To construct the miR-558 expression plasmid, a precursor of the miR-558 sequence amplified from HepG2 genomic DNA was cloned into the pRNA-U6.1/Neo-siFluc vector. The 3′-UTR region of CXCR5 including the rs3922 locus was

amplified and inserted downstream of the luciferase reporter gene in pGL3-Control Vector. The luciferase reporter plasmid carrying an “A” allele in rs3922 was marked as pGL3-3922A-luc, while the pGL3-3922G-luc contains the SNP “G”. HEK 293T cells were seeded into 12-well plates. Twenty-four hours later, the cells were co-transfected with 1.5 μg of miR-558 expression plasmid or U6 control vector and 50 ng pGL3-3922 luciferase ubiquitin-Proteasome degradation vectors. The pRL-TK (25 ng) plasmid was also transfected as a transfection efficiency control. The luciferase activity in each well was quantified 24 h after transfection using a dual luciferase reporter kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. In an additional experiment only luciferase vector (pGL3-3922A-luc or pGL3-3922G-luc) and pRL-TK plasmid were co-transfected, without the miR-558 expression plasmid or U6 control vectors. The majority of most experimental conditions were the same in this case except the quantity of pGL3-3922 vector added was Pictilisib manufacturer 100 ng per well. For each SNP, the association between response statuses to HBV vaccine and various genotypes or allelotypes was estimated

by the chi-square test using SAS version 9.1.3 (SAS Institute, Inc., Cary, NC, USA). The Hardy–Weinberg equilibrium (H–W equilibrium) was calculated based on the control group using Haploview version 4.2 software [23]. Modulators Linkage disequilibrium

(LD) analysis and haplotype construction were carried out with the same software. Specific parameters were set as previously published Ketanserin [4]. P-values, odds ratios (OR), and 95% confidence intervals (95% CIs) were obtained for correlation analysis. A P-value < 0.05 was taken to be statistically significant. A total of 24 SNPs from TfH associated molecules were analyzed in the 20 non-responders and 45 responders. The genotype and allele frequencies of all the SNPs in the study and control groups are listed in Supplementary Table 3. The H–W equilibrium was evaluated in the normal response group and two SNPs (rs3092945, rs715762) in CD40L were excluded from the analysis due to disequilibrium (P < 0.001). Of the remaining 22 SNPs, four (rs3922, rs676925, rs497916 and rs355687) showed significant associations with the immune response triggered by HBV vaccination (P < 0.05, Table 1). Three of these were located in the CXCR5 gene: rs3922 (in 3′-UTR), rs676925 (in 3′-UTR) and rs497916 (in intron), while the fourth one rs355687 was located in the intron of CXCL13. As collected by the international HapMap project, the distributions of these 4 SNPs in different populations were summarized in the Supplementary Table 4.