The regulatory effect of microRNA-21a-3p on the promotion of telocyte angiogenesis mediated by PI3K (p110α)/AKT/mTOR in LPS induced mice ARDS.

BACKGROUND: Telocytes (TCs) are newly identified interstitial cells that participate in tissue protection and repair. The present study investigated the mechanisms underlying the protective effect of TCs in a mouse model of respiratory distress.
METHODS: The mouse model of acute respiratory distress syndrome (ARDS) was established by intratracheal instillation of lipopolysaccharide (LPS). After instillation of TCs culture medium, lung injury was assessed, and angiogenesis markers, including CD31 and endothelial nitric oxide synthase (eNOS), were detected by immunofluorescence. Bioinformatics analysis was used to screen significantly differentially expressed microRNAs (miRNAs) in cultured TCs stimulated with LPS, and the regulation of downstream angiogenesis genes by these miRNAs was analysed and verified. PI3K subunits and pathways were evaluated by using a PI3K p110α inhibitor to study the involved mechanisms.
RESULTS: In ARDS mice, instillation of TCs culture medium ameliorated LPS-induced inflammation and lung injury and increased the protein levels of CD31 and eNOS in the injured lungs. A total of 7 miRNAs and 1899 mRNAs were differentially regulated in TCs stimulated with LPS. Functional prediction analysis showed that the differentially expressed mRNAs were enriched in angiogenesis-related processes, which were highly correlated with miR-21a-3p. Culture medium from TCs with miR-21a-3p inhibition failed to promote angiogenesis in mouse models of LPS-induced ARDS. In cultured TCs, LPS stimulation upregulated the expression of miR-21a-3p, which further targeted the transcription factor E2F8 and decreased Notch2 protein expression. TCs culture medium enhanced hemangioendothelioma endothelial cells (EOMA cells) proliferation, which was blocked by the miR-21a-3p inhibitor. The PI3K p110α inhibitor decreased vascular endothelial growth factor levels in LPS-stimulated TCs and reversed the enhancing effect of TCs culture medium on EOMA cells proliferation.
CONCLUSIONS: TCs exerted protective effects under inflammatory conditions by promoting angiogenesis via miR-21a-3p. The PI3K p110α subunit and transcriptional factor E2F8 could be involved in this process.

Introduction

Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterised by acute progression of respiratory failure. According to an international multi-centre research, the prevalence of ARDS was 10.4% of ICU admissions [1]. Inflammatory responses destroy underlying vascular endothelial cells and respiratory epithelial cells and impair the lungs’ ability to exchange oxygen and carbon dioxide [2]. Therefore, decreasing inflammation and accelerating blood vessel repair are two key factors in the prevention and treatment of ARDS. Since its severity and lack of effective pharmacologic treatments [3], it is of great significance to explore novel therapeutic strategies for ARDS. Recently, cell therapy have been shown to have promising therapeutic potential. Mesenchymal stem cells ameliorated ARDS due to paracrine mechanism [4].

Telocytes (TCs) are newly identified mesenchymal cells that play a role in providing nutrition to surrounding cells by cell–cell communication and have post-injury repair and regeneration functions [5-7]. TCs contribute to angiogenesis within the myocardium [8]. Transplantation of cardiac TCs promotes post ischaemic myocardial repair [9]. Pulmonary TCs also assist with angiogenesis since they participate in forming the structure of the air–blood barrier [10]. Intratracheal administration of activated TCs has been reported to alleviate ventilator-induced lung injury in a mouse model by releasing angiogenic factors [11]. However, the underlying mechanism remains unclear.

Class I Phosphoinositide-3-kinases (PI3Ks) or the four subtypes of catalytic subunit—p110α, p110β, p110γ and p110δ—are expressed in all mammalian cells. The catalytic subunits bind to p85 regulatory subunits, activate receptor tyrosine kinases (RTKs), and transmit a variety of cell surface receptor signals, such as those from the epidermal growth factor receptor (EGFR) or fibroblast growth factor receptor (FGFR), to promote cell growth [12]. The PI3K subunits p110α and p110δ were demonstrated to be associated with tissue repair; however, this function is mediated by different mechanisms. The activity of PI3K p110α can be enhanced by tyrosine kinase ligands, such as vascular endothelial growth factor (VEGF) A, and can induce angiogenesis and vascular remodelling [13]. Moreover, p110α regulates endothelial cell migration through the small GTPase RhoA, mediated by PI3KCG, a gene encoding a p110γ subunit, which has a protective effect on hypoxic-reoxygenated cardiomyocytes mediated by activation of the PI3K/AKT signalling pathway and inhibition of apoptosis [14]. PI3K (p110δ)/AKT/mammalian target of rapamycin (mTOR) signalling pathway mediates interferon-γ (IFN-γ) induced airway epithelial cell growth and proliferation through interaction with CEACAM1 [15].

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the expression of target genes via posttranscriptional degradation of mRNA and/or translational inhibition of protein expression. MiR-135a can influence cell proliferation, migration, invasion, apoptosis and tumour angiogenesis through the IGF-1/PI3K/AKT signalling pathway in non-small cell lung cancer (NSCLC) [16]. Mature miR-21a-5p was found to be secreted by lipopolysaccharide (LPS)-activated macrophages in small vesicles, which were endocytosed and internalised by renal fibroblasts, thereby promoting the expression of fibrosis and inflammation markers in a mouse model of chronic renal allograft dysfunction (CAD) in allogeneic kidney transplantation [17]. Antagonism of miR-21a-5p ameliorated CAD in mouse model following kidney transplantation [17]. In patients with renal allograft, elevation of urinary [18] and plasma [19] miR-21 level was correlated with interstitial fibrosis and tubular atrophy.

The TCs line was established by transfection with simian vacuolating virus 40 (SV40) and identified to maintain TCs morphology and immune characteristics [20]. TCs proliferation was demonstrated to be regulated by transforming growth factor-β (TGF-β) and mediated by the PI3K p110α subunit and the PI3K/AKT/mTOR signalling pathway [21]. The present study was designed to investigate the underlying protective effect of TCs in a mouse model of respiratory distress. Bioinformatics approaches were applied to analyse gene expression profiles in TCs challenged with LPS. Particular attention was devoted to the angiogenesis-related process. The protective mechanisms mediated by the PI3K subunit in TCs were further examined in hemangioendothelioma endothelial cells (EOMA cells) in vitro. The current study presents the theoretical bases of an alternative new potential therapeutic strategy for ARDS.

Methods

Animal models

Eight-week-old male C57BL/6 mice, 22 to 25 g, were purchased from Shanghai Jiesijie Company (Shanghai, China). Mice were randomly divided into four groups: Control, ARDS, ARDS with negative control (NC) TCs treatment, and ARDS with miR-21a-3p inhibited TCs treatment. Under anaesthesia (60 mg/kg sodium pentobarbital, Sinopharm Chemical Reagent Co. Shanghai, China), mice were intratracheally instilled with phosphate-buffered saline or LPS (5 mg/kg, Sigma, Germany) via 20-gauge catheters. Mice in the ARDS treatment groups were also instilled with 20 μL of TCs culture medium from TCs treated with the NC or miR-21a-3p inhibitor in the presence of LPS. Twenty-four hours later, animals were sacrificed, and the lungs were collected.

The study protocol was approved by the Animal Ethics Committee of Zhongshan Hospital, Fudan University.

TCs

Mouse primary pulmonary TCs were a kind gift from Dr. Dongli Song. TCs were cultured in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, Hyclone, Boston, MA) supplemented with 5% foetal bovine serum (FBS, Cellsera, Australia). Experiments with LPS (0.1 μg/mL) were performed in DMEM/F12 without FBS. TCs culture medium was collected from culture dishes after LPS stimulation for 48 h. The p110α inhibitor HS-173 (Selleck, Shanghai, China) was applied 2 h before LPS stimulation.

MiRNA transfection

Both the miR-21a-3p inhibitor and NC were purchased from China Ribobio (Ribobio, Guangzhou, China). TCs were transfected with the miR-21a-3p inhibitor and NC at a final concentration of 50 nmol/L using a lipofectamine RNAiMAX transfection system (ThermoFisher Scientific, Carlsbad, CA) according to the manufacturer’s protocol. Cells were incubated with siRNA in serum-free and antibiotic-free medium for 6 h and then in normal growth medium for another 24 h before the experiments were performed.

Gene expression profiling analysis

Gene expression profiling analysis of both miRNA and mRNA were performed with Agilent Microarray Scanner (Cat # G2565CA, Agilent technologies, Santa Clara, CA). The data were normalised with the AgiMicroRna package [22]. The gene expression files were analysed with R-3.4.1 software. Differentially expressed genes (DEGs) were defined as those with an adjusted P-value of less than 0.05. DEGs were further analysed with the limma package [23]. Heat maps were generated with the ggplot2 package [24].

The online databases miRWalk [25] and TargetScan [26] were used to screen potential miRNA target genes. Overlapping genes in the two databases were selected for further analysis. The online database STRING [27] and the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 [28] were used to analyse gene function. The relationship between DEGs and miRNAs was further visualised with Cytoscape 3.7.1 [29].

Quantification of mRNA and miRNA

Total RNA was extracted from cultured TCs with TRIzol (Takara, Shiga, Japan) according to the provided instructions. MiRNAs were reverse transcribed with a Bulge-Loop miRNA qRT-PCR Starter Kit (Ribobio, Guangzhou, China), and mRNAs were reverse transcribed to complementary DNA (cDNA) with a PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan). The expression levels of miR-21a-3p, miR-221-5p and mRNAs were measured by quantitative real-time polymerase chain reaction (qPCR) on a Bio-Rad IQ5 real-time PCR instrument, with U6 and GAPDH used as the housekeeping genes for miRNAs and mRNAs, respectively. MiRNA PCR was performed with the Bulge-Loop miRNA qRT-PCR Starter Kit, Bulge-Loop mmu-miR-21a-3p Primer Set and Bulge-Loop mmu-miR-221-5p Primer Set (Ribobio, Guangzhou, China). MRNA primers were synthesised by Sangon (Shanghai, China). The following mouse-specific primers were used: GAPDH sense primer: 5′-GTTCAACGGCACAGTCAAG-3′, antisense primer: 5′-GCCAGTAGACTCCACGACAT-3′; E2F8 sense primer: 5′-CTGTTT GCACGAACACTTATCAG-3′, antisense primer: 5′-GTACCGCGCTAGGAATTTGTG-3′; Acvrl1 sense primer: 5′-TGATTCCTGTTGCCGGCCT-3′, antisense primer: 5′-CAGTGTGGGCTCTCACAAGT-3′; Rbpj sense primer: 5′-TGGCGAGAGTTTGTGGAAGA-3′, antisense primer: 5′-AGCACTGTTTGATCCCCTCG-3′; Notch1 sense primer: 5′-TGTGGCTTCCTTCTACTGCG-3′, antisense primer: 5′-CTTTGCCGTTGACAGGGTTG-3′; Flt1 sense primer: 5′-GTGAGCACTGCGGCAAAAAG-3′, antisense primer: 5′-ACTCATTTTGGGAGGAGCGT -3′; EFNB2 sense primer: 5′-CGAGGTGGCAACAACAATGG-3′, antisense primer: 5′-ATAGTCCCCGCTGACCTTCT -3′; Thbs1 sense primer: 5′-CTGCCAATCATAACCAGCG-3′, antisense primer: 5′-TTCGTTAAAGGCCGAGTGCT-3′; EPAS1 sense primer: 5′-CTGAGGAAGGAGAAATCCCGT-3′, antisense primer: 5′-TGTGTCCGAAGGAAGCTGATG-3′; hypoxia inducible factor-1α (HIF-1α) sense primer: 5′-ACCTTCATCGGAAACTCCAAAG-3′, antisense primer: 5′-CTGTTGGCTGGGAAAAGTTAGG-3′; PIK3CA sense primer: 5′-CCACGACCATCTTCGGGTG-3′, antisense primer: 5′-ACGGAGGCATTCTAAAGTCACTA-3′; PIK3CB sense primer: 5′-CTATGGCAGACAACCTTGACAT-3′, antisense primer: 5′-CTTCCCGAGGTACTTCCAACT-3′; PIK3CD sense primer: 5′-GTAAACGACTTCCGCACTAAGA-3′, antisense primer: 5′-GCTGACACGCAATAAGCCG-3′; and VEGF sense primer: 5′-GTACCTCCACCATGCCAAGT-3′, antisense primer: 5′-TCCTATGTGCTGGCTTTGGT-3′.

Western blotting

Total protein was extracted from cultured TCs with lysis buffer (150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L NaF, 1 mmol/L dithiothreitol, 10 μg/μL aprotinin, 10 μg/μL leupeptin, 0.1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 0.5% NP-40). Protein extracts (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany). After blocking with 5% non-fat milk/Tris-buffered saline containing 0.1% Tween 20 at room temperature for one hour, membranes were incubated with primary antibodies [specific for GAPDH (60004-1-Ig, Proteintech, Wuhan, China), E2F8 (ab109596, Abcam, Cambridge, UK), Delta-like 4 (DLL4)(ab7280, Abcam, Cambridge, UK), Notch1 (sc-373891, Santa Cruz, Dallas, TX), Notch2 (sc-5545, Santa Cruz, Dallas, TX), Notch4 (sc-5594, Santa Cruz, Dallas, TX), phosphatase and tensin homolog deleted on chromosome ten (PTEN)(ab32199, Abcam, Cambridge, UK), PI3K (4257T, CST, Boston, MA), p-PI3K (4228T, CST, Boston, MA), mTOR (2983T, CST, Boston, MA), p-mTOR (5536T, CST, Boston, MA), AKT (9272S, CST, Boston, MA), p-AKT (9271S, CST, Boston, MA), and p110α (4249T, CST, Boston, MA)] overnight at 4 °C. Protein expression levels were normalised to those of GAPDH with ImageJ (NIH, Bethesda, MD).

EOMA cells proliferation assay

EOMA cells proliferation was assessed with a colorimetric assay—Cell Counting Kit-8 (CCK8, Yeasen, Shanghai, China)—following the manufacturer’s protocol. Approximately 4000 EOMA cells/well were seeded in a 96-well plate. After adhesion, EOMA cells were incubated for 24 h with culture medium from TCs transfected with the miR-21a-3p inhibitor or NC in the presence of LPS.

Dual luciferase assay

The pGL3 reporter vector (Promega, Madison, WI) was used to generate the plasmids pGL3-WT-E2F8-3′-UTR and pGL3-Mut-E2F8-3′-UTR. Human embryonic kidney cells were co-transfected with pGL3-E2F8-3′-UTR (WT or Mut) and the miR-21a-3p mimic or NC with Lipofectamine 2000 reagent (ThermoFisher Scientific, Carlsbad, CA). After incubation for 24 h, luciferase activity was assessed by the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) according to the manufacturer’s protocol.

Enzyme-linked immunosorbent assay (ELISA)

The concentration of VEGF in the TCs culture medium was measured by a commercial VEGF ELISA kit (Westang, Shanghai, China) according to the manufacturer’s protocol.

Dynamic real-time cell observation

Live observation of EOMA cells was performed with a Cell-IQ cell culture platform (Chip-Man Technologies, Tampere, Finland) equipped with a phase contrast microscope (Nikon CFI Achromat phase contrast objective with 10 magnification) and a camera (Nikon, Fukasawa, Japan). The equipment was controlled by Imagen software (Chip-Man Technologies). Each group contained 16 replicates of visual fields. Images were acquired at 1-h intervals for 48 h.

Tissue preparation and immunofluorescence examination

Lung tissues were fixed with 10% formalin solution and embedded in paraffin. Each tissue was sectioned at 5 μm and stained with haematoxylin–eosin (HE, Beyotime, Shanghai, China) according to the manufacturer’s protocol. For immunofluorescence staining, an antigen retrieval protocol was carried out with incubation in 0.3% H2O2 for 30 min and heating to boiling in a microwave in citrate buffer for 10 min. After blocking with 5% goat serum in Tris-buffered saline, sections were incubated with diluted primary antibodies [CD31 (1:500, ab24590, Abcam, Cambridge, UK), endothelial nitric oxide synthase (eNOS) (1:500, Cat610296, BD Biotechnology, San Jose, CA)] overnight at 4 °C and then with secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI), separately.

Statistical analysis

Data are expressed as the means ± SDs and were analysed by one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test. A P-value of < 0.05 was considered statistically significant. All statistical analyses were performed with GraphPad Prism 7.04 (GraphPad, San Diego, CA).

Results

Protective effects of TCs in ARDS

The ability for TCs protection was first estimated in ARDS mouse models. LPS stimulation caused inflammatory infiltration, alveolar wall widening, and vessel destruction (Fig. 1a). The production of inflammatory cytokines was elevated in ARDS mice (Fig. 1b). Since substances, including molecules and exosomes, released by TCs could be important factors affecting adjacent cells, the effect of TCs culture medium was assessed. Instillation of TCs culture medium reduced the inflammatory infiltration, reduced the alveolar interstitial width and decreased the levels of inflammatory cytokines. Bio-behaviours of TCs were recorded by Cell-IQ to show the typical morphology of cultured cells (Additional file 1: Figure S1).

Fig. 1

Protective effects of TCs in ARDS mouse models. a HE staining of mice lung tissue in lungs of control mice (Control), ARDS mice (LPS), ARDS mice treated with TCs supernatant (LPS/TC), and ARDS mice treated with miR-21a-3p inhibited TCs supernatant [LPS/TC (miR-21 inhibited)]. b The mRNA expression of inflammatory cytokines in mice lungs in the above four groups. *P < 0.05 vs Control, P < 0.05 vs LPS, **P < 0.05 vs LPS/TCs, n = 6. IL-1β interleukin-1β, IL-6 interleukin-6, TNF-α tumour necrosis factor-α

TCs promoted angiogenesis in ARDS

As angiogenesis is essential in tissue repair, the induction of angiogenic factors in TCs after stimulation with LPS was assessed. In ARDS mice, the expression of the angiogenesis-related marker CD31 and eNOS was downregulated. However, an increase in CD31 and eNOS expression was observed in the WT TCs treatment group but not in the group treated with medium from TCs with miR-21a-3p inhibition (Fig. 2).

Fig. 2

TCs promoted angiogenesis in ARDS mouse models. The expression of CD31 and eNOS in lungs of control mice (Control), ARDS mice (LPS), ARDS mice treated with TCs supernatant (LPS/TC), and ARDS mice treated with miR-21a-3p inhibited TCs supernatant [LPS/TC (miR-21 inhibited)] was shown in green fluorescence, dapi was blue. *P < 0.05 vs Control, P < 0.05 vs LPS, **P < 0.05 vs LPS/TCs, n = 6. Original magnification: 200x

MiRNA and mRNA profiles in LPS-stimulated TCs

To identify the critical miRNAs in the regulation of angiogenesis by TCs, miRNA and mRNA profiles were generated, and the relationship of differentially expressed miRNAs with downstream angiogenesis factor-associated mRNAs were analysed in LPS-stimulated TCs. In LPS-stimulated TCs, six miRNAs, including miR-155-5p, miR-21a-3p, miR-5100, miR-221-5p, miR-7a-3p and miR-146a-5p, were upregulated, and one miRNA (miR-188-5p) was downregulated with an absolute fold change > 2 (Fig. 3a and Table 1). By referring these results to two online databases (miRWalk and TargetScan), 4368 target genes were predicted to be downstream targets of the differentially expressed miRNAs.

Fig. 3

Differentially expressed genes in TCs with LPS treatment. a Heat map of differential expressed miRNAs in cultured TCs stimulated with LPS. b Heat map of differential expressed genes in cultured TCs stimulated with LPS. c Relationship between differential expressed miRNAs and their differential expressed target mRNAs. Yellow indicated miRNAs, green indicated downregulated mRNAs, and red indicated upregulated mRNAs. d Interaction of the angiogenesis-related proteins in STRING. Different colours indicated the involvement of proteins in different processes. Red indicated angiogenesis, pink indicated blood vessel morphogenesis, purple indicated vasculature development, brown indicated blood vessel remodelling, blue indicated sprouting angiogenesis, cyan indicated venous blood vessel sprouting, orange indicated venous blood vessel morphogenesis, green indicated regulation of angiogenesis, and yellow indicated positive regulation of angiogenesis. e Angiogenesis related miRNAs and their downstream genes. Red indicated upregulated miRNAs, green indicated downregulated miRNAs, yellow indicated genes that were enriched in more than three processes in STRING, blue indicated other mRNAs

Table 1

Summary of differentially expressed miRNAs in TCs treated with LPS

Gene symbols	Fold change	Gene symbols	Fold change	Gene symbols	Fold change
mmu-miR-146a-5p	8.761	mmu-miR-1231-3p	1.378	mmu-miR-6237	1.067
mmu-miR-7a-5p	2.813	mmu-miR-1948-3p	1.370	mmu-miR-7059-3p	1.065
mmu-miR-221-5p	2.407	mmu-let-7i-5p	1.350	mmu-miR-881-3p	1.064
mmu-miR-5100	2.330	mmu-miR-29b-3p	1.347	mmu-miR-3074-5p	1.063
mmu-miR-21a-3p	2.258	mmu-miR-3967	1.337	mmu-miR-7216-5p	1.063
mmu-miR-155-5p	2.067	mmu-miR-487b-3p	1.333	mmu-miR-770-5p	1.063
mmu-miR-33-5p	1.947	mmu-miR-1981-3p	1.300	mmu-miR-6901-5p	1.062
mmu-miR-96-5p	1.930	mmu-miR-6948-5p	1.299	mmu-miR-3092-5p	1.062
mmu-miR-532-3p	1.901	mmu-miR-6395	1.292	mmu-miR-302c-5p	1.061
mmu-miR-129-5p	1.845	mmu-miR-21a-5p	1.287	mmu-miR-6988-3p	1.060
mmu-miR-532-5p	1.837	mmu-miR-6367	1.263	mmu-miR-3109-3p	1.058
mmu-miR-211-3p	1.771	mmu-miR-7002-5p	1.251	mmu-miR-1668	1.057
mmu-miR-19a-3p	1.768	mmu-miR-27a-3p	1.219	mmu-miR-3572-3p	1.055
mmu-miR-6951-3p	1.766	mmu-miR-5623-5p	1.203	mmu-miR-451b	1.054
mmu-miR-210-3p	1.763	mmu-miR-23b-5p	1.190	mmu-miR-7007-3p	1.051
mmu-miR-126b-3p	1.756	mmu-miR-302b-5p	1.186	mmu-miR-6957-5p	1.048
mmu-miR-221-3p	1.738	mmu-miR-669g	1.181	mmu-miR-219b-5p	1.045
mmu-miR-6369	1.713	mmu-miR-1b-5p	1.167	mmu-miR-377-5p	1.043
mmu-miR-92a-3p	1.712	mmu-miR-147-3p	1.158	mmu-miR-6921-5p	1.043
mmu-miR-18a-5p	1.675	mmu-miR-6994-3p	1.123	mmu-miR-7040-3p	1.043
mmu-miR-802-5p	1.662	mmu-miR-7680-3p	1.114	mmu-miR-7068-3p	1.042
mmu-miR-1897-5p	1.631	mmu-miR-7673-5p	1.101	mmu-miR-376c-5p	1.040
mmu-miR-129-2-3p	1.615	mmu-miR-6908-3p	1.090	mmu-miR-7059-5p	1.039
mmu-miR-7211-5p	1.611	mmu-miR-3572-5p	1.088	mmu-miR-7684-3p	1.038
mmu-miR-210-5p	1.594	mmu-miR-106a-3p	1.084	mmu-miR-181d-3p	1.037
mmu-miR-362-3p	1.576	mmu-miR-6964-5p	1.083	mmu-miR-6942-3p	1.036
mmu-miR-218-5p	1.540	mmu-miR-6407	1.082	mmu-miR-511-3p	1.034
mmu-miR-6396	1.537	mmu-miR-6954-5p	1.081	mmu-miR-741-3p	1.034
mmu-miR-17-5p	1.517	mmu-miR-344h-3p	1.081	mmu-miR-465d-5p	1.029
mmu-miR-1897-3p	1.512	mmu-miR-467g	1.080	mmu-miR-1955-3p	1.029
mmu-miR-1956	1.479	mmu-miR-154-3p	1.080	mmu-miR-6930-5p	1.027
mmu-miR-20a-5p	1.475	mmu-miR-5625-5p	1.080	mmu-miR-7013-5p	1.025
mmu-miR-34a-5p	1.413	mmu-miR-6389	1.076	mmu-miR-3109-5p	1.025
mmu-miR-222-5p	1.401	mmu-miR-466n-5p	1.071	mmu-miR-7089-3p	1.022
mmu-miR-19b-3p	1.390	mmu-miR-326-3p	1.071	mmu-miR-375-5p	1.018
mmu-miR-7115-3p	1.378	mmu-miR-7672-5p	1.069

In total, 1899 mRNAs—901 upregulated and 998 downregulated—were differentially expressed in TCs after LPS stimulation (Fig. 3b and Table 2). A total of 519 genes overlapped with those from the online prediction (Fig. 3c).

Table 2

Summary of differentially expressed mRNAs in TCs treated with LPS

Gene symbol	Fold change	Gene symbol	Fold change	Gene symbol	Fold change
Saa3	88.57679	Sh3kbp1	1.714612	Aldoa	1.358346
Steap4	39.69958	Gm10382	1.713579	Bcl2l11	1.358341
C3	27.54466	Sphk1	1.711903	Wnt5a	1.358042
Cxcl1	21.9035	Tnfsf10	1.709923	Sdc4	1.357917
Lcn2	18.11751	Fosl2	1.70942	Pttg1	1.357846
Cp	13.17588	Arhgap24	1.703888	Eps8	1.356005
Ccl2	9.407872	Rspo3	1.702523	Parp8	1.355285
Ccl7	9.313701	Rasl11a	1.701653	AI413582	1.355264
Lbp	9.248471	Snhg11	1.699314	H2afj	1.35191
Slpi	8.496525	Pnp2	1.69892	Tapbpl	1.350347
Hp	7.020443	9930111J21Rik2	1.697292	Fam46a	1.349368
Casp4	6.491574	F730043M19Rik	1.696927	Gpr162	1.349043
Oas3	6.472296	Gstt1	1.694392	Itpr2	1.347899
Slc16a2	6.293287	Tor3a	1.694311	Fendrr	1.347418
Kng2	6.13421	Tnfrsf14	1.693478	Eif2ak2	1.346754
Neurl3	6.123261	Pcdhgc5	1.692342	Bcam	1.346359
Lgi2	5.548539	Usp18	1.690192	Tmem192	1.346301
Zbp1	5.500857	Icam1	1.685666	Atxn7l1	1.346144
Cebpd	5.490325	Vnn1	1.684905	Tcirg1	1.346103
Tmem176a	5.352542	Isg20	1.684825	H6pd	1.34602
Tmem176b	5.292897	Cdh23	1.682662	Acad10	1.345811
Cxcl5	5.105818	Hivep2	1.678786	Ggta1	1.344527
Serpina3i	5.062072	Serpinb9	1.676066	Ago4	1.343551
Ly6a	4.890107	Ier3	1.670507	Rpl39	1.343105
Ms4a4d	4.778005	Spidr	1.66764	Arsj	1.342469
Kcnj15	4.627825	Parp10	1.667283	Kif21a	1.341944
Slfn2	4.500739	Jak2	1.667282	Grtp1	1.340769
Nfkbiz	4.420487	Lnx1	1.665956	Hook2	1.340696
Oas1g	4.352056	Dtx3l	1.665121	Pisd-ps1	1.340025
Gm8995	4.258631	Hopx	1.661591	Hbp1	1.33937
Zc3h12a	4.118584	Rsl1	1.6613	Mlkl	1.338835
Mt2	4.050379	Insig2	1.660402	Fbln1	1.338393
H2-Q7	4.021978	Syt17	1.659707	Ern1	1.338122
Kank4	3.966313	Ppp1r3b	1.659039	Unc93b1	1.338107
Gm16685	3.952599	Junb	1.658968	Oplah	1.337688
Phf11b	3.899771	Lhfpl2	1.658507	Dhrs9	1.337432
H2-Q5	3.876445	Aqp3	1.658025	Ank3	1.336823
Gbp5	3.860209	Bst1	1.653379	Hspa1a	1.335089
Gbp3	3.711967	Cd14	1.647933	Fos	1.334711
Xdh	3.706347	Abcb1a	1.64782	Dusp1	1.334023
Sod3	3.705108	Gm43068	1.642165	Tmem53	1.332695
H2-T10	3.615936	Tsc22d1	1.640017	Riok3	1.332508
Adamts7	3.547664	Cfap69	1.639558	Zc2hc1a	1.332429
H2-K1	3.536976	Clca3a1	1.637745	Gata6	1.33174
Ccl5	3.523568	En1	1.637662	Nrp2	1.330772
Lrrc32	3.510921	Pdk1	1.637498	Fam134b	1.330567
Ntn1	3.468896	Pnrc1	1.636296	Dgat2	1.330413
Fas	3.466618	Enpp2	1.635831	Trafd1	1.328525
H2-Q6	3.433254	Abcd2	1.63519	Laptm4b	1.328518
Vcam1	3.350214	Gm16365	1.634922	D930015E06Rik	1.328021
Cd74	3.317107	Fabp4	1.634064	Timp1	1.327406
Ch25h	3.315495	Parp9	1.633709	Itga7	1.327246
Psmb8	3.278491	Serpinb1b	1.63368	Galk2	1.326399
Il6	3.219911	Gm13010	1.633034	Rhbdl3	1.325887
Gm4951	3.2116	Stx6	1.632089	Cdkn2b	1.324984
H2-T23	3.207359	Elf3	1.632065	Psme2	1.324769
Plac8	3.197626	Spp1	1.630536	Sh3bp5	1.32441
Oas2	3.197366	Rnf144a	1.630365	Mif	1.323852
Arrdc4	3.158372	Trp63	1.62831	Pgk1	1.323539
C1ra	3.158361	A330074K22Rik	1.626237	Fam43a	1.322902
Gbp6	3.147647	Sfmbt2	1.625612	Arid3a	1.321845
Slc11a2	3.134662	Gbp11	1.625111	Lars2	1.321181
Ppm1h	3.127213	Ifih1	1.622407	Morc3	1.320638
Ifi47	3.105228	Tnnc1	1.620872	Shb	1.319344
Ccl20	3.098563	Tmem86a	1.61825	Sat1	1.318761
Ifi205	3.098305	Gm15433	1.615772	Acvr1b	1.318652
F830016B08Rik	3.053166	Enpp4	1.615508	Tmem170b	1.317625
Oas1a	3.019199	Trim5	1.614655	Hadh	1.317598
Ifi203	3.016137	Serpinb9b	1.608848	Stat5a	1.315396
Map3k8	3.013409	C130074G19Rik	1.606202	2-Mar	1.314095
Gm12250	3.012671	AI854703	1.605264	Eno2	1.313938
Tgtp2	3.005529	Tuba8	1.604317	Enpp5	1.313706
H2-Q4	2.972546	Serping1	1.603547	Irak4	1.312126
Mx1	2.865782	Il13ra1	1.602487	Rsrp1	1.31211
Uba7	2.864864	Piwil4	1.602483	Kcnab2	1.311973
Mmp19	2.861314	Rhbdl2	1.601725	Ptgr1	1.311529
Psmb9	2.850848	Fst	1.600533	Elf1	1.311246
Slc7a2	2.846346	Trim34a	1.600318	Ablim1	1.311022
Tspan11	2.844123	Amigo2	1.599439	Tnfaip6	1.310247
Tnn	2.822897	Hcn1	1.597937	Socs2	1.309989
H2-D1	2.809787	Egfr	1.592054	Pisd-ps2	1.308864
AI607873	2.803398	Hpse	1.588624	Traf2	1.308431
Rsad2	2.794983	AW011738	1.587681	Tfrc	1.308415
C1s1	2.784179	Dpep1	1.587215	Nadk	1.308229
Nod2	2.775693	Pydc3	1.583759	Acacb	1.306549
Sod2	2.762665	Tnfaip2	1.583296	Fbxl5	1.305723
Apol6	2.760061	Irgm1	1.583039	Slc2a1	1.304708
Ifi44	2.759968	Rnd1	1.582149	Zeb2	1.304438
Nfkbia	2.75616	Aldoc	1.581229	Ada	1.302207
Irf7	2.751557	Lrp1	1.579985	Rpl38	1.30147
Bmp3	2.745035	Ninl	1.579937	Plod2	1.30112
Kng1	2.741277	Mgarp	1.579404	Itm2c	1.300462
Cxcl10	2.723592	Gm26669	1.577867	Galnt18	1.300344
Olfr56	2.707222	Rasl11b	1.577414	Cdkn2a	1.300124
Sp100	2.654866	N4bp2l1	1.577031	Jade2	1.299487
Scube1	2.653081	Ikbke	1.573942	Cd320	1.297584
Ak4	2.651385	E230016K23Rik	1.573215	A430105I19Rik	1.296067
B2m	2.641611	Nsun7	1.57274	Cir1	1.295174
Bcl3	2.637123	Fam162a	1.568999	Rnaset2b	1.295007
Gch1	2.620331	Col18a1	1.566809	Pnpt1	1.29442
Angpt1	2.617582	Oas1b	1.563128	Eif3e	1.293846
Pdzrn4	2.608996	Bid	1.561949	Lamp2	1.292359
Ifit3	2.605192	Lipa	1.559201	Itm2b	1.291908
Serpina3h	2.599538	Dock10	1.558234	Enah	1.291837
Dram1	2.576206	Tnfsf13b	1.556325	Pou6f1	1.289991
D030025P21Rik	2.575076	Smim4	1.555893	Fibin	1.289861
Trim30a	2.570312	Gdap10	1.555756	Rgs3	1.289133
Gm5345	2.547477	Gm16217	1.554982	Btg2	1.289051
Phf11d	2.537169	Gng12	1.554866	Naa25	1.288593
Rac3	2.52761	Ddx58	1.554735	Notch3	1.286227
Cxcl3	2.52009	Fam129c	1.553522	Pcmtd2	1.28613
Pik3r5	2.519794	Dhx58os	1.550817	Tacc1	1.284319
Klf15	2.498912	Tsku	1.546568	Arfgef2	1.283314
Gbp9	2.49656	Heatr9	1.541554	Nqo2	1.283077
Wisp2	2.49624	Il6st	1.540291	Dnajb6	1.282975
Angptl4	2.493561	Stat1	1.538849	Ksr1	1.282116
Parp14	2.487984	Stap2	1.537892	Rictor	1.281955
Npy1r	2.487878	Tnip1	1.536153	Azi2	1.281312
Ecscr	2.486491	Junos	1.535682	Narf	1.280799
Tcp11l2	2.482509	Gm43050	1.534956	Aebp1	1.280756
Bst2	2.478323	Parp12	1.534759	Scarb2	1.279926
Lrig1	2.464502	Medag	1.534548	Rras	1.279648
Repin1	2.457953	Ifnlr1	1.534026	Zfp322a	1.279547
Mgst1	2.451896	Il18	1.533373	Renbp	1.279486
Ltbp2	2.441692	Adar	1.532874	Zfp263	1.278938
Fmo1	2.437275	Shisa5	1.532173	Cd302	1.278793
Mndal	2.437132	Rarres2	1.530833	Uaca	1.2784
Ifitm3	2.433955	Mitf	1.530693	Plgrkt	1.278267
Serpinb1a	2.429138	Hif1a	1.52939	Ptges	1.278224
Lgals3bp	2.420976	Znfx1	1.528807	Ezh1	1.277255
Ifi204	2.394185	Pik3r1	1.52783	Ifnar1	1.275384
Gbp2	2.374173	Grem1	1.526552	Slc25a37	1.275033
Ddx60	2.365483	Gm12216	1.520431	Arel1	1.274603
Ifit3b	2.349228	Igfbp7	1.519077	Zfp36	1.274392
Gm4070	2.333254	AI429214	1.517825	Rab11fip1	1.273259
Zmynd15	2.311957	Susd1	1.517214	Fbxl20	1.27301
Slc15a3	2.298627	Pamr1	1.516791	Usp25	1.272639
4930512H18Rik	2.294978	Gas7	1.515823	Mycbp2	1.272037
A530020G20Rik	2.291215	A230050P20Rik	1.515791	Abca2	1.271611
Abcc3	2.277936	Cd274	1.515474	Ctsb	1.27127
Tap1	2.275735	Gm24187	1.512612	Sfi1	1.271049
Il7	2.274442	Slfn10-ps	1.511997	Capg	1.269608
Micall2	2.266222	Serpinb6b	1.508674	Msi2	1.268826
H2-Ab1	2.255115	H2-M3	1.507956	Adam17	1.267733
Slco3a1	2.249391	Pcdh17	1.507897	2810474O19Rik	1.266183
Ly6c1	2.247399	Pnp	1.506862	Cnp	1.266005
Apol9b	2.243974	Errfi1	1.506135	Rhoj	1.265107
Slfn8	2.237092	Psen2	1.504761	Fbn1	1.265104
Serpina3g	2.223564	Bmper	1.503315	Plekha2	1.264872
Trim30d	2.222229	Rassf2	1.502351	Qsox1	1.264705
Macrod1	2.216982	Rnf150	1.502349	Il4ra	1.262579
Susd6	2.216522	Foxred2	1.502222	Zfp862-ps	1.26213
Rab32	2.208063	Nfkb1	1.500361	Abhd4	1.262
RP24-118K20.1	2.203351	Gm26797	1.499863	Apobec3	1.261504
Islr	2.202374	Cebpb	1.499767	Cryzl1	1.26039
Tnfrsf9	2.201074	Gdnf	1.499677	Snx18	1.259801
Mx2	2.1992	Erap1	1.499261	Snx10	1.259016
Dhx58	2.197596	Phactr1	1.498501	Psme1	1.258257
Mgst2	2.196355	Acy3	1.498313	Prdx5	1.256944
Nlrc5	2.19327	Pde1a	1.497383	Rpl19	1.254924
Ifi27l2a	2.185136	Gm16675	1.4961	Fbxw17	1.254918
Atp8b4	2.184018	Pced1b	1.49569	Ahnak2	1.254603
Dcxr	2.173452	Fndc3a	1.494781	Pgm2	1.254392
Gbp7	2.172556	Sik1	1.48986	Lgals8	1.254212
Nos2	2.172346	Pax5	1.489642	Dusp16	1.254097
Trpc3	2.172073	Rbm47	1.489147	Fdps	1.253066
Col24a1	2.166567	Rhbdf2	1.488124	Zswim4	1.25276
A4galt	2.160477	Gla	1.48683	Tmem9	1.252663
Sp110	2.153144	Mt1	1.486484	Ext1	1.252343
Iigp1	2.150941	Helz2	1.484205	Ldha	1.252201
Bdkrb1	2.144746	Adarb1	1.483657	Ccng1	1.251981
Glrx	2.144381	Manba	1.481685	Rps23	1.251225
Oasl2	2.142329	Ssbp2	1.481023	Traf3	1.251037
Gypc	2.141326	Cd47	1.480263	Tbc1d2b	1.251012
Mark1	2.137578	Gpr176	1.48	Pan2	1.250188
Pdgfra	2.130591	Peli3	1.479421	Ip6k1	1.249537
Tgfbr3	2.126753	Parp11	1.479263	Vegfa	1.248657
Gm20559	2.124888	Agpat9	1.475738	Prrx1	1.248548
Tnfaip3	2.124389	Clip1	1.475604	Nfe2l1	1.247093
Ifit1bl2	2.121353	Pcx	1.475162	Ago1	1.246536
Il6ra	2.115453	Mov10	1.475059	Fgfr1op	1.246307
Cyp7b1	2.114831	Mvp	1.473965	Tnfaip8	1.245604
H2-T22	2.112472	Vdr	1.473286	Appl2	1.245541
Tlr2	2.108417	Ampd3	1.472815	Acaa1a	1.245346
Apol9a	2.107259	Mfsd7c	1.470604	Phip	1.244919
Txnip	2.102973	Ifngr2	1.470338	Rev1	1.244257
Cbr2	2.094355	Nampt	1.47029	Lpin1	1.243059
Ptpn13	2.091855	Stat2	1.469684	Hacl1	1.24284
Isg15	2.084784	Klhl24	1.468335	Abtb1	1.242142
Serpina3f	2.082629	Irak3	1.468302	Zfp281	1.241946
Selp	2.067573	Socs3	1.464374	Pkdcc	1.240434
Gvin1	2.043168	Car11	1.462349	Arhgap12	1.239317
Cmpk2	2.030401	Flt1	1.462052	Malat1	1.23865
Trim12c	2.030228	Ypel3	1.460792	Baiap2	1.236936
Grb14	2.02788	Wdyhv1	1.460703	Sh3d19	1.236615
Gm4841	2.026738	2310001H17Rik	1.458497	Igf2bp2	1.236566
Mnda	2.026163	Slc16a3	1.456228	Fbxo38	1.236407
Igfbp3	2.023331	Cdon	1.455779	Zswim6	1.235488
Gm9574	2.0163	3-Mar	1.455144	Rnf115	1.235458
Tgtp1	2.013841	Psmd10	1.454387	Ubr4	1.233944
Ly6e	1.998818	Cntnap1	1.452562	Calcoco1	1.233454
C4b	1.993132	H2-K2	1.451514	Insr	1.233242
Gfra2	1.985483	Trim25	1.451455	Rps15a	1.233115
Gm2619	1.982496	Scamp1	1.450926	Hexim1	1.233029
Slc39a4	1.982118	Tnfrsf1b	1.449087	Aplp2	1.232116
Osmr	1.97891	Acadsb	1.447878	Ankrd17	1.231804
Ifit1	1.966789	Procr	1.447607	Maff	1.231303
Rrad	1.959059	Pla2g16	1.444745	Foxo4	1.230508
Herc6	1.953162	Atp8a1	1.442774	Urod	1.2304
Clec2d	1.95126	Rbpj	1.441469	Nfib	1.230388
Epas1	1.950642	Neat1	1.440341	Zmynd8	1.229542
9330175E14Rik	1.950302	Il18bp	1.435413	Rsbn1l	1.229463
Lifr	1.947789	Arntl2	1.435176	Mapkapk2	1.228835
Hap1	1.946544	Runx1	1.434923	Lgmn	1.228287
Cfap100	1.939772	BC051226	1.433817	Rasa3	1.228205
Cfh	1.939442	Pvrl2	1.433681	Rps20	1.228122
Slc6a2	1.931558	Zfp874b	1.431978	Chmp4b	1.227714
C1rl	1.930971	Acsl1	1.431187	Prkar2b	1.227104
Abca1	1.922383	Mfsd7a	1.43055	Jun	1.225972
Agrn	1.91988	Mitd1	1.428555	Mmp2	1.225692
Sbno2	1.916865	Ctsh	1.425118	Sumo1	1.225557
Tnip3	1.913463	Zfp874a	1.42474	Tor1aip1	1.225418
Ugcg	1.910687	Mtss1	1.424259	Lacc1	1.225387
Spib	1.907491	Perm1	1.423781	Kdm3a	1.224975
Kcnn3	1.898432	Gsdmd	1.422904	Flnb	1.224811
Ripk2	1.89521	Rspo2	1.422633	Ktn1	1.224573
Ptpn5	1.894565	Gm36936	1.420322	Hspa1b	1.223939
Nod1	1.889504	Dpy19l1	1.419975	Psd	1.223572
Gm4955	1.887092	Spry2	1.418837	Nt5dc2	1.222385
Gm43196	1.885374	Fam3c	1.417203	Usp12	1.222266
Kcnq5	1.876112	Gfpt2	1.4161	Axl	1.222093
Xaf1	1.874808	Ifitm2	1.410051	Akr1b8	1.220794
Lyz2	1.874716	Trim21	1.409367	Gaa	1.219884
C920025E04Rik	1.873125	Pnpla7	1.40874	Ptprj	1.219624
Slc2a6	1.870162	Ociad2	1.408322	Mmab	1.216943
Cxcl16	1.869931	Mkx	1.406854	Osbpl3	1.216658
Foxo3	1.869316	Il10rb	1.406503	Ticam1	1.216655
Relb	1.863299	Vmp1	1.405888	Nub1	1.21658
Ifitm1	1.857611	Spsb1	1.40294	Ogfr	1.216222
Ctps	1.85722	Zfpm2	1.402674	Add3	1.215534
Trim12a	1.851346	Ifi35	1.402586	Slc29a1	1.215147
Ell2	1.849304	Tmem154	1.402112	Nfil3	1.214777
Psmb10	1.849216	Oasl1	1.40142	Parp3	1.21461
Adtrp	1.846019	Irf1	1.401037	Nab1	1.214327
Gm16464	1.843872	Kank1	1.400088	Rpl34	1.214249
Cdk6	1.843573	Traf3ip2	1.399787	Naaa	1.21421
Bnip3	1.840519	Trib1	1.399217	Map1lc3b	1.21098
Plscr1	1.832872	Fbxo32	1.398883	Zfos1	1.210827
Rnf213	1.830593	Dtnbp1	1.398525	Irf9	1.210808
Plscr2	1.824617	Dclk1	1.396242	Vps26a	1.210646
Cgn	1.818312	Gatsl2	1.394958	Col5a3	1.210558
Nek6	1.816836	Irf2	1.39425	Kdm5a	1.209736
Gm43197	1.816332	Dnajc12	1.392043	Tor1aip2	1.209517
P2rx4	1.810723	Ctso	1.391962	Gnptab	1.208163
Rbpms	1.809021	Grina	1.388872	Rab8b	1.207975
Sp140	1.804443	Daam1	1.388466	Spred2	1.207248
Lgals9	1.804377	Cxadr	1.387017	Gdf11	1.206992
Il16	1.803712	Arid5b	1.386992	Pak3	1.206941
Camp	1.801329	Stx11	1.386314	Nlgn2	1.206715
Ube2l6	1.800442	Tcn2	1.385764	Dst	1.206062
Pfkl	1.797849	Ppl	1.38494	Nr1d2	1.205401
Gpr88	1.794428	Aftph	1.383957	Daxx	1.204017
Gm5970	1.793526	Ctsl	1.38209	Uvrag	1.203409
Nfkbie	1.79343	Slc16a1	1.379145	Tnfrsf1a	1.203158
Il20ra	1.793392	B4galt5	1.378838	Cmtm6	1.202344
Rgs16	1.789903	Acvrl1	1.378201	Cstb	1.202249
Ccl9	1.789024	Cx3cl1	1.376893	Il17ra	1.201293
Mettl20	1.78797	Podnl1	1.376677	Stat3	1.200716
Cgnl1	1.781933	Txndc16	1.376549	Sgk1	1.199554
Col6a4	1.781322	Aldh1l1	1.375993	Cldn12	1.197903
Gm19684	1.778757	Crebrf	1.375601	Dync1h1	1.197443
Npc2	1.777478	Ptpre	1.375528	Gabarapl1	1.197238
Igtp	1.776729	Flrt2	1.375148	Tbk1	1.196546
Tapbp	1.776018	Dtwd1	1.374774	Myo18a	1.196491
Slc10a6	1.77264	Il1rl1	1.374317	G3bp2	1.195278
Rtp4	1.772625	Pml	1.373905	Rbm33	1.192694
Itih5	1.77129	Ifit2	1.373384	Eml4	1.192421
Gm12185	1.769756	Rnf114	1.372865	Zmiz1	1.19149
Adhfe1	1.765856	Fth1	1.372822	Psma6	1.19122
Ifnar2	1.760615	H2-T24	1.372506	Csf1	1.189881
Slco1a6	1.753534	Pygl	1.37231	Srsf5	1.1887
Cxcl2	1.752155	Phyh	1.371721	Lmo4	1.187609
Negr1	1.751596	Pik3c2b	1.370604	Pip5k1a	1.182817
Gng2	1.751401	Ttc39c	1.370202	Mlxip	1.181411
Fgf7	1.750411	Myrf	1.369639	Uhrf1bp1l	1.18067
Samd9l	1.750223	Slirp	1.368745	Foxp1	1.175338
Tlr3	1.749767	Mef2a	1.367217	Notch2	1.174766
Tap2	1.73925	Nfkb2	1.366624	N4bp1	1.174303
Irgm2	1.738998	Asah2	1.366535	D17Wsu92e	1.170473
Tifa	1.735914	Ndrg2	1.366351	Prkaa1	1.16978
Tgm1	1.734849	Bnip3l	1.365928	Zc3hav1	1.168237
Birc3	1.728337	Fyco1	1.365796	Abcc1	1.167684
Gm26809	1.725966	Gm6548	1.365763	Paip2	1.164378
Il34	1.725927	Gpr146	1.36386	Bsg	1.161778
Thbs2	1.722317	Plekhn1	1.362388	P4ha1	1.160257
Ppm1k	1.720243	Ghr	1.360096	Pld3	1.160076
Casp12	1.719866	Cnnm2	1.359865	Lamc1	1.159628
Arhgdib	1.719733	Arid5a	1.359164	Ece1	1.15875
Stab 1	1.719107	Car13	1.358995	Dcaf8	1.154963
Nmi	1.7184	Jak3	1.358706	Psap	1.148899
Ptgir	1.71638

Pulmonary TCs were reported to promote angiogenesis in a mouse model of ARDS [11]; thus, particular attention was devoted to angiogenesis in the gene ontology (GO) functional analysis. According to the DAVID online database, 28 DEGs were enriched in the processes of blood vessel formation, angiogenesis, blood vessel morphogenesis, blood vessel remodelling, and sprouting angiogenesis. For further analysis, the DEGs were enriched in the STRING database. According to the STRING database, the DEGs were enriched in 9 angiogenesis-related processes: angiogenesis, blood vessel morphogenesis, vasculature development, blood vessel remodelling, sprouting angiogenesis, venous blood vessel sprouting, venous blood vessel morphogenesis, regulation of angiogenesis, and positive regulation of angiogenesis (Fig. 3d). As most genes participated in at least 3 biological processes, those involved in more than three processes—i.e. E2F8, Notch1, EPAS1, Rbpj, Flt1, ACVRL1, EFNB2 and Thbs1—were selected for further research. MiR-21a-3p, miR-221-5p, miR-146a-5p and miR-188-5p regulated these 8 genes (Fig. 3e).

Validation of miRNAs and their target mRNAs in TCs

We next assessed the mRNA levels of angiogenesis factors. The mRNA expression of E2F8, Notch1, EPAS1, Rbpj, Flt1, ACVRL1, EFNB2 and Thbs1 was measured in TCs after LPS stimulation. After LPS stimulation, E2F8, EFNB2, and EPAS1 were significantly downregulated, while Flt1 was upregulated. Given that miRNAs usually negatively regulate downstream genes, E2F8, EFNB2, and EPAS1 were further studied. LPS stimulation significantly increased miR-21a-3p and miR-221-5p expression in TCs compared with that in cells under control conditions. To clarify the relationship between miRNAs and mRNAs, miRNA inhibitors were applied. MiR-221-5p inhibition restored the expression of EPAS1 but not EFNB2, and miR-21a-3p inhibition restored the expression of E2F8 but not EPAS1. MiR-21 had been reported to increase proliferation, migration and tube formation of Human Umbilical Vein Endothelial Cells (HUVECs) and induce angiogenesis by directly targeting PTEN [30, 31]. Moreover, miR-21a-3p and its downstream target E2F8 were further studied. After 24 h, the protein expression of E2F8 was decreased in TCs challenged with LPS and was restored by inhibition of miR-21a-3p. The dual luciferase reporter assay indicated that E2F8 was the direct target of miR-21a-3p (Fig. 4).

Fig. 4

Expression of angiogenesis-related genes in TCs with LPS treatment. a mRNA levels of E2F8, Notch1, EPAS1, Rbpj, Flt1, ACVRL1, EFNB2, Thbs1, FLT1, miR-21a-3p and miR-221-5p in TCs treated with LPS and/or miR-21a-3p inhibitor or miR-221-5p inhibitor. *P < 0.05 vs Control, #P < 0.05 vs LPS. b Protein levels of E2F8 in TCs treated with LPS and/or miR-21a-3p inhibitor. *P < 0.05 vs Control, #P < 0.05 vs LPS. c MiR-21a-3p led to a significant reduction of the luciferase activity of reporter with the wildtype 3′ UTR but not that of the mutant reporter. *P < 0.05 vs E2F8-WT/miRNA miR-21a-3p mimic. n = 6

MiR-21a-3p regulated angiogenesis under inflammatory conditions

The transcription factors E2F7/8 were reported to regulate vessel branching via DLL4-Notch approaches [32] or HIF-1α/VEGFA signalling [33]. In the present study, LPS stimulation reduced the protein expression of Notch 2 but not Notch 1, Notch 4 or DLL4. Inhibition of miR-21a-3p restored Notch 2 protein expression in TCs in the presence of LPS. LPS did not affect HIF-1α expression. However, LPS increased the expression of VEGFA at the mRNA level, and this increase was reversed by miR-21a-3p inhibition in cultured TCs (Fig. 5).

Fig. 5

MiR-21a-3p regulated angiogenesis associated signalling in TCs induced with LPS. a, b Protein levels of DLL4, Notch1, Notch2, and Notch4 in TCs treated with LPS and/or miR-21a-3p inhibitor. c Expression of HIF-1α and VEGF on mRNA level. *P < 0.05 vs Control, #P < 0.05 vs LPS, n = 6

PI3K signalling might participate in angiogenesis

PI3K, especially the Class I catalytic isoforms, plays an important role in angiogenesis. To study the mechanisms underlying the effect of miR-21a-3p in TCs on angiogenesis induction, PI3K subunit expression was first examined. The mRNA levels of the Class I PI3K isoforms PIK3CA, PIK3CB, and PIK3CD did not significantly change with LPS stimulation. However, the protein level of p110α in TCs was significantly increased with LPS stimulation and decreased with miR-21a-3p inhibitor co-treatment. The PI3K signalling molecules AKT, mTOR, and PTEN were unaffected by either LPS or miR-21a-3p. These results indicated that PI3K signalling might participate in angiogenesis via the p110α isoform (Fig. 6).

Fig. 6

Expression of PI3K signalling in TCs induced with LPS. a mRNA levels of PIK3CA, PIK3CB, and PIK3CD in TCs treated with LPS and/or miR-21a-3p inhibitor. b, c Protein levels of p110α, p-PI3K, PI3K, p-mTOR, mTOR, PTEN, p-AKT, AKT in TCs treated with LPS and/or miR-21a-3p inhibitor. *P < 0.05 vs Control, #P < 0.05 vs LPS, n = 6

MiR-21a-3p and p110α in TCs promoted the proliferation of EOMA cells

The proliferation of EOMA cells was then estimated after co-culture with TCs pre-treated with the miR-21a-3p or PI3K p110α inhibitor. Culture medium from TCs stimulated with LPS promoted EOMA cells proliferation, as determined by the CCK8 assay. Compared with medium from NC TCs, culture medium from TCs with miR-21a-3p inhibition significantly reduced EOMA cells proliferation (Fig. 7a). The effect of p110α was examined by dynamic real-time cell observation. The proliferation assay indicated that EOMA cells proliferation decreased with LPS stimulation but was restored by co-culture with TCs. Inhibition of miR-21a-3p or p110α (with its inhibitor HS-173) weakened the protective effect of TCs (Fig. 7b, d). The scratch assay showed similar results (Fig. 7c, e). VEGF protein expression was significantly elevated with LPS stimulation, and this increase was reversed by inhibition of either miR-21a-3p or p110α (Fig. 7f). The results above indicated that VEGF is regulated by both miR-21a-3p and p110α.

Fig. 7

MiR-21a-3p and p110α mediated the promotion of TCs on EOMA proliferation induced by LPS. a Cells proliferation rate of EOMA treated with LPS and/or TCs and miR-21a-3p inhibitor measured by CCK8 assay. *P < 0.05 vs Control, #P < 0.05 vs LPS/TC. b, d Cell proliferation of EOMA treated with LPS and/or TCs and miR-21a-3p inhibitor or p110α inhibitor measured by Cell-IQ. *P < 0.05 vs Control, #P < 0.05 vs LPS, **P < 0.05 vs LPS/TC, n = 6. c, e Cell movement of EOMA treated with LPS and/or TCs and miR-21a-3p inhibitor or p110α inhibitor measured by Cell-IQ. f VEGFA levels secreted by TCs treated with LPS and/or miR-21a-3p inhibitor or p110α inhibitor measured by ELISA. *P < 0.05 vs Control, #P < 0.05 vs LPS, n = 6

Discussion

This study reports that TCs culture medium can alleviate ARDS in mice probably via angiogenesis-associated factors regulated by miR-21a-3p. TCs exposed to LPS exhibited increased miR-21a-3p expression and VEGF production, which further promoted vascular endothelial cell proliferation. The protective effects of TCs mediated by miR-21a-3p might be regulated through PI3K (p110α)/AKT/mTOR signalling and the expression levels of the downstream targets E2F8 and Notch 2 (Fig. 8).

Fig. 8

Schematic representation of the working model. TCs induced by LPS promoted endothelial regeneration and angiogenesis through miR-21a-3p-PI3K (p110α)/AKT/mTOR and VEGF signalling in TCs. The E2F8/Notch2 signalling pathway might also participates in this procession

Endotoxin-induced ARDS has been reported to affect both respiratory epithelial cells and the underlying vascular endothelial cells [34]. In the present study, LPS stimulation induced severe vascular damage in the lungs, as shown by the reduced levels of CD31 and eNOS. TCs are distinct from mesenchymal stem cells and fibroblasts and have been reported to have specific roles in cell signalling, tissue remodelling and angiogenesis [35]. In the present study, TCs culture medium exhibited great potential to reverse the angiogenic signalling that was reduced by LPS-induced inflammation, supporting the observation that TCs alleviate LPS-induced lung injury in mice by releasing angiogenic factors [11].

Non-coding miRNAs are involved in several pathological processes, including angiogenesis [36, 37]. MiR-221-5p [38], miR-146a-5p [39], and miR-21a-3p [40-42] are reported to be associated with the angiogenesis process. MiR-21a-3p and miR-221-5p were demonstrated to be involved in the promotion of angiogenesis in TCs. As miR-21a-3p was more frequently reported on angiogenesis, it was further studied. MiR-21a-3p knockdown in TCs reduced CD31 and eNOS expression in the lungs of ARDS mice in vivo. MiR-21a-3p exerts its protective effects on injury repair by inducing angiogenesis-associated signalling pathways. For instance, miR-21a-3p activates the AKT pathway and increases matrix metalloproteinase-2 (MMP-2) expression to reduce the extent of the infarcted region in heart ischaemia/reperfusion injury [41], inhibits PTEN and sprouty homolog 1 (SPRY1) to heal soft tissue wounds [40], and upregulates VEGF and activates the Ang-1/Tie-2 axis in traumatic brain injury [42]. In the current study, the p110α isoform in PI3K/AKT/mTOR signalling pathway was demonstrated to be involved in miR-21a-3p-mediated angiogenic factor induction in TCs. However, the alteration of other protein levels and HIF-1α in TCs treated with LPS and the miR-21a-3p inhibitor indicated that more complex signalling pathways were involved in regulating the angiogenic function of TCs. Culture medium from LPS-induced TCs promoted EOMA cells proliferation in vitro, accompanied by elevated levels of VEGF mRNA and secretion, which further demonstrated that the functional miR-21a-3p was generated by TCs. These data support the hypothesis that miR-21a-3p plays a role in angiogenesis and profoundly demonstrate the mechanisms mediated by PI3K p110α.

The E2F family was first reported to induce cell proliferation [43], and E2F family members are essential transcriptional regulators of cell cycle progression [44], as well as apoptosis, metabolism and angiogenesis [45]. E2F8 is an atypical transcriptional repressor in the E2F family since it contains domains that differ from the canonical domains [46]. By forming homodimers or heterodimers with E2F7, E2F8 reduces the excessive and destructive activation of E2F1 [47]. However, reports of E2F8 in angiogenesis in the literature are controversial. E2F7/8 has been reported to positively regulate the formation of blood vessels during embryonic development via HIF-1α/VEGFA signalling [33]. On the other hand, E2F7/8 suppresses tumour angiogenesis via the induction of DLL4 [32]. In the present study, E2F8 expression was reduced after LPS stimulation in TCs and restored with miR-21a-3p inhibition, indicating that E2F8 plays a negative role in angiogenesis under inflammatory conditions.

The Notch family, which contains several receptors and ligands, is fundamental in the regulation of blood vessel branching [48]. DLL4, a Notch ligand, has an inhibitory function in blood vessel branching [49] that is compromised by Jagged 1 activation [50]. Notch1 positively regulates angiogenesis [51], while Notch2 negatively regulates cell proliferation [52] and angiogenesis [53]. In the initial stage of angiogenesis, inhibition of Notch 2 promotes vascular endothelial cell proliferation, while activation of Notch 2 reduces endothelial cell responses to VEGF [54, 55]. In the present study, Notch2 expression was mediated by miR-21a-3p. However, the relationship between the transcription factor E2F8 and Notch2 was not illustrated. Further experiments should be conducted to confirm the signalling pathway of E2F8/Notch2 in angiogenesis.

Conclusion

TCs have been reported to be important in tissue repair and healing processes. Via mouse models, bioinformatics approaches and molecular biological methods, the present study shows that activated TCs promote endothelial regeneration and angiogenesis through miR-21a-3p-PI3K (p110α)/AKT/mTOR signalling and further demonstrates the key roles of VEGF in TCs. The E2F8/Notch2 signalling might also participates in this process. These findings shed light on miR-21a-3p in TCs as a new therapeutic target for vessel protection.

Additional file 1: Figure S1. The morphology of TCs. The pictures were gathered by Cell-IQ every 8 h. The white arrow showed the typical telopode.