BMPR1B up-regulation via a miRNA binding site variation defines endometriosis susceptibility and CA125 levels

Abstract

Background: Bone morphogenetic protein receptor I B (BMPR1B) is a transmembrane receptor mediating TGF-β signal transduction. Recent studies indicate a tumor suppressor role for BMPR1B in ovarian cancer. Polymorphism at BMPR1B 3'UTR within the miR-125b binding site alters its binding affinity toward the miRNA, which may result in insufficient post-transcriptional repression.

Methods: Single-nucleotide polymorphisms rs1970801, rs1434536, and rs11097457 near the miR-125b binding site in BMPR1B were genotyped by Taqman assay on 193 endometriosis patients and 202 healthy controls. BMPR1B and CA125 levels in ectopic endometrial tissues were evaluated by quantitative PCR and immunohistochemistry. Luciferase reporter assay was utilized to verify regulatory roles of BMPR1B 3'UTR with allelic variants of rs1434536 in a cell line model. Cell proliferation and migration were recorded, while expression of BMPR1B, CA125, glucocorticoid receptor (GCCR) and IL-1β were measured by quantitative PCR in endometrial cells transfected with wild-type or mutated miR-125b.

Results: This study found two endometriosis-associated SNPs, rs1434536 (P = 0.010) and rs1970801 (P = 0.0087), located within and next to a miR-125b binding site on BMPR1B. Interestingly, patients with homozygous variant alleles at rs1434536 showed significantly lower serum CA125 levels. Immunohistochemistry staining further confirmed inverse correlation between BMPR1B and CA125 levels in three rs1434536 genotypes. Cell assays demonstrated the variant allele of rs1434536 up-regulating BMPR1B at both mRNA and protein levels, which negatively correlated with CA125 and IL-1β levels. Disruption of the binding between miR-125b and BMPR1B hampered abnormal cell proliferation.

Conclusions: SNPs of BMPR1B within and next to the miR-125b binding site manifested strong correlation with endometriosis development in a Taiwanese cohort. Disrupting the binding of miR-125b toward BMPR1B would increase protein expression, diminishing abnormal cell proliferation as well as serum and cellular CA125 levels. Genetic variation at the miR-125b binding site may play functional roles to protect against endometriosis progression.

Figure 1. Genotyping and pair-wise LD measures for SNPs in BMPR1B.

Genotyping was performed by Taqman method using DNA extracted from peripheral blood leukocytes. A. Representative fluorescent signal plots from genotyping of SNPs in BMPR1B. B. Through linkage disequilibrium evaluation, D' and r² values were plotted at top right and bottom left triangles, respectively, for normal controls (upper panel) and endometriosis patients (lower panel). Scales beneath the charts show the relative location of each SNP on chromosome 4. SNP1: rs1970801; SNP2: rs1434536; SNP3: rs11097457.

Figure 2. Genotypic variants presented at miR-125b binding site correlated with varied CA125 levels in patients.

A. Serum CA125 levels in endometriosis patients were grouped by their rs1434536 genotypes. Bold lines indicate median values of CA125 levels in each genotype group. Notch regions define 95% confidence interval estimations of median values. B. Gene expression levels of BMPR1B and CA125 (MUC16) in ectopic endometrial tissues were measured by quantitative PCR for correlation study. C. Immunohistochemistry (IHC) staining was performed on ectopic endometrial tissues from patients. Anti-CD10 staining, a stroma cell-specific marker, serves as the reference to normalize immunointensity. Ectopic endometrium samples represented patients with rs1434536 genotype of CC, CT; and TT, as labeled on top of each column. A total of 6, 8 and 12 samples from tissue microarrays could be defined as TT, CT and CC genotype groups, respectively, and their expression levels for BMPR1B and CA125 were scored as described in method section (right panel). Box plot and ANOVA P value calculation were performed in R software suite.

Figure 3. Variation disrupting recognition between miR-125b and 3′UTR of BMPR1B increases BMPR1B expression and inhibits abnormal cell growth in vitro.

A. Luciferase reporter assay was employed to evaluate repression of BMPRIB in endometrial cell lines with C (HEC-1-A) or T (RL95-2) allele at rs1434536. Luciferase reporter pGL4.73 vectors, containing the reference C or variant T bearing sequence of BMPR1B 3′UTR, were transiently introduced into both cell lines. At 48h post-transfection, luciferase activities were measured and normalized to phosphatase activities. B. Cell growth of HEC-1-A cells transfected with empty vector or vector containing miR-125b-wt or miR-125b-mt with a mutated base complementary to the minor allele at rs1434536. At different time intervals, cells were counted using hemocytometer. C. HEC-1-A cells transfected with vectors containing miR-125b-wt or miR-125b-mt were counted and rated with reference to total positively transfected (GFP+) cells. D. Expression levels of known BMPR1B responsive genes were analyzed in HEC-1 cells transfected with miR-125b-wt or miR-125-mt constructs, using empty vector-treated cells as references. E. Expression levels of IL-1β in clinical samples were measured by quantitative PCR. Statistical significance is calculated by Student t-test: *, P<0.05; **, P<0.01.

Figure 4. Schematic representation of a proposed model depicting functional influences of genetic variations in BMPR1B 3′UTR on endometriosis development.

Mature miRNAs including miR-125b are produced by several processing steps where the microprocessor complex (Drosha/DGCR8), exportin 5 and Dicer/TRBP/PACT complex have been known to involve in. The resulting miR-125b forms a RISC complex with Ago and silences BMPR1B gene by binding to the complementary sequence in BMPR1B 3′UTR. Impaired recognition due to genetic variations in the mir-125b seed region reduces suppressive effect of mir-125b, resulting in up-regulation of BMPR1B. The CA125 level, a biomarker for endometriosis and ovarian cancer, was found reversely correlated with BMPR1B in endometrium cells. Elevated BMPR1B levels in endometrium cells have been proven to reduce cell proliferation and migration activity via down-regulation of IL-1β, indicating a lower risk to develop endometriosis.