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Page 129 Wang et al. J Transl Genet Genom 2023;7:126-40 https://dx.doi.org/10.20517/jtgg.2023.07

were collected and divided into the four groups (30 fetal membrane samples per group) of pregnancies, as
described above.

Discovery study with microarray
The discovery study was performed as we have reported elsewhere . Differentially expressed lncRNAs and
[3]
lncRNA-overlapped mRNAs and their involvement in pathogenic pathways were identified in human
placentas. These pathways, which were constructed by the highly bioinformatic-enriched scores of DEPs of
lncRNAs, included the ECM-A pathway [3,21,22] .

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from human tissues of the placenta, fetal membrane, or cultured HTR8 cells by
using TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcription was performed using a Thermo
reverse transcription kit (Thermo Fisher, Waltham, MA, USA), and qRT-PCR using SYBR Green Master
Mix (Thermo Fisher). The primers used are listed in Table 1. Among them, three lncRNAs (lncADAM9,
LncPCDH10, lncUSP46) and 19 mRNAs, which are involved in the ECM-A pathway [25-27] , were selected as
the targets for quantitative analysis. RNA of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was
used as an internal control, and the expression values of lncRNAs and mRNAs were normalized.
Comparisons were performed inter-group either individually (such as A vs. B) or combined (A + C vs.
B + D). The data were subjected to one-way analysis of variance (one-way ANOVA) followed by an
unpaired, two-tailed t-test. Differences were considered statistically significant at P < 0.05.

Overexpression of incRNAs in HTR8 cells mediated by lentivirus
PSE3569 was the lncADAM9-overexpressing plasmid constructed with an RNA-expressing lentiviral vector
PMT (BoSe Biotech, Shanghai, China) inserted with a cDNA that had been generated from reverse-
transcription from lncADAM9 sequence. Similarly, PSE3570, used as an experimental control, was
constructed with a cDNA of lncPCDH10 inserted into PMT. The lentiviral vector PMT without insertion of
lncRNA was used as a control of “empty skeleton plasmid” in transfection. Packaging and transfection of
[28]
lentiviral plasmids followed a standard procedure . The lentivirus packaging cell line 293T was cultured in
DMEM medium with 10% FBS and antibiotics for 72 h. Lentiviral constructs containing cDNA sequence of
lncADAM9, confirmed in the orientation of 5′ → 3′ transcription via PCR-sequencing, was packed with
@
TM
[29]
293T cells and transfected into HTR8 (ATCC CRL-3271 ) that we have reported in studying IUGR . The
HTR8 cells were cultured in RPMI medium supplemented with 10% FBS and antibiotics. After transfection,
the culture of lncADAM9-TGHTR8 cells was replaced with a fresh medium for another 72 h at 37 °C with
5% CO . The overexpressed lncADAM9 and mRNA-ADAM9 levels were tested by qRT-PCR. Lentiviral
[29]
2
plasmids without insertion were used as controls in transfection studies.
Transcriptomic profile
The differentially expressed genes between lncRNA-overexpressed samples and their blank control were
identified as we have reported earlier , with the filter criteria |log2FC| ≥ 2 and P value < 0. 05. The target
[3]
gene set was clustered with KEGG and GO functional enrichment. Fisher's exact test was carried out to
calculate whether the GO/KEGG pathway function set was significantly enriched in the list of target genes,
and the acquired P values were corrected to obtain FDR (false discovery rate).

Quantitative analysis of ECM-A proteins
The fetal membrane tissues were washed twice with ice-cold distilled PBS and lysed in RIPA protein
extraction buffer (Sigma-Aldrich, Allentown, PA) containing protease inhibitors. Protein concentration was
determined by the BCA protein determination method. The lysates (20 ng protein) were separated by SDS-
PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA). The primary