Down‐regulation of human long non‐coding RNA LINC01187 is associated with nephropathies

FIGURE 1. Expression analysis of selected lncRNAs in human renal biopsies. (A) Human Genotype‐Tissue Expression analysis (GTEx Portal) indicates that LINC01187 is predominantly expressed in kidney tissue (cortex and medulla). TPM: transcripts per million. (B–K) Expression profiles of 9 lncRNAs with real‐time RT‐qPCR analysis in renal biopsies from kidney patients (Pt.) and healthy controls (HC). The lncRNA genes that were examined in human samples have been found deregulated in the kidney of UUO mouse model (conserved lncRNA genes). (J,K) LINC01187 lncRNA is significantly down‐regulated in individuals with renal disorders compared to HC. The lncRNA levels of each lncRNA were normalized to GAPDH and expressed as relative fold of expression to the sample with the minimal fold of expression. *p < 0.05, **p < 0.01, ns: p ≥ 0.05 (t‐test 2;2)

FIGURE 2. Analysis of Gm12121 lncRNA at both RNA and chromatin organization levels. (A) Visualization of RNA‐seq peaks for Gm12121 lncRNA using the UCSC Genome Browser in representative sham‐operated (SO), 2 days ligated (2D) and 8 days ligated (8D) UUO renal samples. Conservation analyses indicate that both promoter and RNA sequences of Gm12121 lncRNA are conserved among many species including mice and humans. (B) Expression analysis of Gm12121 in UUO mouse model. (C) Chromatin immunoprecipitation experiments with antibodies against RNA polymerase II (RNA pol) and H3K4m3 (as indicated) in conjunction with real‐time RT‐qPCR analysis. Schematic representation of the relative genomic position of the primers used for RT‐qPCRs experiments is indicated at top panel. For these experiments, chromatin samples were prepared from kidneys of SO n = 3 and 8D mice n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 (t‐test 2;2). (D) Mouse Genotype‐Tissue Expression analysis (GTEx Portal) shows that Gm12121 lncRNA is predominantly expressed in adult kidney

FIGURE 3. Changes in the expression levels of Gm12121 lncRNA gene in NTS‐induced Glomerulonephritis and I/R animal models of kidney diseases. Real‐time RT‐qPCR analysis of the expression profile of Gm12121 in the: (A) model of glomerulonephritis, induced by intravenous administration of nephrotoxic serum (NTS). Treatment with anti‐sense (AS) oligonucleotides against periostin is protecting kidneys from the development of CKD 31 and is sufficient to restore Gm12121 expression levels. PBS: animals injected with PBS; NTS + SCR Post: animals injected with NTS and treated with scrambled oligonucleotides; NTS + As Post: animals injected with NTS and treated with anti‐sense oligonucleotides against periostin. (B) Ischemia/reperfusion (I/R) model of acute kidney injury. SO: sham operated; I/R 24 and 72 h: tissues are dissected 24 or 72 h after ischemia/reperfusion, respectively. Primer sets 1, 2, 3 are described in Materials and Methods section. The RNA levels of Gm12121 were normalized to Hprt. *p < 0.05; **p < 0.01, ***p < 0.005, ns: p ≥ 0.05 (t‐test 2;2), n = 4 animals per group of treatment

FIGURE 4. Gene expression analysis of LINC01187 gene in the glomerular and tubulointerstitial compartment of manually micro‐dissected kidney biopsies from patients with different kidney diseases. Values are expressed as log2‐fold change compared to controls (living donors, LD). A q‐value below 5% was considered to be statistically significant. ns: p ≥ 0.05. MCD, minimal change disease; FSGS, focal segmental glomerulosclerosis; DN, diabetic nephropathy; RPGN, rapidly progressive glomerulonephritis

FIGURE 5. LINC01187 lncRNA expression is down‐regulated in renal tissue from patients with diabetic nephropathy and rapidly progressive glomerulonephritis. (A) Schematic representation of the relative position of LINC01187 lncRNA (dark blue) in the genome and the riboprobe (light blue) used for the RNA in situ hybridization analysis. (B‐E) RNA in situ hybridization analysis with anti‐sense (experimental) and sense probes (internal control) of LINC01187 on renal tissues from nephrectomies and biopsies. Data from healthy renal tissues from nephrectomies, due to cancer, from four individuals (H1, H2, H3, H4) are presented in (B). Data from biopsies of diabetic nephropathy (DN) from 3 individuals (D1, D2, D3) are presented in (C). Data from biopsies of lupus nephritis (LN) from 2 individuals (L1, L2) are presented in (D). Data from biopsies of rapidly progressive glomerulonephritis (RPGN) from 3 individuals (R1, R2, R3) are presented in (E). Representative scale bar 0.08 mm on first picture

FIGURE 6. LINC01187 overexpression in renal cells reduces apoptotic rate without affecting proliferation. (A) Representative flow cytometry gating analysis of transfected cells (GFP+) in control condition (co‐transfection of GFP plasmid with empty pcDNA3.1 vector) and LINC01187 overexpression condition (co‐transfection of GFP plasmid with pcDNA3.1 containing LINC01187 sequence insert) for Annexin V and 7‐AAD markers. (B) Apoptotic rate in control condition and LINC01187 overexpression condition. Three biological replicates per condition; t‐test; *p < 0.05. (C) Representative flow cytometry gating analysis of transfected cells (GFP+) in control condition (co‐transfection of GFP plasmid with empty pcDNA3.1 vector) and LINC01187 overexpression condition (co‐transfection of GFP plasmid with pcDNA3.1 containing LINC01187 sequence insert). (D) Mean fluorescent intensity (MFI) of Ki‐76 in control condition and LINC01187 overexpression condition. Three biological replicates per condition; t‐test; p = 0.58 (ns)