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Transcription factor and microRNA interactions in lung cells: an inhibitory link between NK2 homeobox 1, miR-200c and the developmental and oncogenic factors Nfib and Myb.

Tagne JB, Mohtar OR, Campbell JD, Lakshminarayanan M, Huang J, Hinds AC, Lu J, Ramirez MI - Respir. Res. (2015)

Bottom Line: Mouse lungs lacking functional phosphorylated Nkx2-1 showed increased expression of miR-200c and alterations in the expression of other top regulated miRNAs.Moreover, chromatin immunoprecipitation assays showed binding of NKX2-1 protein to regulatory regions of these miRNAs.Promoter reporter assays indicated that 1kb of the miR-200c 5' flanking region was transcriptionally active but did not mediate Nkx2-1- repression of miR-200c expression. 3'UTR reporter assays support a direct regulation of the predicted targets Nfib and Myb by miR-200c.

View Article: PubMed Central - PubMed

Affiliation: The Pulmonary Center, Boston University School of Medicine, Boston, USA. tagne@bu.edu.

ABSTRACT

Background: The transcription factor NK2 homeobox 1 (Nkx2-1) plays essential roles in epithelial cell proliferation and differentiation in mouse and human lung development and tumorigenesis. A better understanding of genes and pathways downstream of Nkx2-1 will clarify the multiple roles of this critical lung factor. Nkx2-1 regulates directly or indirectly numerous protein-coding genes; however, there is a paucity of information about Nkx2-1-regulated microRNAs (miRNAs).

Methods and results: By miRNA array analyses of mouse epithelial cell lines in which endogenous Nkx2-1 was knocked-down, we revealed that 29 miRNAs were negatively regulated including miR-200c, and 39 miRNAs were positively regulated by Nkx2-1 including miR-1195. Mouse lungs lacking functional phosphorylated Nkx2-1 showed increased expression of miR-200c and alterations in the expression of other top regulated miRNAs. Moreover, chromatin immunoprecipitation assays showed binding of NKX2-1 protein to regulatory regions of these miRNAs. Promoter reporter assays indicated that 1kb of the miR-200c 5' flanking region was transcriptionally active but did not mediate Nkx2-1- repression of miR-200c expression. 3'UTR reporter assays support a direct regulation of the predicted targets Nfib and Myb by miR-200c.

Conclusions: These studies suggest that Nkx2-1 controls the expression of specific miRNAs in lung epithelial cells. In particular, we identified a regulatory link between Nkx2-1, the known tumor suppressor miR-200c, and the developmental and oncogenic transcription factors Nfib and Myb, adding new players to the regulatory mechanisms driven by Nkx2-1 in lung epithelial cells that may have implications in lung development and tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

NKX2-1 binds to miRNA’s regulatory regions and regulates their transcriptional activity. (A) Scheme representing 1 kb of the 5′ flanking region of each miRNA. The core of the NKX2-1 consensus binding site (CAAG/CTTG) is represented as a green circle. (B) Chromatin immunoprecipitation assays using NKX2-1 antibody or IgG control followed by qPCR analysis of the 5′ flanking regions of miR-200c, miR-221 and miR-1195. Data represents percentage of the input in log scale. (*) p ≤ 0.05. (C) Binding of NKX2-1 to miR-200c 5′ flanking region in E11.5 and E19.5 mouse lung previously determined by ChIP-chip analysis [12]. Data represents binding signal (y axis) for each probe on the promoter tiling array (x axis) corresponding to the 5′ flanking region of the pre-miR-200c. miR-221 and miR-1195 5′ flanking regions were not represented in the array. Red, E11.5; blue, E19.5; black line, transcript. N = 3 (D) Promoter-luciferase analysis of the transcriptional activity of the 1 kb 5′ flanking regions of different miRNAs in MLE15 transduced with NKx2-1 shRNA or control. Data represents Firefly luciferase normalized to Renilla luciferase. Values are relative to the 0-Luciferase (pGL3) control vector in each cell line. (*) p < 0.05 in 0-Luc vs. miR-200c –Luc; (**) p < 0.05 in miR-200c-Luc + scrambled vs. miR-200c.
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Fig3: NKX2-1 binds to miRNA’s regulatory regions and regulates their transcriptional activity. (A) Scheme representing 1 kb of the 5′ flanking region of each miRNA. The core of the NKX2-1 consensus binding site (CAAG/CTTG) is represented as a green circle. (B) Chromatin immunoprecipitation assays using NKX2-1 antibody or IgG control followed by qPCR analysis of the 5′ flanking regions of miR-200c, miR-221 and miR-1195. Data represents percentage of the input in log scale. (*) p ≤ 0.05. (C) Binding of NKX2-1 to miR-200c 5′ flanking region in E11.5 and E19.5 mouse lung previously determined by ChIP-chip analysis [12]. Data represents binding signal (y axis) for each probe on the promoter tiling array (x axis) corresponding to the 5′ flanking region of the pre-miR-200c. miR-221 and miR-1195 5′ flanking regions were not represented in the array. Red, E11.5; blue, E19.5; black line, transcript. N = 3 (D) Promoter-luciferase analysis of the transcriptional activity of the 1 kb 5′ flanking regions of different miRNAs in MLE15 transduced with NKx2-1 shRNA or control. Data represents Firefly luciferase normalized to Renilla luciferase. Values are relative to the 0-Luciferase (pGL3) control vector in each cell line. (*) p < 0.05 in 0-Luc vs. miR-200c –Luc; (**) p < 0.05 in miR-200c-Luc + scrambled vs. miR-200c.

Mentions: To evaluate NKX2-1 protein binding to regulatory regions of the top regulated miRNAs we performed ChIP assays using a NKX2-1 antibody or the corresponding IgG followed by quantitation by qPCR of fragments within the miRNA’s 5′ flanking regions (Figure 3A). The NKX2-1 antibody significantly immunoprecipitated 5′regions of the tested miRNAs compared to IgG (Figure 3B). For miR-200c, the enrichment of the IP fractions with the Nkx2-1 antibody and IgG were 28 and 0.4% of input while for miR-1195, were 129% and 1.3% of input respectively. NKX2-1 also binds to the 5′flanking region of miR-221. These data indicate a potential regulatory link between NKX2-1 and miR-200c, miR-221, or miR-1195. In support of these results, a search in a ChIP-chip dataset of NKX2-1 target genes in mouse lung development that we previously published [12] indicates a significant binding of NKX2-1 within the 1kb 5′ flanking region of the miR-200c gene (Figure 3C).Figure 3


Transcription factor and microRNA interactions in lung cells: an inhibitory link between NK2 homeobox 1, miR-200c and the developmental and oncogenic factors Nfib and Myb.

Tagne JB, Mohtar OR, Campbell JD, Lakshminarayanan M, Huang J, Hinds AC, Lu J, Ramirez MI - Respir. Res. (2015)

NKX2-1 binds to miRNA’s regulatory regions and regulates their transcriptional activity. (A) Scheme representing 1 kb of the 5′ flanking region of each miRNA. The core of the NKX2-1 consensus binding site (CAAG/CTTG) is represented as a green circle. (B) Chromatin immunoprecipitation assays using NKX2-1 antibody or IgG control followed by qPCR analysis of the 5′ flanking regions of miR-200c, miR-221 and miR-1195. Data represents percentage of the input in log scale. (*) p ≤ 0.05. (C) Binding of NKX2-1 to miR-200c 5′ flanking region in E11.5 and E19.5 mouse lung previously determined by ChIP-chip analysis [12]. Data represents binding signal (y axis) for each probe on the promoter tiling array (x axis) corresponding to the 5′ flanking region of the pre-miR-200c. miR-221 and miR-1195 5′ flanking regions were not represented in the array. Red, E11.5; blue, E19.5; black line, transcript. N = 3 (D) Promoter-luciferase analysis of the transcriptional activity of the 1 kb 5′ flanking regions of different miRNAs in MLE15 transduced with NKx2-1 shRNA or control. Data represents Firefly luciferase normalized to Renilla luciferase. Values are relative to the 0-Luciferase (pGL3) control vector in each cell line. (*) p < 0.05 in 0-Luc vs. miR-200c –Luc; (**) p < 0.05 in miR-200c-Luc + scrambled vs. miR-200c.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: NKX2-1 binds to miRNA’s regulatory regions and regulates their transcriptional activity. (A) Scheme representing 1 kb of the 5′ flanking region of each miRNA. The core of the NKX2-1 consensus binding site (CAAG/CTTG) is represented as a green circle. (B) Chromatin immunoprecipitation assays using NKX2-1 antibody or IgG control followed by qPCR analysis of the 5′ flanking regions of miR-200c, miR-221 and miR-1195. Data represents percentage of the input in log scale. (*) p ≤ 0.05. (C) Binding of NKX2-1 to miR-200c 5′ flanking region in E11.5 and E19.5 mouse lung previously determined by ChIP-chip analysis [12]. Data represents binding signal (y axis) for each probe on the promoter tiling array (x axis) corresponding to the 5′ flanking region of the pre-miR-200c. miR-221 and miR-1195 5′ flanking regions were not represented in the array. Red, E11.5; blue, E19.5; black line, transcript. N = 3 (D) Promoter-luciferase analysis of the transcriptional activity of the 1 kb 5′ flanking regions of different miRNAs in MLE15 transduced with NKx2-1 shRNA or control. Data represents Firefly luciferase normalized to Renilla luciferase. Values are relative to the 0-Luciferase (pGL3) control vector in each cell line. (*) p < 0.05 in 0-Luc vs. miR-200c –Luc; (**) p < 0.05 in miR-200c-Luc + scrambled vs. miR-200c.
Mentions: To evaluate NKX2-1 protein binding to regulatory regions of the top regulated miRNAs we performed ChIP assays using a NKX2-1 antibody or the corresponding IgG followed by quantitation by qPCR of fragments within the miRNA’s 5′ flanking regions (Figure 3A). The NKX2-1 antibody significantly immunoprecipitated 5′regions of the tested miRNAs compared to IgG (Figure 3B). For miR-200c, the enrichment of the IP fractions with the Nkx2-1 antibody and IgG were 28 and 0.4% of input while for miR-1195, were 129% and 1.3% of input respectively. NKX2-1 also binds to the 5′flanking region of miR-221. These data indicate a potential regulatory link between NKX2-1 and miR-200c, miR-221, or miR-1195. In support of these results, a search in a ChIP-chip dataset of NKX2-1 target genes in mouse lung development that we previously published [12] indicates a significant binding of NKX2-1 within the 1kb 5′ flanking region of the miR-200c gene (Figure 3C).Figure 3

Bottom Line: Mouse lungs lacking functional phosphorylated Nkx2-1 showed increased expression of miR-200c and alterations in the expression of other top regulated miRNAs.Moreover, chromatin immunoprecipitation assays showed binding of NKX2-1 protein to regulatory regions of these miRNAs.Promoter reporter assays indicated that 1kb of the miR-200c 5' flanking region was transcriptionally active but did not mediate Nkx2-1- repression of miR-200c expression. 3'UTR reporter assays support a direct regulation of the predicted targets Nfib and Myb by miR-200c.

View Article: PubMed Central - PubMed

Affiliation: The Pulmonary Center, Boston University School of Medicine, Boston, USA. tagne@bu.edu.

ABSTRACT

Background: The transcription factor NK2 homeobox 1 (Nkx2-1) plays essential roles in epithelial cell proliferation and differentiation in mouse and human lung development and tumorigenesis. A better understanding of genes and pathways downstream of Nkx2-1 will clarify the multiple roles of this critical lung factor. Nkx2-1 regulates directly or indirectly numerous protein-coding genes; however, there is a paucity of information about Nkx2-1-regulated microRNAs (miRNAs).

Methods and results: By miRNA array analyses of mouse epithelial cell lines in which endogenous Nkx2-1 was knocked-down, we revealed that 29 miRNAs were negatively regulated including miR-200c, and 39 miRNAs were positively regulated by Nkx2-1 including miR-1195. Mouse lungs lacking functional phosphorylated Nkx2-1 showed increased expression of miR-200c and alterations in the expression of other top regulated miRNAs. Moreover, chromatin immunoprecipitation assays showed binding of NKX2-1 protein to regulatory regions of these miRNAs. Promoter reporter assays indicated that 1kb of the miR-200c 5' flanking region was transcriptionally active but did not mediate Nkx2-1- repression of miR-200c expression. 3'UTR reporter assays support a direct regulation of the predicted targets Nfib and Myb by miR-200c.

Conclusions: These studies suggest that Nkx2-1 controls the expression of specific miRNAs in lung epithelial cells. In particular, we identified a regulatory link between Nkx2-1, the known tumor suppressor miR-200c, and the developmental and oncogenic transcription factors Nfib and Myb, adding new players to the regulatory mechanisms driven by Nkx2-1 in lung epithelial cells that may have implications in lung development and tumorigenesis.

No MeSH data available.


Related in: MedlinePlus