Peroxisome Proliferator-Activated Receptors (PPAR), fatty acids and microRNAs: Implications in women delivering low birth weight babies.

ABSTRACT

Low birth weight (LBW) babies are associated with neonatal morbidity and mortality and are at increased risk for noncommunicable diseases (NCDs) in later life. However, the molecular determinants of LBW are not well understood. Placental insufficiency/dysfunction is the most frequent etiology for fetal growth restriction resulting in LBW and placental epigenetic processes are suggested to be important regulators of pregnancy outcome. Early life exposures like altered maternal nutrition may have long-lasting effects on the health of the offspring via epigenetic mechanisms like DNA methylation and microRNA (miRNA) regulation. miRNAs have been recognized as major regulators of gene expression and are known to play an important role in placental development. Angiogenesis in the placenta is known to be regulated by transcription factor peroxisome proliferator-activated receptor (PPAR) which is activated by ligands such as long-chain-polyunsaturated fatty acids (LCPUFA). In vitro studies in different cell types indicate that fatty acids can influence epigenetic mechanisms like miRNA regulation. We hypothesize that maternal fatty acid status may influence the miRNA regulation of PPAR genes in the placenta in women delivering LBW babies. This review provides an overview of miRNAs and their regulation of PPAR gene in the placenta of women delivering LBW babies.Abbreviations: AA - Arachidonic Acid; Ago2 - Argonaute2; ALA - Alpha-Linolenic Acid; ANGPTL4 - Angiopoietin-Like Protein 4; C14MC - Chromosome 14 miRNA Cluster; C19MC - Chromosome 19 miRNA Cluster; CLA - Conjugated Linoleic Acid; CSE - Cystathionine γ-Lyase; DHA - Docosahexaenoic Acid; EFA – Essential Fatty Acids; E2F3 - E2F transcription factor 3; EPA - Eicosapentaenoic Acid; FGFR1 - Fibroblast Growth Factor Receptor 1; GDM - Gestational Diabetes Mellitus; hADMSCs - Human Adipose Tissue-Derived Mesenchymal Stem Cells; hBMSCs - Human Bone Marrow Mesenchymal Stem Cells; HBV - Hepatitis B Virus; HCC - Hepatocellular Carcinoma; HCPT - Hydroxycamptothecin; HFD – High-Fat Diet; Hmads - Human Multipotent Adipose-Derived Stem; HSCS - Human Hepatic Stellate Cells; IUGR - Intrauterine Growth Restriction; LA - Linoleic Acid; LBW - Low Birth Weight; LCPUFA – Long-Chain Polyunsaturated Fatty Acids; MEK1 - Mitogen-Activated Protein Kinase 1; MiRNA - MicroRNA; mTOR - Mammalian Target of Rapamycin; NCDs - NonCommunicable Diseases; OA - Oleic Acid; PASMC - Pulmonary Artery Smooth Muscle Cell; PLAG1 - Pleiomorphic Adenoma Gene 1; PPAR - Peroxisome Proliferator-Activated Receptor; PPARα - PPAR alpha; PPARγ - PPAR gamma; PPARδ - PPAR delta; pre-miRNA - precursor miRNA; RISC - RNA-Induced Silencing Complex; ROS - Reactive Oxygen Species; SAT - Subcutaneous Adipose Tissue; WHO - World Health Organization

KEYWORDS:

• Peroxisome proliferator-activated receptor (PPAR)
• placenta
• epigenetics
• microRNA
• low birth weight (LBW)

Introduction

Low birth weight (LBW), defined as the weight of babies less than 2500 g at birth, affects 15% to 20% of all births worldwide. As per the World Health Organization (WHO), globally in a year, more than 20 million babies are born LBW (WHO 2014). Increased perinatal mortality and morbidity, as well as risk of noncommunicable diseases in later life, are often reported to be associated with LBW (Alexander et al. 2014). LBW can be due to preterm birth or intrauterine growth restriction (IUGR) (Valero De Bernabé et al. 2004). Fetal growth is largely dependent on the placenta which is exposed to a number of changes in the maternal compartment like altered nutrition and insufficient uteroplacental blood flow (Brett et al. 2014). It has been reported that IUGR and preterm births are associated with abnormal placental vascularization (Junaid et al. 2014; Kim et al. 2003; Sundrani et al. 2014). Increasing evidence suggests that alterations in placental epigenetic patterns are associated with improper placental development and maternal-fetal exchange resulting in aberrant fetal growth (Koukoura et al. 2012).

Epigenetic regulation controls gene transcription either by methylation of DNA and histone  modifications or by noncoding RNAs like microRNAs (miRNA). miRNAs are small noncoding single-stranded RNAs that inhibit the target expression either by mRNA degradation or translational repression (Morales Prieto and Markert  2011). They are known to participate in all essential cell processes and regulate almost 30–80% of the genes in the genome. miRNAs are differentially expressed in many tissues and are influenced by a number of external factors and disease conditions (Maccani et al. 2010; Nana-Sinkam 2014; Boon 2015; Issler and Chen 2015). They are reported to be expressed in the placenta (Östling et al. 2019) and are suggested to be crucial regulators of placental development since, they are involved in trophoblast cell invasion, migration and angiogenesis (Hayder et al. 2018).

Various growth factors and transcription factors are known to regulate the process of placental development. Peroxisome proliferator-activated receptors (PPAR) belonging to the ligand-activated nuclear hormone receptor family are transcription factors, critical for placental angiogenesis and development (Fournier et al. 2007; Biscetti et al. 2008; Parast et al. 2009; Nadra et al. 2010). These transcription factors are activated by natural ligands like polyunsaturated fatty acids (PUFA) and are involved in a number of cellular and molecular processes (Jump 2002, 2008). PPAR has the capability to accommodate and bind a variety of natural and synthetic lipophilic acids, such as essential fatty acids (EFA). Along with EFA, various eicosanoids are natural ligands of PPAR (Grygiel-Górniak 2014).

Reviews and studies have independently examined the influence of nutrients like fatty acids, vitamins (folate, vitamin D and vitamin E), minerals (selenium and zinc), proteins, as well as dietary flavonols on the miRNA expression in different in vivo and in vitro models (animal models, cell culture and humans) (Ross and Davis  2011; Burdge and Lillycrop  2014; Yang et al. 2014; Xu et al. 2014; Köpke et al. 2015; van Dijk et al. 2016; Boddicker et al. 2016; Quintanilha et al. 2017; Cui et al. 2017; Park et al. 2019). We have earlier demonstrated lower maternal and placental LCPUFA levels particularly omega-3 fatty acids (DHA) in women delivering LBW babies (Meher et al. 2016a). It is likely that these alterations in maternal fatty acids may influence the expression of miRNAs targeting key genes involved in placental development. In our earlier study on women delivering LBW babies, placental PPAR gamma (PPARγ) mRNA levels were lower and associated with placental DHA and baby weight. (Meher et al. 2016b). However, the underlying molecular mechanism associated with these associations is not clear. We propose the hypothesis that altered maternal fatty acid status is associated with disturbed placental development possibly via alterations in the miRNA regulation of PPAR genes in women delivering LBW babies. The current review provides an overview of the role of miRNAs in placental development and implications of the proposed hypothesis.

MicroRNAs (miRNA) and their biogenesis

MicroRNAs (miRNAs) are single-strand small noncoding RNA molecules of 20 to 24 nucleotides that work as epigenetic gene expression regulators. miRNAs are known to be important regulators of post-transcriptional events by binding to the mRNA thereby leading to its degradation or inhibiting the process of protein synthesis.

The miRNA genes are mostly intragenic, i.e., present within the introns and few miRNA genes are present in the exons of protein-coding genes (de Rie et al. 2017). These miRNA genes undergo a multistep regulatory process to form a mature functional miRNA. The majority of the miRNAs follow the common biogenesis pathway (Kim et al. 2016) that involves the following steps. In the nucleus, the miRNA gene is transcribed to form the primary miRNA (pri-miRNA) with the help of the RNA polymerase II (Lee et al. 2004). This pri-miRNA has self-complementary sequences and therefore it folds back to form a hairpin like structure. The pri-miRNA then undergoes endonucleolytic cleavage by the enzyme Drosha (RNA endonuclease III) and RNA-binding protein DGCR8 to form a stem-loop structure, i.e., precursor miRNA (pre-miRNA) (Lee et al. 2002; Denli et al. 2004). The pre-miRNA is then transported to the cytoplasm by the protein exportin 5 (Perron and Provost 2009) and undergoes endonucleolytic cleavage by the enzyme Dicer (RNA endonuclease III) and RNA-binding protein TPBP to form the mature form of miRNA duplex (Grishok et al. 2001; Hutvagner et al. 2001). This miRNA duplex then binds to the RNA-induced silencing complex (RISC) containing the protein Argonaute2 (Ago2) and other regulatory proteins (Sontheimer 2005). The miRNA duplex then unwinds and the strand with the lowest internal stability at the 5ʹ end, known as the guide strand is retained to form the mature RISC complex. The other strand called the passenger strand is discarded (Khvorova et al. 2003). The mature miRNA RISC complex then binds to the target mRNA resulting in translational repression or mRNA degradation depending on the degree of complementary sequence similarity.

miRNA and placenta

The placenta is a transient organ that forms a connection between the mother and the fetus and helps in the exchange of nutrients, gases and waste products between the two compartments. The human placenta is said to express more than 600 miRNAs (Chen and Wang 2013) and exhibit a specific miRNA expression profile (Mouillet et al. 2011a). miRNA regulation is suggested to be one of the regulatory mechanisms by which in utero exposures alter containing placental development and function (Carreras-Badosa et al. 2017). Normal placental development involves trophoblast proliferation, invasion and migration. Lately, a number of studies discussed the emerging role of miRNAs in pathways associated with trophoblast function (Ali et al. 2020).

A study in mice (Cheloufi et al. 2010) and an in vitro study in placental trophoblast cells (Forbes et al. 2012) indicate that inactivation of miRNA machinery is associated with abnormal placental development and altered cytotrophoblast proliferation thereby indicating the important role of miRNAs in placental growth and development (Frazier et al. 2020). It has been demonstrated that several miRNAs are upregulated or downregulated in the placenta thereby influencing placental development and function (Chen and Wang 2013).

Human trophoblasts express unique miRNAs, termed as ‘trophomiRs’, that have specialized functions during pregnancy (Sadovsky et al. 2015). Evidence from a number of in vitro studies using immortalized trophoblast and choriocarcinoma cell lines, primary cultures of trophoblasts, animal models and human studies indicates that miRNAs are involved in regulating trophoblast differentiation, migration, invasion, proliferation, apoptosis, vasculogenesis/angiogenesis and cellular metabolism, thereby playing an important role in placental growth and development (Hayder et al. 2018; Hu and Zhang 2019). It is suggested that there is a need to develop methods to identify placental miRNAs particularly specific to placental cells that may help to use miRNAs for treatment of placental disorders (Frazier et al. 2020). Thus, understanding the functions of miRNAs in the placenta can significantly improve our understanding of the molecular mechanisms associated with placental pathologies.

miRNA and low birth weight

Birth weight is known to be influenced by placental health and function (Roland et al. 2012), thereby affecting the health of the newborn and increasing the risk of diseases in later life. It is suggested that placental dysfunction is a major cause of fetal growth restriction (Krishna and Bhalerao 2011). Therefore, factors determining fetal growth and the underlying molecular mechanisms are important and worthy of attention.

The human placental villous trophoblasts express a large number of miRNAs and their genes are present in two large clusters, the chromosome 14 miRNA cluster (C14MC) and the chromosome 19 miRNA cluster (C19MC) (Morales-Prieto et al. 2012). The C19MC cluster is primate-specific and is expressed exclusively in the placenta with most of the miRNAs found in the exosomes released from human trophoblast cells thereby playing an important role in placental-maternal signalling (Bentwich et al. 2005; Zhang et al. 2008; Donker et al. 2012). This cluster is of about 100 kb in size on the human chromosome 19 and contains 46 intragenic miRNA genes that give rise to 58 miRNAs (Bortolin-Cavaillé et al. 2009). C19MC miRNAs are reported to be expressed in higher levels in the villous trophoblasts as compared to extravillous trophoblasts and are involved in the migration and invasion of trophoblast cells (Xie et al. 2014).

Studies report that different miRNAs are altered in placenta from fetal growth-restricted pregnancies and are discussed below. A genome-wide expression profile study in placental tissue indicates that a number of miRNAs involved in collagen and growth factor signalling are associated with the birth weight of the infant (Payton et al. 2020). Another genome-wide analysis study in IUGR placenta reports 37 differentially expressed miRNAs of which 4 belong to the C19MC (520a-3p, 520 f-5p, 515–5p, 519–5p) and 6 belong to the C14MC (299–3p, 494–3p, 376a-5p, 382–3p, 154–3p, 369–3p) that are involved in the regulation of cell migration and proliferation (Awamleh et al. 2019). Lower expression of several miRNAs like miR-515-5p, miR-516b, miR-518b, miR-519d, miR-520 h, miR-526b, miR-1323 from the chromosome 19 miRNA cluster and miR-194 has been shown to be associated with fetal growth restriction (Higashijima et al. 2013; Guo et al. 2013; Hromadnikova et al. 2015b). Similarly, a study by Wang et al. reports that the expression of miR-518b is decreased in IUGR cases and expression of another miRNA involved in placental functions, miR-519a, is increased in IUGR cases (Wang et al. 2014). Taken together, these studies suggest that the altered expression of these miRNAs is associated with disturbed placental trophoblast function leading to fetal growth restriction.

Human studies have demonstrated low placental expression of miR-16 and miR-21 (involved in the regulation of cell growth and developmental pathways) in women delivering low birth weight babies at term (Maccani et al. 2011). In contrast, a study in growth-restricted pregnancies at 32 weeks of gestation reported increased placental expression of miR-21 which negatively regulates the cystathionine γ-lyase (CSE) gene involved in the endogenous production of hydrogen sulfide, a placental vasodilator, thereby resulting in increased vascular resistance (Cindrova-Davies et al. 2013). It is likely that this difference could be associated with physiological effects seen at different gestational ages. Further, it is suggested that supplementation of vitamin B₁₂ to pregnant Wistar rats up-regulates the placental expression of miR-16 and miR-21 thereby improving birthweight (Shah et al. 2017).

It has been reported that increased placental expression of miRNAs, miR-424 and miR-141 contributes to the pathogenesis of fetal growth restriction by targeting genes mitogen-activated protein kinase 1 (MEK1) and fibroblast growth factor receptor 1 (FGFR1) by miR-424 (Huang et al. 2013) and E2F transcription factor 3 (E2F3) and pleiomorphic adenoma gene 1 (PLAG1) by miR-141 (Tang et al. 2013). Increased maternal serum and placental miR-517a expression has been observed in pregnancies with LBW newborns. It is suggested that this overexpression of miR-517a is involved in inhibition of trophoblast invasion thereby leading to LBW in the newborn (Song et al. 2013). MiR-424, a hypoxia-regulated miRNA has been shown to be upregulated in fetal growth-restricted placenta and is suggested to be associated with the pathogenesis of fetal growth restriction (Huang et al. 2013) by regulating the trophoblast-derived cell line proliferation and invasion (Zou et al. 2019).

Further, increased expression of miR-10b and miR-363 targeting genes associated with angiogenesis and amino acid transport has been reported in IUGR placenta as compared to controls (Thamotharan et al. 2017). It is suggested that upregulation of miR-210-3p (involved in trophoblast proliferation and invasion) in IUGR placenta may contribute to impaired placentation (Li et al. 2019a). Altered expression of miRNAs associated with cardiovascular and cerebrovascular disease has also been reported in the placenta of IUGR pregnancies (Hromadnikova et al. 2015a).

Thus, identifying the miRNAs associated with the progression of fetal growth restriction is critical and this will help in early diagnosis and management of the disease (Chiofalo et al. 2017).

Extracellular miRNAs

Recently, studies have shown that miRNAs are released in the extracellular space like plasma and extracellular fluids. These are called as extracellular miRNAs and placental extracellular miRNAs have been detected in the plasma of pregnant women across gestation, that rapidly decline after delivery (Chim et al. 2008). These placental extracellular miRNAs are derived primarily from the trophoblast layer, which lines the human placental villi (Mouillet et al. 2015). During pregnancy, these extracellular miRNAs mediate a cross-talk between the mother and the feto-placental unit. They are released from the trophoblast layer in two forms: nonvesicular miRNAs that are bound to proteins like Ago2, nucleophosmin1, or high-density lipoproteins or vesicular miRNAs that are packaged within the extracellular vesicles, such as exosomes, microvesicles, and apoptotic bodies (Ouyang et al. 2014).

Altered expression of circulating placental miRNAs in maternal blood has been reported in IUGR pregnancies (Tsochandaridis et al. 2015) and are suggested to be associated with fetal growth (Rodosthenous et al. 2017). Thus, circulating miRNAs that can be easily found in the maternal blood samples are suggested to provide a potential for noninvasive prenatal diagnostic tests.

The expression of placental miRNAs in trophoblasts is reported to be influenced by various environmental factors like hypoxia (Kulshreshtha et al. 2007), exposure to bisphenol A (Avissar-Whiting et al. 2010) or cigarette smoking (Maccani et al. 2010) thereby regulating the expression of genes involved in trophoblast function. Further, miRNA expression is also suggested to be altered in response to maternal nutrient availability (Cui et al. 2017). Therefore, the next section discusses nutrients particularly fatty acids influencing miRNA regulation.

Influence of fatty acids on MiRNA regulation in pregnancy

Maternal nutrition during pregnancy and postnatal periods are known to influence developmental processes via changes in the epigenetic mechanisms. Establishment of these epigenetic patterns in the placenta and the fetus have a profound influence on the development of disease and disorders in the offspring in their later life (Li 2018).

Evidence suggests that various nutrients both macro – and micro-nutrients can modulate miRNA expression. Animal and in vitro studies indicate that nutrients like folate (Li et al. 2019a), vitamin B₁₂ (Adaikalakoteswari et al. 2017) and flavonols like quercetin (Park et al. 2019) influence the expression of miRNAs. A study in female C57BL/6 mice indicates that maternal omega-3 fatty acid supplementation helps in fetal brown adipogenesis in synergy with miRNA and histone modifications thereby providing long-term health benefits to the offspring (Fan et al. 2018). Other studies on mice and calf also indicate that supplementation of one-carbon metabolites like choline and methionine can affect the expression profile of miRNAs in the placenta (Kwan  et al. 2018) and polymorphonuclear leukocytes (Jacometo et al. 2018) respectively. Maternal low-protein diet in mice is also shown to alter liver expression of miRNAs targeting the inflammatory pathway genes thereby influencing the metabolic health in the offspring (Zheng et al. 2017).

A study carried out in rats demonstrates that fish oil (containing omega-3 fatty acids) supplementation protects the rat colon from carcinogen-induced miRNA dysregulation (LA et al. 2009). A study in pregnant rats reported that maternal intake of different fatty acids from soybean, olive, fish, linseed or palm oil  resulted in the genome wide differential expression of miRNAs in the maternal and offspring liver and adipose tissue suggesting that these effectsfor its functions in placental development particularly may help in understanding the long-term changes in the offspring (Casas-Agustench et al. 2015). Similarly, another animal study suggests that maternal high-fat diet influences offspring lipid metabolism by altering the expression of hepatic β-oxidation-related genes and miRNA targeting these genes (Benatti et al. 2014).

Thus, it is likely that maternal fatty acid status can influence the expression of miRNAs that targets key genes involved in the process of placental development.

Peroxisome proliferator-activated receptors (PPAR) and miRNA in placental development

Peroxisome proliferator-activated receptors (PPAR)

PPARs are nuclear transcription factors that regulate anti-inflammatory, metabolic and tissue developmental processes. Three PPAR isotypes have been identified in mammals: PPAR alpha (PPARα), PPAR delta (PPARδ) and PPAR gamma (PPARγ) (Lee et al. 2009). All these isotypes of PPAR (PPARα, PPARδ and PPARγ) are reported to be expressed in the placenta (Fournier et al. 2007) and they are suggested to play an important role in placental angiogenesis and development (Nadra et al. 2010; Meher et al. 2015).

PPARα

Animal studies indicate that PPARα plays a regulatory role in pathways associated with lipid peroxidation, lipid metabolism and production of nitric oxide associated with angiogenesis (Martínez et al. 2008, 2011). An animal study indicates that maternal obesity decreases placental PPARα expression thereby modulating fatty acid oxidation pathway and maternal supplementation of adiponectin increased the expression of PPARα in the placenta and reversed the negative effects of maternal obesity on placental function (Aye et al. 2015). Similarly, maternal adiponectin has been reported to activate placental PPARα in human primary trophoblast cells (Aye et al. 2014). In addition, a study in rodent reports that paternal obesity can also affect the expression of PPARα in the placenta particularly in a sex-specific manner. The authors suggest that there is a need to explore more on the effect of paternal obesity on placental function (Binder et al. 2015).

PPARδ

PPARδ is also known to play an important role during implantation and decidualization (Ding et al. 2003a; Ding et al. 2003b). A PPARδ null mice study indicates that maternal and embryonic PPARδ is crucial for successful pregnancy since it connects different processes like implantation, decidualization and placentation (Wang et al. 2007). This is supported by another study in the mouse which demonstrates that activation of PPARδ causes changes in the placental metabolic activities thereby influencing placental development during early pregnancy (Ding et al. 2014). In contrast, another study in a rat model indicates that a different dosage of the PPARδ agonist induces placental malformation (Nishimura et al. 2013). These differences in the studies could be attributed to the different dosages of the PPARδ agonists and different animal models used. Further, a study on pregnant rats injected with inhibitors of PPARγ and PPARδ indicates that PPARγ, PPARδ and mammalian target of rapamycin (mTOR) signalling pathways interact synergistically to regulate early placental development (Roberti et al. 2018).

PPARγ

In the human placenta, PPARγ is known to be expressed in the villous and extravillous cytotrophoblast cells, thereby regulating the process of placental development. Considerable evidence indicates that PPARγ plays a role in trophoblast invasion (Fournier et al. 2008; Parast et al. 2009), differentiation of cytotrophoblast into syncytiotrophoblast (Schaiff et al. 2000; Tarrade et al. 2001; Capparuccia et al. 2002) and develops a vascular network between the maternal and fetal compartments (Parast et al. 2009). In addition, PPARγ also plays an important role in trophoblast maturation which is required to establish the maternal-fetal transport (Asami-Miyagishi et al. 2004). PPARγ is known for its functions in placental development particularly spatiotemporal regulation of trophoblast cell genes that are involved in trophoblast differentiation (Shalom-Barak et al. 2012).

This is supported by a number of animal studies indicating that deficiency of PPARγ is associated with placental abnormalities and fetal demise (Barak et al. 1999; Kubota et al. 1999; NI et al. 2013). The PPARγ gene knockout study by Barak et al. provided the first evidence that PPARγ deficiency affects trophoblast differentiation and placental vascularization thereby resulting in embryonic lethality (Barak et al. 1999). In vitro and animal studies indicate that PPARγ regulates angiogenic factors – vascular endothelial growth factor (VEGF) and angiopoietin and hypoxia-inducible factor thereby influencing trophoblast invasion and placental angiogenesis (Garnier et al. 2015; Zhang et al. 2017). A mouse model of hypoxia during pregnancy resulted in the development of IUGR and reduced placental PPARγ expression. Pharmacological activationof PPAR by a PPARγ agonist, pioglitazone, prevented the hypoxia-induced fetal growth restriction (Lane et al. 2019). The authors suggest that this protection of fetal growth restriction could be the due role of PPARγ in regulating placental vascularization. Further, PPARγ is known to have anti-inflammatory effects by inhibiting the production of reactive oxygen species (ROS) and lowering inflammatory markers through down-regulation of NF-kappaB pathway (Zhang et al. 2018), thereby mediating placental vascularization. This is supported by studies during pregnancy which demonstrates that PPARγ activation by rosiglitazone reduces the macrophage-mediated pro-inflammatory response, thereby preventing preterm birth (Xu et al. 2016) and prevents trophoblast-associated inflammation by inhibition of NF-kappaB (Kadam et al. 2019).In vitro studies on extravillous trophoblast cells and placental explants indicate that the heterodimer of PPARγ/RXRα binds to the promoter region of angiopoietin-like protein 4 (ANGPTL4). ANGPTL4 is an angiogenic marker involved in the process of angiogenesis in various tissues and thus is suggested to be a potential target gene of PPARγ (Liu et al. 2017). As in vitro study on human placental choriocarcinoma cells suggests that the PPARγ signalling pathway is associated with progesterone regulation thereby influencing fetal growth and development (Hu et al. 2017). A study in cultured human term villous cytotrophoblast cells suggests that phthalates affect that transcriptional activity of PPARγ thereby influencing lipid metabolism and differentiation of cytotrophoblast cells (Shoaito et al. 2019). Thus, considerable evidence indicates that alterations in the placental expression of PPARγ is associated with placental abnormalities.

Fatty acids and PPARs in pregnancy

Fatty acids are important for the developing fetus and play a critical role in determining the duration of gestation and pregnancy outcome (Cetin et al. 2010; Coletta et al. 2010). Polyunsaturated fatty acids (PUFA) are classified into two main families based on the presence of the first double bond at the n-6 or n-3 position: omega-6 and omega-3 fatty acids. Linoleic acid (LA) (omega-6) and alpha-linolenic acid (ALA) (omega-3) are the dietary essential fatty acids and precursors of highly unsaturated members of their family, i.e., long-chain polyunsaturated fatty acids (LCPUFA) – arachidonic acid (AA) and docosahexaenoic acid (DHA) respectively (Benatti et al. 2004).

Maternal omega-3 fatty acid supplementation during pregnancy has been reported to be associated with length of gestation and birth weight (Larqué et al. 2012; Middleton et al. 2018). Fatty acids are also known to affect gene expression by regulating transcription factors (Deckelbaum et al. 2006). One of the widely studied transcription factor during pregnancy is PPAR.

Evidence from various animal studies suggests that dietary treatments supplemented with monounsaturated and polyunsaturated fatty acids influence the expression of PPARγ, thereby regulating the PPAR signalling (Capobianco et al. 2008, 2018; Jawerbaum and Capobianco 2011; Martinez et al. 2012). Different types of maternal dietary fat have been shown to differentially influence the expression of PPAR isoforms. Cows fed LA diets report increased expression of placental PPARα as compared to those fed oleic acid (OA) whereas, PPARγ and PPARδ expression were decreased in the placenta of cows fed with LA compared to cows fed with OA (Salehi and DJ 2017).

In vitro studies on human placental trophoblasts indicate that PPARs are involved in the regulation of fatty acid storage and transport (Schaiff et al. 2005). Incubation of fatty acids like OA, EPA, DHA, AA, conjugated linoleic acid (CLA) has been shown to increase the expression of PPARγ in placental choriocarcinoma (BeWo) cells (Duttaroy et al. 2003).

A study in obese pregnant women reports higher placental PPARγ mRNA and protein expression whereas lower placental PPARα mRNA expression as compared to lean women. It is suggested that this higher PPARγ stimulates lipid esterification and storage in the obese placenta and the lower PPARα expression may influence the fatty acid oxidation in obese placenta (Calabuig-Navarro et al. 2017). Another study by the same group reported that supplementation of omega-3 fatty acids during pregnancy lowered the total lipid content and expression of PPARγ and other related genes involved in lipid esterification and storage. In contrast, omega-3 fatty acid supplementation had no significant effect on the expression of PPARα and other genes associated with fatty acid oxidation pathway (Calabuig-Navarro et al. 2016).

These observations indicate an important role of fatty acids in regulating key transcription factors like PPAR in the placenta thereby influencing placental lipid metabolism.

placenta

microRNA regulation of PPAR

PPAR genes are suggested to be regulated by epigenetic changes (Lendvai et al. 2016; Kitsiou-Tzeli and Tzetis 2017; Capobianco et al. 2018). Various review articles have summarized that several miRNAs target different forms of PPAR, thereby suggesting their role in the transcriptional regulation of different pathological conditions like metabolic disorders (inflammation and cancer) (Portius et al. 2017), mesenchymal stem cell differentiation (Huang et al. 2016a) and hepatocellular carcinoma (Hsu and Chi 2014).

Evidence from independent review articles and studies in different health conditions has suggested that the different isoforms of PPAR are regulated by different miRNAs summarized in Table 1. These studies indicate their regulatory role in various processes like adipogenic differentiation, pulmonary hypertension, vascular dysfunction associated with disease conditions like cardiovascular diseases, heart diseases and obesity-related diseases.

Table 1. MicroRNAs Targeting PPAR

Target	miRNA	Species	Sample type	Function	Reference
PPARα	miR-27b	Human	Human Hepatocyte Cell Line	Controlling Lipid Metabolism	Kida et al. 2011
PPARα	miR-21	Human	Human Hepatocyte Cell Line	Controlling Lipid Metabolism	Kida et al. 2011
PPARα	miR-22	Human	Human Osteoarthritic Chondrocytes	Blocking Inflammatory and Catabolic Changes in Osteoarthritic Chondrocytes	Iliopoulos et al. 2008
PPARα	miR-10b	Human	Human Hepatocyte Cell Line	Regulation of Hepatocyte Steatosis	Zheng et al. 2010
PPARα	miR-519d	Human	Subcutaneous Adipose Tissue (SAT) of Obese Human Adults	Metabolic Imbalance and Subsequent Adipocyte Hypertrophy in SAT during Obesity	Martinelli et al. 2010
PPARα	miR-21	Human	Hypoxia-Induced Pulmonary Artery Smooth Muscle Cell (PASMC)	Regulation of PASMC Cell Proliferation and Migration	Sarkar et al. 2010
PPARα	miR-519d	Human	Human Adipocytes	Regulating Adipogenesis	McGregor and Choi 2011
PPARα	miR-21	Human	Cultured Human Umbilical Vein Endothelial Cells	Endothelial Inflammation	Zhou et al. 2011
PPARα	miR-506	Human	Hydroxycamptothecin (HCPT)-Resistant Human Colon Cancer Cells	Chemotherapeutic Drug Resistance	Tong et al. 2011
PPARα	miR-141	Human	Hepg2 Cells (Human Liver Carcinoma Cells)	Suppress Hepatitis B Virus (HBV) Replication	Hu et al. 2012
PPARα	miR-17/92 cluster	Human	Prostate Cancer Cells	Regulates Lipogenesis	Wang et al. 2013
PPARα	miR-9-5a	Human	Hepatocellular Carcinoma (HCC) Cells	Involved in Progression Of HCC	Cai et al. 2019
PPARα	miR-33a	Human	Human Hepatic Stellate Cells (HSCS)	Regulates Hepatic Fibrosis	Li et al. 2014
PPARα	miR-518d	Human	Human Placenta Tissue	Associated With Pathogenesis of GDM	Zhao et al. 2014
PPARα	miR-21	Mouse	Mouse Kidney Fibrosis Models	Promotes Fibrosis of the Kidney	Chau et al. 2012
PPARα	miR-27	Mouse	Mouse Primary Adipocytes	Regulates Brown Adipogenesis	Sun et al. 2014
PPARα	miR-21	Mouse	Retina of mice model of type 2 diabetes	Regulates Diabetic Retinopathy	Chen et al. 2017
PPARα	miR-540	Mouse	Hepatocytes	Regulation of Hepatic Steatosis	Kumar et al. 2019
PPARα	miR-21	Rat	Rat left ventricle	Involved in chronic kidney disease-related cardiac dysfunction	Chuppa et al. 2018
PPARγ	miR-27b	Human	Primary Human Macrophages	Increases Inflammation	Jennewein et al. 2010
PPARγ	miR-130a	Human	Human Acute Monocyte THP-1 Leukemia Cell Line	Macrophage Polarization	Lin et al. 2015
PPARγ	miR-146b	Human	Human Visceral Preadipocyte	Visceral Obesity and Metabolic Dysfunction	Chen et al. 2014b
PPARγ	miR-27b	Human	Human Multipotent Adipose-Derived Stem (Hmads)	Impairing Human Adipocyte Differentiation	Karbiener et al. 2009
PPARγ	miR-138	Human	Human Adipose Tissue-Derived Mesenchymal Stem Cells (hADMSCs)	Inhibiting Adipogenic Differentiation	Yang et al. 2011
PPARγ	miR-130	Human	Human Adipose Tissue	Reduced Adipogenesis	Lee et al. 2011
PPARγ	miR-20a	Human	Human Mesenchymal Stem Cells	Promoting Osteogenesis	Zhang et al. 2011
PPARγ	miR-27b	Human	Neuroblastoma Cells	Inhibit Growth, Tumor Progression and Inflammatory Response	Lee et al. 2012
PPARγ	miR-128a	Human	Human Muscle Stem Cells	Regulates Proliferation and Differentiation in Muscle Stem Cells	Motohashi et al. 2012
PPARγ	miR-155	Human	Human Adipose Tissue Macrophages	Influences Adipocyte Metabolism	Tryggestad et al. 2019
PPARγ	miR-27	Human	Adipocytes	Regulating Adipogenesis	McGregor and Choi 2011
PPARγ	miR-548d-5p	Human	Human Bone Marrow Mesenchymal Stem Cells (hBMSCs)	Suppresses the Adipogenic Differentiation of hBMSCs	Sun et al. 2014
PPARγ	miR-130a and miR-27b	Human	Human Mesenchymal Stem Cells (MSCs)	Promote Osteogenesis in Human MSCs	Seenprachawong et al. 2018
PPARγ	miR-130b	Human	Human Glioma Cells	Regulates Cell Proliferation and Invasion	Gu et al. 2016
PPARγ	miR-130b	Human	Human Glioma Cells	Promotes Glioma Proliferation, Migration and Invasion	Li et al. 2017
PPARγ	miR-130b	Human	Systemic Sclerosis (Ssc) Skin Biopsy Samples	Regulates Scleroderma Fibrosis	Luo et al. 2015
PPARγ	miR 34a and miR 34 c	Human	Human Hepatic Stellate Cells	Promotes Activation of Human Hepatic Stellate Cells	Li et al. 2015
PPARγ	miR-130b	Human	Human Hepatocellular Carcinoma	Promotes Cell Aggressiveness	Tu et al. 2014
PPARγ	miR-27b	Human	Human Pulmonary Artery Endothelial Cells (HPAECs)	Role in Pulmonary Arterial Hypertension	Bi et al. 2015
PPARγ	miR-942	Human	Human Primary Hepatic Stellate Cell (HSCs)	Regulates Hepatic Fibrosis	Tao et al. 2020
PPARγ	miR-27a and miR-27b	Human	Primary Orbital Fibroblast Cultures	Inhibits Adipogenic Differentiation	Jang et al. 2019
PPARγ	miR-146a	Mouse	RAW264.7 Monocyte/Macrophage Like Cell Linage	Macrophage Polarization	Huang et al. 2016a
PPARγ	miR-27a	Mouse	Adipose Tissue Obese Mice Model	Increases Inflammation	Yao et al. 2017
PPARγ	miR-130b	Mouse	Peritoneal Macrophages Type 2 Diabetes Mice Model.	Macrophage Polarization	Zhang et al. 2016
PPARγ	miR-27	Mouse	Fat Tissue of Obese Mice	Inhibits Adipogenesis	Lin et al. 2009
PPARγ	miR-27a	Mouse	Mice Adipocytes	Reduced Adipocyte Differentiation	Kim et al. 2010
PPARγ	miR-27b	Mouse	Mice Cardiomyocyte	Regulation of Cardiac Hypertrophy	Wang et al. 2012
PPARγ	miR-27a	Mouse	High-Fat Diet (HFD)-Fed Obese Mouse Model and Mature 3T3-L1 Adipocytes	Promotes Insulin Resistance and Mediates Glucose Metabolism	Chen et al. 2019
PPARγ	miR-183	Mouse	Mouse Fibroblast Cell Line 3T3-L1	3T3-L1 Differentiation	Chen et al. 2014b
PPARγ	miR-302a	Mouse	3T3-L1 Adipocytes	Regulation of Adipocyte Differentiation	Jeong et al. 2014
PPARγ	miR-9	Mouse	3T3-L1 Adipocytes	Inhibits Adipocyte Differentiation	Xu et al. 2020
PPARγ	miR-128	Mouse	Brain tissue	Influences Development of Alzheimer’s Disease	Liu et al. 2019
PPARγ	miR-130b	Porcine	Meishan Pig Subcutaneous Fat	Regulates Lipid Metabolism	Pan et al. 2013
PPARγ	miR-33b	Porcine	Porcine Preadipocytes	Regulates Adipocyte Differentiation	Taniguchi et al. 2014
PPARγ	miR-145	Porcine	Porcine Preadipocytes	Inhibit Adipogenesis	Guo et al. 2012
PPARγ	miR-128	Cattle	Calves Rumen Tissue	Regulates Rumen Development	Xue et al. 2019
PPARδ	miR-9	Human	Primary Human Monocytes	Inhibits Inflammatory Response	Thulin et al. 2013
PPARδ	miR-122	Mouse	Mice Liver	Hepatic Circadian Metabolism	Gatfield et al. 2009
PPARδ	miR-199a∼214 cluster	Rat	Cardiomyocyte Cultures of Rat Heart	Impairs Mitochondrial Fatty Acid Oxidation	El Azzouzi et al. 2013

microRNA regulation of PPAR during pregnancy

Studies have reported the role of different miRNAs as epigenetic regulators of PPARs in various tissues and cell lines during pregnancy and are summarized below. An animal study by Sarr et al. indicates that adverse in utero environmental conditions alter the expression of lipid-synthesis genes including PPARγ and miRNAs like miR-24 and miR-103-2 factors in the adipose tissue of IUGR offspring (Sarr et al. 2014). Another study using a rat model of gestational diabetes reported increased expression of PPARγ and reduced expression of miR-130 that targets PPARγ in the liver of only male fetuses. In contrast, increased expression of PPARδ and reduced expression of miR-9 that targets PPARδ was observed only in the liver of female fetuses. The authors conclude that there are sex-dependent changes in the miRNAs that targets different isoforms of PPAR in the liver of rats with gestational diabetes (Fornes et al. 2018).

It is well known that alterations in the maternal nutrients during pregnancy epigenetically programs the fetus. A rat model of maternal food restriction resulting in intrauterine growth-restricted offspring reported that miRNAs promoting adipogenesis like miR-30d, let-7a and miR-103 were increased in the bone marrow-derived mesenchymal stem cells as compared to control offspring. However, the miRNAs, miR-27 and miR-130 targeting PPARγ were not significantly different. Therefore, it is likely that in these growth-restricted offspring as a result of maternal food restriction, selective adipogenic pathways are activated (Gong et al. 2016). Another study in Meishan pigs reported that maternal low-protein diet throughout gestation and lactation increased the expression of miR-130b in the subcutaneous fat of the offspring. Thus, miR-130b targeting PPARγ gene regulates offspring lipid metabolism in response to maternal dietary protein status (Pan et al. 2013).

A study in IUGR rat offspring reported that increased expression of miR-29a in the skeletal muscle cell line inhibited the expression of its target gene PPARδ thereby inducing insulin resistance (Zhou et al. 2016). Another study by Adaikalakoteswari et al. observed that the expression of miRNAs (miR-27b, miR-23a, miR-130b) targeting PPARγ gene was downregulated in the human adipocyte cell line deficient in vitamin B₁₂. This was also validated in the subcutaneous adipose tissue and maternal serum collected at the delivery of pregnant women deficient in vitamin B₁₂. The authors suggest that miRNAs can regulate the expression of genes involved in the process of adipogenesis (Adaikalakoteswari et al. 2017).

The above suggests a miRNA mediated mechanism of PPAR regulation. To the best of our knowledge, there is limited literature on the placenta-specific miRNAs targeting the different PPAR isoforms in pregnancy outcomes. Only one study on women with gestational diabetes mellitus (GDM) has reported that increased placental expression of miRNA-518d is associated with reduced placental PPARα protein expression suggesting that upregulation of miR-518d may be associated with the pathogenesis of GDM (Zhao et al. 2014). Therefore, there is a need to explore miRNAs in the human placenta that target different isoforms of PPAR thereby regulating placental development and function.

Implications for women delivering LBW Babies

LBW is a public health problem and India is reported to have the largest number of LBW babies. Alterations in the maternal nutritional status is known to alter placental and fetal growth and development, resulting in LBW babies. These LBW babies are at increased risk of developing NCDs in later life. However, the underlying molecular mechanisms are not clear. Over a decade, growing evidence suggests that epigenetic factors during the early intrauterine, perinatal and postnatal periods can influence the health of the offspring during infancy and later in adult life. These effects may possibly be mediated by epigenetic modifications like miRNA regulation.

Emerging evidence indicates a potential role for nutrients like fatty acids in influencing epigenetic mechanisms. Fatty acids, particularly LCPUFA play an important role during placental as well as fetal growth since they are involved in number physiological processes associated with cellular growth and function. These LCPUFA are ligands for a transcription factor, PPAR and reports indicate that reduced placental PPAR expression is associated with disturbed placental development. Based on this we hypothesize that altered maternal fatty acid status is associated with disturbed placental development possibly via alterations in the miRNA regulation of PPAR genes in women delivering LBW babies (Figure 1). The literature\described in this review indicates the importance of PPARs in placental development; however, the role of these transcription factors in women delivering LBW essentially remains to be explored. Increased mRNA and protein expression of PPARγ has been reported in the placental tissue of fetal growth-restricted pregnancies as compared to controls (Chui et al. 2013). Similarly, another study by Holdsworth–Carson et al. reported increased protein levels of PPARα and PPARγ in placentas from intrauterine growth restriction (IUGR) pregnancies (Holdsworth-Carson et al. 2010). However, using immunocytochemistry, a study by Rodie et al. demonstrated no difference in the PPARδ and PPARγ expression in IUGR and control placenta (Rodie et al. 2005). Thus, these reports are variable and inconclusive with some showing lower, higher or no change in the levels of PPAR. The discrepancy in these studies could be due to a difference in the methodology used and the difference in patient selection particularly with respect to the mode of delivery or the status of labor. Further, these studies have limited sample size.

Figure 1. Maternal fatty acids epigenetically regulate PPAR expression through miRNA regulation in the placenta thereby influencing fetal outcome. Fatty acids are ligands for transcription factor PPAR, which are expressed in the placenta and are involved in placental angiogenesis and development. Alterations in the maternal fatty acids status can epigenetically regulate PPAR in the placenta by influencing the miRNAs targeting the PPAR gene thereby resulting in defective placentation. This may result into adverse pregnancy outcome like LBW and increased risk for noncommunicable diseases in the offspring.PPAR: Peroxisome Proliferator-Activated Receptor; RISC: RNA-induced Silencing Complex; LBW: Low Birth Weight; NCDs: NonCommunicable Diseases

Considering the important role of PPARs in the process of implantation, placental development as well as the initiation of labor, PPAR could be considered as a potential therapeutic target for placental disorders. However, the underlying molecular mechanisms associated with PPAR regulation need to be explored. There are no studies examining the expression of miRNAs targeting PPARs in placenta from women delivering LBW babies and their association with maternal fatty acid status. Elucidation of these regulatory mechanisms will provide a better understanding of the molecular pathways leading to altered fetal growth. This will also identify placental epigenetic markers (differentially regulated miRNAs) associated with LBW. miRNAs are considered as valuable circulating biomarkers and therapeutic targets. The differentially regulated miRNAs identified can be validated in the maternal plasma. This may lead to the identification of epigenetic biomarkers associated with fetal growth during pregnancy. In addition, studies examining the influence of maternal nutrition on miRNA expression and function may provide clues for supplementation to prevent adverse pregnancy outcomes.

To summarize, based on the above literature, investigating the miRNAs regulating fetal growth by targeting the miRNAs regulating transcription factors like PPAR is warranted. Animal studies can help to understand the effect of fatty acid supplementation on the placental expression of transcription factors (PPAR) and miRNAs targeting PPAR. In human studies, the expression of predicted miRNAs regulating PPAR genes can be examined in the placenta of women delivering LBW babies and compared with women delivering normal birth weight (NBW) babies. Further, the association of these miRNAs with maternal and placenta fatty acid status can be examined. In vitro studies on placental trophoblast cells from LBW and NBW samples can also be used. The above-mentioned examinations will provide insights into the molecular mechanisms related to epigenetic programming of diseases in LBW babies.

Disclosure of interest

The authors report no conflict of interest.

Author Contributions

Contributed to the conception and design of the review: DPS, SRJ; wrote the review: DPS, ARK, SRJ. All authors reviewed the final manuscript.

Acknowledgments

The authors acknowledge the Department of Biotechnology (DBT), Government of India for funding the project “Epigenetic regulation of placental peroxisome proliferator activated receptor (PPAR) in women delivering low birth weight babies” (Grant number: BT/PR30592/BIC/101/1081/2018).

Additional information

Funding

This work was supported by the Department of Biotechnology (DBT), Government of India [BT/PR30592/BIC/101/1081/2018].