Objective
MicroRNAs (miRNAs) are noncoding RNAs involved in posttranscriptional regulation of target genes. The objective of this study was to determine the miRNA expression profile of the human uterine cervix after spontaneous term labor (TL).
Study Design
The miRNA expression pattern of cervical tissue was characterized using microarrays. Samples were collected at term from patients with (n = 8) and without (n = 9) TL. Moderated t tests and false discovery rate correction were applied. Results were confirmed using quantitative reverse transcriptase–polymerase chain reaction.
Results
A total of 226 miRNAs were expressed in human cervical tissue. miR-223, miR-34b, and miR-34c were overexpressed in cervical tissue of patients with TL compared to those without. Quantitative reverse transcriptase-polymerase chain reaction assays confirmed these findings (miR-223 [fold change {FC} = 5.7], miR-34b [FC = 4.5], miR-34c [FC = 6.2]; P < .001).
Conclusion
This is the first report of miRNA expression in the human uterine cervix in pregnancy. Cervical remodeling after TL and delivery was associated with changes in miR-223, miR-34b, and miR-34c.
Cervical biology is central to the understanding of term parturition, cervical insufficiency, preterm labor, and functional disorders of term labor (TL) such as arrest of dilatation. Cervical tissue has been examined for decades to understand the biology of cervical change during pregnancy. High-throughout biology techniques have been employed to begin to decipher the complex mechanisms involved in physiological cervical change in pregnancy. Thus far, transcriptomics and pathway analysis have been employed to describe an unbiased and comprehensive gene expression profile of the uterine cervix before and after labor and the uterine cervical transcriptome before and after labor at term has been characterized. Examination of potential regulatory mechanisms that may affect these gene expression patterns, such as microRNAs (miRNAs), is the next step to better understand the role of the uterine cervix in human parturition.
MicroRNA is a newly discovered class of small, single-stranded, noncoding regulatory RNA molecules that are responsible for posttranscriptional regulation of target genes, and are found ubiquitously in all living organisms such as bacteria, viruses, and humans. Through binding of complementary sites in the 3′ untranslated region (3′UTR) of a messenger RNA (mRNA), a specific miRNA reduces the expression of target genes through either: (1) steric inhibition of translation; or (2) promotion of mRNA degradation. More than 500 human miRNAs have been identified. Although studies elucidating the function of these miRNAs are few, existing research suggests critical roles for miRNAs in multiple processes of human physiology and pathologic conditions. MicroRNAs play a role in embryonic development, granulocyte differentiation, and oncogenesis. MicroRNA expression profiles have been used to distinguish between malignant and benign B cells in chronic lymphocytic leukemia, and unique miRNA expression profiles are associated with prognostic factors and disease progression. Moreover, microRNA expression profiles have shown promise for the diagnosis, assessment of prognosis, and prediction of outcome in patients with breast, hepatocellular, lung, pancreatic, and colon cancer. For example, in patients with colon adenocarcinoma, higher miR-21 expression was noted in adenomas and in tumors with more advanced staging than in nontumorous tissue. In addition, high miR-21 expression was associated with poor survival and poor therapeutic outcome in patients with colon adenocarcinoma. Furthermore, in cervical cancer, miR-143 and miR-145 are suppressive to cell growth and miR-146a was found to promote cell proliferation when introduced into cell lines.
MicroRNAs have only recently been investigated in obstetrics and gynecology. We reported that distinct subsets of miRNAs are differentially expressed in the human placentas of patients with preeclampsia and chorioamnionitis. Recently, the gestational age-dependent gene expression patterns of miRNAs in the chorioamniotic membranes have been described. The objective of this study was to determine whether cervical dilatation and remodeling after term human parturition is associated with changes in miRNA expression profiles.
Materials and Methods
Study design
A cross-sectional study was performed in patients undergoing elective cesarean section with an unripe cervix (term not in labor [TNL], n = 9) and in patients after spontaneous vaginal delivery (TL, n = 8). MicroRNA gene expression profiling was performed using microarrays. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to confirm differential expression of miRNAs. Computational biology techniques were then used to identify the targets of differentially expressed miRNAs.
Cervical biopsy collection
Patient inclusion criteria were: (1) term gestation (≥37 weeks); (2) no prostaglandin, oxytocin, or antibiotic administration; (3) absence of histologic chorioamnionitis; (4) negative Neisseria gonorrhoeae and Chlamydia trachomatis cervical secretion assays during the current pregnancy; and (5) normal Pap smear. Patients underwent cervical biopsy following spontaneous vaginal delivery after TL or after elective cesarean section without signs of labor or cervical ripening. Biopsy specimens (0.5 cm) were obtained transvaginally from the anterior lip of the cervix at the 12 o’clock position and immediately snap frozen in liquid nitrogen or placed in RNA later (Ambion Inc, Austin, TX) and stored at –70°C.
Patient clinical and demographic data, obstetric and gynecologic history, as well as pregnancy outcome were recorded. All biopsies were performed after patients gave written informed consent, according to a protocol approved by the Wayne State University Human Investigation Committee.
MicroRNA gene expression profiling
The miRCURY locked nucleic acid array (v.8.0; Exiqon, Vedbaek, Denmark) was used to examine the expression of 455 miRNAs. A detailed description of the methodology and analysis has been previously described. After background correction, the quantified signals were normalized using the global LOWESS (Locally Weighted Scatterplot Smoothing) regression algorithm. Moderated t tests were used to assess differential miRNA expression between the 2 groups. The false discovery rate algorithm was used to adjust for multiple testing and control the fraction of false-positive results. A significance threshold of P < .05 on the false discovery rate-corrected P values was used to select the differentially expressed miRNAs.
Real-time qRT-PCR assays
qRT-PCR assays of the miRNAs found to be differentially expressed in the microarray analysis were performed on a set of cervical biopsy samples obtained from subjects different than those used in the microarray analysis. The biopsy samples were obtained from women undergoing elective cesarean section with an unripe cervix (n = 10) as well as patients after spontaneous vaginal delivery (n = 9). Details of the RNA isolation and qRT-PCR assays have been previously described.
Target analysis (computational technique)
MicroRNA target analysis was performed to identify potential mRNA targets of the differentially expressed miRNAs using the miRBase targets database. MicroRNA target prediction begins with identification of miRNA sequences and sequences of the 3′ UTR of mRNAs, where miRNAs most frequently bind. The miRANDA algorithm used by miRBase scans these sequences for homologous sites and applies rules and weighting criteria such as conservation across species, number of miRNA binding sites in a 3′ UTR, stability of the bound complex, and other factors known to affect in vitro and in vivo miRNA-target binding.
Computational predictions combined with experimental evidence
The targets of the miRNAs up-regulated after spontaneous labor were computationally predicted. The uterine cervix transcriptome after term spontaneous labor has been previously described. We hypothesized that the genes that were down-regulated in patients after TL would contain targets of the up-regulated miRNAs. The list of down-regulated genes was intersected with the targets of the miRNAs to identify the set of genes that the miRNAs may be targeting in cervical dilatation after TL.
Results
Demographic and clinical characteristics of the study population are depicted in Tables 1 and 2 .
| Characteristic | Term no labor n = 9 | Term labor n = 8 | P |
|---|---|---|---|
| Age, y | 28 (21–35) | 25 (18–27) | NS |
| Parity | 1 (1–2) | 1 (0–3) | NS |
| GA delivery, wk | 39 (38–40) | 39 (37–40) | NS |
| Time ROM, min | 0 | 167 (62–617) | < .001 |
| Biopsy time, min | 61 (30–78) | 12 (9–24) | < .001 |
| Labor duration, min | 0 | 300 (76–471) | < .001 |
| African American ethnicity | 6 (67%) | 6 (75%) | NS |
| Characteristic | Term no labor n = 10 | Term labor n = 9 | P |
|---|---|---|---|
| Age, y | 30 (24–35) | 26 (18–36) | NS |
| Parity | 2 (1–5) | 1 (0–4) | NS |
| GA delivery, wk | 39 (38–40) | 38 (33–41) | NS |
| Time ROM, min | 0 | 105 (6–317) | < .001 |
| Biopsy time, min | 62 (30–72) | 12 (7–21) | < .001 |
| Labor duration, min | 0 | 205 (61–362) | < .001 |
| African American ethnicity | 5 (50%) | 7 (78%) | NS |
Microarray analysis
Microarray analysis revealed that 226 miRNAs were expressed in human cervical tissue at term. miR-223, miR-34b, and miR-34c were up-regulated in the cervical tissue of women who underwent spontaneous labor compared to those not in labor (false discovery rate <0.05) ( Table 3 ).
| Variable | Fold change term labor vs term no labor | P value |
|---|---|---|
| miR-223 | 4.8 | < .0001 |
| miR-34b | 3.3 | < .0001 |
| miR-34c | 8.2 | < .0001 |
Because the delivery-to-biopsy interval was different between the TL and TNL groups (median, 61 minutes [30-78] vs 12 [9-24]; P < .001), we performed an additional analysis to evaluate the relationship between the delivery-to-biopsy interval and the miRNA expression. The log2 miRNA expression levels in each group (TL and TNL) were fit against the delivery-to-biopsy interval using a linear model. There was no significant association between the expression of miR-223, miR-34b, or miR-34c with delivery-to-biopsy interval (adjusted P value > .54 for all 3 miRNAs). In addition, the direction of change in miRNA expression in relationship with time for all 3 miRNAs was not the same in TL as in TNL, providing further evidence that these changes in miRNA expression seen between groups is not due to differences in the delivery-to-biopsy interval.
qRT-PCR
Targeted qRT-PCR assays confirmed the microarray findings (miR-223 [fold change {FC} = 5.7; P < .00004], miR-34b [FC = 4.5; P < .00008], miR-34c [FC = 6.2; P < .0001]) ( Figure 1 ).
