Supplementary Material for: miR-181a Improved Renal Inflammation by Targeting TNF-α in a Diabetic Nephropathy Animal Model

<b><i>Introduction:</i></b> Recent studies have elucidated that miR-181a plays a vital role in various inflammation-associated diseases; however, its role in the pathogenesis of diabetic nephropathy (DN) has not been elucidated clearly. Hence, we investigated the involvement of miR-181a in the pathogenesis of DN in patients and animal models. <b><i>Methods:</i></b> miR-181a levels were measured in the kidneys of DN patients and a DN mouse model (db/db mice) by RT-PCR. The interaction of miR-181a with the 3′-UTR of potential target transcripts was investigated using luciferase reporter assays. miR-181a-GFP-AAV and miR-181a inhibitor were used to overexpress or downregulate miR-181a in the kidney of db/db mice, respectively. The expression of inflammatory proteins and target genes was measured in the kidneys of the animals. <b><i>Results:</i></b> In DN patients and DN mouse models, expression analyses showed a significant decrease in miR-181a levels in the kidney. In addition, a negative linear correlation between miR-181a and proinflammatory gene (IL-1β and TNF-α) levels was observed in the kidneys of DN patients. Overexpression of miR-181a reduced IL-1β and TNF-α expression, whereas suppression of miR-181a worsened these changes, which was attenuated by combined treatment with TNF-α neutralizing antibody in db/db mice. Moreover, luciferase assays revealed that TNF-α is a direct target of miR-181a. <b><i>Conclusion:</i></b> Our data emphasized the anti-inflammatory actions of miR-181a by targeting TNF-α in the context of renal inflammation in DN. Hence, we concluded that miR-181a could be a potential therapeutic option for minimizing renal inflammation in DN.