Transcriptome interrogation of human myometrium identifies differentially expressed sense-antisense pairs of protein-coding and long non-coding RNA genes in spontaneous labor at term

Abstract

Objective: To identify differentially expressed long non-coding RNA (lncRNA) genes in human myometrium in women with spontaneous labor at term.

Materials and methods: Myometrium was obtained from women undergoing cesarean deliveries who were not in labor (n = 19) and women in spontaneous labor at term (n = 20). RNA was extracted and profiled using an Illumina® microarray platform. We have used computational approaches to bound the extent of long non-coding RNA representation on this platform, and to identify co-differentially expressed and correlated pairs of long non-coding RNA genes and protein-coding genes sharing the same genomic loci.

Results: We identified co-differential expression and correlation at two genomic loci that contain coding-lncRNA gene pairs: SOCS2-AK054607 and LMCD1-NR_024065 in women in spontaneous labor at term. This co-differential expression and correlation was validated by qRT-PCR, an experimental method completely independent of the microarray analysis. Intriguingly, one of the two lncRNA genes differentially expressed in term labor had a key genomic structure element, a splice site, that lacked evolutionary conservation beyond primates.

Conclusions: We provide, for the first time, evidence for coordinated differential expression and correlation of cis-encoded antisense lncRNAs and protein-coding genes with known as well as novel roles in pregnancy in the myometrium of women in spontaneous labor at term.

Keywords: Genomics; lncRNA; parturition; pregnancy; preterm birth.

Figure 1

Correlation and co-differential expression of mRNA-lncRNA sense-antisense pairs based on microarray data. (A) Log (base 2) microarray expression of SOCS2 gene (probed by Illumina^® probe ILMN_2131861) are plotted against the expression of lncRNA AK054607 (probed by Illumina^® probe ILMN_1699188). Each sample is represented by one filled circle (black: term not in labor; red: term in labor), while the solid lines give the linear fit to the data in each group separately. (B) Log (base 2) microarray expression of LMCD1 gene (probed by Illumina^® probe ILMN_1754969) are plotted against the expression of lncRNA NR_024065 (probed by Illumina^® probe ILMN_2052135). Each sample is represented by one filled circle (black: term not in labor; red: term in labor), while the solid lines give the linear fit to the data in each group separately.

Figure 1

Correlation and co-differential expression of mRNA-lncRNA sense-antisense pairs based on microarray data. (A) Log (base 2) microarray expression of SOCS2 gene (probed by Illumina^® probe ILMN_2131861) are plotted against the expression of lncRNA AK054607 (probed by Illumina^® probe ILMN_1699188). Each sample is represented by one filled circle (black: term not in labor; red: term in labor), while the solid lines give the linear fit to the data in each group separately. (B) Log (base 2) microarray expression of LMCD1 gene (probed by Illumina^® probe ILMN_1754969) are plotted against the expression of lncRNA NR_024065 (probed by Illumina^® probe ILMN_2052135). Each sample is represented by one filled circle (black: term not in labor; red: term in labor), while the solid lines give the linear fit to the data in each group separately.

Figure 2

Correlation and co-differential expression of mRNA-lncRNA sense-antisense pairs based on qRT-PCR data. (A) The −ΔCT values, surrogate for log (base 2) expression of SOCS2 gene (probed by Taqman primer Hs0091962_m1) are plotted against the −ΔCT values of lncRNA AK054607 (probed by Taqman primer Hs04274293_g1). (B) −ΔCT values of LMCD1 gene (probed by Taqman primer Hs00205871) are plotted against the −ΔCT values of lncRNA NR_024065 (probed by Taqman primer Hs00705981_s1). Each sample is represented by one filled circle (black: term not in labor; red: term in labor), while the solid lines give the linear fit to the data in each group separately.

Figure 2

Correlation and co-differential expression of mRNA-lncRNA sense-antisense pairs based on qRT-PCR data. (A) The −ΔCT values, surrogate for log (base 2) expression of SOCS2 gene (probed by Taqman primer Hs0091962_m1) are plotted against the −ΔCT values of lncRNA AK054607 (probed by Taqman primer Hs04274293_g1). (B) −ΔCT values of LMCD1 gene (probed by Taqman primer Hs00205871) are plotted against the −ΔCT values of lncRNA NR_024065 (probed by Taqman primer Hs00705981_s1). Each sample is represented by one filled circle (black: term not in labor; red: term in labor), while the solid lines give the linear fit to the data in each group separately.

Figure 3

Genomic structure and evolutionary conservation of mRNA-lncRNA sense-antisense pairs. (A) Sense-antisense transcriptional units and cognate Illumina^® probe positions at the SOCS2-AK054607 locus. Arrows indicate: the differentially-expressed lncRNA probe (bottom), and genomic boundaries of the antisense and sense transcriptional units (middle and top, respectively). (B) Multi-species sequence alignment of a splice donor in an intron of AK054607. The spliced side is on the antisense strand, corresponding to sense strand nucleotides CT. Obtained from the UCSC Genome Browser. (C) The LMCD1 transcriptional unit and its downstream sense-strand neighbor, lncRNA NR_024065 (synonym: LINC00312). Arrow indicates the differentially-expressed Illumina^® probe corresponding to this lncRNA.

Figure 3

Genomic structure and evolutionary conservation of mRNA-lncRNA sense-antisense pairs. (A) Sense-antisense transcriptional units and cognate Illumina^® probe positions at the SOCS2-AK054607 locus. Arrows indicate: the differentially-expressed lncRNA probe (bottom), and genomic boundaries of the antisense and sense transcriptional units (middle and top, respectively). (B) Multi-species sequence alignment of a splice donor in an intron of AK054607. The spliced side is on the antisense strand, corresponding to sense strand nucleotides CT. Obtained from the UCSC Genome Browser. (C) The LMCD1 transcriptional unit and its downstream sense-strand neighbor, lncRNA NR_024065 (synonym: LINC00312). Arrow indicates the differentially-expressed Illumina^® probe corresponding to this lncRNA.

Figure 3

Genomic structure and evolutionary conservation of mRNA-lncRNA sense-antisense pairs. (A) Sense-antisense transcriptional units and cognate Illumina^® probe positions at the SOCS2-AK054607 locus. Arrows indicate: the differentially-expressed lncRNA probe (bottom), and genomic boundaries of the antisense and sense transcriptional units (middle and top, respectively). (B) Multi-species sequence alignment of a splice donor in an intron of AK054607. The spliced side is on the antisense strand, corresponding to sense strand nucleotides CT. Obtained from the UCSC Genome Browser. (C) The LMCD1 transcriptional unit and its downstream sense-strand neighbor, lncRNA NR_024065 (synonym: LINC00312). Arrow indicates the differentially-expressed Illumina^® probe corresponding to this lncRNA.