Association of polymorphisms in pre-miRNA with inflammatory biomarkers in rheumatoid arthritis in the Chinese Han population

Human Immunology 73 (2012) 101-106

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Association of polymorphisms in pre-miRNA with inflammatory biomarkers in rheumatoid arthritis in the Chinese Han population Bin Yang a,†, Jie Chen a,†, Yi Li a, Junlong Zhang a, Dongdong Li a, Zhuochun Huang a, Bei Cai a, Lixin Li a, Yunying Shi b,*, Binwu Ying a,*, Lanlan Wang a,* a b

Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China Department of Nephrology, West China Hospital, Sichuan University, Chengdu 610041, China

A R T I C L E

I N F O

Article history: Received 9 February 2011 Accepted 3 October 2011 Available online 12 October 2011

Keywords: SNPs MicroRNA Rheumatoid arthritis (RA) C-reactive protein (CRP) Erythrocyte sedimentation rate (ESR) Inflammatory cytokine

A B S T R A C T

The aim of this study was to detect the association between 2 single nucleotide polymorphisms (SNPs), rs2910164 G⬎C and rs3746444 T⬎C, in pre-miRNA (hsa-mir-146a and hsa-mir-499) and the chronic inflammation in the Chinese Han population with rheumatoid arthritis (RA). Two hundred sixty-two Han Chinese patients with RA were recruited in this study. The SNPs were genotyped by polymerase chain reaction restriction fragment length polymorphism. C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and the plasma concentrations of interleukin (IL)-6, tumor necrosis factor ␣ (TNF-␣), and transforming growth factor ␤1 (TGF-␤1) were measured. There was a significant difference in the levels of CRP and ESR among different genotypes in rs3746444 (p ⫽ 0.031 and p ⫽ 0.047, respectively). The heterozygote CT had significantly higher levels of CRP and ESR compared with homozygotes CC and TT. No significant association was observed between the SNP rs2910164 and the levels of CRP, ESR, IL-6, TNF-␣, and TGF-␤1 (all p ⬎ 0.05). The results of this study provided the first evidence that the SNP rs3746444 in pre-miR-499 could affect the inflammatory reaction in patients with RA. The findings were significant and might contribute to the clinical assessment of inflammatory activity, which in turn may influence therapeutic decision making. 䉷 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by persistent chronic inflammation of the synovial tissue, which results in irreversible joint damage [1]. Inflammatory cytokines, including tumor necrosis factor ␣ (TNF-␣) and interleukin (IL)-1␤, play an important role in the pathogenesis and development of RA, and inhibition of these cytokines can ameliorate disease in some patients [2,3]. Persistent chronic inflammation resulted in tissue or joint damage. The higher the level of in vivo inflammation, the more serious the tissue damage and the poorer the prognosis. MicroRNAs (miRNAs) are small noncoding RNAs, which are approximately 19 –23 nucleotides long and negatively regulate gene expression at the posttranscriptional level [4,5]. They can cause the degradation or translational repression of their target mRNA [6]. miRNAs participate in the regulation of most biologic processes and are involved in the pathogenesis of human diseases, including chromosome architecture, cell proliferation, apoptosis, stress resistance, and stem cell maintenance [7]. * Corresponding authors. E-mail addresses: [email protected] (L. Wang); [email protected] (B. Ying); and [email protected] (Y. Shi). † These authors contributed equally to this work and should be considered cofirst authors.

mir-146 could regulate the expression of IL-1 receptor– associated kinase and TNF receptor–associated factor 6, which are regulators of the TNF-␣ signaling pathway [8]. The targets of mir499 include IL-17 receptor B (IL-17RB), IL-23a, IL-2 receptor B (IL-2R␤), IL-6, IL-2, B and T lymphocyte attenuator, IL-18 receptor (IL-18R), IL-21, peptidyl arginine deiminase type 4, and regulatory factor X 4 (influences human leukocyte antigen class II expression) (http://www.targetscan.org). All of the inflammatory cytokines and genes play an important role in the pathogenesis of RA [9 –15]. Therefore, mir-146a and mir-499 may affect the inflammatory reaction process in vivo of RA patients. The genetic background also has an important role in the occurrence and development of RA. The genetic variants, which may result in biologic and functional alterations, have been demonstrated in many genes/loci to be associated with the development of RA in many studies [16 –19]. The single nucleotide polymorphisms (SNPs), the most common type of genetic variation in the human genome that can contribute to human phenotypic differences, can affect the functions of miRNA and in turn influence individual susceptibility to disease [20]. Sequence variations have been observed to affect the processing and/or target selection of human miRNAs. For example, Calin et al. [21] observed that a germline mutation, located 7 bp downstream of the miR-16-1 precursor, resulted in dramatically reduced expression of mature miR-

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16-1. Jazdzewski et al. reported that the polymorphisms within the miR-146a sequence caused the additional generation of mature microRNAs from the passenger strands of the miRNA precursor, which can be functionally important because of different target selection [22]. Previous studies demonstrated that the SNPs rs2910164 and rs3746444 at the pre-miR-146a (G⬎C) and premiR-499 (T⬎C) had a strong relation with many diseases, including prostate cancer, papillary thyroid carcinoma, and hepatocellular carcinoma [22–24]. Therefore, we hypothesized that the SNP of mir-146a and mir-499 may affect the chronic inflammation of RA. However, the association between pre-miRNA SNP and the chronic inflammation of RA is completely unknown. C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were the commonly used indicators for evaluating the degree of chronic inflammation in vivo in RA patients. CRP expression would increase rapidly following an inflammatory stimulus, leading to serum levels that may exceed 500 times baseline, making CRP an ideal marker for the diagnosis and monitoring of inflammatory processes [25]. ESR had become less widely used because of its slow response following an inflammatory stimulus; however, this was not important in chronic inflammation where both CRP and ESR were at a steady state. IL-6 and TNF-␣ are 2 well-known inflammatory mediators and critical players in regulating tissue inflammation. The plasma concentrations of IL-6 and TNF-␣ can reflect the degree of inflammation in vivo. TGF-␤1 is an anti-inflammatory cytokine that can inhibit inflammation. We generated a clinically interpretable estimate of the effect of the different genotypes on chronic inflammation by analyzing the levels of CRP, ESR, IL-6, and TNF-␣. In this study we genotyped 2 selected SNPs (rs2910164 G⬎C and rs3746444 T⬎C) located at the pre-miRNA regions of hsa-mir146a and hsa-mir-499 and compared the levels of CRP, ESR, IL-6, TNF-␣, and TGF-␤1 among different genotypes.

each primer (Takara, Osaka, Japan), 1.5 mM MgCl2, 200 ␮M dNTPs (Promega, Madison, WI), and 0.7 U Taq DNA polymerase (Promega). The reaction mixture was initially heated at 94⬚C for 8 minutes to activate the polymerase and DNA amplification was achieved by 45 cycles of 94⬚C for 30 seconds, annealing for 45 seconds (rs2910164: 60⬚C; rs3746444: 62⬚C), and 72⬚C for 45 seconds in an ABI 9700 cycler. The final elongation step was 72⬚C for 8 minutes. After PCR amplification, 4 ␮L of PCR products was added to a 2% agarose gel, stained with ethidium bromide, and subjected to electrophoresis at 110 V for 1 hour. In the meantime, 2.5 ␮L of the PCR product was digested by the required enzyme (rs2910164, 5U SacI [MBI, Canada] in a final volume of 7.5 ␮L; rs3746444, 2.5 U BcII [MBI, Canada] in a final volume of 8 ␮L) in the presence of the accompanying buffer, and then incubated (rs2910161, 37⬚C; rs3746444, 56⬚C) with optimal activity of the enzyme overnight. Genotype calling was made by separating the DNA in an 8% polyacrylamide gel and then staining it with silver and subjecting it to electrophoresis at 110 V for 1 hour. The gels were photographed under ultraviolet light with Gel Doc (Bio-Rad). Some PCR products were randomly selected for DNA sequencing through an ABI 3130 sequencing system (ABI, Foster City, CA). 2.3. Measurement of CRP and ESR CRP was measured by rate nephelometry (immage 800, Beckman Coulter, Miami, FL), whereas ESR was examined by photometric capillary stopped-flow kinetic analysis (Test-1 automated analyzer, Alifax S.p.A., Polverara, Italy). 2.4. Enzyme-linked immunosorbent assay (ELISA) detection of plasma IL-6, TNF-␣, and TGF-␤1

2. Subjects and methods

The plasma concentrations of IL-6, TNF-␣, and TGF-␤1 were measured by ELISA following the manufacturer’s instructions (all ELISA kits from Bender MedSystems, Burlingame, CA). All samples were measured in duplicate.

2.1. Study subjects

2.5. Statistical analysis

This study had been approved by the Institutional Review Boards of Sichuan University in China. Informed consent was obtained from all participants or their representatives if direct consent was unavailable. A total of 262 patients with RA were recruited in this study and diagnosed according to the 1987 American College of Rheumatology criteria [26]. The mean age of these RA patients was 48 ⫾ 13 years; 17.94% (47/262) were male, whereas 82.06% (215/262) were female, and the ratio of female to male was 4.6:1 (female 215, male 47). Patients’ clinical laboratory indexes included CRP and ESR, which were valuable to estimate the activity of RA. The median concentration of CRP was 8.69 mg/L (3.08 to 28.05 mg/L)and the median concentration of ESR was 24 mm/H (15 to 40 mm/H). The plasma concentrations of IL-6, TNF-␣, and TGF-␤1 of 27 of these 262 patients were also measured.

Hardy–Weinberg equilibrium for each SNP polymorphism was tested by ␹2 test with df ⫽ 1. The difference in data for CRP and ESR among different genotypes was analyzed using the Kruskal–Wallis and Mann–Whitney tests. The difference in frequency distribution was analyzed by ␹2 test and a p value ⬍ 0.05 was considered statistically significant. All analyses were performed using SPSS software (version 17.0, SPSS, Austin, TX).

2.2. DNA extraction and genotyping assay DNA was extracted from the peripheral blood using Chelex-100 (Bio-Rad, Hercules, CA) and SNP genotyping was performed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Primers described previously were used [27]. A 147-bp fragment containing the pre-miR-146a region and polymorphism site (rs2910164) was amplified using the following primers: 5=-CATGGGTTGTGTCAGTGTCAGAGCT-3= and 5=-TGCCTTCTGTCTCCAGTCTTCCAA-3=. A 146-bp fragment containing the pre-miR-499 region and polymorphism site (rs3746444) was amplified using the following primers: 5=-CAAAGTCTTCACTTCCCTGCCA-3= and 5=- GATGTTTAACTCCTCTCCACGTGATC-3=. Briefly, the standard PCR and restriction fragment length polymorphism protocols were performed as follows: PCR was performed in a total of 25 ␮L PCR reaction mix, including 0.1 ng genomic DNA, 5 pmol of

3. Results 3.1. The relationship between CRP and ESR and the frequency distribution of genotypes and alleles in RA patients Regarding rs2910164, the median levels of CRP in genotype CC, CG, and GG were 6.2 mg/L (1.98 to 19.9), 9.5 mg/L (2.5 to 34.4), and 5.8 mg/L (3.3 to 17.3), respectively, among which there was no significant difference (p ⫽ 0.436). However, a significant difference was observed in the levels of CRP among different genotypes in rs3746444 (p ⫽ 0.031; Fig. 1). The median level of CRP in genotype CT was 16.9 mg/L (3.7 to 67.4), whereas that in genotype CC in rs3746444 and CT was 6.4 mg/L (3.0 to 20.1) and 5.7 mg/L (2.3 to 19.5), respectively. There was a significant difference in the CRP level between genotype TT and genotype CT (p ⫽ 0.015), but no significant difference was observed either between genotype CC and genotype CT or between genotype CC and genotype TT (p ⫽ 0.092 and p ⫽ 0.76, respectively). The CRP level of genotype CT was higher than that of genotype CC and genotype TT in rs3746444. Regarding ESR in rs2910164, the median levels of ESR in genotype CC, CG, and GG were 22 mm/H (13.8 to 40.5), 25 mm/H (14.5 to 43.5), and 27 mm/H (12 to 32), respectively, among which no significant difference was observed (p ⫽ 0.797). However, there

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A

C

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B

D

Fig. 1. The levels of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), interleukin (IL)-6, tumor necrosis factor ␣ (TNF-␣), and transforming growth factor ␤1 (TGF-␤1) in rs3746444 in RA patients. (A). The relationship between the plasma concentrations of IL-6 and hsa-mir-499 rs3746444 (p ⬎ 0.05). (B) The levels of CRP and ESR in hsa-mir-499 rs3746444. (C) The relationship between the plasma concentrations of TNF-␣ and hsa-mir-499 rs3746444 (p ⬎ 0.05). (D) The relationship between the plasma concentrations of TGF-␤1 and hsa-mir-499 rs3746444 (p ⬎ 0.05).

was a significant difference in the levels of ESR among different genotypes in rs3746444 (p ⫽ 0.047; Fig. 1). The median level of ESR in genotype CT in rs3746444 was 28.5 mm/H (17.5 to 44.5), whereas that for genotype CC and TT was 24.5 mm/H (16 to 46.1) and 22 mm/H (12 to 34.7), respectively. There was a significant difference between genotype TT and genotype CT (p ⫽ 0.042), but no significant difference was observed either between genotype CC and genotype CT or between genotype CC and genotype TT (p ⫽ 0.483 and p ⫽ 0.375, respectively). The level of ESR of genotype CT in rs3746444 was higher than that in genotype CC and genotype TT. We also compared the frequency distribution of alleles in rs2910164 and observed no statistically significant difference (p ⫽ 0.984, OR ⫽ 0.996 [95% confidence interval (95% CI) 0.696 –1.427], and p ⫽ 0.637, OR ⫽ 1.092 [0.758 –1.571], respectively) in different

groups of CRP and ESR. A similar result was also observed in rs3746444 (p ⫽ 0.979, OR ⫽ 1.006 [0.654 –1.546], and p ⫽ 0.582, OR ⫽ 0.884 [0.570 –1.371], respectively). The above results are presented in Tables 1 and 2 and Fig. 1. 3.2. The relationship between active/inactive state of RA and frequency distribution of genotypes and alleles in RA patients We divided the RA patients into an active group and an inactive group according to the 28-Joint Disease Activity Score (DAS28), which was used to assess disease activity [28]. There was no significant difference in the distribution of genotype in rs2910164 between the 2 groups (p ⫽ 0.693). However, regarding the distribution of genotype in rs3746444, a significant difference was

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Table 1 The relationship between the levels of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) and frequency distribution of genotypes in rheumatoid arthritis patients Frequency (n ⫽ 262) rs2910164 CC CG GG rs3746444 CC CT TT

98 (37.40%) 137 (52.29%) 27 (10.31%)

CRP (n ⫽ 262) (mg/L)

6.2 (1.98–19.9) 9.5 (2.5–34.4) 5.8 (3.3–17.3)

12 (4.58%) 86 (32.82%) 164 (62.60%)

ESR (n ⫽ 262) (mm/H)

pa

6.4 (3.0–20.1) 16.9 (3.7–67.4) 5.7 (2.3–19.5)

pa

0.436b NS

22 (13.8–40.5) 25 (14.5–43.5) 27 (12–32)

0.797b NS

0.031b

24.5 (16–46.1) 28.5 (17.5–44.5) 22 (12–34.7)

0.047b

Kruskal–Wallis test; NS, not significant; p ⬍ 0.05 is significant. There was no significant difference in age and sex among different genotypes in rs2910164 (p ⬎ 0.05) or in rs37464444 (p ⬎ 0.05).

a

b

observed between the 2 groups (p ⫽ 0.044). The ratio of genotype CT in the active group was higher than that in the inactive group. There was no significant difference in the frequency distribution of alleles in rs2910164 between the 2 groups (p ⫽ 0.742, OR ⫽ 0.940, and 95% CI 0.650 –1.360). Although a similar result was observed in rs3746444 (p ⫽ 0.061, OR ⫽ 1.490, and 95% CI 0.980 –2.265), interestingly, the ratio of the C allele in the active group was higher than that in the inactive group in rs3746444. Data are presented in Table 3. 3.3. The relationship among IL-6, TNF-␣, and TGF-␤1 and frequency distribution of genotypes in RA patients There was no significant difference in the plasma concentrations of IL-6, TNF-␣, and TGF-␤1 among different genotypes in rs2910164 (p ⫽ 0.889; p ⫽ 0.736, and p ⫽ 0.633, respectively) or in rs37464444 (p ⫽ 0.823; p ⫽ 0.979, and p ⫽ 0.146, respectively). Regarding rs2910164, the plasma concentrations of IL-6, TNF-␣, and TGF-␤1 in genotype CC (IL-6, 27.54 [5.27–162.40] pg/mL; TNF-␣, 916.79 [57.01–1,854.85] pg/mL; TGF-␤1, 4,738.95 [2,011.02–7,612.83] ng/mL), genotype CG (IL-6, 58.73 [6.45– 192.78] pg/mL; TNF-␣, 904.25 [0 –1,833.46] pg/ml; TGF-␤1, 3,999.33 [3,030.66 – 6,101.61] ng/mL), and genotype GG (IL-6, 37.64 [3.88 –212.0] pg/mL; TNF-␣, 1,281.52 [288.24 –3,217.71] pg/ mL; TGF-␤1, 7,238.94 [2,751.04 – 8,884.36] ng/mL) were not significantly different from each other. However, the plasma concentrations of IL-6 and TNF-␣ in genotype CT in rs3746444 (IL-6, 37.64 [7.69 –229.82] pg/mL; TNF-␣, 1,042.94 [0 –3,217.71] pg/ml) were higher than that in genotype CC (IL-6, 25.18 [3.88 –76.30] pg/mL; TNF-␣, 853.03 [288.24 –1,417.81] pg/mL) and genotype TT (IL-6, 31.32 [5.27–179.50] pg/mL; TNF-␣, 904.25 [57.01–1,854.85] pg/ mL). The plasma concentrations of TGF-␤1 in genotype CT in rs3746444 (3,256.31 [2,042.06 –7,238.94] ng/mL) were lower than that in genotype CC (9,038.68 [8,884.36 –9,193.0] ng/mL) and genotype TT (4,148.32 [2,651.71– 6,340.51] ng/mL). Results are presented in Fig. 1.

4. Discussion In this study, significant differences were observed in the levels of CRP and ESR among different genotypes of hsa-mir-499 rs3746444 T⬎C. There was a significant difference in the distribution of genotype in rs3746444 between the active group and the inactive group. The rate of genotype CT in the active group was higher than that in the inactive group. However, various genotypes of hsa-mir-146a rs2910164 G⬎C were not associated with the level of CRP, ESR, IL-6, TNF-␣, and TGF-␤1 in RA patients in the Han Chinese population. There was no significant difference in the plasma concentrations of IL-6, TNF-␣, and TGF-␤1 among different genotypes in rs3746444. This study provided the first evidence that SNP rs3746444 in pre-miR-499 could affect the inflammatory reaction of RA. We believe that the association observed between SNP in pre-microRNA and the levels of CRP, ESR, IL-6, TNF-␣, and TGF-␤1 is strong enough to contribute to the clinical assessment of the inflammatory disease activity of disease, which in turn may influence therapeutic decision making. A characteristic of RA was persistent chronic inflammation leading to synovial tissue damage, which resulted in irreversible joint damage. The clinical treatment was to control the inflammation and the excessive immune response. But patients with different genetic backgrounds may have different degrees of inflammation, which was very important for RA patients [29 –31]. miRNAs participate in the regulation of many biologic processes and are involved in the pathogenesis of many human diseases. There have already been a number of studies regarding microRNA regulating the development of RA. miR-146a, which belongs to the family of microRNA, was suggested to play an important role in RA development [8,32]. miR146a could act as negative feedback during the activation of immune responses and thus may function in physiologic immunity and autoimmunity. Increased expression of miR-146a could reduce the expression of IL-1 receptor–associated kinase 1 and TNF recep-

Table 2 The relationship between the levels of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) and frequency distribution of alleles in rheumatoid arthritis patients pa

rs2910164

CRP ⬍10 mg/L (n ⫽ 148) High ⬎10 mg/L(n ⫽ 114) Odds ratio ESR ⬍20 mm/H (n ⫽ 105) ⬎20 mm/H (n ⫽ 157) Odds ratio

C (%)

G (%)

188 (63.51%) 145 (63.60%) 0.996 (0.696–1.427)

108 (36.49%) 83 (36.40%)

136 (64.76%) 197 (62.74%) 1.092 (0.758–1.571)

74 (35.24%) 117 (37.26%)

␹ test; NS, not significant; p ⬍ 0.05 indicates a significant difference.

a 2

pa

rs3746444 C (%)

T (%)

0.984 NS

60 (20.27%) 46 (20.18%) 1.006 (0.654–1.546)

236 (79.73%) 182 (79.82%)

0.979 NS

0.637 NS

40 (19.05%) 66 (21.02%) 0.884 (0.570–1.371)

170 (80.95%) 248 (78.98%)

0.582 NS

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Table 3 The relationship between the active/inactive state of rheumatoid arthritis (RA) and frequency distribution of genotypes and alleles in RA patients rs3746444 (n ⫽ 200) CC (%) Active (n ⫽ 95) Inactive (n ⫽ 167) Alleles Active (n ⫽ 95) Inactive (n ⫽ 167) odds ratio

CT (%)

4 (4.21%) 43 (45.26%) 8 (4.79%) 50 (29.94%) C (%) 51 (26.84%) 66 (19.76%) 1.490(0.980–2.265)

Pa TT (%) 48 (50.53%) 109 (65.27%) T (%) 139 (73.16%) 268 (80.24%)

rs2910164 (n ⫽ 200) CC (%)

0.044

0.061 NS

Pa CG (%)

33 (34.74%) 53 (55.79%) 65 (38.92%) 84 (50.30%) C (%) 119 (62.63%) 214 (64.07%) 0.940 (0.650–1.360)

GG (%) 9 (9.47%) 18 (10.78%) G (%) 71(37.37%) 120 (35.93%)

0.693 NS 0.742 NS

␹ test; NS, not significant.

a 2

tor–associated factor 6, which are regulators of the TNF-␣ signaling pathway [8]. miR-146a, at least in part, could control the production of proinflammatory cytokines/chemokines. Thus, miR-146a appears to be an important regulator in RA development as an effector in driving a negative feedback mechanism to prevent excess inflammation [33]. However, in this study we did not observe an association between the SNP of hsa-mir-146a rs2910164 and levels of CRP, ESR, and TNF-␣. The SNP rs2910164 would not affect mir-146a regulating the TNF-␣ signaling pathway. Interestingly, we observed a close relationship between the SNP of hsa-mir-499 rs3746444 and the inflammation of RA. The targets of mir-499 involved IL-17RB, IL-23a, IL-2R␤, IL-6, IL-2, and IL-18R. IL-6 can trigger the synthesis of CRP and fibrinogen through the liver [34] and IL-17RB, IL-23a, IL-2R␤, IL-6, IL-2, and IL-18R play important roles in the pathogenesis of RA [9 –12]. Therefore, mir-499 can affect the production of CRP and inflammation in RA. CRP and ESR can reflect the degree of inflammation and disease activity of RA. In this study we observed that the heterozygote CT in rs3746444 had significantly higher levels of CRP and ESR compared with homozygotes CC and TT. We also demonstrated that the rate of genotype CT in rs3746444 in active group was higher than that in the inactive group. Meanwhile, we also observed the tendency that heterozygote CT exhibited higher IL-6 and TNF-␣, but lower TGF-␤1 compared with homozygote CC and TT, although there was no significant difference among them. IL-6 and TNF-␣ are 2 wellknown inflammatory mediators and critical players in regulating tissue inflammation. TGF-␤1 is an anti-inflammatory cytokine. Although there is no statistically significant difference that may be caused by the small sample size, this result indicates that heterozygote CT had higher levels of proinflammatory cytokine and lower levels of anti-inflammatory cytokine. All results demonstrated that the heterozygote in rs3746444 could lead to more serious inflammation than the homozygote. What might account for this strange phenomenon? Jazdzewski et al. [22] reported that because each mature miR binds to a distinct set of target genes, different target genes can be affected by the miRs produced by the GG or CC homozygote (miR-146a and miR146a*G or -*C, respectively) and a third set can be produced by the GC heterozygote (miR-146a and both miR-146a*G and miR146a*C). Therefore, heterozygosis was the only state in which the set of target genes comprised all 3 subsets. The production of distinct miRs and the regulation of different target genes by heterozygote compared with homozygote may explain the predisposition to papillary thyroid carcinoma displayed by heterozygous individuals. Jazdzewski et al. [22] drew conclusions from the SNP rs2910164 of hsa-mir-146a; thus, the findings of SNP rs3746444 of hsa-mir-499 in this study may be because of the same reason. Further studies are therefore required to rule out specific reasons. The heterozygote in rs3746444 in RA exhibited higher levels of CRP and ESR than the homozygote; therefore, the heterozygote needed more effective treatment than the homozygote. The joint damage was heavier when the patient had persistent chronic in-

flammatory response. CRP and ESR plays an important role in a clinician’s decision-making processes of diagnosing inflammatory disease and choosing treatment options. If a patient presents with the CT heterozygote in rs3746444, he or she may need more effective treatment than a patient who presents with the CC or TT homozygote. In summary, this study first demonstrated that genotype CT in rs3746444 greatly increased the levels of CRP and ESR compared with genotypes CC and TT. A significant difference existed in the distribution of genotype in rs3746444 between the active group and the inactive group. The rate of genotype CT in the active group was higher than that in the inactive group. All results suggest that SNP rs3746444 may play an important role in RA inflammation. Researchers are just beginning to understand the relationship between miRNA expression patterns and functions in different diseases. Therefore, further characterization of the SNPs of miRNAs would improve our understanding of miRNA biogenesis and the potential contribution of these SNPs to disease etiology and progression. This study provided the first evidence that the SNP rs3746444 in pre-miR-499 can affect the inflammatory reaction of RA. We investigated the association between the SNP in premicroRNA and the inflammatory cytokines. The findings were significant in that it might contribute to the clinical assessment of inflammatory activity, which in turn may influence therapeutic decision making. The mechanism of how the SNP of mir-499 affects the inflammation of RA requires further functional studies. Acknowledgments We thank the participating RA patients and their families. The authors acknowledge grant support from the National Natural Science Foundation of China (Grants 30670819, 30900658, 30772051, and 30950010). References [1] Smolen JS, Aletaha D, Koeller M, Weisman MH, Emery P. New therapies for treatment of rheumatoid arthritis. Lancet 2007;370:1861–74. [2] Lipsky PE, van der Heijde DM, St Clair EW, Furst DE, Breedveld FC, Kalden JR, et al. Infliximab and methotrexate in the treatment of rheumatoid arthritismor: Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study Group. N Engl J Med 2000;343:1594 – 602. [3] Lee UJ, Choung SR, Prakash KV, Lee EJ, Lee MY, Kim YJ, et al. Dual knockdown of p65 and p50 subunits of NF-kappaB by siRNA inhibits the induction of inflammatory cytokines and significantly enhance apoptosis in human primary synoviocytes treated with tumor necrosis factor-alpha. Mol Biol Rep 2008;35: 291– 8. [4] Fabbri M, Croce CM, Calin GA. MicroRNAs. Cancer J 2008;14:1– 6. [5] Lodish HF, Zhou B, Liu G, Chen CZ. Micromanagement of the immune system by microRNAs. Nat Rev Immunol 2008;8:120 –30. [6] Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of posttranscriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 2008;9:102–14. [7] Zamore PD, Haley B. Ribo-gnome: the big world of small RNAs. Science 2005; 309:1519 –24. [8] Pauley KM, Satoh M, Chan AL, Bubb MR, Reeves WH, Chan EK, et al. R-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients. Arthritis Res Ther 2008;10:1–10. [9] Hillyer P, LarchÊ MJ, Bowman EP, McClanahan TK, de Waal Malefyt R, Schewitz LP, et al. Investigating the role of the interleukin-23/-17A axis in rheumatoid arthritis. Rheumatology (Oxford) 2009;48:1581–9.

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