Mesenchymal Stem Cell-Derived Exosomal Mir-146A Reverses

He et al. Stem Cell Research & Therapy (2021) 12:449 https://doi.org/10.1186/s13287-021-02371-0

RESEARCH Open Access Mesenchymal stem cell-derived exosomal miR-146a reverses diabetic β-cell dedifferentiation Qin He1, Jia Song1, Chen Cui1, Jinbang Wang1, Huiqing Hu1, Xinghong Guo1, Mengmeng Yang1, Lingshu Wang1, Fei Yan1, Kai Liang1, Zhaojian Liu2, Fuqiang Liu1, Zheng Sun1, Ming Dong1,3,4, Xinguo Hou1,3,4 and Li Chen1,3,4,5*

Abstract Background: Mesenchymal stem cells (MSCs) show promising therapeutic potential in treating type 2 diabetes mellitus (T2DM) in clinical studies. Accumulating evidence has suggested that the therapeutic effects of MSCs are not due to their direct differentiation into functional β-cells but are instead mediated by their paracrine functions. Among them, exosomes, nano-sized extracellular vesicles, are important substances that exert paracrine functions. However, the underlying mechanisms of exosomes in ameliorating T2DM remain largely unknown. Methods: Bone marrow mesenchymal stem cell (bmMSC)-derived exosomes (bmMDEs) were administrated to T2DM rats and high-glucose-treated primary islets in order to detect their effects on β-cell dedifferentiation. Differential miRNAs were then screened via miRNA sequencing, and miR-146a was isolated after functional verification. TargetScan, reporter gene detection, insulin secretion assays, and qPCR validation were used to predict downstream target genes and involved signaling pathways of miR-146a. Results: Our results showed that bmMDEs reversed diabetic β-cell dedifferentiation and improved β-cell insulin secretion both in vitro and in vivo. Results of miRNA sequencing in bmMDEs and subsequent functional screening demonstrated that miR-146a, a highly conserved miRNA, improved β-cell function. We further found that miR-146a directly targeted Numb, a membrane-bound protein involved in cell fate determination, leading to activation of β- catenin signaling in β-cells. Exosomes derived from miR-146a-knockdown bmMSCs lost the ability to improve β-cell function. Conclusions: These findings demonstrate that bmMSC-derived exosomal miR-146a protects against diabetic β-cell dysfunction by acting on the NUMB/β-catenin signaling pathway, which may represent a novel therapeutic strategy for T2DM. Keywords: Exosome, Mesenchymal stem cell, Type 2 diabetes mellitus, β-cell dedifferentiation, miR-146a

* Correspondence: [email protected] 1Department of Endocrinology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, No. 107 Wenhua Xi Road, Jinan 250012, Shandong, China 3Institute of Endocrine and Metabolic Diseases of Shandong University, Jinan 250012, China Full list of author information is available at the end of the article

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Background involved in the development of diabetic vascular compli- Type 2 diabetes mellitus (T2DM) is characterized by in- cations [21]. However, it is unknown whether bmMSC- sufficient β-cell mass and insulin resistance and has be- derived exosomal miRNAs affect β-cell dedifferentiation. come a worldwide disease due to its increasing To elucidate the effects of bmMSCs on β cells, in the morbidity [1]. This T2DM-induced decline in β-cell present study, we first demonstrated that bmMSC- mass is attributed to a decrease in both β-cell number derived exosomes (bmMDEs) reversed β-cell dedifferen- and function [2]. It has been suggested that reduced β- tiation and ameliorated β-cell function both in vitro and cell mass is the result of apoptosis [3]. However, recent in vivo. miRNA sequencing was then used to further ex- data challenge the β-cell apoptosis hypothesis. These plore the role of bmMDEs. Finally, we showed that data indicate that dedifferentiation represents an alterna- beneficial effects of bmMDEs on β-cell dedifferentiation tive mechanism explaining the loss of functional β-cell were mainly achieved via the miR-146a-5p/Numb/β-ca- [4, 5]. Dedifferentiated β-cells lose their insulin secretion tenin pathway. Taken together, our findings shed light capacity and transform into endocrine progenitor cells on the potential of an MSC-derived exosome-based with multi-directional differentiation potential, charac- therapeutic approach for treating diabetes. terized by the re-expression of Ngn3, thereby causing the total amount of functional β-cells to decline [6, 7]. Methods Interventions of β-cell dedifferentiation can reverse Cell culture and treatments hyperglycemic phenotypes and slow the progression of Rat primary bmMSCs were isolated as previously de- diabetes. Therefore, safe and effective treatments that re- scribed [22]. Briefly, femurs and tibiae were obtained verse β-cell dedifferentiation may represent novel and from Sprague-Dawley rats after euthanasia via an promising therapeutic strategies for T2DM. anesthetic overdose. The bone marrow was harvested by Recently, mesenchymal stem cell (MSC)-based therapy flushing the cavities of rats with DMEM/F12 medium has offered a promising strategy for treating diabetes. (Gibco, Invitrogen, Carlsbad, CA) supplemented with Some clinical studies have found that MSCs and their 20% fetal bovine serum (FBS, depletion of exosomes by derivatives can significantly improve fasting blood glu- ultracentrifugation; Gibco). The media were changed cose, C-peptide levels, HbA1c levels, and insulin secre- after 24 h, and cells were at approximately 80% con- tion [8–10], with no significant side effects having yet fluency when they were passaged. DMEM/F12 with 10% been observed. Among MSCs, bone marrow MSCs FBS was used in the subsequent cultures. The third to (bmMSCs) have shown significant effects in regulating fourth generations of cells were used for experiments. β-cell mass and in ameliorating chronic high-glucose- The INS-1 cell line was obtained from Nanjing Med- induced β-cell injury [11, 12]. Our previous research has ical University, PR China. Cells were cultured in the also confirmed that bmMSCs can improve diabetic islet RPMI-1640 medium (Gibco) supplemented with 15% microcirculation via Wnt4-β-catenin signaling [13]. Al- FBS (depletion of exosomes by ultracentrifugation), 10 though these studies have documented improved β-cell mM of Hepes (Sigma-Aldrich, St. Louis, MO), 1 mM of function after MSC infusion, whether decreased β-cell sodium pyruvate (Sigma-Aldrich), 2 mM of L-glutamine dedifferentiation is involved in improved β-cell mass and (Gibco), and 50 μmol/L of β-mercaptoethanol (Sigma- insulin secretion has received less intention. In the Aldrich) at 37°C with 5% CO2. Cells were inoculated in present study, we directly address this question. a six-well plate and were cultured in a medium contain- Exosomes are extracellular nanoparticles with lipid- ing 11.1 mmol/L (control) or 35 mmol/L of glucose bilayer membranes [14]. Exosomes can directly deliver (high glucose, HG) for 72 h with or without INS-1 cell- effectors—such as proteins, mRNAs, and microRNAs— derived exosomes (IDEs, 30 μg/mL, normal control exo- from secretory cells to recipient cells [15, 16]. MSCs can somes) or bmMDEs (30 μg/mL). Mannitol (MA, 35 secrete larger amounts of exosomes compared to those mmol/L, SM8120, Solarbio) was used as an osmotic of terminally differentiated cells [17]. MSC-derived exo- pressure control. somes can reverse peripheral insulin resistance and re- lieve cellular destruction [18, 19]. Compared to MSCs, Isolation and characterization of exosomes exosomes have lower immunogenicity and lower risk of Isolation and purification of exosomes were performed aneuploidy and can be more easily mass-produced, all of according to standard methods as described previously which make them more attractive therapeutic agents. [23]. Briefly, the bmMSC culture medium was collected MicroRNAs are small, non-coding RNAs that exert after being replaced with an exosome-free medium for many biological roles by negatively regulating mRNA ex- 48 h. The conditioned medium was centrifuged at 300g pression at the post-transcriptional level [20]. Recent for 10 min and was then centrifuged a second time at studies have demonstrated that specific miRNAs play a 2000g for 20 min to remove cells and apoptotic bodies. vital role in regulating β-cell activities and are also Next, samples were centrifuged at 10,000g for 30 min He et al. Stem Cell Research & Therapy (2021) 12:449 Page 3 of 16

and were then filtered through a 0.22-μm filter to re- exosomes were delivered into T2DM rats via tail-vein in- move cellular debris. The final supernatant was centri- jections, and each rat and its harvested organs were im- fuged at 120,000g for 70 min at 4°C to pellet exosomes. aged using an IVIS200 imaging system (Xenogen Corp., Ultracentrifugation experiments were performed with an Alameda, CA, USA) at 24 h after injection. During the Optima MAX-XP (Beckman Coulter, USA). All of the experiments, no animals became severely ill or died. All exosome-containing pellets were resuspended in PBS or animal experimental protocols were approved by the were lysed in RNA lysis buffer for further analysis. Exo- Animal Ethics Committee of Shandong University. somes were observed using a transmission electron microscope (TEM; JEM-1400, JEOL, Japan) and were Analysis of metabolic parameters quantified by a Nano-Sight NS300 (Malvern Instruments Rats were fasted for 3 h before measuring blood glucose Ltd, UK). levels. Body weight (BW) and food intake were moni- tored weekly. Intraperitoneal glucose tolerance tests Tracing of exosomes in INS-1 cells (IPGTTs) and intraperitoneal insulin tolerant tests (IPIT Exosomes were labeled with a PKH67 Green Fluorescent Ts) were performed after the last intervention. IPGTTs Cell Linker Kit (PKH67, Sigma-Aldrich) according to the were performed following overnight fasting. Blood glu- manufacturer’s protocol. First, 1 mL of Diluent C was cose levels were detected at 0, 30, 60, 120, and 180 min added to the exosomes diluted in PBS. Then, 4 μLof after intraperitoneal injections of glucose (1.5 g/kg, PKH67 dye was added to 1 mL of Diluent C and was Sigma-Aldrich). For IPITTs, rats were provided free ac- then incubated with exosomes (protected from light) for cess to food and water. Blood glucose levels were mea- 4 min. To bind excess dye, 2 mL of 1% BSA was added sured at 0, 30, 60, 120, and 180 min after intraperitoneal to the reaction system. Re-extraction of labeled exo- injections of insulin (2 IU/kg, Wanbang Pharmaceutical, somes was performed by ultracentrifugation and subse- Jiangsu, China). quent dilution in 100 μL of PBS. The labeled exosomes were incubated with INS-1 cells. Fluorescence signals Isolation and intervention of islets from rats were detected by an Olympus BX53 fluorescent Pancreatic tissues from rats were isolated after microscope. anesthesia. A syringe was pumped with 1 mg/mL of Col- lagenase V (C9263; Sigma-Aldrich) and was then re- Animals and diets placed with a fine syringe needle inserted into the Four-week-old male Sprague-Dawley rats (60–70 g) were pancreas. The outflow of the enzymatic solution was re- purchased from Synergy Pharmaceutical Bioengineering injected 2–3 times. The harvested pancreas was then Co., Ltd (Nanjing, China, Ethical number: DWLL-2015- placed into a digestive bottle, and it was then digested at 005). After 1–2 weeks of adaptive feeding, the rats were 37°C for 15 min. Subsequently, the sample was removed given a 45% high-fat diet (HFD) for 4 weeks, whereas from the water bath and was lightly shaken 12 times. the control group rats were fed with a normal chow diet. Ice-cold Hanks solution (with calcium and magnesium) These HFD rats were then injected intraperitoneally with was added to the digestive bottle and was shaken to stop STZ (30 mg/kg in 0.1 M citrate-buffered saline, pH=4.5, collagenase digestion, after which the sample was placed single dose, S0130; Sigma-Aldrich) after fasting for 12 h. on ice three times. Finally, the clean islets were hand- T2DM rats were identified as having fasting glucose ≥ picked under a stereoscopic microscope. After preincu- 16.7 mmol/L. STZ-treated rats continue HFD for an- bation overnight, the islets from Sprague-Dawley rats other 13 weeks. All animal experimental protocols were were exposed to 5.0 mmol/L (control) or 35 mmol/L of approved by the Animal Ethics Committee of Shandong glucose (high glucose, HG) with or without IDEs (30 μg/ University. mL) or bmMDEs (30 μg/mL) for 72 h. The islets from control, T2DM, and T2DM + bmMDE rats were pre- Treatment with bmMDEs in T2DM rats cultured overnight, after which they directly proceeded T2DM rats were divided into the following five groups to the next experiment. MA (35 mmol/L) was used as an (n = 7/group): PBS, bmMSCs, bmMDEs, bmMDEsmiR-NC osmotic pressure control. KD, and bmMDEsmiR-146a KD. bmMSCs (5×106 cells/rat) were suspended in PBS and injected via the tail vein Glucose-stimulated insulin secretion (GSIS) every 2 weeks for five cycles after STZ injections. Exo- After the corresponding intervention was completed, somes (10 mg/kg) were injected into rats via the tail vein glucose-starved INS-1 cells and islets (50–60 islets in every 3 days for 10 weeks. The normal control group each group) were assessed in Krebs–Ringer bicarbonate (control, n=7) was injected intravenously with an equal solution (KRBS) containing 2.5 mmol/L of glucose at volume of PBS (Fig. S7 for a more detailed experimental 37°C for 1 h. The supernatant was collected before being procedure). For exosome-tracking in vivo, Cy7-labeled incubated in KRBS containing 25 mmol/L of glucose for He et al. Stem Cell Research & Therapy (2021) 12:449 Page 4 of 16

another 1 h at 37°C. The supernatant was collected and Luciferase reporter assay insulin levels were detected using a commercial kit (Blue To identify the binding site between miR-146a and Gene, Shanghai, China) to evaluate stimulated insulin re- Numb, INS-1 cells were transfected with a luciferase lease. The final insulin content was normalized to the construct containing Numb with the wild-type or a mu- protein concentrations of cells and islets. tated version of the binding site and then co-transfected with miR-NC or miR-146a mimics (GenePharma, Immunofluorescence staining Shanghai, China). Luciferase activities were detected INS-1 cells and paraffin sections of pancreatic tissue after 48 h of transfection by using a Dual-Luciferase kit were incubated with the following antibodies: insulin (Promega, Madison, USA) according to the manufac- ’ (ab7842; Abcam), glucagon (ab10988; Abcam), PDX1 turer s instructions. (5679; Cell Signaling Technology), FOXO1 (2880; Cell Signaling Technology), neurogenin3 (NGN3, sc- RNA interference and plasmids 3744042; Santa Cruz, USA), OCT4 (GTX101497; Gene- siRNAs (siNC, siNUMB), plasmid vectors (vector con- Tex, CA, USA), and NUMB. After incubated with the taining Numb and empty vector), mimics/inhibitors of fluorescence-conjugated secondary antibodies, the cell indicated miRNAs, and lentiviral vectors (vectors con- nuclei were stained with DAPI. Digital images were cap- taining miR-146a sponge inhibitor and control) were tured using an Olympus BX53 fluorescence microscope. prepared by GenePharma (Shanghai, China). The target Immunofluorescence staining statistics are measured sequence of Rattus-Numb (NM_133287.1) was 1799 bp, from 3 to 5 rats per group, or 4–5 pancreas sections per the vector was pcDNA3.1(+), and the restriction site was rat for 15–20 islets. Quantitative analysis was performed NheI-NotI. The sequences of siRNAs and miRNA using the CytoNuclear count function of the Image Pro- mimics/inhibitors referred above are listed in the Sup- Plus software. Mean β-cell percentage per islet = average porting Information, namely in Tables S1 and S2. (β-cell number/total islet cell number) × 100%. Densi- tometry of staining was performed using ImageJ. Images Statistical analysis were separated by color and inverted to black and white, Three independent experiments were performed. The and the integrated density was calculated for each stain results are expressed as the mean ± the standard devi- ’ using the tracing function. ation (SD). Data were compared using paired Student s t tests or one-way analyses of variance (ANOVAs) followed by Bonferroni tests in GraphPad Prism 8 soft- Real-time quantitative PCR (RT-qPCR) ware (San Diego, CA, USA). A P-value < 0.05 was con- Total RNAs were extracted from INS-1 cells and islets sidered statistically significant (*P<0.05; **P<0.01; ***P< using MicroElute Total RNA Kit (Omega Bio-Tek, Dora- 0.001). ville, GA, USA). Total miRNAs were extracted by a miR- cute miRNA isolation kit (TIANGEN Biotech, Beijing, Results ’ China) according to the manufacturer s instructions. bmMDEs improve pancreatic islet functions in T2DM rats The sequences of above primers are listed in Supple- bmMSCs were identified by the expression of surface mentary Table 1. Real-time PCR was conducted with the markers (negative for CD45 and CD34, and positive for SYBR Green PCR kit (catalog no. RR820B; Takara), and CD105, CD73, and CD90) (Fig. S1c), as well as by differ- β quantification was achieved by normalization using - entiation capacities toward adipocytes confirmed by Oil- actin or U6 as the control. Red-O staining (Fig. S1a) and toward osteoblasts confirmed by Alizarin Red staining (Fig. S1b). bmMDEs Small RNA library construction and sequencing were isolated using standard differential centrifugation Total RNA was extracted using Trizol reagent (Invitro- from cell culture supernatants. The cup-shaped mem- gen, CA, USA) following the manufacturer’s procedure. branous structure, size, and number of exosomes were The total RNA quantity and purity were analyzed with a identified by transmission electron microscopy (TEM) Bioanalyzer 2100 and an RNA 6000 Nano LabChip Kit (Fig. 1a) and with a mode diameter around 100 nm via a (Agilent, CA, USA) with RIN number >7.0. Approxi- nanoparticle tracking system (Fig. 1a, b). In addition, iso- mately 1 μg of total RNA was used to prepare a small lated exosomes were characterized using Western blot- RNA library according to the protocol of TruSeqTM ting to determine the presence of the established Small RNA Sample Prep Kits (Illumina, San Diego, exosomal protein markers, CD9 and TSG101. Calnexin, USA). Additionally, single-end sequencing (36 bp) was an endoplasmic reticulum marker, was present in cells performed on an Illumina Hiseq2500 Lianchuan Bio- but not in exosomes (Fig. 1c). To address the biodistri- technology (LC-BIO, Hangzhou, China) following the bution of exosomes in rats, PBS and Cy7-labeled vendor’s recommended protocol. bmMDEs were injected into each rat via the tail vein. He et al. Stem Cell Research & Therapy (2021) 12:449 Page 5 of 16

Fig. 1 (See legend on next page.) He et al. Stem Cell Research & Therapy (2021) 12:449 Page 6 of 16

(See figure on previous page.) Fig. 1 bmMDEs ameliorate islet functions in T2DM rats. a Transmission electron microscopic images of bmMDEs (scale bar 50 nm). b Nano-sight tracking analysis was used to measure the size distribution and concentration of bmMDEs. c Western blotting was performed to detect protein markers (CD9, TSG101) of bmMDEs and protein markers (Calnexin) of cells. d Representative IVIS images of rats at 24 h after tail-vein injection with either PBS (left) or Cy7-bmMDEs (10 mg/kg, right). e Representative IVIS images of organs (heart, lung, liver, pancreas, spleen, kidney, and gastrointestinal tract) from rats (left: PBS, right: Cy7-bmMDEs). Blood glucose after 3 h starved (f) and body weight (g) in control + PBS, T2DM + PBS, T2DM + bmMSC, and T2DM + bmMDE rats. IPGTT (h) and IPITT (i) were performed after the last intervention of bmMDEs. j Fasting serum insulin of control + PBS, T2DM + PBS, and T2DM + bmMDEs rats as indicated. k Representative sections of pancreases stained with insulin and glucagon (scale bar 20 μm). l The quantification of Insulin+ cells per islet is shown as mean ± SEM of 3 to 5 rats per group, or 4–5 pancreas sections per rat for 15–20 islets. Data are presented as the mean ± SEM (n=7). ∗P < 0.05, ∗∗P < 0.01, ***P < 0.001 (compared with the control group); #P < 0.05, ##P < 0.01, ###P < 0.001 (compared with the T2DM group)

Rats were imaged at 24 h after injections using an significantly reduced in islets of diabetic rats and were in vivo imaging system (IVIS) (Fig. 1d). Organs were dramatically upregulated after bmMDE intervention. then harvested and imaged ex vivo to clarify fluorescent NGN3 and OCT4, which are markers for islet progeni- signals and minimize interference (Fig. 1e). tor cells, were almost undetectable in islets from control To evaluate the therapeutic effects of bmMDEs on rats compared with those in islets from diabetic rats. In T2DM, we used a rat model of T2DM based on a high- addition, the expression levels of NGN3 and OCT4 in fat diet combined with STZ injections. Blood glucose diabetic islets were markedly decreased after bmMDE was significantly reduced after bmMSC intervention treatment. Glucose-stimulated insulin secretion (GSIS) (5×106 cells/rat, once every 2 weeks) compared to that results showed that bmMDEs improved glucose- in the T2DM group (Fig. 1f). Additionally, bmMDEs (10 stimulated insulin release in diabetic islets (Fig. 2c). The mg/kg, once every 3 days) also greatly ameliorated mRNA levels of Pdx1, Foxo1, Ngn3, and Oct4 were also hyperglycemia, and blood glucose levels continued de- consistent with the results of immunofluorescent stain- creasing after the last infusion (Fig. 1f). Moreover, the ing (Fig. 2d). body weight of T2DM rats was lower than that of the High-glucose-induced β-cell loss commonly leads to normal control group but was significantly increased islet dysfunction. Therefore, we investigated the effects after bmMSC or bmMDE treatment (Fig. 1g). Import- of bmMDEs on β-cell dysfunction in primary islets and antly, T2DM rats treated with bmMSCs and bmMDEs in the INS-1 cell line. First, we used mannitol (MA) as (Fig. 1h, i; Fig. S1d, e) showed significant improvement an osmotic pressure control to eliminate the effects of in glucose tolerance and insulin sensitivity. Serum insu- osmotic pressure in HG environments. The results lin levels were dramatically decreased in the T2DM showed that MA had no significant effect on insulin se- group and were elevated after bmMDE intervention cretion or β-cell dedifferentiation in islets and INS-1 compared to that in the control group (Fig. 1j). T2DM cells, while exposure to HG environment led to a signifi- rats had a lower β-cell ratio, and the α-and-β-cell imbal- cant decrease in glucose-stimulated insulin release (Fig. ance was reversed by bmMDEs (Fig. 1k, l). These data S2-2a–d). The expression levels of Pdx1 and Foxo1 were suggest that bmMDEs exerted therapeutic effects on islet reduced after HG stimulation in islets and INS-1 cells, function in bmMSCs in vivo. and the levels of Ngn3 and Oct4 were obviously elevated In view of the safety assessment of exosomes, previous in islets but not significantly different in INS-1 cells studies have carried out relevant reports. There was no from those in the normal control group (Fig. S2-2c, d), evidence that MSC-derived exosomes can cause tumori- which indicated that HG-induced INS-1 dedifferenti- genesis. There was no obvious hemolysis, systemic aller- ation was relatively insufficient. Next, bmMDEs were la- gic reactions, and liver and kidney toxicity in different beled with PKH67 green and were co-cultured with kinds of animals, and exosome infusion did not affect INS-1 cells, after which cytoskeletons were visualized the body weight, liver, or kidney function of normal rats. using incubation in rhodamine phalloidin for 24 h to de- Our results also showed that the tail-vein infusion of tect exosomal uptake in INS-1 cells (Fig. S2-2e). GSIS bmMDEs for 10 weeks had no significant effect on the results showed that bmMDEs improved the reduction of liver and kidney of normal rats (Fig. S1f, g). glucose-stimulated insulin release in islets (Fig. 2e) and INS-1 cells (Fig. S2-2f). Real-time quantitative PCR (RT- bmMDEs reverse β-cell dedifferentiation in T2DM rats qPCR) analysis showed that bmMDEs could upregulate Dedifferentiation caused by high glucose can lead to β- the expression of Pdx1, Foxo1 in HG-induced islets and cell dysfunction [24]. We therefore investigated whether INS-1 cells, and downregulate the expression of Ngn3 bmMDEs could alleviate the dedifferentiation level in β- and Oct4 in islets (Fig. 2f, Fig. S2-2g). cells. As shown in Fig. 2a, b and Fig. S2-1, PDX1 and In all of the above results, normal control exosomes FOXO1, which are markers of mature β-cells, were (IDEs) had no effects in HG-treated islets or INS-1 cells. He et al. Stem Cell Research & Therapy (2021) 12:449 Page 7 of 16

Fig. 2 bmMDEs reverse β-cell dedifferentiation. a Paraffin-embedded sections from control + PBS, T2DM + PBS, and T2DM + bmMDE rats were immunolabeled for insulin along with mature β-cell markers (PDX1, FOXO1) and islet progenitor cells (NGN3, OCT4) (scale bar 20 μm). b Fluorescent-intensity expression analysis of the above indicators. c GSIS assays to determine insulin secretion of islets isolated from the indicated groups. RT-qPCR analyses for Pdx1, Foxo1, Ngn3, and Oct4 in islets (d) isolated from the indicated groups. Insulin secretion in islets (e) of control + PBS, HG + PBS, HG + IDEs, and HG + bmMDE groups. RT-qPCR analyses for Pdx1, Foxo1, Ngn3, and Oct4 in islets (f) from the above groups. Data are presented as the mean ± SEM (∗P < 0.05; ∗∗P < 0.01; ***P < 0.001)

These data suggest that treatment with bmMDEs re- therefore conducted high-throughput sequencing of versed β-cell dedifferentiation both in vivo and in vitro. miRNA profiles in IDEs, HG-treated INS-1-cell-derived exosomes (HIDEs), and bmMDEs. A heat map was cre- MiR-146a in bmMDEs reverses β-cell dedifferentiation ated to identify the differentially abundant miRNAs (Fig. We next explored how bmMDEs reversed dedifferenti- 3a), and 10 of the most upregulated miRNAs in ation of β-cells. Exosomes can regulate physiological ac- bmMDEs were subjected to validation by RT-qPCR. The tivities through exosomal miRNAs [25, 26]. We expression levels of all 10 miRNAs in bmMDEs were He et al. Stem Cell Research & Therapy (2021) 12:449 Page 8 of 16

Fig. 3 MiR-146a in bmMDEs reverses β-cell dedifferentiation. a Heat map of miRNA sequencing showing the differently expressed miRNAs in IDEs, HG IDEs, and bmMDEs. b RT-qPCR verification of the expression levels of the top 10 differential miRNAs. c A GSIS assay was performed to detect insulin stimulation in HG-treated INS-1 cells transfected with the top 10 differential miRNA mimics. Levels of miR-146a in INS-1 cells (d) and islets (e) treated with bmMDEs. RT-qPCR analyses for Pdx1, Foxo1, Ngn3, and Oct4 in islets (f–i) transfected with an miR-146a mimic or miR-NC mimic as indicated. mRNA expression levels of Pdx1, Foxo1, Ngn3, and Oct4 in islets (j–m) transfected with an miR-146a inhibitor or miR-NC inhibitor as indicated. Data are presented as the mean ± SEM (∗P < 0.05; ∗∗P < 0.01; ***P < 0.001) higher than those in IDEs and HIDEs (Fig. 3b). We then namely in Fig. S3a. In addition, miR-146a mimics also performed a screen for the functional miRNA using the ameliorated deleterious effects of HG in islets (Fig. S3b, GSIS experimental setup in INS-1 cells. After pretreat- c). RT-qPCR analysis further confirmed that treatment ment with top 10 miRNA mimics, the cells were then in- with bmMDEs elevated the expression level of miR-146a cubated with HG medium for 72 h. As shown in Fig. 3c, in both INS-1 cells and primary cultured islets (Fig. 3d, miR-146a-5p (miR-146a) mimics significantly increased e). Furthermore, expression levels of Pdx1 and Foxo1 in- insulin secretion that was otherwise inhibited by high- creased, while those of Ngn3 and Oct4 decreased in the glucose environments compared to those of the other HG + miR-146a-mimic group, compared with those in nine miRNA mimics. The overexpression efficiencies of the HG + miR-NC-mimic group in primary cultured is- miRNAs are shown in the Supporting information, lets (Fig. 3f–i). To further investigate the role of miR- He et al. Stem Cell Research & Therapy (2021) 12:449 Page 9 of 16

146a, INS-1 cells (Fig. S3d) and islets (Fig. S3e) were is the direct downstream target of miR-146a-5p in the transfected with an miR-146a inhibitor. As expected, the regulation of β-cell function and dedifferentiation. effect of bmMDEs on islets was abolished by an miR- 146a inhibitor (Fig. 3j–m). Collectively, these data dem- MiR-146a/Numb improves β-cell function through β- onstrate that miR-146a in bmMDEs mediated the rever- catenin nuclear translocation sal of the dedifferentiated state of β-cells. β-catenin is a known signaling molecule that is downstream of Numb [31]. β-catenin has been shown to Exosomal miR-146a directly targets Numb in β-cells modulate pancreatic ontogenesis and islet function [32, To explore the mechanism of how miR-146a regulates 33]. Therefore, we explored whether β-catenin mediates β-cell function, TargetScan (https://www.targetscan.org) the ameliorative effects of bmMDEs in our T2DM model was used to predict the potential targets of miR-146a. in vitro. First, we examined the protein levels of β- Among all of the predicted targets with conserved sites catenin under different intervention conditions. We of miR-146a, NUMB, a human homolog membrane- found an increase in nuclear protein levels of β-catenin bound protein [27], was noteworthy. It is known that in islets treated with bmMDEs, whereas the protein level NUMB is upregulated in diabetic patients and positively of β-catenin in the cytoplasm was not changed (Fig. 5a, regulates the expression of NGN3, a marker of islet pro- Fig. S5a). Similar results were examined in the miR- genitor cells [28–30]. We found that the expression level 146a-mimic group (Fig. 5b; Fig. S5b). The effect of of NUMB was significantly upregulated in islets within bmMDEs increasing the nuclear protein level of β- T2DM rats compared with that in the control group, catenin protein was attenuated when the expression of and that bmMDE treatment decreased NUMB expres- miR-146a was inhibited (Fig. 5c; Fig. S5c). To confirm sion (Fig. 4a; Fig. S4a). We then confirmed that Numb activation of the β-catenin signaling pathway, XAV-939, expression was enhanced after HG treatment and was a β-catenin inhibitor, was used to promote β-catenin downregulated by an miR-146a mimic at the mRNA degradation. GSIS was impaired after XAV-939 interven- level in islets (Fig. 4b), as well as at the protein level in tion, and miR-146a mimics effectively attenuate this INS-1 cells (Fig. 4c; Fig. S4b). Subsequently, we found damage (Fig. 5d). We then found that miR-146a mimics that bmMDEs reduced the expression of Numb post-HG elevated the expression levels of Pdx1 and Foxo1 and de- treatment, whereas an miR-146a inhibitor attenuated creased expression levels of Ngn3 and Oct4 in HG- this effect of bmMDEs in terms of mRNA in islets (Fig. treated islets. However, the effect of miR-146a mimics 4d) and at the protein level in INS-1 cells (Fig. 4e; Fig. was partially restored when combined with XAV-939 S4c). Through bioinformatic analysis, we found that the (Fig. 5e–h). Then, we verified the relationship between 3′UTR of the Numb gene contained putative binding Numb and β-catenin in primary islets and INS-1 cells, sites for miR-146a (Fig. 4f). To further verify the associ- respectively. The nuclear protein levels of β-catenin de- ation between Numb and miR-146a, luciferase reporter creased after Numb overexpression, while these levels plasmids containing the wild-type 3′UTR (Numb 3′UTR were elevated after knock down of Numb (Fig. 5i, j and WT) and mutated 3′UTR (Numb 3′UTR MUT) of Fig. S5d-g). These results suggest that Numb/β-catenin Numb were generated. We then found that miR-146a may be downstream effectors of miR-146a in the regula- mimics significantly reduced luciferase activity in INS-1 tion of islets and β-cell function and dedifferentiation. cells containing Numb 3′UTR WT but had no significant effect on Numb 3′UTR MUT cells (Fig. 4g). Fur- MiR-146a knockdown abolishes bmMDE-mediated thermore, to determine the function of NUMB on islets, reversal of β-cell dedifferentiation in vivo we overexpressed full-length Numb (Fig. S4d, e) and To test whether miR-146a is required for the efficacies knocked down Numb expression (Fig. S4f, g) with small of bmMDEs in vivo, we knocked down miR-146a ex- interfering RNAs (siRNAs) in islets. GSIS experiments pression via lentiviruses in bmMSCs (Fig. S6a). As ex- showed that Numb overexpression reduced GSIS, and pected, the level of miR-146a in exosomes derived from inhibition of Numb attenuated the inhibition of GSIS by miR-146a-knockdown bmMSCs (miR-146a KD the HG environment (Fig. 4h, m). Finally, we examined bmMDEs) was decreased significantly compared with the effect of NUMB on the dedifferentiation of islet β- that from exosomes derived from miR-NC-knockdown cells in vitro. RT-qPCR results showed that the expres- bmMSCs (miR-NC KD bmMDEs) (Fig. S6b). Exosomes sion levels of Pdx1 and Foxo1 were significantly de- were then injected into T2DM rats to detect their effects creased after Numb overexpression and that the on β-cell function in vivo. Levels of blood glucose rose expression levels of Ngn3 and Oct4 were significantly in- to ~18 mmol/L at week 5 in all rats after STZ injection. creased (Fig. 4i–l). The effect achieved after suppressing However, blood glucose of rats decreased dramatically Numb was similar to that of miR-146a mimics shown in after bmMDE intervention in the T2DM + bmMDEs- Fig. 3f–i (Fig. 4n–q). These data demonstrate that Numb miR-NC KD group (~10 mmol/L at week 10), as compared He et al. Stem Cell Research & Therapy (2021) 12:449 Page 10 of 16

Fig. 4 Exosomal miR-146a directly targets Numb in β-cells. a Immunofluorescent staining for NUMB expression in pancreatic sections (scale bar 20 μm). RT-qPCR analyses of the mRNA levels of Numb in islets transfected with miR-146a mimics (b) or treated with an miR-146a inhibitor combined with bmMDEs (d). Western blotting for NUMB in INS-1 cells transfected with miR-146a mimics (c) or treated with an miR-146a inhibitor combined with bmMDEs (e), β-actin was used as the loading control. f Schematic of the sequence that miR-146a targets in the WT or mutated 3′ UTR of Numb mRNA. g Luciferase activity of Numb WT or mutated 3′UTR reporter plasmids in INS-1 cells transfected with miR-146a mimics or miR-NC mimics as indicated. A GSIS assay was performed to detect insulin stimulation in islets transfected with Numb overexpression (h) or siRNA (m). RT-qPCR analyses for Pdx1, Foxo1, Ngn3, and Oct4 in islets transfected with Numb overexpression (i–l) or siRNA (n–q). Data are presented as the mean ± SEM (∗P < 0.05; ∗∗P < 0.01; ***P < 0.001) He et al. Stem Cell Research & Therapy (2021) 12:449 Page 11 of 16

Fig. 5 MiR-146a/Numb reverses β-cell dedifferentiation and improves β-cell function through β-catenin nuclear translocation. a Nuclear translocation of β-catenin detected after bmMDE treatment in primary islets, as indicated by Western blotting analysis. b, c Western blotting for nuclear translocation of β-catenin in primary islets transfected with miR-146a mimics or treated with an miR-146a inhibitor combined with bmMDEs. Glucose-stimulated insulin secretion (d) in islets transfected with miR-146a mimics combined use XAV-939. e–h RT-qPCR analyses for Pdx1, Foxo1, Ngn3, and Oct4 in islets transfected with miR-146a mimics combined use XAV-939. Confirmation of the changes of β-catenin nuclear translocation of Numb overexpression (i) and siRNA (j). β-actin and GAPDH were used as loading controls. Data are presented as the mean ± SEM (∗P < 0.05; ∗∗P < 0.01; ***P < 0.001) with that in the T2DM + PBS group (~18 mmol/L at expected, the effect of bmMDEs was abolished via miR- week 10) and T2DM + bmMDEsmiR-146a KD group 146a knockdown (Fig. 6b–e). Moreover, serum insulin (~16.5 mmol/L at week 10, Fig. 6a). The body weight of levels in the T2DM + bmMDEsmiR-NC KD group were T2DM rats was significantly increased after bmMDEs- significantly higher than those in the T2DM + PBS miR-NC KD treatment, but there was no statistical differ- group and T2DM + bmMDEsmiR-146a KD group (Fig. 6f). ence compared with that in the bmMDEsmiR-146a KD- Next, immunofluorescent staining was performed in treated group (Fig. S6c). T2DM rats treated with pancreatic tissue sections from bmMDE-treated rats to bmMDEsmiR-NC KD showed significant improvement in examine dedifferentiation of islets in vivo. The expres- glucose tolerance and insulin sensitivity, and, as sion levels of PDX1 and FOXO1 were increased in islets, He et al. Stem Cell Research & Therapy (2021) 12:449 Page 12 of 16

Fig. 6 MiR-146a-knockdown bmMSC-derived exosomes fail to reverse β-cell dedifferentiation in vivo. a Blood glucose in control + PBS, T2DM + PBS, T2DM + bmMDEmiR-NC KD, and T2DM + bmMDEmiR-146a KD rats. b–e IPGTT and IPITT were performed after the last intervention of bmMDEsmiR-NC KD and bmMDEsmiR-146a KD. Values of the areas under the curve in IPGTT and IPITT were measured. f Fasting serum insulin of control + PBS, T2DM + PBS, T2DM + bmMDEmiR-NC KD, and T2DM + bmMDEmiR-146a KD rats as indicated. g Paraffin-embedded sections from the indicated groups were immunolabeled for insulin along with PDX1, FOXO1, NGN3, and OCT4 (scale bar 20 μm). h–k RT-qPCR analyses for Pdx1, Foxo1, Ngn3, and Oct4 in islets from the above groups. Data are presented as the mean ± SEM (∗P < 0.05; ∗∗P < 0.01; ***P < 0.001) while those of NGN3 and OCT4 were decreased in levels of Pdx1 and Foxo1 and decreased expression levels T2DM rats after bmMDEmiR-NC KD treatments com- of Ngn3 and Oct4, while the effects of bmMDEs were pared with those in the T2DM + PBS group and T2DM abolished by miR-146a knockdown in T2DM rats (Fig. + bmMDEsmiR-146a KD group (Fig. 6g; Fig. S6d). RT- 6h–k). These data indicate that exosomes without miR- qPCR results of corresponding primary cultured islets 146a attenuated their ability to improve β-cell function demonstrated that bmMDEs upregulated the expression and reverse dedifferentiation. He et al. Stem Cell Research & Therapy (2021) 12:449 Page 13 of 16

Fig. 7 Working model of bmMSC-derived exosomal miR-146a in reversing diabetic β-cell dedifferentiation. Exosomes derived from mesenchymal stem cells reverse diabetic β-cell dedifferentiation via the miR-146a/Numb/β-catenin signaling pathway

Discussion dedifferentiation markers were suppressed, indicating The declining function of pancreatic β-cells is central to that bmMDEs may alleviate the dedifferentiation of β- the progression of T2DM, and it has been confirmed cells. These findings suggest novel treatment effects of that β-cell dedifferentiation is an important mechanism bmMDEs in mitigating hyperglycemia-induced β-cell leading to functional β-cell reduction [5, 34, 35]. In re- dysfunction. cent years, MSC-based therapies have been demon- Exosomes regulate many physiological activities strated as potential approaches for treating diabetes and through exosomal miRNAs [37, 45]. In our present other diseases [36, 37]. However, some disadvantages of study, miR-146a emerged from our non-biased sequen- MSCs—such as their tumorigenic potential, issues in in- cing and subsequent functional screen. Interestingly, ducing thrombosis, and low survival times in vivo—have miR-146a has previously been characterized in the con- limited their clinical applications. Increasing evidence text of T2DM [46, 47]. For example, miR-146a is signifi- has suggested that exosomes, an important manifest- cantly lower in diabetic patients than in healthy controls ation of paracrine actions, are the underlying substances and is significantly overexpressed in T2DM patients that mediate the therapeutic effects of MSCs [38–40]. treated with metformin [48]. Liu et al. found that miR- Our present results also confirmed that bmMDEs had 146a mimics improve diabetic retinopathy by regulating similar therapeutic effects to those of bmMSCs on islet IRAK1/TRAF6 and their downstream pro-inflammatory functions. gene expressions [47]. Beatrix et al. discovered that miR- Hyperglycemia-induced dedifferentiation is an import- 146 is a potential therapeutic target for the treatment of ant mechanism that leads to β-cell failure [35, 41–43], diabetic retinopathy through its inhibition of NF-κB acti- and we found that β-cells in diabetic rats and primary is- vation [49]. Roggli et al. indicated that miR-146a was up- lets with high glucose interventions lost their identities regulated in human pancreatic islets after exposure to and turned into pancreatic endocrine progenitors that pro-inflammatory cytokines, and inhibition of miR-146a featured by high levels of Ngn3 and Oct4, as well as sig- leads to improved β-cell function [50]. In addition, Anna nificantly decreased expression of Pdx1 and Foxo1. et al. showed that compared with human pancreatic is- These results are consistent with previous reports [6, lets (24 h), INS-1 cells have a rapid (<6 h) expression of 44]. However, in the bmMDE treatment group in our inflammatory genes induced by cytokine signaling, and present study, β-cell markers were restored and the expression of miR-146a is downregulated upon He et al. Stem Cell Research & Therapy (2021) 12:449 Page 14 of 16

exposure in INS1 cells after 24 h. Transfection of INS1 T: Intraperitoneal glucose tolerance test; IPITT: Intraperitoneal insulin cells with miR-146a-5p also downregulates islet inflam- tolerance test; AUC: Area under the curve; FBS: Fetal bovine serum; β κ PBS: Phosphate-buffered saline; HFD: High-fat diet; HG: High glucose; mation and -cell death in part by impaired NF- B and MA: Mannitol; NC: Negative control; TEM: Transmission electron microscopy; MAPK signaling [51]. In our study, high glucose inter- GSIS: Glucose-stimulated insulin secretion; PDX1: Pancreatic duodenal vention for 72 h is a chronic stimulus, and the decrease homeobox-1; FOXO1: Forkhead box-containing protein O1; NGN3: Neurogenin-3; OCT4: Octamer-binding transcription factor-4 in miR-146a-5p expression may be a combination of alterations secondary to changes in the transcription of early immediate response genes and compensatory/re- Supplementary Information storative responses. However, further verification in hu- The online version contains supplementary material available at https://doi. man pancreatic islets or β-cell derived from hiPSC/hESC org/10.1186/s13287-021-02371-0. is still needed to make the conclusion more convincing. Additional file 1. Next, we discussed the specific role and mechanism of Additional file 2. miR-146a in regulating β cell dedifferentiation. After Additional file 3. consulting the TargetScan database and previous studies, we found that the miR-146a downstream target gene, Numb, is upregulated in diabetic patients and positively Acknowledgements regulates the expression of the islet progenitor cell We thank LetPub (www.letpub.com) for its linguistic assistance during the marker, NGN3. Here, we then indicated that miR-146a preparation of this manuscript. negatively regulated β-cell dedifferentiation by directly binding to 3′UTR of Numb mRNA and inhibited its ex- Authors’ contributions β QH performed the research, data analysis, and manuscript writing. JS, CC, pression in -cells. Even though the association between JBW, HQH, XHG, and MMY participated in the research and data collection. miR-146a and Numb has been reported previously [52– LSW, FY, and KL helped with the sample collection. ZJL and FQL contributed 54], our study demonstrated, for the first time, that to the data analysis. ZS provided technical support and edited the paper. β XGH and MD contributed to the design of the study. LC supervised the bmMSC-derived exosomal miR-146a rescued diabetic - overall study design. All authors read and approved the final manuscript. cell dysfunction via Numb inhibition. β-catenin has been reported to be a downstream target Funding gene that is negatively regulated by Numb and is linked This work was supported by the National Natural Science Foundation of to the regulation of islet function [54, 55]. To further China (grant numbers 82070800, 81670706, 81873632, 81770818, 81800736, understand the mechanism underlying Numb-mediated 81800727, 81900756), the National Key R&D Program of China (grant β numbers 2016YFC0901204, 2018YFC1311801), the Taishan Scholars responses, we investigated -catenin signaling in the Foundation of Shandong Province (grant number ts201712089), and the context of diabetic β-cell dysfunction. We demonstrated Natural Science Foundation of Shandong Province (grant number that bmMDE treatments or miR-146a mimics enhanced ZR2019BH018, ZR2019PH078). β-catenin nuclear translocation. Moreover, miR-146a mimics rescued the inhibition of β-catenin, islet dysfunc- Availability of data and materials tion, and dedifferentiation caused by XAV-939. We also The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. verified the targeting relationship between Numb and β- catenin. These results highlight that the miR-146a/ Numb/β-catenin signaling pathway is a crucial mediator Declarations of bmMDE-induced amelioration of hyperglycemia- Ethics approval and consent to participate induced β-cell dysfunction. All animal experiments were conducted in accordance with the Animal Ethics Committee of Shandong University (ethical number DWLL-2015-005). Conclusion In summary, our present findings elucidate a novel Consent for publication Not applicable. mechanism by which bmMDEs regulate islet function and β-cell dedifferentiation. Results from T2DM rats Competing interests and primary islets indicated that the beneficial effects of The authors declare that they have no competing interests. bmMDEs were mainly achieved via the miR-146a-5p/ Numb/β-catenin pathway (Fig. 7). Our findings shed Author details 1Department of Endocrinology, Qilu Hospital, Cheeloo College of Medicine, light on the potential of MSC-derived exosome-based Shandong University, No. 107 Wenhua Xi Road, Jinan 250012, Shandong, therapeutic approaches for treating diabetes. China. 2Department of Cell Biology, Cheeloo College of Medicine, Shandong University, Jinan 250012, China. 3Institute of Endocrine and Metabolic Abbreviations Diseases of Shandong University, Jinan 250012, China. 4Key Laboratory of MSCs: Mesenchymal stem cells; bmMSC: Bone marrow mesenchymal stem Endocrine and Metabolic Diseases, Shandong Province Medicine & Health, cell; bmMDEs: Bone marrow mesenchymal stem cell-derived exosomes; Jinan 250012, China. 5Jinan Clinical Research Center for Endocrine and IDEs: INS-1-derived exosomes; T2DM: Type 2 diabetes mellitus; IPGT Metabolic Diseases, Jinan 250012, China. He et al. Stem Cell Research & Therapy (2021) 12:449 Page 15 of 16

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