Fig 1: LIN28A enhanced BMAL1 expression induced by H. pylori. a and b, qRT-PCR and Western blot analysis of BMAL1 in AGS and BGC-823 cells infected with H. pylori. c and d, qRT-PCR and Western blot analysis of BMAL1 with LIN28A suppression on the basis of H. pylori infection. e and f, IHC staining for LIN28A in 37 cases of superficial gastritis(SG), atrophic gastritis(AG) and Intestinal metaplasia(IM) patients respectively with or without H. pylori infection, and the relative values of IHC optical density. Scale bars, 100 μm (insets 50 μm).
Fig 2: LIN28A directly regulated the transcription level of BMAL1, independent of let-7a. a, binding site of let-7a-3p on the 3′UTR region of BMAL1. b, qRT-PCR analysis of let-7a-3p and BMAL1 in AGS and BGC-823 cells transfected with mimics or inhibitor of let-7a-3p. c, Western blot analysis of BMAL1 and LIN28A in AGS and BGC-823 cells transfected with mimics or inhibitor of let-7a-3p. d, the dual luciferase assay detected the transcriptional regulation of let-7a-3p on the 3′UTR region of BMAL1. e and f, qRT-PCR and Western blot analysis of BMAL1 in AGS and BGC-823 cells transfected with LIN28A siRNA or overexpression plasmid. g, the binding sites of LIN28A in the BMAL1 promoter region, and the corresponding base mutation. h, ChIP was used to detect the binding between LIN28A and the two sites of the BMAL1 promoter region with and without LIN28A depletion. i, the transcriptional activation of the BMAL1 promoter region by LIN28A was detected by using a dual luciferase assay in case of binding sites mutation.
Fig 3: Schematic model of the study. H. pylori up-regulated the expression of LIN28A, and LIN28A directly bound to the BMAL1 promoter, activating the transcription of BMAL1 to increase its expression. BMAL1 in turn promoted transcription of TNF-α by directly binding to the E-box elements on its promoter to increase its secretion. Therefore, our study revealed the mechanism through which disorder of circadian rhythm aggravated the inflammatory response induced by H. pylori.
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