A single-nucleotide polymorphism of miR-146a and psoriasis: an association and functional study
a functional SNP of rs2910164 in miR-146a and the risk of psoriasis in the Chinese Han population
a significantly increased risk of psoriasis was associated with the rs2910164 miR-146a CG and GG genotypes (adjusted OR, 1.38; 95% CI, 1.06–1.80).
the rs2910164G allele in miR-146a attenuated its inhibitory regulation on the expression of EGFR as well as the proliferation of human keratinocytes, and lowered the level of miR-146a in the psoriatic lesions.
These findings indicate that the rs2910164G allele in miR-146a weakens its suppression on the proliferation of keratinocytes probably through the decreased inhibition of the target gene, EGFR, which may account for the increased risk of psoriasis in this study population.
MIR146A gene位置 & rs2910164疾病相关性；
研究层面：医院case-control study，healthy controls and psoriasis patients；in vitro experiments；
#Materials and methods
DNA samples from 521 psoriasis cases and 582 healthy controls
外周血DNA抽提：a DNA isolation kit (Tiangen, Beijing, China).
SNP基因分型：polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis.
PCR扩增引物：5′-CATGGGTTGTGTCAGTGTCAGAGCT-3′ and reverse 5′-TGCCTTCTGTCTCCAGTCTTCCAA-3′ (product of 147 bp).
PCR体系：95°C for 5 min.; 32 cycles of 95°C for 30 sec., annealing temperature of 56°C for 30 sec. and 72°C for 40 sec.; and final extension step at 72°C for 10 min.
##Collection of Skin Specimens and RNA extraction
##Quantitative real-time polymerase chain reaction (qRT-PCR)
PrimeScript RT reagent Kit (Takara, Ohtsu, Japan)；
- qRT-PCR for pre-miR-146a：
SYBR Premix Ex Taq II (TaKaRa) with a BIO-RAD Multicolour Real-time -
PCR Detection System (iQTM5)；
pre-miR-146a (forward 5′-TGAGAACTGAATTCCATGGGTT-3′ and reverse 5′-CTGAAGAACTGAATTTCAGAGG-3′),
β-actin (forward 5′-AGAAAATCTGGCACCACACC-3′and reverse 5′-AGAGGCGTACAGGGATAGCA-3′).
- 体系：95°C for 2 min., followed by 45 cycles of denaturation at 95°C for 5 sec., annealing at 57°C for 10 sec. and extension at 72°C for 15 sec
- qRT-PCR for miR-146a：
miRNA SYBR qRT-PCR Kit (Qiagen, Valencia, CA, USA) with iQTM5；
- The primers of RUNU6, and hsa-miR-146a：
designed and synthesized by Tiangen Company based on miRBase (http://www.mirbase.org).
DNA was amplified for 40 cycles of denaturation for 20 sec. at 95°C and annealing for 60 sec. at 60°C.
- HaCaT cells
- Normal human keratinocytes
##Construction of the plasmids
To express the different precursors of miR-146a, the pri-miRNA sequence was amplified by PCR (oligonucleotide primer sequences: for Pre-mir-146a C allele, forward 5′-GGCAACAAGAAACTGcCTGAGTTACATCAGTC-3′ and reverse 5′-GACTGATGTAACTCAGGCAGTTTCTTGTTGCC-3′; for Pre-mir-146a G allele, forward 5′-CCCAAGCTTGTCTACTCTCTAGTCCTTAGGGAGGTTG-3′ and reverse 5′-CCGGAATTCCACTCACAGCTTGTCCTCCTTGG-3′) and was cloned into pEX-5 plasmids with HindIII and EcoRI digestion (Invitrogen). Human EGFR plasmid using pCMV6-ENTRY vector was a commercial product from Origene Company (Rockville, MD, USA). All the constructs used in the study were restriction mapped and sequenced to confirm their authenticity.
Transfections of the plasmids were performed with HaCaT cells and normal human keratinocytes by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommendations. All the transfections were carried out in triplicate.
Null vector transfection was used as the control.
##Cell growth assay
- CCK-8 assay
The effect of the SNP in miR-146a on the growth of keratinocytes was determined by using the CCK-8 assay with some modifications.
- HaCaT cells or normal human keratinocytes
- miR-146a expression plasmids (C or G allele) or control plasmids pCDNA3.1; the EGFR expression plasmids ;
##Western blotting assay
48h later，the cells were first lysed with RIPA lysis buffer (Beyotime, Shanghai, China) containing a cocktail of protease inhibitors and phosphatase inhibitors for 30 min. on ice and were then centrifuged at 10,303 g for 20 min.
The total protein concentration was measured by using a Bradford assay. Equal amounts of the protein samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane (Millipore, Bedford, MA, USA). The blot was blocked for 1 hr and then incubated overnight with a primary antibody against EGFR (Abcam, Cambridge, UK) and β-actin (Santa Cruz, CA, USA). After extensive rinsing, the blot was incubated with HRP-conjugated secondary antibodies (Zhongshan Biotechnology, Beijing, China) for 2 hrs at room temperature (RT). The immunoreactive bands were detected with an enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA).
Genotype distribution of the rs2910164 SNP in miR-146a in cases and controls
Stratification analysis of the rs2910164 SNP in miR-146a and risk of psoriasis by selected variables
The rs2910164 SNP can affect the level of mature miR-146a
The rs2910164G allele in miR-146a impairs its regulation on the expression of EGFR in human keratinocytes
The rs2910164G allele in miR-146a weakens its inhibitory effect on the proliferation of human keratinocytes
Re-introduction of EGFR rescues the inhibition on the proliferation of keratinocytes induced by miR-146a
Association of the miR-146a G/C polymorphism with the mature miR-146a level in psoriatic and normal skin