Emerging role of miRNAs, lncRNAs, and circRNAs in pregnancy-associated diseases

Abstract

Accumulating studies have demonstrated that non-coding RNAs (ncRNAs), functioning as important regulators of transcription and translation, are involved in the establishment and maintenance of pregnancy, especially the maternal immune adaptation process. The endometrial stromal cells (ESCs), trophoblast cells, and decidua immune cells that reside at the maternal–fetal interface are thought to play significant roles in normal pregnancy and pregnancy-associated diseases. Here, we reviewed the up-to-date evidence on how microRNA, long non-coding RNA, and circular RNA regulate ESCs, trophoblast cells, and immune cells and discussed the potential applications of these ncRNAs as diagnostic and therapeutic markers in pregnancy complications.

Introduction

Pregnancy is the process that embryo and fetus grow and develop normally in the maternal uterus. It is well established that a successful pregnancy is reliant on normal embryo implantation and maintenance of maternal–fetal interface.^[1] Uterine receptivity, embryo attachment, and embryo invasion constitute the decisive factors for successful implantation. The maternal–fetal interface is a complex system containing various cells, among which endometrial stromal cells (ESCs), trophoblast cells, and immune cells are key populations.^[2] In early pregnancy, ESCs are transformed into decidual cells (DCs) through decidualization, which promotes embryo implantation. The degree of decidualization of ESCs determines the degree of invasion of trophoblast cells.^[3] The differentiation, migration, and invasion of trophoblast cells are crucial for a successful pregnancy. In early pregnancy, decidual immune cells constitute 30% to 40% of the total cells in the uterus. Among them, approximately 70% of cells are decidual natural killer cells (dNKs); macrophages, T cells, and DCs account for 20%, 10%, and 1% to 2%, respectively, B cells can hardly be measured; furthermore, differentiation and functional status of immune cell subsets are essential for maintaining maternal–fetal tolerance and normal pregnancy.^[4] In summary, successful embryo implantation and maternal–fetal immunotolerance are necessary for the establishment and maintenance of pregnancy.^[2] Once the balance of the maternal–fetal interface is broken, pregnancy-associated diseases will occur consequently [Figure 1]. Maintaining the functional stability of the maternal–fetal interface is a complex and delicate regulatory process, and multiple mechanisms are involved. Therefore, further exploring the causes of maternal–fetal imbalance will provide effective ways for clinical diagnosis and therapy.

Figure 1: Critical processes of normal pregnancy. Decidualization prepares for the successful implantation in endometrium. Trophoblast cells migrate into the vascular lumen, replace endothelial cells, and thus facilitate blood flow into the space surrounding the placental villi tree, which is essential for nutrient and gas exchange between conceptus and mother. Decidual immune cells play critical roles in maintaining maternal–fetal tolerance. NK: Natural killer.

Non-coding RNAs (ncRNAs) are widely expressed in various tissues including decidua and profoundly affect diverse cellular processes.^[5] ncRNAs can be classified into two main categories based on its length: those with length under 200 ribonucleotides are short ncRNAs, such as microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs), while over 200 ribonucleotides are long ncRNAs (lncRNAs). Besides, in view of covalently circularization or not, the lncRNAs can be further divided into linear or circular ncRNAs (circRNAs).^[6] At present, an increasing number of studies of ncRNAs focused on miRNAs, lncRNAs, and circRNAs.^[7] miRNAs, 18 to 25 nucleotides length, mainly participate in mRNA silencing and post-transcriptional regulation.^[7] lncRNAs and circRNAs often serve as miRNA sponges to regulate gene expression at the transcriptional level. Emerging evidence has clearly indicated clinical and pathological associations between these ncRNAs and pregnancy-associated diseases. We therein summarized the recent findings concerning the roles of miRNAs, lncRNAs, and circRNAs in normal pregnancy and pregnancy-associated diseases, which is beneficial to clinical implications on diagnosis and treatment [Figure 2].

Figure 2: Biogenesis and functions of miRNAs, lncRNAs, and circRNAs. miRNAs are transcribed into pri-miRNAs in the nucleus. Then, pri-miRNAs are processed by Drosha to form pre-miRNAs, which are transported by exportin 5 to cytoplasm and are cleaved by Dicer into mature double-stranded miRNAs. Subsequently, the mature miRNA binds the 3′UTR, and leads to the degradation of the target mRNA or its translation inhibition. lncRNAs regulate gene transcription in the nucleus, and negatively regulate the expression of related miRNAs in the cytoplasm as ceRNAs. circRNAs are primarily generated by back-splicing of various transcripts, and can serve as a molecular sponge for miRNA to inhibit its function. Besides, circRNAs could also act as protein scaffold, RBP sponge, or even lead to the yield of related proteins. 3′UTR: 3′-untranslated region; ceRNAs: Competitive endogenous RNAs; circRNAs, Circular RNAs; ciRNA: Circular intronic RNA; ecircRNA: Exonic circRNA; EIciRNA: Exon-intron circRNA; lncRNA: Long non-coding RNAs; miRNA: MicroRNA; pre-miRNAs: Precursor miRNAs; pri-miRNAs: Primary miRNAs; RBP: RNA-binding protein; TF: Transcription factor; U1 snRNP: U1 small nuclear ribonucleoprotein.

miRNAs, lncRNAs, and circRNAs in the Establishment and Maintenance of Pregnancy

Successful embryo implantation represents the most important factor in the establishment of pregnancy, which requires reciprocal interactions between the placenta and endometrium. Growing evidence has demonstrated the important roles of ncRNAs in this maternal–fetal interface in the establishment and maintenance of pregnancy.^[8] Both expression changes and biological functions of miRNAs, lncRNAs, and circRNAs in the processes of normal pregnancy and pregnancy-associated diseases are introduced here [Figure 2].

miRNAs in pregnancy

miRNAs are differentially expressed in cells at the maternal–fetal interface and regulate the target genes’ expression.^[7] A recent study profiled miRNome of the first- and third-trimester human placenta by next-generation sequencing; such a miRNA atlas is fundamental for using miRNAs as biomarkers to monitor maternal–fetal health throughout pregnancy.^[9] Furthermore, many miRNAs’ functions were explored in pregnancy-related cellular and animal models. Downregulation of miR-542-3p in human ESCs facilitated ESCs decidualization by activating integrin-linked kinase (ILK)/transforming growth factor (TGF)-β1/SMAD family member 2 signaling pathway and induced loss of embryo implantation in a mouse model of early pregnancy.^[10] Conversely, a decreased expression of miRNA-155-5p in decidua tissue not only promoted proliferation but also inhibited apoptosis of decidua stromal cells by inhibiting the nuclear factor kappa B signaling pathway, which could maintain normal pregnancy.^[11] Li et al^[12] found that miR-21a was elevated in human or mouse decidua tissues compared with endometrial tissues, which could inhibit DC apoptosis through targeting programmed cell death 4 (Pdcd4). In addition, it was found that downregulated miR-346, and miR-582-3p in human placental tissues increased the migration and invasion of trophoblast cells in early pregnancy by promoting the expression of matrix metalloproteinase (MMP)-2 and MMP9.^[13] Furthermore, it has been identified that decreased miR-590-3p in trophoblast cells could directly target low-density lipoprotein receptor-related protein 6 and promote the ability of migration, invasion, and tube formation of trophoblast cells by activating wingless/integrated (wnt)/β-catenin signaling pathway.^[14] Additionally, upregulated miR-218-5p in the placenta could promote differentiation and invasion of trophoblast cells and spiral artery remodeling by suppressing TGF-β2.^[15] These studies clearly indicated that miRNAs play an important role in normal pregnancy by regulating decidualization and the function of trophoblast cells.

From the perspective of immunology, normal pregnancy is similar to a successful allograft, and it is necessary for the immune system to maintain tolerance to embryo and at the same time eliminate pathogenic microorganisms effectively. ^[16] Studies have demonstrated that miRNAs could regulate maternal–fetal immunotolerance. A clinical study on full-transcriptome high-throughput detection of miRNAs in healthy pregnant women identified 36 differentially expressed miRNAs in dNKs compared with peripheral blood natural killer cells (pbNKs), ingenuity pathway analysis detected that the dNKs-differential miRNAs were enriched in the processes of cell differentiation.^[17] In addition, upregulation of miR-30e in peripheral blood and decidua tissue of normal pregnant women in comparison to non-pregnant women was examined to be able to reduce the cytotoxicity of pbNKs and dNKs by inhibiting perforin expression, thus contributing to a micro-immune tolerance environment of the maternal–fetal interface.^[18] However, Fu and Wei^[19] reported that dNKs are the key cells for maintaining immune tolerance and dNKs should be properly activated during clinical treatment for recurrent spontaneous abortion (RSA).

In addition to NK cells, miRNAs are also involved in the regulation of other immune cells at the maternal–fetal interface, such as macrophages and T cells. Previous studies have demonstrated that increased miR-103 in human decidua inhibited the classical M1 polarization of macrophage, which helped maintain the maternal–fetal immunotolerance.^[20] Moreover, it was identified that decreased miR-25, miR-93, and miR-106b in T cells isolated from normal pregnant women could maintain immunotolerance during pregnancy by activating the TGF-β pathway.^[21] Recently, a high-throughput sequencing and real-time polymerase chain reaction (RT-PCR) validation identified 36 differentially expressed circulating miRNAs in plasma and serum during three time points of a normal and healthy pregnancy, which will help the understanding of how miRNAs could be potential non-invasive biomarkers for gestation complications during pregnancy progression.^[22]

lncRNAs in pregnancy

Studies have elucidated that lncRNAs were involved in maintaining normal pregnancy by regulating the function of trophoblast cells. For instance, it was found that increased lnc-LOXL1-AS1 in the human placenta inhibited the expression of miR-515-5p by acting as a molecular sponge and increased the expression of recombinant cyclin D1, MMP2, and MMP9, which subsequently enhanced the proliferation and migration of trophoblast cells.^[23] Moreover, it has been demonstrated that upregulated lnc-H19 in human placenta could promote the expression of integrin β3 and enhance the invasion ability of trophoblast cells by sponging miR-let-7 in early pregnancy.^[24]

From the perspective of maternal–fetal immunity, studies demonstrated that downregulated lnc-DC in human DCs reduced tissue inhibitor of metalloproteinase (TIMP)1/MMP9 and TIMP2/MMP2 ratios in trophoblast cells and promoted the invasion of trophoblast cells; however, further mechanism is greatly anticipated.^[25] Furthermore, the expression of lnc-MEG3 was reduced in villi and maternal decidua during the human first trimester, which enhanced trophoblast cells invasion and improved uterine spiral artery remodeling by activating the phosphoinositide 3-kinase/Akt pathway.^[26] Besides, it has been identified that lnc-49a in human primary dNKs could apparently alleviate the toxicology activity of dNKs by positively regulating CD49a expression, which helps maintain the normal pregnancy.^[27] In conclusion, lncRNAs are important in maintaining normal pregnancy but their regulatory mechanisms need to be further explored.

circRNAs in pregnancy

circRNA could regulate the function of trophoblast cells via sponging miRNA. For instance, downregulated circPAPPA in normal pregnant women's placenta and plasma contributed to the proliferation of trophoblast cells by activating the miR-384/signal transducer and activator of transcription (STAT) 3 axis.^[28] Moreover, increased circ_0005243 in human placenta facilitated trophoblast cells migration and invasion by promoting β-catenin expression.^[29] These results collectively indicated the regulatory role of circRNAs in the proliferation and invasion of trophoblast cells. However, the role of circRNAs in maternal and fetal immune interface is rarely investigated, and further exploration is needed in the future.

miRNAs, lncRNAs, and circRNAs in Pregnancy-associated Diseases

The abnormal expression and dysfunction of miRNAs, lncRNAs, and circRNAs have been associated with a series of pregnancy-related diseases, which endanger normal pregnancy and include recurrent implantation failure (RIF), RSA, pre-eclampsia (PE), fetal growth restriction (FGR), and preterm birth (PTB), we thus discussed herein.

miRNAs, lncRNAs, and circRNAs in RIF

RIF is usually considered as two or more implant failures in the embryo, which is the main cause of in vitro fertilization. There are multiple risk factors for RIF including advanced maternal age, elevated body mass index, and stress levels.^[30] Currently, the roles of ncRNAs in RIF have become a research hotspot [Table 1].

Table 1 - miRNAs, lncRNAs, and circRNAs in RIF.

Status	ncRNAs	Species	Source	Mechanism	Gestational age (days)	Reference
Upregulated	miR-22	Mice	Endometrium	Suppress embryo implantation	2–7	[31]
	miR-145	Human	Endometrium	Inhibit endometrial receptivity, embryo attachment	20–24	[32]
	miR-542-3p	Human	Endometrium	Suppress ESCs decidualization	NA	[10]
	miR-326	Human	PBMCs	Promote the proliferation of Th17 cells	NA	[33]
	lnc-TUNAR	Human	Endometrium	Suppress ESCs decidualization	10–12	[34]
Downregulated	circ_0038383	Human	Endometrium	Impair endometrial receptivity	28–31	[35]

circRNAs: Circular RNAs; ESCs: Endometrial stromal cells; lncRNA: Long non-coding RNA; miRNA: MicroRNA; NA: Not applicable; ncRNAs: Non-coding RNAs; RIF: Recurrent implantation failure; PBMCs: Peripheral Blood Monocytes.

Emerging evidence has demonstrated that dysregulated miRNAs could cause RIF by regulating the decidualization process, trophoblast cells function and immune cells at maternal–fetal interface. It has been demonstrated that miR-22 was increased in the endometrium from mice and patients of RIF, which impair decidualization by inhibiting the T lymphoma invasion and metasis 1/Rac family small GTPase 1 signaling pathway.^[31] Moreover, studies found that elevated miR-145, miR-192-5p, and miR-542-3p in the endometrium of RIF patients could, respectively, reduce the expression of insulin-like growth factor 1 receptor, plasminogen activator inhibitor-1, e-cadherin, mucin 1, and ILK, which inhibited decidualization and embryo attachment.^[10,32] In addition to the studies mentioned above, it was reported that upregulated miR-326 in peripheral blood Th17 cells of RIF patients compared with normal pregnant women could promote the differentiation of Th17 cells by inhibiting the expression of ETS proto-oncogene 1 (Ets1), a negative regulator of Th17 differentiation, thus leading to the imbalance of Th17/Treg ratio.^[33]

Meanwhile, recent studies have shown that lncRNAs and circRNAs are also involved in RIF. Elevated lnc-TUNAR were identified in the endometrial tissues in women suffering from RIF, which impaired embryo adhesion and implantation through downregulating the expressions of prolactin, and insulin-like growth factor binding protein 1.^[34] In addition, decreased circ_0038383 in the endometrium of RIF patients compared to healthy pregnant women could impair endometrial receptivity and induce RIF by targeting miR-196b-5p/homeoboxA (HOXA)-9.^[35]

miRNAs, lncRNAs, and circRNAs in RSA

RSA refers to two or more consecutive miscarriages, which is one of the serious complications of pregnancy that threaten maternal health worldwide. The etiology of RSA is multifactorial, including genetics, anatomy, endocrine, placental abnormalities, infections, smoking, and stress. However, there are still about 50% of the causes are unclear, called unexplained recurrent spontaneous abortion (URSA).^[36] Here, we discussed the functional roles of ncRNAs in RSA [Table 2].

Table 2 - miRNAs, lncRNAs, and circRNAs in RSA.

Status	ncRNA	Species	Source	Mechanism	Gestational age	Reference
Upregulated	miR-365	Human	Decidua	Promote trophoblast cells apoptosis	6–8 weeks	[37]
	miR-6875-5p	Human	PBMCs	Decrease the differentiation of pDCs	6–10 weeks	[38]
	miR-184	Human	Decidua	Promote trophoblast cells apoptosis	6–8 weeks	[41]
	miR-516a-5p, miR-517a-3pmiR-519a-3p, miR-519d	Human	Decidua/villi	Participate in trophoblast cells death, apoptosis, proliferation	8.33 ± 1.80 weeks	[39]
	miR-153-3p	Human	Macrophage	Attenuate the proliferation and migration	7.63 ± 1.22 weeks	[42]
	miR-34a, miR-141	Human	dNKs	Diagnostic and prognostic biomarkers	7–10 weeks	[43]
	lnc-SLC4A1-1	Human	Villi	Promote trophoblast apoptosis, inflammatory reaction	Before 20 weeks	[44]
Downregulated	miR-103	Human	N/A	Promote M1 macrophage polarization	6–10 weeks	[20]
	lnc-SNHG1	Mice	Placenta and decidua	Restrain proliferation, migration, and invasion of trophoblast cells	NA	[45]
	lnc-MEG3	Human	Villi	Inhibit the implantation, proliferative, and invasive capacities of trophoblasts	49–63 days	[46]

circRNAs: Circular RNAs; DCs: Decidual cells; dNKs: Decidual natural killer cells; lncRNAs: Long non-coding RNA; miRNAs: MicroRNAs; NA: Not applicable; ncRNA: Non-coding RNA; RSA: Recurrent spontaneous abortion; PBMCs: Peripheral Blood Monocytes; pDCs: Plasmacytoid dendritic cells.

It was demonstrated thatupregulated miR-365 in the decidua of RSA patients compared to that of normal pregnant women could cause DCs apoptosis throughsuppressing serum and glucocorticoid-regulated kinase-1, which may be a potential underlying mechanism of early RSA.^[37] Our recent research demonstrated increased miR-6875-5p in plasmacytoid DCs of RSA patients inhibited the cell differentiation via the STAT3/transcription factor 4 (Tcf4/E2-2) signaling.^[38] Additionally, a study distinguished 32 upregulated miRNAs in the decidua of RSA patients compared to normal pregnant women through deep sequencing; Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses predicted that these miRNAs are mainly involved in the regulation of trophoblast cells apoptosis and death.^[39] The latest study revealed the expression profiles of mRNA, lncRNA, miRNA, and circRNA in villi tissues between URSA and elective termination of pregnancy via whole-transcriptome sequencing.^[40] Further study found that enhanced miR-184 in the decidua of RSA repressed invasion and spiral artery remodeling of trophoblast cells and promoted trophoblast cells apoptosis by suppressing wild-type p53-induced gene 1, thus inducing early RSA.^[41]

At the same time, studies found that miRNAs are also involved in RSA by breaking the maternal–fetal immunotolerance. For instance, it was demonstrated that increased miR-153-3p in decidual macrophages of RSA patients attenuated the proliferation and migration of trophoblast cells by suppressing the indoleamine 2,3-dioxygenase/STAT3 pathway.^[42] In addition, it was identified that miR-34a and miR-141 were upregulated while miR-24 was downregulated in dNKs of RSA patients, these 3 miRNAs are involved in 140 pathways including phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) and mitogen-activated protein kinases (MAPK).^[43] Moreover, our previous research has proved that downregulated miR-103 was involved in RSA by promoting M1 macrophage polarization by enhancing the activity of STAT1/interferon regulatory factor (IRF) 1 signaling.^[20]

In addition to miRNAs, lncRNAs and circRNAs have also been identified to be involved in RSA. For example, increased lnc-SLC4A1-1 in villi from RSA patients facilitated the expression of interleukin-8 and repressed Wnt/β-catenin signaling, which promoted inflammatory reaction and trophoblast apoptosis as well as inhibited the migration and invasion of trophoblast cells in RSA.^[44] In RSA mouse models, downregulated lnc-SNHG1 in placental and decidual tissues could restrain proliferation, migration, and invasion of trophoblast cells via the miR-183-5p/Zinc finger E-box bongding homeobox 2 and Wnt/β-catenin signaling.^[45] The downregulated lnc-MEG3 in URSA villus led to the activation of RAS P21 protein activator 1 (RASA1) and silence of the RAS-MAPK pathway in trophoblast cells, thereby inhibiting the implantation, proliferative, and invasive capacities of trophoblast cells.^[46] Nevertheless, further research is essential for translating the bench discoveries into clinical diagnosis and treatment.

miRNAs, lncRNAs, and circRNAs in PE

PE is considered to be one of the leading causes of maternal and fetal morbidity and death worldwide, especially in developing countries, affecting 2% to 8% of pregnant women.^[47] Generally, PE emerges after 20 weeks of gestation, with high blood pressure, high proteinuria, and some complications, including hemolysis, visual disturbances, and so on.^[48] Accumulating studies have shown that ncRNAs play important roles in the occurrence and development of PE [Table 3].

Table 3 - miRNAs, lncRNAs, and circRNAs in PE.

Status	ncRNA	Species	Source	Mechanism	Gestational age	Reference
Upregulated	miR-210	Human	Placenta	Suppress invasion, migration, vascular remodeling	255.62 ± 5.72 days	[49]
	miR-106b, miR-326	Human	PBMCs	Increase Th17/Treg cells ratio	30–40 weeks	[52]
	miR-31-5p, miR-155-5p, miR-214-3p	Human	Serum	Impair vascular remodeling	After 20 weeks	[53]
	miR-125b	Human	Placenta, serum	Induce inflammatory reaction	20–34 weeks	[51]
	lnc-SPRY4-IT1, lnc-SH3PXD2A-AS	Human	Placenta	Impair spiral artery remodeling	35.84 ± 3.29 weeks	[56,57]
	lnc-DC	Human	DCs	Broke immune balance	34.9 ± 2.41 weeks	[58]
	circZDHHC20	Human	Placenta	Imped trophoblast cells proliferation, migration	NA	[59]
	circTNRC18	Human	Placenta	Impair trophoblast cells invasion	NA	[60]
	circ_0111277	Human	Placenta	Suppress trophoblast cells invasion, migration	After 20 weeks	[61]
	circ_0001438	Human	Villi	Prohibit trophoblast cells proliferation, migration	NA	[62]
	circ_0011460	Human	Placenta	Inhibit trophoblast cells migration	249.33 ± 2.08 days	[63]
Downregulated	miR-221-3p	Human	Placenta	Inhibit trophoblast cells migration, invasion	30.28 ± 0.36 weeks	[50]
	miR-31, miR-21	Human	Plasma	Act as diagnostic biomarker of PE	20–34 weeks	[54]
	lnc-TUG1	Human	Placenta	Inhibit trophoblast cells migration, invasion, angiogenesis	NA	[55]

circRNAs: Circular RNAs; DCs: Decidual cells; lncRNAs: Long non-coding RNA; miRNAs: MicroRNAs; NA: Not applicable; ncRNA: Non-coding RNA; PE: Pre-eclampsia; PBMCs: Peripheral Blood Monocytes.

Studies have reported that aberrantly expressed miRNAs in trophoblast and immune cells participated in the pathogenesis of PE. For example, increased miR-210 in the placenta and maternal serum from PE patients suppressed invasion, migration, and vascular remodeling capability of trophoblast cells and promoted inflammation by inhibiting ephrin-A3, HOXA-9 levels.^[49] Moreover, it was demonstrated that downregulated miR-221-3p in the placenta of PE patients inhibited trophoblast cells migration and invasion through targeting thrombospondin 2 in PE.^[50] These studies interlinked the impaired invasion of trophoblast cells with poor arterial remodeling through complex miRNA networks, which in turn causes various pathological reactions in PE.

Notably, elevated miR-125b in the placenta and plasma from PE patients promoted IL-8 secretion and produced an inflammatory reaction by directly targeting sphingosine-1-phosphate lyase 1, which could induce PE.^[51] Additionally, it was reported that upregulated miR-106b and miR-326 in peripheral blood monocytes from PE patients increased the Th17/Treg cells ratio, which contributed to the development of inflammation, finally leading to PE, but the mechanism is not clear.^[52] In addition, clinical studies verified three circulating miRNAs (miR-31-5p, miR-155-5p, and miR-214-3p) were upregulated, and one (miR-1290-3p) was downregulated in the serum of PE patients through the receiver operating characteristic curves analysis.^[53] However, miR-31 was contradictorily reported to be decreased in the plasma of early onset PE by an independent study.^[54] Collectively, the complexity of miRNAs in PE needs to be deciphered.

Similarly, growing evidence has shown that numerous lncRNAs are differentially expressed in patients with PE. Studies have shown that decreased lnc-TUG1 in the placenta from PE patients induced the occurrence of PE by inhibiting the migration, invasion, and angiogenesis of trophoblast cells through miR-204-5p/MMP9 pathway.^[55] In addition, it has been demonstrated that upregulated lnc-SPRY4-IT1 and lnc-SH3PXD2A-AS in PE placenta prohibited trophoblast cells migration, invasion, and impaired spiral artery remodeling and led to the occurrence of PE by inhibiting the Wnt/β-catenin pathway and chemokine receptor 7 expression.^[56,57] Moreover, upregulated lnc-DC in PE patients enhanced p-STAT3 expression and broke immune balance via promoting the proliferation of Th1 cells, leading to PE^[58]; however, the underlying mechanism has not been fully elucidated.

Recent studies have indicated that dysregulated circRNAs were also involved in PE. It was reported that increased circTNRC18, circ_0111277, and circZDHHC20 in the placenta of PE patients in comparison to normal pregnant women could impair trophoblast invasion, resulting in PE by suppressing the miR-762/grainyhead 12 (Grh12), miR-494-3p/high-temperature requirement A serine peptidase 1 (HTRA1)/notch homolog 1 (Notch-1), and miR-144/grainyhead-like 2 (GRHL2) axis.^[59-61] In addition, upregulated circ_0001438 in villous trophoblast cells of PE patients prohibited proliferation, migration, and invasion ability and promoted inflammatory response through sponging miR-942 and upregulating NLRP3, which could cause the occurrence of PE.^[62] Moreover, one study suggested that upregulated circ_0011460 in the placenta of PE were involved in the pathogenesis of PE by directly interacting with prostaglandin transporter (PGT) to inhibit the migration of trophoblast cells, although the molecular mechanism needs to be further studied.^[63]

miRNAs, lncRNAs, and circRNAs in FGR

FGR, also known as intrauterine growth restriction (IUGR), is defined as impaired fetal growth capacity and fetal weight below the 10th percentile of the same gestational age. FGR poses a serious threat to maternal reproductive health and increases maternal mortality and also affects fetal neural development. FGR may be related to placental hypoplasia, insufficient nutritional supply, and heredity, but the specific mechanism has not been fully elucidated.^[64]

Studies have shown that miRNAs participate in the development of FGR [Table 4]. For instance, it was reported that increased miR-210-3p in trophoblast cells repressed fibroblast growth factor 1, therefore obstructing the migration and invasion of trophoblast cells, resulting in FGR.^[65] Additionally, studies found that upregulated miR-141-3p and miR-200a-3p in mouse could inhibit placental development via downregulating insulin-like growth factor 2 and ultimately cause FGR.^[66] Moreover, increased miR-424 in FGR trophoblast cells impeded proliferation, migration, and invasion of trophoblast cells and led to FGR via reducing the expressions offibroblast growth factor receptor 1.^[67] miR-148b-3p and miR-25-3p in umbilical vein plasma are significantly elevated in FGR patients compared with normal pregnancy.^[68] A study analyzed differentially expressed miRNAs in plasma samples of patients from FGR and normal pregnancy by qRT-RCR, found miR-16-5p, miR-103-3p, and miR-27b-3p were remarkably upregulated in FGR.^[69]

Table 4 - miRNAs, lncRNAs, and circRNAs in FGR and PTB.

Diseases	Status	ncRNA	Species	Source	Mechanism	Gestational age	Reference
FGR	Upregulated	miR-210-3p	Human	Trophoblast cells	Obstruct trophoblast cells migration, invasion	After 37 weeks	[65]
		miR141-3p, miR-200a-3p	Mice	Trophoblast cells	Inhibit placental development	12.5 days	[66]
		miR-424	Human	Trophoblast cells	Imped trophoblast cells proliferation, migration, invasion	NA	[67]
		lnc-DC	Human	DCs	Block trophoblast invasion	NA	[25]
	Downregulated	lnc-H19	Human	Placenta	Inhibit trophoblast cells proliferation, invasion	After 37 weeks	[70]
		lnc-H19	Human	Placenta	Suppress trophoblast cells migration, invasion	NA	[71]
		circ_0000848	Human	Placenta	Suppress trophoblast cells migration, invasion, promote apoptosis	∼38 weeks	[72]
PTB	Upregulated	miR-26a, miR-199a	Human	Trophoblast cells	Promote inflammatory response	NA	[74]
		lnc-SNHG29	Human	Placenta	Enhance trophoblast cells senescence	Before 37 weeks	[78]
		miR-200b/c/429, miR-200a/141	Human/mice	Myometrium	Promote uterine contractility	NA	[77]
	Downregulated	miR-548e-5p, miR-146a-5p	Human	Mesenchymal stem cell-derived exosome	Promote inflammatory response, inhibit trophoblast cell migration	NA	[75]
		miR-146a, miR-155	Human	DCs	Accelerate inflammatory response	NA	[76]

NcircRNAs: Circular RNAs; DCs: Decidual cells; FGR: Fetal growth restriction; lncRNAs: Long non-coding RNA; miRNAs: MicroRNAs; NA: Not applicable; ncRNA: Non-coding RNA; PTB: Preterm birth.

At present, studies demonstrated that lncRNAs and circRNAs are involved in the regulation of FGR [Table 4]. For example, it was identified that downregulated lnc-H19 in the placenta from FGR patients could inhibit trophoblast cells proliferation and invasion, and promote autophagy of trophoblast cells by the miR-18a-5p/IRF2, PI3K/AKT/mammalian target of rapamycin (mTOR), and MAPK/extracelluar sigal-regulated kinase/mTOR pathways.^[70] Moreover, lnc-H19 knockdown could promote miR-let-7 to impede type III TGF-β receptor expression, which suppressed trophoblast cells migration and invasion and induced FGR.^[71] In addition, increased lnc-DC in DCs from FGR patients participated in FGR by blocking trophoblast invasion through impeding MMP2, MMP9, and p-STAT3 levels.^[25] circ_0000848 was significantly downregulated in the placenta of FGR patients and inhibited trophoblast cells migration and invasion by enhancing miR-6768-5p, which led to FGR; meanwhile, the targets of miR-6768-5p are involved in the pathways of proliferation, apoptosis, and metastasis based on GO and KEGG analysis, especially SMAD7.^[72,73]

miRNAs, lncRNAs, and circRNAs in PTB

PTB is defined as <37 weeks of gestational age and the organs and physiological functions of the fetus are not fully developed, which is a severe threat to maternal–fetal health. There are many reasons for PTB, including infections, smoking, stress, inflammation, and immune alteration.^[71] The premature activation of the inflammatory response often disrupts the immune balance and may promote the occurrence of PTB in severe cases. More and more research concentrates upon the regulatory effects of ncRNAs on PTB [Table 4].

Recently, it was reported that upregulated miR-26a and miR-199a in the PTB placenta promoted inflammation and myometrial contraction by inhibiting the expression of prostaglandin dehydrogenase to induce PTB.^[74] In addition, it was demonstrated that in PTB patients, downregulated miR-548e-5p and miR-146a-5p in the mesenchymal stem cell-derived exosome promoted inflammation and inhibited trophoblast cell migration by activating AKT and MAPK signaling and inhibiting the expression of MMP.^[75] Moreover, the downregulated miR-146a and miR-155 in PTB DCs promoted the expression of pro-inflammatory signals, which accelerated the inflammatory response and induced PTB through the analysis of the inflammatory gene network in human DCs.^[76] The upregulated miR-200 family in mouse and human myometrium could promote the expression of the contraction-associated genes, which promoted myometrial cell contractility and lead to PTB.^[77] Additionally, 1626 differentially expressed mRNAs and 135 differentially expressed proteins were identified in myometrium between non-labor and labor groups via RNA sequencing and proteotranscriptomic analysis.^[78] In summary, the above studies showed that inflammation and contraction in myometrium induced by miRNAs is an important risk factor for PTB.

However, in contrast to the miRNAs, functional studies of lncRNAs in PTB are rather lacking. The latest study revealed 112 differentially expressed lncRNAs in the human myometrium of labor and non-labor groups by RNA sequencing and bioinformatic analysis.^[79] Jiang et al^[80] found that increased lnc-SNHG29 in PTB placenta compared to healthy pregnancy without labor enhanced trophoblast cells senescence and release of inflammatory factor by activating the p53/p21 signaling pathway, finally leading to PTB. Further investigation of the contributions of lncRNAs in PTB is required.

Therapeutic Potentials of miRNAs, lncRNAs, and circRNAs in Pregnancy-associated Diseases

Through reviewing the literature, emerging evidence indicates multiple ncRNAs are expressed differentially in the course of pregnancy-associated diseases and may serve as promising biomarkers. Despite the growing interest of ncRNAs in pregnancy-related diseases, they are still in their infancy as potential clinical therapeutic targets. Kim et al^[81] reported that aspirin could prevent tumor necrosis factor-induced endothelial cell dysfunction via inhibiting the expression of miR-155, which could decrease endothelial nitric oxide synthase/nitric oxide activity to relieve blood pressure in PE. Furthermore, in a PE rat model, ligustrazine (2,3,5,6-tetramethylpyrazine, TMP) impedes trophoblast cell autophagy and promotes its migration to cure PE by negatively regulating the miR-16-5p/IGF-2 axis.^[82] Moreover, metformin treatment in the PE rat model elevated the levels of miR-148-5p and miR-216-3p and inhibited IL-27 protein expression in reducing the incidence of PE.^[83] Our recent study demonstrated that a traditional Chinese medicine Shoutai Pill could treat URSA effectively, which could reduce autophagy of trophoblast cells via negatively regulating the miR-374c-5p/ATG12 axis.^[84] Previous study reported the chromosome 19 miRNA cluster (C19MC), C14MC and miR-371-3 cluster in maternal serum reflected the state of the placenta and might indicate pregnancy disorders.^[85] In summary, ncRNAs have been explored as mechanism-of-action or off-targets to many therapeutic regimens in pregnancy-related diseases, directing the targeting of ncRNAs might be a future direction in this field.

Conclusions

Pregnancy-associated diseases, including RIF, RSA, PE, etc., are not rare, but their pathogenesis remains largely unknown. Extensive studies examined the expression profiles of miRNAs, circRNAs, and lncRNAs in diseased patients compared with normal pregnancy. Emerging evidence indicated the functional roles of these ncRNAs in pregnancy-associated diseases and pinpointed new directions for clinical application. In this review, we mainly discussed miRNAs, lncRNAs, and circRNAs in ESCs, trophoblast cells, and decidua immune cells and their potential target genes, which provide additional diagnostic tools for pregnancy-associated diseases. Although the previous study has reviewed numerous miRNAs, lncRNAs, and circRNAs in the placenta and maternal serum, and differences exist between animals and humans in gestational ages and ncRNAs expression profile, the functions of these ncRNAs are still limited. Future investigation should be focused on the functional studies and molecular mechanism of miRNAs, lncRNAs, and circRNAs in different stages of pregnancy.

Funding

This work was supported by grants from the Natural Science Foundation of China (No. 81873337), the Natural Science Foundation of Shandong Province (No. ZR2019MH039), the Central Government Guides Local Science and Technology Development Fund Projects of Shandong Province (No. YDZX20203700001407), the Research Leader Studio Project of Jinan Science and Technology Bureau (No. 2020GXRC050), and Taishan Scholars (No. Tsqn201812125).

Conflicts of interest

None.