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MicroRNA-3148 modulates allelic expression of toll-like receptor 7 variant associated with systemic lupus erythematosus.

Deng Y, Zhao J, Sakurai D, Kaufman KM, Edberg JC, Kimberly RP, Kamen DL, Gilkeson GS, Jacob CO, Scofield RH, Langefeld CD, Kelly JA, Ramsey-Goldman R, Petri MA, Reveille JD, Vilá LM, Alarcón GS, Vyse TJ, Pons-Estel BA, Argentine Collaborative GroupFreedman BI, Gaffney PM, Sivils KM, James JA, Gregersen PK, Anaya JM, Niewold TB, Merrill JT, Criswell LA, Stevens AM, Boackle SA, Cantor RM, Chen W, Grossman JM, Hahn BH, Harley JB, Alarcόn-Riquelme ME, BIOLUPUS and GENLES networksBrown EE, Tsao BP - PLoS Genet. (2013)

Bottom Line: Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3'UTR segment bearing the C allele (P = 0.0003).Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148.These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta  = 2.0×10(-19), OR = 1.25 [1.20-1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, University of California Los Angeles, Los Angeles, California, USA.

ABSTRACT
We previously reported that the G allele of rs3853839 at 3'untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10(-10), odds ratio (OR) (95%CI) = 1.27 (1.17-1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10(-11), OR = 1.24 [1.18-1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3'UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R(2) = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3'UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta  = 2.0×10(-19), OR = 1.25 [1.20-1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.

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The SLE-risk G allele of rs3853839 confers elevated TLR7 expression through slower mRNA degradation.(A) Association of rs3853839 genotypes with TLR7/8 transcript levels in EA normal PBMCs. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (B) Association of rs3853839 genotypes with TLR7 protein levels in normal PBMCs. FACS histograms show the log MFI values plotted against the cell counts for PBMCs in individuals carrying G or C allele, compared with isotype control. Results are from one representative pair (GG or G vs. CC or C) of 7 in each gender group. MFI of TLR7 expression in PBMCs is graphically depicted. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (C) Verification of the G allele conferring elevated expression of a luciferase reporter in vitro. TLR7 3′UTR segment bearing G or C allele of rs3853839 was cloned into the psiCHECK-2 reporter vector and luciferase activity was determined after 24 hours of transfection. Relative luciferase activity is Renilla/Firefly luciferase ratio. Data show the mean ± SEM and are representative of cumulative data from four independent experiments. (D) The kinetics of the G/C allele ratio in TLR7 transcripts from PBMCs of healthy heterozygous individuals (n = 7) in the absence or presence of actinomycin D (ActD). The G/C allele ratio obtained in TLR7 transcripts was normalized to that measured from gDNA of the same sample. Data are expressed as mean ± SEM at each time point and representative of cumulative data from two independent experiments with seven healthy donors. *P<0.05. (E) Summary of the G/C allele ratio in TLR7 transcripts 4 hours after the addition of ActD or vehicle control (n = 7). Comparisons are between ActD and vehicle control cultures; P = 0.04; paired t test. FACS, Fluorescence-activated cell sorter; MFI, mean fluorescence intensity.
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pgen-1003336-g002: The SLE-risk G allele of rs3853839 confers elevated TLR7 expression through slower mRNA degradation.(A) Association of rs3853839 genotypes with TLR7/8 transcript levels in EA normal PBMCs. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (B) Association of rs3853839 genotypes with TLR7 protein levels in normal PBMCs. FACS histograms show the log MFI values plotted against the cell counts for PBMCs in individuals carrying G or C allele, compared with isotype control. Results are from one representative pair (GG or G vs. CC or C) of 7 in each gender group. MFI of TLR7 expression in PBMCs is graphically depicted. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (C) Verification of the G allele conferring elevated expression of a luciferase reporter in vitro. TLR7 3′UTR segment bearing G or C allele of rs3853839 was cloned into the psiCHECK-2 reporter vector and luciferase activity was determined after 24 hours of transfection. Relative luciferase activity is Renilla/Firefly luciferase ratio. Data show the mean ± SEM and are representative of cumulative data from four independent experiments. (D) The kinetics of the G/C allele ratio in TLR7 transcripts from PBMCs of healthy heterozygous individuals (n = 7) in the absence or presence of actinomycin D (ActD). The G/C allele ratio obtained in TLR7 transcripts was normalized to that measured from gDNA of the same sample. Data are expressed as mean ± SEM at each time point and representative of cumulative data from two independent experiments with seven healthy donors. *P<0.05. (E) Summary of the G/C allele ratio in TLR7 transcripts 4 hours after the addition of ActD or vehicle control (n = 7). Comparisons are between ActD and vehicle control cultures; P = 0.04; paired t test. FACS, Fluorescence-activated cell sorter; MFI, mean fluorescence intensity.

Mentions: Given the convincing evidence for the trans-ancestral association of rs3853839 with SLE susceptibility, we then evaluated its effect on regulation of TLR7/8 expression. Messenger RNA (mRNA) levels of TLR7 and the two alternative TLR8 isoforms were measured by real-time PCR in PBMCs from healthy EA individuals (n = 62). TLR7 mRNA levels were significantly different among women (n = 41) carrying different genotypes of rs3853839 [P = 0.003, one-way analysis of variance (ANOVA)], in which the GG and GC carriers exhibited notably increased TLR7 mRNA levels compared with the CC carriers [P = 0.02 for GG (n = 5) vs. CC (n = 18) and 0.02 for GC (n = 18) vs. CC, respectively, Student's t test; Figure 2A) and the number of rs3853839 risk G allele was significantly correlated with increased TLR7 mRNA levels (R2 = 0.26, P = 8×10−4, linear regression test). Consistently, male G allele carriers (n = 5) also had significantly higher TLR7 mRNA expression than male C allele carriers (n = 16) (P = 0.01, Figure 2A). There was no significant association of rs3853839 genotypes with mRNA levels of two TLR8 isoforms in either women or men (Figure 2A). No sex differences in TLR7 or TLR8 mRNA levels were observed between individuals carrying the same genotype [GG women vs. G men: P = 0.41 (TLR7), 0.63 (TLR8a) and 0.50 (TLR8b); CC women vs. C men: P = 0.10 (TLR7), 0.91 (TLR8a) and 0.65 (TLR8b)]. These results were in accordance with our previous observations in Chinese [5], supporting the importance of rs3853839 in regulating TLR7 rather than TLR8 gene expression.


MicroRNA-3148 modulates allelic expression of toll-like receptor 7 variant associated with systemic lupus erythematosus.

Deng Y, Zhao J, Sakurai D, Kaufman KM, Edberg JC, Kimberly RP, Kamen DL, Gilkeson GS, Jacob CO, Scofield RH, Langefeld CD, Kelly JA, Ramsey-Goldman R, Petri MA, Reveille JD, Vilá LM, Alarcón GS, Vyse TJ, Pons-Estel BA, Argentine Collaborative GroupFreedman BI, Gaffney PM, Sivils KM, James JA, Gregersen PK, Anaya JM, Niewold TB, Merrill JT, Criswell LA, Stevens AM, Boackle SA, Cantor RM, Chen W, Grossman JM, Hahn BH, Harley JB, Alarcόn-Riquelme ME, BIOLUPUS and GENLES networksBrown EE, Tsao BP - PLoS Genet. (2013)

The SLE-risk G allele of rs3853839 confers elevated TLR7 expression through slower mRNA degradation.(A) Association of rs3853839 genotypes with TLR7/8 transcript levels in EA normal PBMCs. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (B) Association of rs3853839 genotypes with TLR7 protein levels in normal PBMCs. FACS histograms show the log MFI values plotted against the cell counts for PBMCs in individuals carrying G or C allele, compared with isotype control. Results are from one representative pair (GG or G vs. CC or C) of 7 in each gender group. MFI of TLR7 expression in PBMCs is graphically depicted. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (C) Verification of the G allele conferring elevated expression of a luciferase reporter in vitro. TLR7 3′UTR segment bearing G or C allele of rs3853839 was cloned into the psiCHECK-2 reporter vector and luciferase activity was determined after 24 hours of transfection. Relative luciferase activity is Renilla/Firefly luciferase ratio. Data show the mean ± SEM and are representative of cumulative data from four independent experiments. (D) The kinetics of the G/C allele ratio in TLR7 transcripts from PBMCs of healthy heterozygous individuals (n = 7) in the absence or presence of actinomycin D (ActD). The G/C allele ratio obtained in TLR7 transcripts was normalized to that measured from gDNA of the same sample. Data are expressed as mean ± SEM at each time point and representative of cumulative data from two independent experiments with seven healthy donors. *P<0.05. (E) Summary of the G/C allele ratio in TLR7 transcripts 4 hours after the addition of ActD or vehicle control (n = 7). Comparisons are between ActD and vehicle control cultures; P = 0.04; paired t test. FACS, Fluorescence-activated cell sorter; MFI, mean fluorescence intensity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585142&req=5

pgen-1003336-g002: The SLE-risk G allele of rs3853839 confers elevated TLR7 expression through slower mRNA degradation.(A) Association of rs3853839 genotypes with TLR7/8 transcript levels in EA normal PBMCs. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (B) Association of rs3853839 genotypes with TLR7 protein levels in normal PBMCs. FACS histograms show the log MFI values plotted against the cell counts for PBMCs in individuals carrying G or C allele, compared with isotype control. Results are from one representative pair (GG or G vs. CC or C) of 7 in each gender group. MFI of TLR7 expression in PBMCs is graphically depicted. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (C) Verification of the G allele conferring elevated expression of a luciferase reporter in vitro. TLR7 3′UTR segment bearing G or C allele of rs3853839 was cloned into the psiCHECK-2 reporter vector and luciferase activity was determined after 24 hours of transfection. Relative luciferase activity is Renilla/Firefly luciferase ratio. Data show the mean ± SEM and are representative of cumulative data from four independent experiments. (D) The kinetics of the G/C allele ratio in TLR7 transcripts from PBMCs of healthy heterozygous individuals (n = 7) in the absence or presence of actinomycin D (ActD). The G/C allele ratio obtained in TLR7 transcripts was normalized to that measured from gDNA of the same sample. Data are expressed as mean ± SEM at each time point and representative of cumulative data from two independent experiments with seven healthy donors. *P<0.05. (E) Summary of the G/C allele ratio in TLR7 transcripts 4 hours after the addition of ActD or vehicle control (n = 7). Comparisons are between ActD and vehicle control cultures; P = 0.04; paired t test. FACS, Fluorescence-activated cell sorter; MFI, mean fluorescence intensity.
Mentions: Given the convincing evidence for the trans-ancestral association of rs3853839 with SLE susceptibility, we then evaluated its effect on regulation of TLR7/8 expression. Messenger RNA (mRNA) levels of TLR7 and the two alternative TLR8 isoforms were measured by real-time PCR in PBMCs from healthy EA individuals (n = 62). TLR7 mRNA levels were significantly different among women (n = 41) carrying different genotypes of rs3853839 [P = 0.003, one-way analysis of variance (ANOVA)], in which the GG and GC carriers exhibited notably increased TLR7 mRNA levels compared with the CC carriers [P = 0.02 for GG (n = 5) vs. CC (n = 18) and 0.02 for GC (n = 18) vs. CC, respectively, Student's t test; Figure 2A) and the number of rs3853839 risk G allele was significantly correlated with increased TLR7 mRNA levels (R2 = 0.26, P = 8×10−4, linear regression test). Consistently, male G allele carriers (n = 5) also had significantly higher TLR7 mRNA expression than male C allele carriers (n = 16) (P = 0.01, Figure 2A). There was no significant association of rs3853839 genotypes with mRNA levels of two TLR8 isoforms in either women or men (Figure 2A). No sex differences in TLR7 or TLR8 mRNA levels were observed between individuals carrying the same genotype [GG women vs. G men: P = 0.41 (TLR7), 0.63 (TLR8a) and 0.50 (TLR8b); CC women vs. C men: P = 0.10 (TLR7), 0.91 (TLR8a) and 0.65 (TLR8b)]. These results were in accordance with our previous observations in Chinese [5], supporting the importance of rs3853839 in regulating TLR7 rather than TLR8 gene expression.

Bottom Line: Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3'UTR segment bearing the C allele (P = 0.0003).Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148.These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta  = 2.0×10(-19), OR = 1.25 [1.20-1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, University of California Los Angeles, Los Angeles, California, USA.

ABSTRACT
We previously reported that the G allele of rs3853839 at 3'untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10(-10), odds ratio (OR) (95%CI) = 1.27 (1.17-1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10(-11), OR = 1.24 [1.18-1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3'UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R(2) = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3'UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta  = 2.0×10(-19), OR = 1.25 [1.20-1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.

Show MeSH
Related in: MedlinePlus