Figure 1.
The effect of miR-197 on proliferation and differentiation of KC.
a) HaCaT cells were transfected with miR-197 expressing plasmid or HTR expressing plasmid as a control. Total RNA was extracted from each cell line, subjected to qPCR analysis and normalized by Rnu48 (*P<0.049988). b–c) Cell Proliferation of HaCaT cells stably expressing hsa-miR-197 or the HTR control RNA was assessed by (b) BrdU incorporation (c) MTT and assays. Proliferation was calculated as the percentage of the reading at seeding time (T = 0) given a value of 100%. For the BrdU assays mean and standard deviation were calculated from 3 independent experiments (*P<0.0232). For the MTT assays mean and standard deviation were calculated from at list 5 independent experiments (*P>0.002638) as calculated by ttest. d–e). Total RNA of HaCaT-miR-197 or HaCaT-HTR was subjected to qPCR analysis for involucrin, keratin 10 (K10) or keratin 14 (K14) and normalized by Rplp0. Y bars are arbitrary units that define fold change *p = 0.05.f). Total RNA of HaCaT-miR-197 or HaCaT-HTR was subjected to RT-PCR analysis for K10 and K14 and normalized by β-Actin expression. *p = 0.01. f) PHK were seeded in high Ca++ medium or low Ca++ medium. After 24 h cells were harvested and subjected to miR-197 qPCR as in *p = 0.05.
Figure 2.
IL-22 enhances miR-197 expression.
IL-22 was added to PHK cells at the indicated concentrations. Cells were harvested and subjected to miR-197 specific qPCR. a) 1 h post IL-22 addition *P = 0.022 b) 48 h post IL-22 addition. *p = 0.01 **P = 0.017 ***p = 0.03. (The mean −/+ SD was calculated from 4 independent experiments).
Figure 3.
STAT3 binds to the putative promoter region of miR-197 after IL-22 treatment.
a) STAT3 inhibitor (final concentration of 100 µM) was added to PHK, 30 min after 5 ng/ml IL-22 was added. a) 1 h afterword miR-197 quantity was measure by qPCR (The mean −/+ SD was calculated from 4 independent experiments) *p = 0.047699) b) Alignment of genomic miR-197 gene region sequence in six Primates. Pri-miR-197 marked in blue; mature miR-197 marked in red; miRNA “seed” marked in blue box. Different bases form consensuses are in green. The putative promoter region has 3 conserved STAT sites and one un-conserved (pink boxes). c) PHK cells were treated or not with 5 ng/ml IL-22 for 30 min, then were subject to ChIP assay using phosphorylated STAT-3 antibody. The results present the amount measured by PCR of immune precipitated DNA with the anti pSTAT3 divided to the amount of measured by PCR of input DNA. All PCR were done with qPCR SYBR Green dye. The mean −/+ SD was calculated from 4 independent experiments (t test *P = 0.016). Primers that were used in the ChIP assay are marked by underline in b.
Figure 4.
miR-197 suppresses the expression of IL22RA1 by binding to its 3′UTR.
a) miR-197 binding site on IL22RA1 3′UTR. b) HEK-293T cells were transfected with psi-CHECK2 vectors encoding luciferase (vector), luciferase fused to the IL22RA1-3′UTR (IL22RA1 wt 3′UTR), or luciferase fused to a mutated in miR-197 binding to the IL22RA1 3′UTR (IL22RA1 mut 3′UTR) together with 2 µg miR-197 expressing plasmid or 2 µg scramble RNA expressing plasmid. Cells transfected with only with vector lacking the IL22RA1 3′UTR was valued as 100%. The error bars are calculated as standard error of at least 6 independent experiments. c) Western blot (WB) analysis of IL22RA1 protein 48 h after transfection with 5/10 nM of scramble control pre-miR or with 5/10 nM of pre-miR-197. d) Densitometry analysis of 4 WBs analysis of IL22RA1 protein 48 h after transfection with 10 nM of scramble control RNA (scramb), or 10 nM pre-miR-197 *p = 0.0000818.
Figure 5.
Feedback loop model between IL-22 signaling and miR-197.
Figure 6.
miR-197 slows proliferation and migration of KC.
HaCaT-miR-197 or control HaCaT–HTR were subject to; a) BrdU incorporation as in figure 1a. 24 h after seeding, at the 0 time point, 5 ng/ml IL-22 was added. Cell Proliferation was calculated as in figure 1a. Mean and standard deviation were calculated from 3 independent experiments (*P<0.0145). b) 24,000 cells were seeded on platypus 96 wells plate to reach 80% confluence. Then cells were serum starved for 24 h, afterward, IL-22 was added as written. 48 h later, cells were washed and fixated. C) Representative experiment of Cell migration: percentage was calculated as the subtraction of the area, as marked, from the area at time 0. Area was determined by imageJ program. (p<0.001 **p<0.000001).
Figure 7.
Cytosine methylation in psoriatic and normal skin.
a) Schematic presentation of miR-197 putative promoter. The 8 circles represent the CpG, of the CpG Island. Sequence from −1544 to −1594 before or after bisulfite conversion is shown. b) Represented sequencing chromatogram of the bisulfite conversion region, analysis by BioEdit, C and T residues at the CpG are marked.
Figure 8.
Evolutionary conservation of miR-197 and its putative binding site on IL22RA13′UTR.
a) Using the miRviewer program, the present of miR-197 was examined in various animals genome. The name of the species is written above each block. The white dot in box indicates that this miRNA are present in miRbase. Grey box indicates that the miRNA was not identified in the genome of the specific species, under stringent parameters. The tone of green defined the degree of conservation b) Alignment of pre-miR-197 gene sequences of few primates, mouse, rat, cow, dog, horse, and guinea pig. c) Alignment of the IL22RA1 3′UTR of some of the above species and the miR-197 putative binding site is marked.
Table 1.
Primer used to generate psiCHECK-IL22RA1-3′UTR WT and mutant.