Table 1.
Association of rs2596542 with the progression from CHC to LC and HCC.
Table 2.
Linkage disequilibrium between 11 candidate SNPs and SNP rs2596542.
Figure 1.
SNP rs2596538 affects the binding affinity of nuclear proteins.
(A) Real-time quantitative PCR (upper) and Western blotting (lower) of MICA before and after heat shock treatment in HLE cells. B2M and β-actin are served as internal and protein loading control. (B) EMSA using 31 bp labeled probes flanking each SNP located within the 4.8 kb region upstream of MICA transcription start site. A black arrow indicates the shifted band specific to G allele of SNP rs2596538. (C) EMSA using the labeled G allele of SNP rs2596538 and nuclear extract from heat treated HLE cells. Non-labeled A or G allele of SNP rs2596538 at different concentrations are used as competitors. Pointed arrow indicates shifted band. *P<0.05 by Student's t-test.
Figure 2.
Binding of transcription factor SP1 to G allele of SNP rs2596538.
(A) Multiple alignment of a GC box and DNA sequence of A or G probe of SNP rs2596538 used in EMSA. (B) EMSA using the labeled G allele of SNP rs2596538 and nuclear extract from heat treated HLE cells. Non-labeled consensus oligonucleotides of seven transcription factors are used as competitors. Pointed arrow indicates shifted band. (C) EMSA using the labeled G allele of SNP rs2596538 and nuclear extract from heat shock treated HLE cells in the presence of anti-SP1 antibody or normal rabbit IgG. Asterisks on the left side indicate the shifted (*) and super-shifted bands (**). Normal rabbit IgG serves as a negative control. (D) ChIP assay using HepG2 and HLE cell lines were ectopically expressed with SP1 protein. DNA-protein complex was immunoprecipitated with anti-SP1 antibody followed by PCR amplification using a primer pair flanking SNP rs2596538. DNAs precipitated without antibody are served as a negative control. PCR primers flanking the 3' UTR region of MICA are served as a negative control. (E) Genotype distribution at SNP rs2596538 in PCR fragment amplified from the input genomic DNA and DNA-protein complex immunopurified from HepG2 cells by using anit-SP1 antibody. *P<0.05 by Student's t-test.
Figure 3.
Transcriptional regulation of MICA by SP1 through genomic region including SNP rs2596538.
(A) Reporter assay using constructs including 3 copies of 31 bp DNA fragment flanking SNP rs2596538. Reporter constructs are transfected into HLE cells with pRL-TK and pCAGGS or pCAGGS-SP1 vector. The value of relative luciferase activity was calculated as the firefly luciferase intensity divided by the renilla luciferase intensity. The data represent the mean ± SD value of 4 independent studies. (*P<0.05, Student's t-test) (B) MICA expression in HLE cells after transfection with pCAGGS or pCAGGS-SP1 vector. β-actin is served as a protein loading control.
Figure 4.
Association between the soluble MICA levels and SNP rs2596538 genotype.
The samples were classified into 3 groups according to rs2596538 genotype. The sMICA levels measured by ELISA are indicated in y-axis. The numbers of samples and the proportion of sMICA positive subjects from each group are shown in x-axis. The percentage of the positive sMICA expression in each group are AA = 10%, AG = 39%, and GG = 42%. Statistical significance was determined by Kruskal-Wallis test.
Table 3.
Association of SNP rs2596542 and SNP rs2596538 with HCV-induced HCC.