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Wang et al. J Transl Genet Genom 2023;7:126-40 https://dx.doi.org/10.20517/jtgg.2023.07 Page 128

sPTB. Despite efforts to understand the genetic and/or genomic mechanisms associated with sPTB, little is
known about the epigenetic regulation of lncRNA involved in sPTB. The insufficiency of a lncRNA
transgenic model limits the ability to address the gene-environmental interactions that may result in the
highly heterogenous sPTB.

Evidence obtained in our earlier studies indicates that lncRNA may be involved in regulating transcription
to produce lncRNA-overlapped mRNA and gene-specific mRNA [3,21,22] . In particular, co-differentially
expressed pairs of lncRNAs and mRNAs sharing the same genomic loci in sPTB were identified in the
ubiquitin-proteasome system (UPS) and found to be related to the ubiquitin-proteasome-collagen (CUP)
pathway . Similarly, a relationship between lncADAM9 and lncADAM9 overlapping mRNA-ADAM9 was
[23]
identified in the fetal membrane that is associated with sPTB [3,20] . ADAM9, a disintegrin and a
metalloproteinase domain (ADAM) 9, is known to be expressed by monocytes and macrophages, and
functions potently as matrix metalloproteinase-9 to degrade several proteins, including fibronectin,
entactin, laminin, and insoluble elastin, which are the molecules involved in the pathways of extracellular
matrix and/or cellular adhesion (ECM-A) . We therefore hypothesize that differentially expressed
[24]
lncADAM9 in human placentas may have a pathogenic impact on the ECM-A pathway. This impact is
likely through its epigenetic regulation on the overlapped mRNA of ADAM9.

MATERIALS AND METHODS
Ethics statement
The parent study design was reviewed and approved by the Ethics Committee of Inner Mongolia Maternal
and Child Health Care Hospital. Written informed consent was obtained from the pregnant women who
participated in this study. All material and data were previously coded and are anonymous to the authors of
this study.

Study design
The parent study was designed as two phases: discovery and confirmation, and its goal was to identify
lncRNA and functionally characterize the potential epigenetic regulatory role. In the discovery study ,
[3]
lncADAM9 and the transcript at the locus where the lncADAM9 overlapped, mRNA-ADAM9, were
identified by microarray analysis and were further studied to determine their differential expression profiles
(DEPs). In the confirmation phase, the identified lncRNA was designed to be subjected to an in vitro study
using transgenic cell culture in human HTR8 cells. Next, variant pathways were determined through RNA
sequencing (RNA-seq). The genetic locus of ADAM9 was selected as our target in the current confirmatory
functional analysis because of that ADAM9 is a membrane-anchored protein that participates in a variety of
physiological functions, primarily through the disintegrin domain for adhesion and the metalloprotease
domain for ectodomain shedding of a wide variety of cell surface proteins. ADAM9 influences the
[25]
developmental process and inflammation . Given that cellular adhesion and the metalloprotease have been
determined to be associated with sPTB in our previous studies [21-23] , we believe that focusing lncADAM9
regulation on the ECM-A pathway would be a logical reason to study the epigenetic impact with in vitro
system.

Specimens
On entry into the study, previously banked specimens were selected according to their clinical outcomes of
birth and GWs at parturition. These groups were defined as A, pPROM ≤ 35 GWs; B, FTB (full-term birth)
at 39-40 GWs without membrane rupture; C, sPTL at ≤ 35 GWs without membrane rupture; and D,
premature rupture of membranes (PROM) at 39-40 GWs. In the discovery study, 40 cases of human
[3]
placentas (10 from each group) were subjected to an array-based assessment, as we have reported .
Additionally, 120 fetal membrane tissue samples (30 in each group) from age-matched (25-35 years of age)