SNP extraction and haplotype construction
Data from 74 SNPs across the entire TLR4 3'-UTR, between positions 120,476,927 and 120,479,769 (GRCh37.p13) of chromosome 9, were downloaded from the 1KG Phase 3 Pipeline using the Ferret version 2.1.1 JAVA tool . The data used in this Analysis from 10 human populations, included 993 Asian individuals as described in 1KG Browser (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/) (Additional file 1: Table S1). The two sequences defined by NCBI (DNA: NG_011475.1, and mRNA: NM_003266.3) were used as references in the study. Haploview version 4.2 software was used to filter the SNPs to ensure that that the MAF was present at least 1% of the time, to select tag SNPs, calculate Hardy Weinberg equilibrium p value (HWp), and analyze haplotype patterns and linkage disequilibrium  among the SNPs . Six SNPs were identified in the TLR4 3'-UTR = using this screening approach (Table 1). The Haploview software tagger option was used to predict values r2 and D' for the remaining SNPs in the dataset. Block-based and block-free approaches were used to increase the accuracy and representativeness of tag SNPs . Haplotypes were evaluated with tag SNPs in 10 populations using Arlequin version 3.5 software .
Total RNA was isolated from cells using TriPure Isolation Reagent (Roche). Reverse transcription was performed using the PrimeScript™ RT reagent Kit (TAKARA, Japan). Taq Plus DNA Polymerase (TianGen, China) was used for RT-PCR, and TB Green® Premix Ex Taq™ II (TAKARA) was used to perform the qRT-PCR in a CFX96 Real-Time thermocycler platform (Bio-Rad). Relative quantifications were determined by the comparative 2−ΔΔCt method . All primers used are listed in Additional file 2: Table S2.
Western blot assay
Total protein was extracted separately for five cell cultures using RIPA buffer (Beyotime, China) with added phenylmethylsulfonyl fluoride and phosphatase inhibitor (Beyotime). Equal amounts of proteins were separated by SDS-PAGE on 6% or 12% polyacrylamide separating gels, and then transferred onto polyvinylidene-diﬂuoride-fluoride membranes (PVDF; Roche). The membranes were blocked with 5% Non-Fat powdered milk in TBS-T 1 h at room temperature (15-25°C), then incubated with the primary antibody at 4°C for 12 hours. The antibodies used were: mouse anti-TLR4 (1:500; Santa Cruz, USA), rabbit anti-MyD88 (1:1000, Beyotime, China), rabbit anti- NF-κB p65 (1:1000, Abcam, UK), rabbit anti- NF-κB p-p65 (phospho S536; 1:10000, Abcam, UK), and mouse anti-beta-Tubulin (1:5000, Invitrogen, USA). After washing, the PVDF membranes were incubated with goat anti-mouse IgG or goat anti-rabbit IgG conjugated to horseradish peroxidase (1:10000, Abcam, UK) secondary antibodies at room temperature for 1 hour. Subsequently, an enhanced chemiluminescence kit (CwBio, China,) was used to capture protein signals. A chemiluminescent Western Blot imaging system (Amersham Imager 600, GE, USA) was used to visualize the protein signals with image.
The human TLR4 promoter (-3494 to +235) was amplified from normal human genomic DNA using Cobuddy Super Fidelity DNA Polymerase (CwBio, China) with primer pair TPprimer1/TPprimer2 (Additional file 2: Table S2). The thermal cycling conditions were 2 min at 98°C, 35 cycles of 10 s at 98°C, 30 s at 67°C, 2 min at 72°C, and 5 min at 72°C. The full length TLR4 3'-UTR sequence was amplified by Cobuddy Super Fidelity DNA Polymerase with the primer set TPprimer3/TPprimer4 (Additional file 2: Table S2). The thermal cycling conditions were 2 min at 98°C, 35 cycles of 10 s at 98°C, 30 s at 62°C, 1.5 min at 72°C, and 5 min at 72°C. Amplicons were electrophoresed and the products were gel purified using a Gel Extraction Kit (Omega Bio-Tek, USA). The pGL3-Basic luciferase vector (Promega, USA) was digested with Xba I and Nco I (TAKARA, Japan), and the linearized vector fragments (1656 bp and 3162 bp) were purified from the electrophoresis gel. Gibson assembly mix was prepared following the one-step isothermal DNA assembly protocol . Approximately 10 ng of the 1656 bp linearized vector fragment, ~15 ng TLR4 3'UTR fragment, ~20 ng 3162 bp linearized vector fragment, and the TLR4 promoter (-3493 to +234) were mixed with 10 μL of Gibson assembly mix, and the final reaction volume adjusted to 20 μL. The mixture was incubated at 50℃ for 1 hour, and the entire volume was transformed into 200 μL of DH5α competent cells. Plasmid DNA extracted using an Endo-free Plasmid Mini Kit (Omega Bio-Tek, USA) and validated by sequencing.
To simulate the physiological state of SNPs, PCR-mediated site-directed mutagenesis for circular macromolecules was performed using pGL3-3493-3UTR. For rs41426344, a mutagenic reverse primer (mut344_G/C_R) was designed to introduce the necessary base (Additional file 2: Table S2, see the underlined base) substitutions to convert guanine (G) to cytosine (C). The forward primer (mut344_G/C_F) was designed in the opposite direction from mut344_G/C_R. The annealing positions of the two primers were back-to-back closed to each other. The primers underwent 5' phosphorylation using T4 Polynucleotide Kinase (NEB, USA) to allow for subsequent ligation. PCR was performed with the pGL3-3493-3UTR template to introduce mutation using the Cobuddy Super Fidelity DNA Polymerase system (CwBio, China). the amplification conditions were 2 min at 98°C, followed by 35 cycles of 10 sec at 98°C, 30 sec at 55°C, and 5 min at 72°C, and a 5 min final extension at 72°C. The PCR products were recovered, purified using the Omega Gel Extraction Kit, and self-ligated following the T4 DNA Ligase Protocol (NEB). The constructs were sequenced by the Beijing Genomics Institute for confirmation. After digestion with BglII, the constructs were religated to generate pGL3-684, pGL3-684-3UTR, and seven mutated constructs (-684 to +235).
Cell culture and transfection
The THP-1 human leukemia monocytic and HEK embryonic kidney 293T cell lines were purchased from the Conservation Genetics CAS Kunming Cell Bank (Kunming, China). THP-1 was maintained in L-glutamine-containing RPMI 1640 (GIBCO, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (GIBCO), 1% non-essential amino acids, 1% sodium pyruvate, and penicillin/streptomycin as previously described . The SiHa human cervical carcinoma cell line, C33A, and HeLa cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured at 37℃ in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) containing 10% fetal bovine serum in an incubator with 5% CO2. THP-1 and SiHa cells were transiently transfected with luciferase constructs. Exponentially growing THP-1 and SiHa cells were plated onto 24-well plates at a density of 5×105 and 0.5×105 cells/well, respectively. Twenty-four hours after plating, THP-1 cells were transfected using DNAfectin™ Plus Transfection Reagent (ABM, Canada) and SiHa cells were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Luciferase reporter assay
THP-1 cells were transiently co-transfected with 350 ng of constructs and 35 ng renilla luciferase plasmid pRL-CMV (Promega, USA), as an internal control for normalizing luciferase activity. After 36 h, the cells were treated with serum starvation for 12 h, and 1 ng/ml recombinant human Interleukin-6 (IL-6; Sangon Biotech, China) or 1 µg/ml LPS (Sigma-Aldrich, USA) was added 6 h before harvesting. SiHa cells were co-transfected with 350 ng of constructs and 17.5 ng of pRL-CMV, and stimulated with 50 µg/ml LPS and 50 ng/ml IL-6. Firefly and renilla luciferase activities were measured following the Dual-Luciferase® Reporter Assay System (Promega, USA) protocol using a Molecular Devices SpectraMax iD5 multi-mode microplate reader. Experiments were performed at least in triplicate.
SPSS version 22 software (IBM Corp, USA) was used for statistical analyses. The unpaired Student’s t test was used to compare relative luciferase activities between two groups. Frequency differences among haplotypes were assessed using the Chi-square statistical method. Statistical analyses were expressed as mean ± SD of three or more independent experiments. P < 0.05 was considered statistically significant.