Single nucleotide polymorphism of SREBF-1 gene associated with an increased risk of endometrial cancer in Chinese women.

AIM: Elevated levels of sterol regulatory element-binding protein-1 (SREBP-1) have been found in endometrial cancer (EC), suggesting that it is essential to the development of EC. Obesity and diabetes have been established as known risk factors of EC, while SREBF-1 gene polymorphisms have also been found to be associated with obesity and type II diabetes. Therefore, we hypothesize that single nucleotide polymorphism (SNP) in SREBF-1 gene may be associated with increased risk of EC. METHOD: We analyzed the sequence of SREBF-1 in tissue samples from 30 EC cases and 6 benign controls using high throughput method. Based on the primary results, we selected one SNP (rs2297508) as a genetic marker to conduct a hospital-based case-control study with 139 EC cases and 129 benign controls. The samples were examined under the microscope to determine their histopathology prior to the SNP analysis using RT-PCR. RESULTS: Through sequence analysis, we found 10 SNPs of SREBF-1 associated with EC, including 3 new SNPs. Fourteen percent of EC showed the rs2297508 SNP with C allele, while only 7% had the C allele was present in benign controls (p = 0.027, OR = 1.983). Additionally, the C allele was associated with cancer differentiation (p CONCLUSION: Our study indicates that SNP (rs2297508) of SREBF-1 may serve as a genetic predisposition factor for the development of EC and screening of such genetic marker may be helpful in its early detection

CCL20 Secretion from the Nucleus Pulposus Improves the Recruitment of CCR6-Expressing Th17 Cells to Degenerated IVD Tissues.

Studies elucidated that Th17 cells are important contributors to the pathogenesis of many immune-mediated diseases, and IL-17A is present in pathologic intervertebral disc (IVD) tissues. However, the mechanisms, how these cells traffic into the degenerate discs are not clear.The samples collected from 53 patients had been divided into 3 groups: Group P (annulus fibrosus was intact), Group E (annulus fibrosus was reptured) and normal control. Immunohistochemistry was used to detect the expression of CCL20, CCR6, IL-17A, TNF-α and CD4 in IVD tissues. Moreover, nucleus pulposus (NP) cells had been cultured in the presence and absence of Th17 associated cytokines. The supernatants were detected for CCL20 concentrations by ELISA, and the NP cells for the expression of CCL20 mRNA. Additionally, peripheral blood (PB) samples had undergone detection for the expression of CCR6 mRNA and the proportion of IL-17-producing cells, including the surface expression of CCR6.Immunohistochemistry revealed that CCL20 and TNF-α were expressed in degenerated NP cells. Double-labeled immunofluorescence elaborated, IL-17-producing cells (CD4(+)IL-17A(+) and CD4(+)CCR6(+)) appeared in the Group E samples, but no traces or expression in Group P and normal control. IL-17A and TNF-α, alone or combined, could enhance CCL20 secretion in a dose-dependent manner, which was obtained through RT-PCR results. There was a notable difference of CCR6 mRNA expression between patients and normal controls. In comparison to controls, flow cytometry data indicated that the proportion of IL-17-producing cells and the CCR6 expression in PB were significantly increased.Our results provide a potential explanation for involvement of the CCL20-CCR6 system in the trafficking of IL-17-producing cells to degenerated IVD tissues. Additionally, our results explain the contribution of Th17 associated cytokines to the development of degenerated discs via the up-regulation of CCL20 secretion from NP cells, which forms a positive chemotactic feedback loop

Figure 1

<p>A: SNPs in SREBF-1 gene. The whole SREBF-1gene includes 22 exons. Ten SNPs (SNP1 to SNP10) were identified within the SREBF-1 in our study. FIG. 1 B: SNP ID. SNP1 to SNP10 are listed from 5′-3′ of the SREBF-1. “CHR-ID” shows chromosome ID of ten SNPs in SREBF-1. Listed “RS ID” has been reported in ucsc or NCBI website, “-” expresses newly detected SNP. “Function” column listed altered genetically coded function of the SNPs. “Ref SNP” shows SNP form in original reference sequence. In “mRNA location”, corresponding mRNA positions are showed in consistent with each SNP. (For details:Sequence IDs included in CCDS 32583.1). NCBI db is available from <a href="http://www.ncbi.nlm.nih.gov/SNP" target="_blank">http://www.ncbi.nlm.nih.gov/SNP</a>.</p

GG, CC and GC genotypes in the samples.

<p>Fig. 2 A, B and C show GG, CC and GC genotype respectively as shown by the results obtained by RT-PCR.</p

Genotypic and allelic frequencies of the 10 SNPs in the 6 controls and 30 EC patients.

<p>Genotypic and allelic frequencies of the 10 SNPs in the 6 controls and 30 EC patients.</p

Genotype/allele distribution of SREBF-1(rs2297508) in EC patients with different characteristics.

<p>Statistical analysis:Pearson χ2 test.</p><p>Age ≤50 years old, endometrioid type, low grade, stage I and myometrial invasion <1/2 were considered as references for comparison.</p>a<p>Odds ratio(OR) of the GC/CC against the GG genotypes.</p>b<p>Odds ratio(OR) of the C against the G alleles.</p>c<p>Comparison between stage I and II.</p>d<p>Comparison between stage I and III–IV.</p

Genotype/allele frequency of SREBF-1(rs2297508) in EC patients and normal controls.

<p>OR odds ratio Cl confidence limit.</p

Presence of IL-17-producing cells in the degenerated IVD tissues.

<p>Fifty IVD tissue samples (Group P, n = 20 and Group E, n = 30) from disc degeneration patients and 3 from scoliosis patients were analyzed. In Group P or the control group, there were few or no positive cells (data not shown). The representative results of group E are shown. (a), IL-17-producing cells were detected by a rabbit anti-IL-17 polyclonal antibody and a mouse anti-CD4 monoclonal Ab, followed by secondary staining with an Alexa 488-conjugated donkey anti-rabbit and an Alexa 568-conjugated donkey anti-mouse IgG. DAPI mounting medium was used for nuclear staining. (b), surface CCR6 expression on the cells was detected with a rabbit anti CD4 monoclonal Ab and a mouse anti-CCR6 monoclonal Ab, followed by secondary staining with an Alexa 488-conjugated donkey anti-rabbit and an Alexa 568-conjugated donkey anti-mouse IgG. DAPI mounting medium was used for nuclear staining. In the top panel, green and red represents the expression of IL-17 and CD4, respectively, and the double-stained cells represent the IL-17-producing cells. In the bottom panel, green and red represents the expression of CD4 and CCR6, respectively, and the double-stained cells demonstrate the surface expression of CCR6 on T lymphocytes.</p