Sirtuin 1 is a potential therapeutic candidate gene for fetal growth restriction via insulin-like 4

Abstract Objective Insufficient placental development causes various obstetric complications, including fetal growth restriction (FGR). The Sirtuin 1 (SIRT1) and insulin-like 4 (INSL4) protein-coding genes have been demonstrated to play an important role in placental development. However, no treatment for FGR is available due to placental dysfunction. Therefore, this study aimed to examine the potential of the SIRT1–INSL4 axis as a treatment candidate for FGR caused by insufficient placental development. Methods Twenty patients were enrolled, including 10 with FGR and 10 full-term controls. FGR and control placental samples were collected. Quantitative real-time polymerase chain reaction, immunohistochemical analysis, and western blotting were used to analyze INSL4 and SIRT1 expression. An in-vitro loss-of-function approach with the human choriocarcinoma cell line BeWo was applied for functional analyses of SIRT1 in placental development. BeWo cells were differentiated into syncytiotrophoblasts by silencing SIRT1 using small interfering RNA. SIRT1 activator was added during differentiation of SIRT1-knockdown BeWo cells into syncytiotrophoblasts. Results The FGR samples had lower INSL4 and SIRT1 mRNA and protein expression levels than the control samples. Immunohistochemistry showed that both SIRT1 and INSL4 were expressed mainly in syncytiotrophoblasts. In-vitro analyses showed that SIRT1 knockdown decreased INSL4 expression; however, SIRT1 activator restored SIRT1 expression in SIRT1-silenced BeWo cells. Conclusions SIRT1 and INSL4 are downregulated in the placenta of FGR, and INSL4 is regulated by SIRT1. These findings indicate that the SIRT1–INSL4 axis may be a potential therapeutic target for FGR.


Introduction
The placenta is a temporary but crucial organ for intrauterine fetal development during pregnancy, which controls fetal growth and development by facilitating the exchange of metabolic products and gases between the pregnant mother and fetus, termed maternal-fetal interaction [1,2].A functional placenta ensures adequate fetal growth; however, its insufficient development reduces fetal nutrient and oxygen availability, leading to impaired somatic growth and fetal growth restriction (FGR).FGR is a significant contributor to preterm birth, stillbirth, and neonatal mortality [3,4]; it also induces neonatal complications, such as pulmonary hypertension, hypoglycemia, pulmonary hemorrhage, and neurological disorders in childhood and cognitive delay, hypertension, and respiratory diseases in adulthood [5][6][7][8][9].
FGR-associated placentas have several distinct pathological features, including scattered infarcts, syncytiotrophoblast (ST) nodules, cytotrophoblast (CT) thickening, fibrinoid deposition, pathological inflammation, and a decreased vascular bed, villi volume, and villous space [10].Furthermore, gene expression patterns in FGR-associated placentas differ from those of healthy placentas, suggesting that the placenta contributes to FGR [11].Numerous genes are associated with placental development, including insulin-like 4 (INSL4) and sirtuin 1 (SIRT1) genes.INSL4a member of the insulin superfamily, which includes relaxin, insulin growth factors, and insulinis unique to primates, is highly expressed in placental STs during early gestation, and can participate in placental development [12][13][14].Furthermore, SIRT1, also known as nicotinamide adenine dinucleotide-dependent deacetylase sirtuin-1, is a protein-encoding gene involved in the targeting of many proteins, including histones and transcription factors [15].SIRT1 also contributes to bioregulatory responses such as oxidative stress, inflammatory responses, aging, and autophagy [16,17] In mice, Sirt1 is necessary for appropriate trophoblast differentiation and placental development, with research showing that Sirt1-null embryos develop growth restrictions [18,19].In humans, SIRT1 is expressed in STs and plays an important role in placental development [20].
Although SIRT1 plays an important role in placental differentiation, the direct target genes of SIRT1, especially during placental differentiation to STs, have not been elucidated in detail.We speculate that SIRT1 is involved in the regulation of INSL4 during placental differentiation, especially during differentiation into STs.In this study, the gene expression levels of INSL4 and SIRT1 in normal and FGR full-term placentas were compared, and the association between these genes was analyzed.

Participants and placental sample collection
Ethical approval for this study was granted by the Tokyo Medical University Ethics Committee (approval no: T2021-0257).Twenty patients participated in the study after providing written informed consent.Placental tissue samples were collected after delivery from 10 mothers with normal-sized babies (controls) and 10 mothers with FGR babies (Supplementary Figure S1).No maternal complications were observed in either the control or FGR group.Mean calving weeks were 268.9 ± 5.15 d in the control group and 267.2 ± 5.95 d in the FGR group.Mean birth weight was 2935.6 ± 168.6 g in the control group and 2047 ± 205.5 g in the FGR group.Mean placental weight was 533 ± 51.2 g in the control group and 347 ± 81.7 g in the FGR group.Birth weight and placental weight were significantly lower in the FGR group, although no significant difference was observed in the number of weeks of delivery.Chromosomal abnormalities or malformations were not observed in the offspring of either the control or FGR group.Our approach has been used previously [21].Pieces of placental tissue approximately 2-3 cm in size, excluding the fetal and maternal sides, were collected by cutting near the middle between the umbilical cord insertion site and the margin of the placenta after delivery.
Subsequently, chorionic tissue was removed from a standardized central site and thoroughly washed with Dulbecco's phosphate-buffered saline (Thermo Fisher Scientific, Waltham, MA, USA).Within 4 h of delivery, the obtained tissue samples were frozen and stored at À80 C for further examination.Lastly, a portion of some of the samples was preserved in 4% paraformaldehyde and coated with paraffin for morphological studies.

Cell culture
The National Institute of Health Sciences provided the human choriocarcinoma cell line (BeWo).Cells were cultivated in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific) supplemented with 100 g/ml streptomycin, 100 U/ml penicillin, and 10% fetal bovine serum, at 37 C in a humidified incubator with 5% CO 2 and 95% air.Cells were examined for mycoplasma infection and confirmed as mycoplasma negative using the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland).

Messenger RNA expression analysis
The AllPrep DNA/RNA/Protein Kit (Qiagen, Germantown, MD, USA) was used to extract total RNA from tissues and cell cultures following the manufacturer's protocol.Reverse transcription was performed on 1 mg of RNA per tissue and cell line using the Superscript IV VILO kit (Applied Biosystems, Foster City, CA, USA) to obtain single-stranded complementary DNA (cDNA) according to the manufacturer's protocol.The Power SYBR TM Green polymerase chain reaction (PCR) Master Mix (Thermo Fisher Scientific) and StepOnePlus TM real-time PCR (qPCR) assay (Life Technologies, Waltham, MA, USA) were used to perform reverse transcription-quantitative PCR (RT-qPCR), and each reaction was conducted in triplicate.The qPCR cycle conditions for the SYBR Green system were 10 min at 95 C, followed by 40 cycles of 15 s at 95 C, and 1 min at 60 C. Data were calculated using the 2 ÀDDCt method with Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) as an internal control [22].Table 1 contains a list of the primer sequences used in RT-qPCR.

Immunohistochemistry
FGR tissues were fixed in 10% formalin for 48 h, routinely processed, and sliced into sections of 5 mm in thickness, which were then subjected to immunohistochemical staining.In brief, after deparaffination, heatintroduced antigen retrieval, and peroxidase blocking, incubation with the primary antibody SIRT1 (1:400 dilution, #ab110304, Abcam, Cambridge, UK) and anti-INSL4 (1:50 dilution, #sc-373728, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies was performed overnight at 4 C.The secondary peroxidase-labeled anti-rabbit or anti-mouse antibody (Histofine Simple Stain Max PO; Nichirei) was used.Sections were visualized by adding diaminobenzidine (DAB; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and the cellular nuclei were stained with hematoxylin.Pictures were taken using a whole-slide scanner (Nano Zoomer XR, Hamamatsu Photonics, Hamamatsu, Japan).

Western blotting
FGR tissues and BeWo cells were homogenized using radioimmunoprecipitation (RIPA) lysis containing a 1% Halt protease inhibitor cocktail (Thermo Scientific), followed by centrifugation in a microfuge at 12,000 rpm for 15 min.The protein concentration was analyzed using the Pierce TM BCA protein assay kit (Thermo Fisher Scientific) and a PerkinElmer EnSpire plate reader at 562 nm absorbance.Twenty micrograms of each protein was separated on a 4-12% Bis-Tris-polyacrylamide gel (Invitrogen) and transferred to an iBlot Mini Transfer Stack polyvinylidene fluoride (PVDF) membrane (Invitrogen).After brief incubation with 5% nonfat milk to block nonspecific binding, membranes were exposed overnight at 4 C to anti-INSL4 (1:500 dilution, #ab75061; Abcam) and anti-SIRT1 (1:1000 dilution, #ab189494; Abcam) antibodies.After washing the membranes, they were subsequently exposed to the anti-rabbit IgG-HRP antibody (diluted at 1:2000, identified as NA934V, from GE Healthcare).Signals from the protein bands were visualized using chemiluminescence (EMD Millipore, Cat.No. WBLUF0100, Cat.No. WBLUC0100) and quantified using a ChemiDoc XRS Plus system with the Image Lab software (Bio-Rad, Hercules, CA, USA).
Treatment with small interfering RNA BeWo cells (250,000 cells per well) were plated in sixwell culture plates.The next day, they were transfected with non-targeting small interfering RNAs (siRNAs; catalog No. SIC001-10 NMOL, Universal Negative Control, Sigma-Aldrich, St. Louis, MO, USA), SIRT1 #1 (Cat.No. SASI_Hs01_00153666), and SIRT1 #2 (Cat.No. SASI_Hs01_00153667) using siRNA transfection reagent (Sigma-Aldrich), following the manufacturer's protocols.After treatment with siRNA for 24 h, the medium was changed to a fresh culture medium.After slicing SIRT1, BeWo cells were cultivated with 250 mM db-cAMP to induce differentiation for 3 d [23,24]; this is because cultured CTs transform into STs when exposed to analog 8-bromo-cAMP.

SIRT1 activator and cell treatment
A small-molecule SIRT1 activator, CAY10602 (#ab144416, Abcam), was used to assess the effect of SIRT1 activation on restoring the expression of INSL4.SIRT1knockdown BeWo cells were further exposed to dimethylsulfoxide (control) or 20 mM CAY10602 with the addition of 250 mM db-cAMP for 3 d [25].

Statistical analysis
The unpaired Student's t-test was applied to evaluate group differences using SPSS version 26.0 (IBM Corp., Armonk, NY, USA).Error bars indicate the standard error.A p-value <.05 was considered statistically significant.

SIRT1 and INSL4 were downregulated in fetal growth restriction
FGR samples exhibited a substantial decrease in SIRT1 mRNA expression compared to control samples.Additionally, protein expression levels were notably weaker, as observed in western blotting (mRNA expression decreased by 2.6-fold; Figure 1(a,b)).Furthermore, INSL4 mRNA and protein expression levels showed a significant reduction in FGR samples compared to controls, with mRNA expression decreasing by more than 100-fold (Figure 2(a,b)).Furthermore, although robust signals were observed in the control specimens, immunohistochemical analysis revealed that in FGR placentas, INSL4 and SIRT1 were expressed moderately and faintly, respectively (Figure 1(c,d)).These results indicate that SIRT1 and Table 1.Specific small hairpin (shRNA) and primer sequences.

SIRT1 knockdown repressed INSL4 expression
To elucidate the relationship between SIRT1 and INSL4 in placental development, particularly in syncytialization, we conducted an analysis using SIRT1-knockdown BeWo cells with the addition of db-cAMP.The effectiveness of SIRT1 knockdown was confirmed in the transfected cells, showing a 2.1-fold decrease in mRNA expression with both siRNAs (Figure 3(a,b)).Furthermore, our results revealed that the gene and protein expression levels of INSL4 were significantly reduced in SIRT1-knockdown cells, suggesting that INSL4 is regulated by SIRT1.Specifically, the mRNA expression levels decreased by 1.4-and 1.5-fold in siRNA#1 and #2, respectively.Overall, these findings highlight the potential role of SIRT1 in regulating INSL4 during placental development and syncytialization.

SIRT1 activator improved INSL4 expression
The effect of treatment with a SIRT1 activator on restoring INSL4 expression in vitro was investigated.SIRT1-knockdown BeWo cells were treated with CAY10602 (SIRT1 activator) for 3 d during db-cAMPinduced differentiation.This treatment restored gene expression and protein levels of INSL4 in SRIT1-knockdown BeWo cells to normal levels (Figure 3(c,d)).Overall, these results indicate that SIRT1 activators could be therapeutic targets for FGR.

Discussion
FGR commonly affects approximately 1 in 10 pregnancies, resulting in perinatal and neonatal morbidity and mortality.Babies with FGR have an increased risk of cardiovascular diseases, metabolic disorders, and brain microstructural abnormalities in childhood [8,9,26,27].Moreover, infants with FGR have weak immune responses and are more likely to develop postnatal infections [28].Idiopathic FGR, commonly known as FGR of unknown etiology, is closely associated with placental insufficiency when FGR does not present with congenital malformations or clinical signs of preeclampsia (PE) and infection; low birthweight newborns with idiopathic FGR occur as a result of a poor placental function [29].However, the pathogenesis and pathophysiology of placental development remain unclear.Examining the molecular pathways underlying placental development will aid in developing new therapeutic approaches for FGR patients.
SIRT1 is extensively expressed in murine and human placentas [19,20].SIRT1-null animals present with FGR, poor placental development, and abnormalities in the junctional zones and labyrinth layer of the placenta, as evidenced by the histological assessment of several trophoblast markers [19].In vitro, SIRT1-null trophoblast stem cells grow slowly and more blunted, with a reduction in the labyrinth layer and junctional zone markers [19,30,31].
In STs of the preeclamptic placenta, the levels of senescent extracellular matrix (ECM) proteins are increased, whereas SIRT1 is dramatically downregulated [32].By controlling senescence-and ECM-related proteins, syncytialization of BeWo cells and SIRT1 activation prevented senescence in an in-vitro model.These findings suggest that PE occurred because of SIRT1 downregulation, which may trigger ST senescence through targets regulating the cell cycle and ECM synthesis [33].Additionally, Mishra et al. reported that SIRT1 expression is decreased in trophoblast cells in patients with gestational diabetes mellitus (GDM); this is because decreased SIRT1 reduces docosahexaenoic acid (DHA) transport to the fetus [31].DHA is considered important to fetal neurodevelopment and is thought to cause neurodevelopmental disorders in GDM.However, increasing SIRT1 expression improves DHA transport, thereby alleviating GDM.Therefore, SIRT1 is important to placental development and the pathogenesis of GDM and PE.
Only a few reports have shown that SIRT1 is downregulated in the placenta of FGR patients [34].In one study, decreased SIRT1 expression was suggested to accelerate placental aging, leading to FGR and placental insufficiency.Although our data align with these results, the association between SIRT1 and placental developmental insufficiency causing FGR remains unclear.The downstream targets of SIRT1 and many other related genes and signaling pathways have been investigated [35,36].Among these targets, the serine/threonine kinase LKB1 (liver kinase B1; also known as serine/threonine kinase 11 [STK11]) tumor suppressor gene has been reported to be negatively regulated by SIRT1 in osteosarcoma and endothelial cells [37,38], and its expression is negatively correlated with placental weight [39].Furthermore, the AMP-activated protein kinase (AMPK) family of kinaseswhich regulates many cellular activities, including cell polarity, proliferation, and metabolismis modulated by LKB1 [40].
In non-small cell lung carcinoma, loss of LKB1 promotes the expression of INSL4, which regulates cell proliferation and viability, and modulates phosphatidylinositol 3-kinase-protein kinase B and/or 5 0 -adenosine monophosphate-APK signaling [41].INSL4 plays an important role in the aggressive growth of the normal human placenta by promoting the differentiation of CTs to STs [14].Herein, it was hypothesized that, in FGR placentas, low SIRT1 expression induces high LKB1 expression, which represses INSL4; therefore, CAY10602 restores SIRT1 and may decrease LKB1 expression, which in turn induces INSL4 expression.However, more in-depth research is required to support this theory.
In conclusion, this is the first study to analyze the role of INSL4 in FGR placentas, specifically ST.Using BeWo cells, we found that SIRT1 affected INSL4 expression in the ST.The data also suggested that SIRT1 and INSL4 play an important role in FGR, which is thought to be caused by placental insufficiency.Our results shed light on the possible molecular mechanisms linking fetal outcomes that may be caused by placental insufficiency.However, the function of SIRT1-INSL4 in trophoblast differentiation requires further molecular studies.Large-scale randomized clinical trials are needed to support the feasibility of SIRT1 as a future FGR therapeutic target.

Figure 1 .
Figure 1.Downregulation of SIRT1 expression in full-term fetal growth restriction (FGR) placentas.(a) the mRNA and (b-d) protein expression levels of sirtuin 1 (SIRT1) were analyzed using (a) reverse transcription-quantitative polymerase chain reaction (RT-qPCR), (b) western blotting, and (c) immunohistochemical analysis of full-term FGR placental tissues and (d) full-term normal delivery or cesarean (C-) section placental tissues.Data are presented as the mean ± standard error of the mean (SEM).Asterisks ( Ã ) indicate statistical significance as compared with the control (p < .05).Ctrl: control placenta; FGR: fetal growth restriction; SIRT1: sirtuin 1.

Figure 2 .
Figure 2. Downregulation of INSL4 expression in full-term FGR placentas.(a) mRNA and (b-d) protein expression levels of SIRT1 were analyzed using (a) RT-qPCR, (b) western blotting, and (c) immunohistochemical analysis of full-term FGR placental tissues and (D) full-term normal delivery or C-section placental tissues.Data are presented as the mean ± SEM.Asterisks ( Ã ) indicate statistical significance as compared with the control (p < .05).Ctrl: control placenta; FGR: fetal growth restriction; INSL4: insulin-like 4.

Figure 3 .
Figure 3. Downregulation of SIRT1 and INSL4 expression in SIRT1-knockdown BeWo cells.(a) mRNA and (b) protein expression levels of SIRT1 and INSL4 were analyzed using (a) RT-qPCR and (b) western blotting.SIRT1-knockdown BeWo cells treated with SIRT1 activator (c) mRNA and (d) protein expression levels of SIRT1 and INSL4 were analyzed using (c) RT-qPCR and (D) western blotting.Data are presented as the mean ± SEM.Asterisks ( Ã ) indicate statistical significance as compared with the control (p < .05).si Ctrl: transfected with control siRNA; si Ctrl #1 and #2: BeWo cells transfected with different types of SIRT1 knockdown siRNAs; INSL4: insulin-like 4.