Table 2.

Promoter binding of NF-κB p65 subunit in LPS-stimulation transactivation of miRNA genes in H69 cells

LPS-up-regulated mature miRNAs	Corresponding pri-miRNAs	Potential NF-κB binding site(s) within the promoter region	Promoter p65 binding by ChIP	Conformed by promoter reporter assay
miR-17	pri-miR-17-92	TGGAATTTCC (−1698 to −1689)	−	−
miR-18a		TGGGATTTCC (−1442 to −1433)	−	−
miR-20a		CGGAATTTCC (−827 to −818)	+	+
miR-125b	pri-miR-125b-1	GGGAATTTCA (−2455 to −2446)	−	−
		GGGGCTTTCC (−1059 to −1050)	+	+
miR-21	pri-miR-21	GGGAATTTTC (+1167 to +1176)	−	−
		GGGAATTCTC (+1395 to +1404)	+	+
miR-23b	pri-miR-23b-27b-24-1	GGGACTCTCC (−1263 to −1254)	+	+
miR-27b
miR-24-1
miR-30b	pri-miR-30b	AGGAATTTAC (−347 to −338)	+	+
miR-130a	pri-miR-130a	GGGAATTTGC (−2977 to −2968)	+	+
miR-29a	pri-miR-29b-1-29a	AGGATTTTCC (−89 to −80)*	NS	NS

Putative promoter element for each miRNA gene was based on previous studies as referred in Table 1. Potential NF-κB binding site(s) in the promoter region was identified by the TFSEARCH (http://www.cbrc.ip/research/db/ TFSEARCH.html) and MOTIF (http://motif.genome.jp/). LPS-induced promoter binding of NF-κB p65 subunit to the predicted binding site was confirmed by ChIP analysis and marked as ‘+’; otherwise marked as ‘−’ if p65 binding was not enhanced after LPS stimulation. LPS-induced transactivation of miRNA gene by p65 was confirmed by using luciferase reporter gene constructs that spanned the promoter region of each individual gene. If LPS stimulation increased luciferase activity in cells transfected with the luciferase constructs containing the binding site for p65 and this induction was blocked by SC-514, it was presented as ‘+’; otherwise presented as ‘−’. The putative NF-κB binding site of miR-29a was identified from the start site of pre-miR-29a, which was indicated by asterisk. NS, not selected for further ChIP analysis or luciferase reporter assay in this study. Refer to Supplementary Figures S2–S6 for details.