Greatest overnight delivery overlap was seen between tubulointerstitium-enriched diabetic kidney disease biopsies and the TGF-��1�Cstimulated HK-2 cells, and notable among these were multiple collagen encoding genes and THBS1. Figure 5. Overlap of let-7c targets upregulated by TGF-��1 in HK-2 cells and also upregulated in renal biopsies of CKD patients versus control. (A) Top 20 let-7c targets significantly upregulated (P��0.05) in HK-2 cells stimulated with TGF-�� … let-7c Targets TGF-��1�CInduced COL1A1, COL1A2, and THBS1 Expression Sequence analysis of the 3�� UTRs of COL1A1, COL1A2, and THBS1 identified let-7c miRNA binding sites at positions 789�C795, 378�C385, and 1518�C1524, respectively (Figure 6A). We previously demonstrated that LXA4 attenuates collagen deposition in an animal model for renal fibrosis, and attenuates the upregulation of let-7c target COL1A2.

5 Minimal free energy calculations for let-7c pairing with 3�� UTR recognition sites indicated the formation of stable hybrid structures between let-7c and COL1A1 (��G = ?20.8 kcal/mol?1), COL1A2 (��G = ?20.3 kcal/mol?1), and THBS1 (��G = ?26.9 kcal/mol?1). TGF-��1 stimulation of HK-2 cells (10 ng/ml; 24 hours) resulted in significant upregulation of COL1A1, COL1A2, and THBS1 gene expression, and transfection with let-7c miRNA mimic suppressed TGF-��1�Cmediated upregulation of these genes (Figure 6, B, D, and F). We conclude that suppression of let-7c miRNA by TGF-��1 is critical for TGF-��1�Cinduced COL1A1, COL1A2, and THBS1 expression.

In HK-2 cells transfected with let-7c anti-miR, TGF-��1 induction of COL1A1, COL1A2, and THBS1 remained unperturbed (Figure 6, C, E, and G), and let-7c miRNA silencing did not prevent LXA4-mediated suppression of these genes. Furthermore, protein levels of THBS1 in response to let-7c anti-miR transfection validated what we observed at the mRNA level (Supplemental Figure 7). Our data indicate that although let-7c miRNA upregulation may be a novel mechanism through which LXA4 suppresses COL1A1, COL1A2, and THBS1 expression, it is not the exclusive mechanism. Figure 6. let-7c targets COL1A1, COL1A2, and THBS1. (A) Predicted let-7c target sites (red) of the human COL1A1, COL1A2, and THBS1 3�� UTRs, and minimal free energy (mfe) calculations measuring stability of miRNA/RNA hybrids. TaqMan qRT-PCR measurement of … Discussion TGF-��1 is implicated in multiple fibrotic disorders.

In this study, we assessed the role and mechanism of lipoxins in attenuating profibrotic responses to TGF-��1. We demonstrate that LXA4 potently represses the expression of key markers of renal fibrosis induced by TGF-��1. miRNA profiling identifies let-7c miRNA as being induced by LXA4 and repressed by TGF-��1 in renal epithelia. let-7c targets a key component of the TGF-��1 signaling pathway, namely TGF��R1, and several effectors and markers of renal fibrosis including HMGA2, COL1A1, COL1A2, and Brefeldin_A THBS1.