represent a percentage of luciferase activity (CAGA12LUC) normalized against renilla luciferase (pRLTK) and compared with control. Values in A-C were normalized against GAPDH and are presented as -fold induction versus the non-treated cells. Statistical significant difference ( p ≤ 0.05) is shown as asterisks and p ≤ 0.01 is shown as double asterisks .
FIGURE 4 A , overexpression of miR-155 reduces the TGF𝛃 dependent transcriptional activity. THP1-155 cells were treated with or without doxycycline for 96 h to de-repress or not the expression of the miR-155 transgene. Cells were then treated or not with TGF𝛃 1 (10 ng/ml) for 48 and assayed in RT-qPCR to determine the levels of SERPINE1 mRNA. B , THP-1 cells were transfected with pre-miR-155, pre-miR-221, or pre-miR control oligonucleotides. 48 h after transfection, cells were treated or not with TGF𝛃 1 for an additional 48 h period and then assayed by RT-qPCR to determine the levels of SERPINE1 mRNA. C , THP-1 cells were transfected with different siRNAs: anti-SMAD2#1, anti-SMAD2#2, and siRNA-Control#1. 48 h after transfection cells were treated or not with TGF𝛃 1 for an additional 48 h period and then assayed by RT-qPCR to determine the levels of SERPINE1 mRNA. D , THP1155 cells were treated with doxycycline as in A . Cells were then transfected with the CAGA12 LUC vector and pRLTK vector as a normalizing vector. Cells were then starved for 12 h before the addition or not of TGF𝛃 1 (10 ng/ml). 24 h later cell lysates were collected and subjected to a dual luciferase activity assay. Values in D represent a percentage of luciferase activity (CAGA12LUC) normalized against renilla luciferase (pRLTK) and compared with control. Values in A-C were normalized against GAPDH and are presented as -fold induction versus the non-treated cells. Statistical significant difference ( p ≤ 0.05) is shown as asterisks and p ≤ 0.01 is shown as double asterisks .CC-BY · From the PDF
Image 5
CC-BY · From the PDF
Image 6
CC-BY · From the PDF
Image 7
CC-BY · From the PDF
FIGURE 5 Expression of SMAD2-dependent and -independent genes in THP1-155 cells overexpressing miR-155. THP1-155 cells were treated with or without doxycycline for 96 h, then starved for 12 h before the addition of TGF𝛃 1 (10 ng/ml). 48 h later RNA from these cells was extracted and subjected to RT-qPCR to determine the levels of mRNA of TGF𝛃 1, TGF𝛃 2, TGF𝛃 3, ICAM1, VEGF, IL-1 𝛃 , MMP9, IL-4R, DC-SIGN, and SMAD2. Values were normalized against GAPDH and are presented as -fold induction versus the non-treated cells. RNA from the same sample was subjected to a specific micro-RNA RTqPCR to quantify levels of miR-155. Values were normalized to the housekeeping RNU44. Statistical significant difference ( p ≤ 0.05) is shown as asterisks .
FIGURE 5 Expression of SMAD2-dependent and -independent genes in THP1-155 cells overexpressing miR-155. THP1-155 cells were treated with or without doxycycline for 96 h, then starved for 12 h before the addition of TGF𝛃 1 (10 ng/ml). 48 h later RNA from these cells was extracted and subjected to RT-qPCR to determine the levels of mRNA of TGF𝛃 1, TGF𝛃 2, TGF𝛃 3, ICAM1, VEGF, IL-1 𝛃 , MMP9, IL-4R, DC-SIGN, and SMAD2. Values were normalized against GAPDH and are presented as -fold induction versus the non-treated cells. RNA from the same sample was subjected to a specific micro-RNA RTqPCR to quantify levels of miR-155. Values were normalized to the housekeeping RNU44. Statistical significant difference ( p ≤ 0.05) is shown as asterisks .CC-BY · From the PDF

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Metadata

Authors
Louafi, F., Martinez-Nunez, R. T., & Sanchez-Elsner, T.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2010-12
Volume
285
Issue
53
Pages
41328-41336
ISSN
0021-9258
DOI
10.1074/jbc.m110.146852
Cited by
181
References
1
Language
en
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