Circ-0081343 binds and activates RBM8A nuclear translocation to promote autophagy in Fetal growth restriction

doi:10.21203/rs.3.rs-2600975/v1

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Circ-0081343 binds and activates RBM8A nuclear translocation to promote autophagy in Fetal growth restriction

https://doi.org/10.21203/rs.3.rs-2600975/v1

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Fetal growth restriction (FGR) is a common disease that occurred during pregnancy and is the second leading cause of perinatal death, affecting neonates' short- and long-term prognosis. However, the etiology and pathogenesis of FGR are poorly understood. Recent study found that circ-0081343 is significantly reduced in human FGR placenta and is involved in cell migration, invasion, and apoptosis of trophoblast in vitro. However, the mechanism of how circ-0081343 regulates placental development have not been fully understood. In this study, we aimed to explore the function of circ-0081343 in pathogenesis of FGR placenta using in vitro experiment and FGR mouse model. Overexpression of circ-0081343 in HTR-8/SVneo increased the binding of circ-0081343 to RNA binding motif protein 8A (RMB8A). Furthermore, it also inhibited the nuclear translocation of RMB8A via downregulating the importin 13 (IPO13), which transported RMB8A across the nuclear membrane into the nucleus. Based on the integration of RNA-seq and Chip-seq, we found that phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (AKT) pathway was the target of circ-0081343. circ-0081343 bound to RMB8A and inhibited its nuclear translocation, resulting in inactivation of PI3K/AKT pathway via interaction with PI3K3C3, thereby regulating autophagy in HTR-8/SVneo cells. In FGR mouse model, overexpression of circ-0081343 was able to restore the impaired autophagy through inhibition of PI3K/AKT pathway, improving pathological placenta and bodyweight of pups. These results provided new molecular targets and ideas for the diagnosis and treatment of clinical FGR from the perspective of circRNA affecting the nuclear translocation of binding proteins and leading to abnormal transcriptional control.

Fetal growth restriction

Circ0081343

RMB8A

PI3K-AKT pathway

IPO13

Autophagy

Fetal growth restriction (FGR) is a condition in which an unborn fetus is smaller than expected for gestational age and is a major cause of fetal and neonatal death in the perinatal period [1]. Epidemiological surveys show that the average incidence of FGR in China is high, reaching about 8.77% [2]. Premature FGR babies have an increased risk of mortality and long-term morbidity [1]. Furthermore, they are not only predisposed to premature birth, fetal distress, and stillbirth in utero, but also have long-term adverse outcomes, including perinatal death, perinatal morbidity, long-term neurodevelopmental disability, growth retardation and cognitive retardation [2–4]. Although the causes of FGR vary, they all result in a common causative process: insufficient uteroplacental blood flow [5]. Therefore, a problem in the placenta is the most common cause of FGR.

Autophagy, which plays an important role in the development of the human placenta. Nakashima et al. discover that placental hypoxia enhances that the autophagy in the extravillous trophoblasts, which plays an important role in the invasion of extravillous trophoblasts and the remodeling of maternal spiral arteries [5].

Circular RNAs (circRNAs), which are closed long non-coding RNAs, have a variety of biological functions, including regulating cell proliferation, invasion, apoptosis, etc., but whether circRNAs mediate autophagy is still unknown. Current researches on circRNAs are mainly focused on miRNA sponging, which involves inhibiting miRNAs' ability to act on mRNAs [6, 7]. However, recent studies have found that circRNAs could exclusively bind to a single protein or interact with multiple proteins under different circumstances. The circFoxo3 could bind to the inhibitor of DNA binding 1 (ID1), E2F transcription factor 1 (E2F1), and hypoxia-inducible factor 1 subunit alpha (HIF-1-alpha), preventing their translocation into the nucleus, while knock out of circFoxo3 induces the nuclear translation of these proteins [8]. circFoxo3 also binds cyclin-dependent kinase 2 (CDK2) and cyclin-dependent kinase inhibitor 1A (CDKN1A), result in an inhibition of the binding of CDK2 to Cyclin A and E, inhibiting cell proliferation [9]. In post-myocardial infarction mouse hearts, circFndc3b acts as a binding site for Fused in sarcoma (FUS) proteins rather than as a miRNA sponge; and it increases angiogenesis and cardiomyocyte survival through binding to FUS [10].

It has been reported circ_0081343 is low expressed in human FGR placenta and regulates cell migration, invasion, and apoptosis of trophoblast through hsa-miR-210-5p/DLX3 axis [11]. However, the mechanisms of how the circ_0081343 regulates placental development are not well studied. In this study, we investigated the roles of circ-0081343 in the pathogenesis of FGR placentation using RNA pull-down, mass spectrometry, and RNA binding protein Immunoprecipitation (RIP) and identified that circ-0081343 could bind to RNA Binding Motif Protein 8A (RMB8A) and restrain the protein expression of nuclear transporter Importin 13 (IPO13) [12], inhibiting the transport of RMB8A into nucleus in HTR-8/SVneo cells. Knockout of circ-0081343 could induced nuclear translocation of RBM8A, resulting in insufficient autophagy. Next, we integrated the RNA-seq and Chip-seq data and found that phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (AKT) pathway might be a player in circ-0081343 mediated-autophagy in HTR-8/SVneo cells. via activating phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (AKT) pathway, thereby promoting FGR. Based on ChIP-seq data and ChIP-qPCR result, we identified RBM8A could bind to the promoter of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3, also called Class III PI3K Vps34). Taken together, circ-0081343 restrained the nuclear translation of RBM8A, resulting in up-regulation of PIK3C3, which inactivated PI3K/AKT pathway. We next induced the expression of circ-0081343 in FGR mice model using adenovirus-mediated overexpression technique and found that it improved the pathological placenta bodyweight of pups via restoring the impaired PI3K/AKT-mediated autophagy. These findings provide new insights into the pathogenesis of FGR as well as a novel target for FGR diagnosis and treatment.

Cell culture

HTR-8/SVneo cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured at 37°C in a humidified incubator with 5% CO2 in RPMI 1640 (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco), 1% penicillin/streptomycin, and 1% L-glutamine.

RNA Pull-Down

RNA pull down was employed to examine the circ-0081343-binding proteins. Full-length circ-0081343 probe, NC probe, ARPC1B intron3, ARPC1B intron4, and LacZ probes were synthesized by Guangzhou Reebok Biotechnology Co. The RNA transcripts were labeled by Desthiobiotin at 16°C overnight (Cat#20163, Thermo Scientific, USA). RNA pull-down assays were performed using the Magnetic RNA-Protein Pull-Down Kit (Cat#20164, Thermo Scientific, USA). The protein was detected using a silver staining kit (Thermo Scientific, USA) or standard immunoblotting.

Liquid Chromatography Mass Spectrometry (LC-MS)

The protein was centrifuged for 5 minutes at 12000rpm in 10KDa size ultrafiltration tube, and 20µl of lysis buffer was added to the ultrafiltration tube to re-dissolve. 100µl 100mM ammonium bicarbonate containing 25mM DTT was added and incubated for 30 minutes at 60°C. 100µl 100mM ammonium bicarbonate containing 55 mM IAA was added and incubated for 30 minutes at room temperature. 1/5 volume of mass spectrometry-grade trypsin solution was added the above sample solution and digested overnight at 37°C. The sample was centrifuged at 12000 rpm for 15 minutes at 4°C. The supernatant was transferred to a Zip Tip tube. After centrifugation, the supernatant was collected. After vacuum drying, the pallet was re-solubilized with 30 µl of 30% acetonitrile (ACN) for mass spectrometry analysis.

Silver protein staining

Silver protein staining was used to monochromatically detect proteins in 1D or 2D polyacrylamide gels with metallic silver ions (Ag). The purified proteins were fractionated via SDS-PAGE and were visualized using silver staining by Fast Silver Stain Kit (Beyotime, Nantong, China) as described by the manufacturer. Briefly, the gel was fixed using fixing solution for 1 hour and washed using 30% Ethanol. After rising with water, gel was treated with sensitization reagents to enhance the sensitivity of silver staining for protein. After washing with water, the gel was stained with silver nitrate solution for 10 minutes at room temperature. The gel was briefly washed following silver impregnation step to remove excess silver from the gel surface. And the gel was incubated with the development solution for 10 minutes to reduce silver ion to metallic silver. After washing, the gel was incubated with stopping solution for 10 minutes at room temperature.

RNA Binding Protein Immunoprecipitation (RIP)

RIP was performed as documented previously [13]. The Magna RIP kit (Millipore, Burlington, MA) was used for RIP experiments. Briefly, 1 × 10⁷ HTR-8/SVneo were pelleted and lysed in RIPA buffer. Then, 100 µl of cell lysate was incubated with RIP buffer containing magnetic beads conjugated with human anti-AGO2 antibody or IgG at 4°C overnight. After washing, RNA was purified and assessed the RNA Quality by Nanodrop. qRT-PCR analysis was used for determining the relative expression of circ-0081343. To denature the RNA, it was heated to 85°C for 5 minutes. RNA was reverse-transcripted using ImProm-II™ (Promega, Madison, WI). The primary antibodies used were listed in Table S2.

Quantitative real-time PCR (qRT-PCR)

The total RNA was extracted using Trizol (10296-028, Invitrogen, Waltham, MA) as described by the manufacturer. Briefly, cells or homogenized samples were added 500 µl Trizol reagent. 100 µl chloroform was added to isolated RNA from DNA and protein. The aqueous phase was transferred to a new Eppendorf tube and mixed with the same volume of 2-propanol after centrifugation at 12,000 g for 15 minutes at 4°C. RNA pellet was collected after the mixture was centrifuged at 12,000 g for 15 minutes at 4°C and reverse-transcripted using ImProm-IITM (Promega, Madison, WI) according to the instruction. The qRT-PCR was performed on a LightCycler480 Real-Time PCR System (Roche, Basel, Switzerland) using SYBR GREEN qPCR Super Mix (Invitrogen, Waltham, MA). The primers used in this research was listed in Table S1 and GAPDH was employed as the normalization standard.

Cell transfection

circ-0081343 full length was synthesized by Shanghai Jima Chemical Co (Shanghai, China) and cloned to the pLCDH-ciR vector to construct the circ_0081343 overexpression vector (ov-circ-0081343).

Two small interfering RNAs (siRNAs) targeting circ_0081343 and negative control siRNA (si-NC) were synthesized by Shanghai Jima Chemical Co (Shanghai, China). The sense sequence of the siRNAs was as follow: (1) si-NC: 5-UUCUCCGAACGUGUCACGUTT-3; (2) si-circ-0081343-1: 5-GGAGAAUGACUGGCAUCGATT-3; (3) si-circ-0081343-2: 5- GACUGGCAUCGACUGGGCCTT-3.

The amino acid sequence of RBM8A was identified, and the nuclear localization sequence (NLS) and two amino acid sites of RBM8A (LKEKAKKRKGR and PPKGKRRGGRR) were found using NLS software, and we chose LKEKAKKRKGR that with the higher score, which corresponding base sequence is CTGAAAGAAAAAGCGAAGAAACGGAAGGGTCGC. To examine that whether IPO13 transported RBM8A into the nucleus via binding to the NLS sequence of RBM8A, we constructed the RBM8A-NLS point mutation plasmid and the RBM8A-NLS wild-type plasmid using vectors with HA tags. Both mutated the NLS corresponding base sequence was TGTGAACAACAACCGGAGCAATGGCAGTGTGGC.

Transient transfection was performed to the plasmids or si-RNAs into HTR-8/SVneo cells using lipofectamine 2000 provided by Invitrogen (Carlsbad, CA).

Chromatin immunoprecipitation (ChIP)

ChIP assay was performed using EZ-ChIP kit (Millipore, Burlington, MA) according to the instruction from the manufacturer. Briefly, Eto-treated and untreated cells were crosslinked with 1% formaldehyde for 15 min. The crosslinking reaction was stopped by glycine. The resultant DNA-protein complex was sonicated to produce 200–1000 bp DNA fragments. The anti-RBM8A antibody (Millipore, Burlington, MA) was added to form the antibody-target protein-DNA complex and protein A-Sepharose beads were used to immunoprecipitated complex. After washing and reversing the cross-links, the enriched DNA was purified and then examined by qRT-PCR.

RNA seq and ChIP seq

Total RNA was extracted as described [11]. Illumina TruSeq RNA libraries were constructed and sequenced by Nanjing Vazyme Biotech Co (Nanjing, China). The differential gene expression analysis was carried out using DESeq2. RBM8A ChIP was performed using anti-RBM8Aantibody as described above. Two Illumina True Seq ChIP libraries were constructed from ChIP and input control DNA and sequenced by Nanjing Vazyme Biotech Co (Nanjing, China). ChIP-seq and RNA-seq data were visualized on the UCSC genome browser. Gene ontology (GO) analysis was performed using clusterProfiler (3.8.1) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis was performed using clusterProfiler (3.8.1).

Transmission electron microscopy (TEM)

Cells were fixed with 5% glutaraldehyde and post-fixed with 1% Osmic Acid-1.5% potassium ferricyanide and rinsed with PBS after 2h. After dehydration, ultra-thin sections were stained with uranyl acetate for 15 minutes and lead citrate for 5 minutes. The morphology and number of autophagosomes were observed under transmission electron microscopy.

Subcellular fractionation and Western blot

Nucleus and cytoplasmic fractions were extracted using commercial NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA). Briefly, Cells were harvested with trypsin-EDTA and centrifuged for 5 minutes at 500 xg. After washing with PBS, cells were transferred to a 1.5mL microcentrifuge tube and centrifuged 5 minutes at 500 xg. The cell pellet was incubated with 200 µl Ice-cold cytoplasmic extraction reagent (CER) I on ice for 10 minutes. 11 µl Ice-cold CER II was added to the tube and incubated on ice for 1 minute. The tube was centrifuged for 5 minutes at 16000 xg for 5 minutes. Supernatant (cytoplasmic extract) was collected and stored at -80°C. The insoluble fraction was incubated with 100 µl nuclear extraction reagent (NER) on iced for 40 minutes and centrifuged for 5 minutes at 16000 xg for 10 minutes. The supernatant was the nuclear extract.

Cells were collected with cell scraper and centrifuged for 5 minutes at 500 xg. The protein was lysed using RIPA lysis buffer (Beyotime, Nantong, China), and protein concentration was measured using the bicinchoninic acid (BCA) Protein Assay kit (Keygentic, Nanjing, China). 10 µg of proteins are electrophoretically separated by SDS-PAGE and then electron transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 hour at room temperature and incubated with primary antibodies at 4°C overnight. After washing with tris-buffered saline with tween (TBS-T), The membranes were incubated with the secondary antibody at 37°C for 1 h. The protein band was visualized using ECL (Beyotime, Nantong, China). The primary antibodies used were listed in Table S2.

Co-Immunoprecipitation (Co-IP)

Co-IP assay was performed as previously described [14]. Briefly, the extracted protein was precleared with protein A/G agarose beads. The lysates were incubated with protein A/G agarose beads overnight at 4°C with a negative control IgG or a primary antibody against the indicated protein. The beads were collected and washed with IP buffer, and the eluates and reserved lysates (input) were electrophoresed and transferred to PVDF membranes and probed with indicated antibodies. The primary antibodies used were listed in Table S2.

Immunofluorescence

The cells cultured on cover glass were fixed with 4% PFA for 30 minutes at 4°C, then the cells were washed with PBS for 5 minutes three times. The cells were permeabilized with 0.2% Triton X-100 for 5 minutes and blocked with blocking buffer containing 10% goat serum (Beyotime, Shanghai, China) for 1 hour at 37°C. Cells were incubated with primary antibodies at 4°C overnight. After washing in PBS, cells were incubated with secondary antibodies for 1 hours at room temperature, and then mounted with an antifade mounting medium with 4,6-diamino-2-phenyl indole (DAPI) solution (Beyotime, Shanghai, China). All assays were performed in triplicate. The primary antibodies used were listed in Table S2.

HE staining

Placentas were embedded in paraffin and sectioned followed by standard H&E staining protocol. Sections (5 µm thick) were deparaffinized in xylene, rehydrated through a graded series of ethanol, and washed in water. Next the sections were stained with hematoxylin, and followed by a rinse in a weak acid solution to remove excess staining. After the application of hematoxylin, the tissue was counterstained with eosin Y.

Immunohistochemistry (IHC)

After deparaffinizing, paraffin-embedded sections were microwave heated for 10 minutes in 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval and then cooling to room temperature. Hydrogen peroxide incubation for 10 minutes was performed to inactivate the endogenous horseradish peroxidase. After blocking with 10% goat serum buffer (Beyotime, Shanghai, China) for 1 hour to prevent nonspecific binding, sections were incubated with primary antibodies at 4°C overnight. After washing with PBS, sections were incubated with secondary antibodies for 1 hours at room temperature. DAB detection kit (Boster, Wuhan, China) was used for visualization as a reddish-brown color. The primary antibodies used were listed in Table S2.

FGR mouse model

Mouse (Kunming strain) were housed in an animal room with a controlled environment (22–24°C and 60–70% relative humidity) and maintained on a light/dark cycle (12 h light/12 h dark). All animal procedures were approved by the Animal Care and Use Committee of Southern Medical University. Female mice were mated with fertile male mice. When plugs were detected in the morning, embryos were considered to be day 1 of pregnancy.

Pregnant mice were randomly divided into four groups: (1) control: no treatment; (2) FGR: bilateral partial ligation of the uterine arteries was performed; (3) FGR + Ad-null: bilateral partial ligation of the uterine artery was performed along with caudal vein injection of empty virus 2×10⁸ pfu/litter; (4) FGR + Ad-mmu-circ-0081343: bilateral partial ligation of the uterine artery was performed along with caudal vein injection of Ad-mmu-circ-00813432×10⁸ pfu per litter. Ad-mmu-circ-0081343 was constructed using adenovirus (Ad). All mice were delivered by cesarean section on gestational day 19.

Statistical analysis

SPSS 18.0 software was used to analyze and process the raw experimental data in this experiment, and all experiments were repeated three times. GraphPad Prism 6.0 software was used for graphing. Data were expressed as mean ± standard deviation (SD). One-way ANOVA was used for comparison of multi sample means, and t-test was used for comparison of means between two groups. P < 0.05 indicates a statistically significant difference, and P < 0.01 indicates a highly significant difference. P > 0.05 was considered not significant (ns).

circ-0081343 mediates nuclear translocation of RBM8A

To identify the protein that circ-0081343 interacted with, we performed RNA pull-down using biotinylated circ-0081343 probes and NC probes and applied western blotting to isolate and identify proteins directly bound to circ-0081343 (Fig. S1A). Proteins were analyzed using LC-MS, and a total of 619 proteins were identified (Fig. S1B). 17 circ-0081343 interacted proteins were predicted using catRAPID. After RNA pull down, protein levels of RBM8A, SRSF3, and TARDBP were detected in circ-0081343 probe group but not in NC group by Western blot, indicating circ-0081343 can bind to these proteins in HTR-8/Svneo cells (Fig. S1C). The binding propensity of protein-RNA pairs for RBM8A, SRSF3, and TARDBP were estimated by catRAPID (Table. 1). To examine whether circ-0081343 regulated the expression of RBM8A, SRSF3, and TARDBP, HTR-8/Svneo cells were transfected with ov-circ-0081343 and si-circ-0081343, respectively. The circ-0081343 was significantly induced in HTR-8/Svneo cells transfected with ov-circ-0081343 and reduced in si-circ-0081343 (Fig. S2 A and B). However, qRT-PCR and Western blot results revealed that overexpression and knockout of circ-0081343 had no effect on the mRNA and protein levels of RBM8A, SRSF3 and TARDBP in HTR-8/Svneo cells (Fig. S2 C-D).

We previously found that circ_0081343 was localized in the cytoplasm, while SRSF3 and TARDBP were mainly expressed in the nucleus [15, 16]. RBM8A is widely expressed in both cytoplasm and nucleus [17]. To verify the interaction of RBM8A protein with circ-0081343, RIP experiments were performed on extracts from HTR-8/Svneo cells using an antibody against RBM8A. Circ_0081343 enrichment was observed in the input group, indicating that RBM8A protein could bind circ-0081343 (Fig. 1A). This indicates that RBM8A can directly bind with circ-0081343 in the form of RNA-binding protein (RBP). In HTR-8/SVneo cells, we found overexpression of circ-0081343 induced cytoplasmic RBM8A and reduced nuclear RBM8A, while knockout of circ-0081343 had an opposite effect on RBM8A (Fig. 1B and C), suggesting that circ-0081343 regulated the nuclear translocation of RBM8A through binding to it in the cytoplasm.

Circ-0081343 Mediates Nuclear Translocation Of Rbm8a Via Ipo3

RT-PCR and Western blot results showed that overexpression of circ-0081343 down-regulated the mRNA and protein expression of IPO13, and inhibition of circ-0081343 up-regulated the mRNA and protein expression of IPO13 (Fig. 2A and B), indicating that circ-0081343 inhibited IPO13 mRNA and protein expression. We detected RBM8A and IPO13 protein expression in IP assay using IPO13 or RBM8A antibodies, indicating that there was an interaction between RBM8A and IPO13 protein (Fig. 2C).

To further examine whether circ-0081343 regulates nuclear transport of RBM8A via IPO13, IPO13 was silenced or overexpressed in HTR-8/SVneo cells treated with si-circ-0081343 using si-IPO13 and ov-IPO13 transfection, respectively. Immunofluorescent results showed that silence of IPO13 decreased the nuclear RBM8A expression and overexpression of IPO13 increased the nuclear RBM8A, indicating that circ-0081343 restrained nuclear transport of RBM8A via down-regulation of IPO13 (Fig. 2D).

The current study suggested that IPO13 mainly transports target proteins into the nucleus in an NLS-dependent manner [18, 19], therefore, we used NLS software to identify the NLS sequence and amino acid sites of RBM8A (Fig. S3A). The RBM8A-NLS wild-type plasmid and RBM8A-NLS mutant plasmid were constructed using a vector with HA tag and the NLS amino acid sequence LKEKAKKRKGR was mutated to CEQQPEQWQCG. Immunofluorescent images showed that RBM8A was expressed in both cytoplasm and nucleus in RBM8A-NLS wild-type transfected group, while it was mainly located in the cytoplasm in the RBM8A-NLS mutant transfected group (Fig. S3B). These results suggested that IPO13 transported RBM8A into the nucleus in an NLS-dependent manner.

Integrating of RNA-seq and ChIP-seq data to unravel the potential pathways regulated by circ-0081343-mediated RBM8A nuclear translocation.

We performed RNA-seq of HTR-8/SVneo cells transfected with si-circ-0081343 and Chip-seq of HTR-8/SVneo cells transfected with si-circ-0081343 using RBM8A antibody and integrated these data to explore the potential target pathways regulated by circ-0081343-mediated RBM8A nuclear translocation. The ChIP-seq signal intensity (RPM) in promoter regions of differentially and non-differentially expressed genes from RNA-seq data was shown as a box plot for both differentially and non-differentially expressed genes (Fig. 3A). The heatmaps displayed the log10 (FPKM) of RNA-seq and log10 (RPM) values of ChIP-seq (Fig. 3B). The ChIP-seq signal changes and the gene expression changes were shown as heatmaps based on ChIP-seq data and RNA-seq analysis (Fig. 3C). Scatter plot depicted ChIP-seq log2 versus RNA-seq FPKM for all peak-associated genes (Fig. 3D). After integrating RNA-seq and ChIP-seq data, a total of 1335 genes were identified that could be affected by nuclear translocation of RBM8A (Fig. 3E). GO BP analysis and KEGG signaling pathway analysis of DEGs were performed using clusterProfiler (3.8.1) and clusterProfile (3.8.1), respectively (Fig. 3F and g, Table 2).

Table 1

The binding propensity of protein-RNA pairs
Protein	Sequence Position	Motif	K-mer	Z-score	P-value
RBM8A	126	rygcgb	gcccgu	2.473	0.0067
	133	rygcgb	gcgugg	2.743	0.00304
	135	rygcgb	gugcgu	2.743	0.00304
SRSF3	103	wcwwc	ucauc	2.431	0.00753
	179	cuckuy	cucucu	2.22	0.0132
	181	cuckuy	cucguu	2.58	0.00494
	197	cuckucy	cuguuau	2.22	0.0132
TARDBP	60	ugugug	uacgug	1.789	0.0368
	62	ugugug	cgugug	2.183	0.0145
	64	ugugug	ugugga	1.789	0.0368
	132	ugugug	ugcgug	2.009	0.0368
	160	ugugug	aguuug	2.156	0.0155
	165	ugugug	gcugug	2.147	0.0159
	167	ugugug	uguggg	2.716	0.0033
	183	ugugug	cgugug	2.752	0.00296
	185	ugugug	ugugau	2.183	0.0145
	196	ugugug	ucuguu	1.908	0.0282

Table 2

GO pathway enrichment list
Term			Input number	Background number	P-Value
Cytokine-cytokine receptor interaction	KEGG Pathway	Has0 4060	31	265	1.10751164253e-09
Metabolic pathways	KEGG Pathway	Has0 1100	79	1243	2.13429811159e-09
PI3K-Akt signaling pathway	KEGG Pathway	Has0 4151	31	342	2.37355493534e-07
p53 signaling pathway	KEGG Pathway	Has0 4115	13	69	7.32523767693e-07
NOD-like receptor signaling pathway	KEGG Pathway	Has0 4621	11	57	4.23485239998e-06

Nuclear Translocation Of Rbm8a Inhibits Autophagy Via P13k/akt Pathway

Based on GO enrichment analysis, we examined the regulation of circ-0018343 on P13K/AKT pathway. Western blot results showed p-PI3K and p-AKT protein levels were significantly reduced after overexpression of circ-0081343; but increased after silence of circ-008134 in HTR-8/Svneo cells (Fig. 4A). Western blot analysis revealed that circ-0081343 overexpression increased LC3II/I radio while decreasing p62 protein expression in HTR-8/Svneo cells, indicating that overexpression of circ-0081343 induced autophagy; and autophagy inhibitor (3-MA) inhibited the active autophagy induced by circ-0081343 overexpression (Fig. 4B). Furthermore, TEM images showed the number of autophagosomes was significantly increased after overexpression of circ-0081343, and was significantly reduced by 3-MA (Fig. 4D). Silence of circ-0081343 showed an opposite effect on LC3II/I radio and p62 protein expression, suggesting that blockade of circ-0081343 resulted in insufficient autophagy. The autophagy activator (Rapamycin) induced autophagy that restrained by circ-0081343 overexpression (Fig. 4C) and restored the insufficient autophagosomes caused by silence of circ-0081343 after overexpression of circ-0081343 (Fig. 4D).

We next examined whether the circ-0018343-mediated autophagy was dependent on RBM8A. Western blot analysis revealed that p-PI3K and p-AKT protein levels were significantly increased after RBM8A overexpression, but decreased after RBM8A inhibition (Fig. 5A), suggesting that the circ-0018343 inactivated PI3K/AKT pathway via restraint the nuclear translation of RBM8A in HTR-8/Svneo cells. RBM8A overexpression down-regulated LC3II/I radio while up-regulated p62 protein expression, indicating that RBM8A inhibited autophagy (Fig. 5B); and TEM images showed that Rapamycin restored the insufficient autophagosomes caused by RBM8A overexpression (Fig. 5D). Western blot results showed that silence of RBM8A induced LC3II/I expression but decreased p62 expression and 3-MA inhibited autophagy caused by RBM8A inhibition (Fig. 5C). The increased autophagosomes by silencing RBM8A were reduced after 3-MA treatment (Fig. 5D).

Transfection of the ov-circ-0081343 plasmid alone inactivated P13K/AKT pathway, increased the LC3II/I radio, decreased the expression of p62, and increased autophagosome formation (Fig. 6A and B). Overexpression of RBM8A was able to reverse these changes to normal (Fig. 6A and B). Taken together, reduction of nucleus RBM8A expression that caused by cicr-0081343 overexpression inactivated PI3K/AKT pathway, and induced autophagy.

Nuclear RBM8A interacted with PI3K3C3 promoter and downregulated PI3K3C3.

To elucidate the mechanism coordinating nuclear RBM8A mediated autophagy, ChIP-qPCR was employed to analyze PIK3C3, ULK2, and RPTOR, which were the top 3 upregulated genes in HTR-8/SVneo cells after silencing circ-0081343 according to ChIP-seq data. The percentage of input analysis represents the amount of DNA pulled down in si-circ-0081343 group using RBM8A antibody in the ChIP reaction, comparing to the amount of DNA pulled down in the NC group. We observed that three regions of PIK3C3, one region of ULK2, and one region of RPTOR were significantly enriched in HTR-8/SVneo cells after silencing circ-0081343 (Fig. 7A). Western blot results showed protein level of PI3K3C3 was significantly reduced in HTR-8/SVneo cells after silence of circ-0081343 or overexpression of RBM8A (Fig. 7B and C), suggesting that nuclear RBM8A downregulated PI3K3C3 protein expression through protein-DNA interaction.

PI3K is Class IA PI3K, and PI3K3C3 is Class III PI3K [20, 21]. Here, we used the PI3K inhibitor, LY294002 to explore the effect of PI3K on autophagy in HTR-8/SVneo cells. We observed that the downregulated PI3K3C3 by silence of circ-0081343 or overexpression of RBM8A was significantly increased by LY294002 (Fig. 7C and D), indicating that PI3K had an adverse effect on PI3K3C3 in HTR-8/SVneo cells. Furthermore, consistence with the above results, silence of circ-0081343 or overexpression of RBM8A reduced protein expression of LC3 in HTR-8/SVneo cells, while PI3K inhibitor restrained the adverse effect of silence of circ-0081343 or overexpression of RBM8A on LC3, significantly induced the LC3 expression (Fig. 7E and F), suggesting that inactivation of autophagy by silence of circ-0081343 or overexpression of RBM8A can be induced via inhibition of PI3K.

Circ-0081343 Improves Pregnancy Outcome In Fgr Mouse Model Via Restoring The Impaired Autophagy

circ-0081343 improves pregnancy outcome in FGR mouse model via restoring the impaired autophagy

The EMBOSS Needle comparison tool found no homologous sequences between the human and rat circ-0081343 sequences. A comparison of circ-0081343 sequences from human and mouse placentas revealed 91.50% sequence homology. Mice were undergoing bilateral uterine vein ligation to establish FGR mouse model. We constructed the ad-mmu-circ-0081343 adenovirus and injected it into FGR mouse model. RT-PCR analysis showed that placental circ-0081343 expression was significantly reduced in FGR and FGR + Ad-null groups, which mimicked in vivo placental tissues with low expression of circ-0081343. The ad-mmu-circ-0081343 injection significantly induced circ-0081343 expression in FGR placenta (Fig. 8A). The total number of pups and the average number of pups per litter did not significantly differ between the four groups, but the alive pup’s rate was significantly lower in the FGR, FGR + Ad-null, and FGR + Ad-mmu-circ-0081343 groups (Table 3). FGR pups were defined as pups with body weight less than 1.25g. The incidence of FGR in 4 groups was as follows: control group (8.86%), FGR group (47.30%), FGR + Ad-null group (43.47%) and FGR + Ad-mmu-circ-0081343 group (25.26%) (Table 3). The incident rate of FGR was significantly reduced in FGF mouse injected with Ad-mmu-circ-0081343. The body and size and weight of the pups in the FGR and FGR + Ad-null groups were significantly lower than those in the control and FGR + Ad-mmu-circ-0081343 groups (Fig. 8B and C). The placental weight in FGR + Ad-mmu-circ-0081343 group was significantly higher than in FGR and FGR + Ad-null groups, but it was still lower than in control group (Fig. 8D).

Table 3

The general condition of pregnant mice and fetus
Group(S)	Control		FGR + CAd	FGR + mmu-circ-0081343
Dams (Pregnant mice) (n)	7	7	7	8
Maternal GD19 weight (g)	54.02 ± 1.52	52.94 ± 1.07	52.71 ± 2.06	53.84 ± 0.94
Implants examined, No	81	84	79	93
Live fetuses, No (%)	78(96.2)	68(80.95) ^**/##	69(82.22) ^**/##	85(91.40) ^**
Resorbed fetuses, No (%)	1(1.23)	4(3.57)^**	3(3.80)^**	4(4.30)^**
Death fetuses, No (%)	2(2.47)	10(11.90)^**/##	7(8.86)^**/##	4(4.30)^**
FGR per litter (%)	7.59	37.83^**/##	40.57^**/##	27.50^**

The layers of placenta were labeled as: decidua zone (DZ), junctional zone (JZ) and labyrinth zone (LZ) [22]. In control, the DZ, JZ, and LZ structures were clearly defined, and the LZ was densely distributed with abundant blood flow (Fig. 8E). In FGR and FGR + Ad-null groups, the proportions of DZ and JZ were significantly higher, while LZ was significantly lower. Furthermore, the majority of decidual cells had hyaline degeneration and lysis necrosis, resulting in vacuole-like changes, and the sizes of trophoblast cell in the LZ were various, with scattered apoptotic cells. We also observed the interstitial edema in the vascular zone (Fig. 8E). The placental pathology in FGR + Ad-mmu-circ-0081343 group was comparable to the control group (Fig. 8E).

Western blot results showed that PI3K3C3 and Beclin1 protein levels were significantly reduced and p62 protein level was significantly increased in FGR and FGR + Ad-null groups compared to control, and Ad-mmu-circ-0081343 injection restored protein levels of PI3K3C3 and Beclin1, while decreased p62 expression (Fig. 9A and B). Furthermore, P13/AKT pathway was active in FGR and FGR + Ad-null groups, and inactive in Ad-mmu-circ-0081343 injection groups (Fig. 9C). These results suggested that circ-0081343 improves pregnancy outcome in FGR mouse model via P13/AKT mediated autophagy.

Rbm8a Downregulates Circ-0081343 Expression

Based on JASPAR and PROMO database, 6 transcription factors that bind to the circ-0081343 promoter region were identified (Table 4). qRT-PCR and Western blot results showed that mRNA and protein expression were significantly increased after overexpression of PAX5, YY1, FOXP3, SRY, Hoxd9 and SP1 (Fig. S4 A and B); and they were significantly reduced after transfection with shRNA of PAX5, YY1, FOXP3, SRY, Hoxd9 and SP1 (Fig. S4 C and D). However, overexpression or knockdown of PAX5, YY1, FOXP3, SRY, Hoxd9 and SP1 had no effect on the expression of circ-0081343, suggesting that circ-0081343 was not regulated by these transcription factors (Fig. S4 E and F).

Table 4

Prediction of transcription factors binding to the promoter region of circ-0081343.
Model ID	Model name	Score	Relative score	Start	End	Strand	predicted site sequence
MA0850.1	FOXP3	5.632	0.858740454384751	288	294	-1	gtaaatg
MA0850.1	FOXP3	5.375	0.852274408290388	449	455	1	gtaaagg
MA0850.1	FOXP3	5.501	0.855444532056496	688	694	1	gaaaaga
MA0850.1	FOXP3	5.501	0.855444532056496	984	990	-1	gaaaaga
MA0850.1	FOXP3	9.305	0.951152078138043	1016	1022	-1	gcaaaca
MA0850.1	FOXP3	8.371	0.927652906728957	1088	1094	1	ataagca
MA0850.1	FOXP3	5.482	0.854966497520337	1105	1111	1	gtaaaaa
MA0850.1	FOXP3	7.284	0.900304299318167	1113	1119	-1	gcaagca
MA0850.1	FOXP3	7.226	0.898845035997261	1399	1405	1	aaaaaca
MA0850.1	FOXP3	5.870	0.864728465942955	1403	1409	1	acaaaga
MA0850.1	FOXP3	5.482	0.854966497520337	1510	1516	1	gtcaaca
MA0850.1	FOXP3	5.482	0.854966497520337	1729	1735	1	gtacaca
MA0913.1	Hoxd9	6.829	0.856716206310909	252	261	1	ccaagaaaaa
MA0913.1	Hoxd9	6.614	0.851746266066696	1102	1111	1	tcagtaaaaa
MA0913.1	Hoxd9	13.027	0.999989181351063	1394	1403	1	gcaataaaaa
MA0079.3	SP1	5.828	0.854463593375058	107	117	-1	cctcagccccc
MA0079.3	SP1	12.677	0.940631854380767	139	149	-1	cctccgcctcc
MA0079.3	SP1	6.881	0.867711538891048	308	318	-1	tcctctccccc
MA0079.3	SP1	6.193	0.859055711241474	313	323	-1	tcccttcctct
MA0079.3	SP1	13.766	0.954332721110978	337	347	-1	gcccctcccca
MA0079.3	SP1	9.296	0.898095003678431	413	423	-1	tccccacccta
MA0079.3	SP1	6.930	0.868328014988183	786	796	-1	ccaccacctcc
MA0079.3	SP1	9.866	0.905266256236943	802	812	-1	gcaccaccccc
MA0079.3	SP1	12.677	0.940631854380767	1322	1332	-1	cctccgcctcc
MA0079.3	SP1	12.677	0.940631854380767	1459	1469	-1	cctccgcctcc
MA0079.3	SP1	11.231	0.922439518942856	1465	1475	-1	cgcccgcctcc
MA0079.3	SP1	6.183	0.858929899793079	1788	1798	-1	cctctgcctcc
MA0084.1	SRY	8.914	0.908001454386283	1399	1407	1	aaaaacaaa
MA0084.1	SRY	7.546	0.862201486441934	2030	2038	-1	ggtcacaat
MA0095.2	YY1	10.314	0.874521551254532	25	36	1	caacatggtgaa
MA0095.2	YY1	10.314	0.874521551254532	1512	1523	1	caacatggtgaa
MA0095.2	YY1	9.157	0.857734354137966	1559	1570	1	catgatggcggg

RNA binding proteins bind to circRNA flanking intron sequences and regulate the expression of circRNA [23]. Based on UCSC, the reverse complementary sequence of circ-0081343 flanking sequence was 585bp in length with 83.76% match (490/585). The specific complementation of the flanking sequences was intron 3 complementary sequence combination (upper line) and intron 4 complementary sequence combination (lower line) (Fig. S5A). CircRNAs with RNA-binding protein binding have reverse complementary sequences in their upstream and downstream introns, particularly reverse complementary Alu repeats [24]. Alu repeats are the most abundant moderately repetitive sequences in the genome and the largest family of short dispersed repeats (SINEs), with a length of approximately 300 bp and an Alu sequence occurring every 3–4 kb, accounting for approximately 10% of the genome [24]. We found that the circ-0081343 flanking intron was present with six Alu sequences, three each upstream and downstream (Fig. S5B and C).

To investigate whether the formation of circ-0081343 is related to intron or Alu sequences, we used PCDNA3.1 to construct three vectors, inserting circ-0081343 without intron sequences, circ-0081343 with complete upstream and downstream intron sequences, and circ-0081343 with Alu sequences removed, respectively (Fig. S6A). RT-PCR results showed that vectors without intron sequences were unable to express circ-0081343, while vectors containing introns, with or without Alu sequences, were able to express circ-0081343, suggesting that circ-0081343 expression was independent of the Alu sequence, but dependent on the upstream and downstream introns (Fig. S6B). RNA pull-down and Western blot results showed that the Intron3 and Intron4 of cicr-0018343 can interact with RBM8A, while the interaction of Intro3 and RBM8A was stronger, suggesting that RBM8A might regulate circ-0018343 expression (Fig. S6C and D). Expression of circ-0081343 was decreased after overexpression of RBM8A and increased after silence of RBM8A, implying that RBM8A down-regulated circ-0081343 expression (Fig. S6I and J).

FGR is one of the leading causes of neonatal morbidity and mortality, severely affecting the quality of long-term survival of affected children, but its pathogenesis has not been fully elucidated [1]. Maternal, fetal, or placental factors are thought to be the most important factors in the development of FGR [1]. Although various mechanisms contribute to the development of FGR, they all result in uteroplacental dysperfusion [5]. The proliferation, migration and invasion of extravillous trophoblast cells are important aspects of embryo implantation and placenta formation [25]. Abnormalities invasion and migration abilities trophoblast cells impaired placental development, leading to a reduced blood flow exchange between the mother and the fetus, which is the main cause of FGR [26, 27]. Excessive or insufficient trophoblast autophagy affects the function of the placenta and promotes the development of FGR [28, 29]. Studies have shown that CircRNAs are involved in the regulation of human embryonic development, but whether CircRNAs regulate trophoblast autophagy is still unknown. We previously analyzed the expression profile of circular RNA in FGR and found that circ-0081343 was significantly decreased in human FGR placentas [30]. However, the mechanisms of how the circ_0081343 mediates placental development are not well studied.

In this study, we used RNA pull-down to analyze the proteins that interact with circ-0081343 in trophoblast cells. Based on RNA pull-down and predicting circ-0018343 interacted proteins results, we identified RBM8A as a binding target of circ-0018343. RBM8A, also known as Y14, is a newly discovered RNA-binding motif protein that regulates RNA transcription, translation, cell cycle regulation and apoptosis [31, 32]. RBM8A binds to the transcription factor STAT3 in the nucleus to activate STAT3 pathway [33, 34]. Here, we found that circ-0018343 did not regulate the mRNA and protein expression of RBM8A but mediated the nuclear transport of RBM8A in trophoblast cells. Proteins must be transported efficiently through the nuclear pore complex (NPC) after their synthesis, which necessitates the presence of a nuclear localization signal (NLS) on these cargo proteins [19]. NLS is usually classified into classical NLS and non-classical NLS [19].The classical NLS is divided into two categories: "monopartite" and "bipartite". Monopartite NLS is a single cluster composed of 4–8 basic amino acids and bipartite NLS is two clusters of 2–3 positively charged amino acids [19].Unlike classical NLS, non-classical NLS is characterized by 20–30 amino acids that assume a disordered structure [19]. The classical NLS is recognized by nuclear transport receptors Importin α/β, while non-classical NLS is recognized by Importin β [35]. Importin13 (IPO13) is a member of the importinβ protein family and a rare bidirectional nucleoplasmic transporter protein receptor. It has been reported that IPO13 functions as an RBM8A entry translocation receptor and interacts directly with RBM8A in the RBMS-MGN dimer in HeLa cells [18]. In trophoblast cells, IPO13 was negatively regulated by circ-0018343 and mediated the intracellular localization of RBM8A. Prediction of amino acid sequence showed that RBM8A carries two NLSs, indicating that RBM8A is likely to transport the nucleus through the NLS sequence. The Co-IP assay showed that NLS mutated RBM8A protein were unable to bind to IPO13 and failed to transport to nucleus. Therefore, circ-0081343 restrained RBM8A nuclear transportation through binding to RBM8A in cytoplasm and down-regulation of IPO13.

We next integrated the RNA-Seq and ChIP-Seq data and suggested cytokine receptor interactions, metabolic pathways, PI3K-AKT signaling pathway, p53 signaling pathway and NOD-like receptor signaling pathway might be the potential pathways that regulated by circ-0081343 and RBM8A in trophoblast cells. The PI3K/AKT pathway is a classical autophagic signaling pathway that plays an important role in regulating cellular autophagy [36]. Herein, we found that circ-0081343/RBM8A induced autophagy via inactivating PI3K/AKT pathway in trophoblast cells. However, whether circ-0081343 promoted autophagy in trophoblast cells via induction of autophagosome formation or blockade of autophagosomes degradation was unknown. It has been reported that RBM8A regulates autophagy through PI3K or mTOR signaling pathways [37]. Our results showed that induction of nuclear RBM8A inhibited autophagy via activating PI3K/Akt pathway. Furthermore, circ-0018343-mediated autophagy was restrained by overexpression of RBM8A. Using Chip-Seq analysis, we found that the nuclear RBM8A could bind to PI3K3C3 promoter and downregulated PI3K3C3. The PI3K-III complex formed by PI3K3C3 and Beclin1 protein regulates the formation of bilayer membrane structures in pre-autophagic vesicles [38]. We also found that underexpression of circ-008134 had adverse effect on PI3K3C3 via interaction with nuclear RBM8A, result in insufficient autophagy. Taken together, insufficient circ-0081343 expression increased the nuclear transport of RBM8A, which downregulated PI3K3C3 through binding its promoter, inhibiting autophagy. suggesting that circ-0081343 is likely to affect the FGR process through autophagy.

Currently, FGR animal models are divided into three main categories according to the intervention method: maternal intervention, placental intervention and genetic models [39].The pathogenesis of FGR in animals induced by uterine vascular ligation is similar to that of human disease. In this study, the FGR mouse model of FGR was established by bilateral partial ligation of uterine arterioles using the Wigglesworth method to verify whether circ-0081343 regulated FGR process through autophagy [40]. The incidence of FGR fetuses induced in this study was as high as 47%. We injected circ-0081343 adenovirus to FGR mice and observed that pup birth weight and the number of live pups were significantly increased, suggesting circ-0081343 improved pregnancy outcomes and promote fetal growth and development in FGR mouse model. The uteroplacental circulation starts with the maternal blood flow into the intervillous space [41]. Labyrinth is the site of transport between fetal and maternal blood in the mouse placenta and the labyrinth dysplasia is a major cause of intrauterine growth restriction in fetuses [42]. Bilateral partial uterine artery ligation reduces the area of the placental vagus zone, which impairs the placenta's material transport function, resulting in impaired material exchange between the fetus and the mother and the development of FGR. Overexpression of circ-0081343 was able to reverse the placental dysfunction caused by this ischemic-hypoxic state, increasing the area of the placental vagus zone.

Autophagy is present in the normal human placenta and is associated with placental dysfunction [43]. The autophagy related 16 like 1 (Atg16L1) was found to be significantly reduced in the placenta of preterm birth than that at full term and the insufficient expression could promote inflammatory responses, which might be closely associated with preterm birth [44]. In preeclampsia, impaired autophagy exacerbates the lysosomal biogenesis defect and accumulation of toxic protein aggregates, which may result in poor placentation and the production of pregnancy-incompatible milieus, such as anti-angiogenic factors, extracellular vesicles, and alarmins [45]. Moreover, inhibition of placental autophagy predisposes offspring to obesity, as well as metabolic and cardiovascular diseases [46]. The abnormal expression of Beclin1 and p62 in FGR placenta compared to normal placental suggested that autophagy was impaired in FGR mice. The circ-0018343 could restore the impaired autophagy in FGR placenta via inactivating P13K/AKT pathway through up-regulation of nuclear RBM8A-mediated PI3K3C3, improving pathological placenta and bodyweight of pups.

RBPs play a key role in the regulation of CircRNAs by binding to the flanking intron reverse complementary sequence [47, 48]. Knockout of Alu sequences had no effect on the regulation of circ-0081343, whereas knockout of intron flanking sequences significantly reduced circ-0081343 expression, implying that upstream and downstream intron sequences were important for the regulation of circ-0081343. Furthermore, RNA pull-down results revealed that RBM8A bound to the circ-0081343 flanking intron sequence and downregulated its expression.

In summary, insufficient expression of circ-0081343 induced nuclear translocation of RBM8A by promoting the binding of IPO13 to RBM8A-NLS, and the nuclearized RBM8A inhibited cellular autophagy through down-regulating PI3K3C3 in FGR placenta (Fig. 9D). Moreover, RBM8A also regulated the expression of circ-0081343 by binding to the flanking sequences involved in circ-0081343. Our study explored the mechanism of circ-0081343-mediated nuclear translocation of RBM8A inhibited trophoblast autophagy in vivo and ex vivo, offering new insights into the pathogenesis of FGR.

Acknowledgement

We thank the team of Zhao Liang from the department of pathology in Southern Medical University for valuable suggestion. We acknowledge BioRender software (biorender.com) for creation of figures. This study was supported by research grants from National Natural Science Foundation of China cultivation Project 530（NO. 2021MSXM02）.

Authors’ contributions

Linmei Zheng, Junbo Fang and Fujin Liu planned and performed the in-animal experiments and analyzed the data with Haoyue Hu, such as HE staining, Immunohistochemistry (IHC) and established FGR mouse model. Linmei Zheng, Rong Tang and Haoyue Hu performed and analyzed RNA Pull-Down and RNA Binding Protein Immunoprecipitation experiments. Qiong Tang generated and characterized the shRNA constructs. Linmei Zheng, Rong Tang, Haoyue Hu and Qiong Tang contributed to the cell experiments. The manuscript was written by Linmei Zheng and Rong Tang commented on by all authors.

Funding

Partial financial support was received from National Natural Science Foundation Cultivation 530 Project of Hainan General Hospital

The research leading to these results received funding from Hainan Natural Science Foundation of China under Grant Agreement No. [821QN395 and 822MS167)].

This study was funded by Hainan Natural Science Foundation of China (NO. 821QN395 and NO.822MS167), National Natural Science Foundation Cultivation 530 Project of Hainan General Hospital (2021MSXM02) and Guangdong Natural Science Foundation of China (NO. 2021A1515011845).

This work was supported by Hainan Natural Science Foundation of China (NO. 821QN395 and NO.822MS167) and Guangdong Natural Science Foundation of China (NO.2021A1515011845). Grant numbers 821QN395, 822MS167 and 2021A1515011845.

Availability of data and materials

The materials supporting the conclusion of this review has been included within the article.

Ethics approval and consent to participate

All experimental procedures conformed to the guidelines of Care and Use of Laboratory Animals of China for animal experimentation.

Procedures involving animals and their care were conducted in conformity with NIH guidelines (NIH Pub. No. 85-23, revised 1996) and was approved by Animal Care and Use Committee of the Hainan Medical University

Consent for publication

Not applicable

Conflict of interest

The authors declare no competing interests

Author details

¹Department of Obstetrics, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, Haikou, Hainan, China

²Department of General Surgery, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, Haikou, Hainan, China

³Southern Medical University, Guangzhou, Guangdong China

⁴Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong China

⁵Department of pathology, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, Haikou, Hainan, China

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No competing interests reported.

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Circ-0081343 binds and activates RBM8A nuclear translocation to promote autophagy in Fetal growth restriction

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Abstract

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Introduction

Material And Methods

Cell culture

RNA Pull-Down

Liquid Chromatography Mass Spectrometry (LC-MS)

Silver protein staining

RNA Binding Protein Immunoprecipitation (RIP)

Quantitative real-time PCR (qRT-PCR)

Cell transfection

Chromatin immunoprecipitation (ChIP)

RNA seq and ChIP seq

Transmission electron microscopy (TEM)

Subcellular fractionation and Western blot

Co-Immunoprecipitation (Co-IP)

Immunofluorescence

HE staining

Immunohistochemistry (IHC)

FGR mouse model

Statistical analysis

Result

circ-0081343 mediates nuclear translocation of RBM8A

Circ-0081343 Mediates Nuclear Translocation Of Rbm8a Via Ipo3

Nuclear Translocation Of Rbm8a Inhibits Autophagy Via P13k/akt Pathway

Circ-0081343 Improves Pregnancy Outcome In Fgr Mouse Model Via Restoring The Impaired Autophagy

Rbm8a Downregulates Circ-0081343 Expression

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1