MSCs-EXOs containing miR-21a-5p promoted cell proliferation in the rat retinal Müller cell rMC-1 via targeting LATS1

Abstract

Background

Diabetic retinopathy (DR) is a leading cause of blindness in adults, which is characterized by neurovascular dysfunction. Retinal neurodegeneration was involved in the pathogenesis of early-stage DR. This research aims to reveal the therapeutic effects and potential molecule mechanism of Mesenchymal stem cells-derived exosomes (MSCs-EXOs) in Müller cells at the early stage of DR. More specifically, we investigated in the rat retinal Müller cell line rMC-1.

Methods

We cultured rMC-1 in high glucose (HG) medium mixed with MSCs-EXOs to observe the changes in cell proliferative capacity and function. EdU assay, Immunofluorescence staining and Cell viability were used to assess cell proliferative capacity and function. Western blot and Quantitative Real-Time polymerase chain reaction (qRT-PCR) were used to assess the expression of cell proliferation-related proteins and cell cycle-related protein. Finally, dual-luciferase reporter assay, miRNA sequencing and cell transfection were used to assess the relationships between MSCs-EXOs and binding site.

Results

Here our results displayed that MSCs-EXOs promoted HG-induced cell proliferation in rMC-1. Furthermore, MSCs-EXOs protected HG-induced function of rMC-1. Mechanistically, MSCs-EXOs promoted the proliferation of rMC-1 by inhibiting the Hippo pathway to decrease the expression of Large tumor suppressor 1 (LATS1) and p-YAP and increase the expression of cell proliferation-related proteins Yes-associated protein (YAP), EGFR and cell cycle-related protein CYCLIN D1. In addition, LATS1 knockdown inhibited p-YAP expression and promoted HG-induced cell proliferation and YAP, EGFR and CYCLIN D1 expression in rMC-1. We subsequently performed bioinformatics sequencing analysis of MSCs-EXOs and confirmed that LATS1 was the target of miR-21a-5p.

Conclusion

Our research proved that MSCs-EXOs containing miR-21a-5p increased the expression of YAP via targeting LATS1, and ultimately promoted the cell proliferation in rMC-1. Our current study clarified the molecular mechanism of MSCs-EXOs-regulated cell proliferation and function protection in rMC-1 and provided a novel strategy for the treatment of DR.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04639-1.

Introduction

DR is one of the most common complications of diabetes mellitus. DR is generally considered a microvascular disorder. In addition, the pathogenesis of DR is also associated with retinal neuroglial degeneration. Previous studies have shown that retinal neurodegeneration occurs earlier than retinal microangiopathy and affects the progression of microangiopathy [1]. Studies have found that existing treatments for microangiopathy such as retinal photocoagulation can cause a decrease in the amplitude of both the ERG a- and b-waves [2]. This is one of the reasons why the vision of DR patients cannot be restored or even continues to decline after the microvascular lesions are controlled. There is no effective treatment to reverse the progression of DR. Therefore, it is very important to study the pathogenesis of DR, with the hope of providing a new therapeutic strategy for the treatment at early stage of DR.

Müller cells are the main glial cells in the retina that span from the inner limit of the retina to the outer retinal membrane. Müller cells not only act to maintain the integrity of the retinal structure, but they are essential for retinal homeostasis and physiological functions [3]. In research in the field of stem cells, Müller cells have been found to have stem cell characteristics, which offer hope for the induction of endogenous retinal regeneration [4]. In zebrafish, the response of Müller glia to retinal injury involves a reprogramming event that imparts retinal stem cell characteristics and enables them to produce a proliferating population of progenitors that can regenerate all major retinal cell types and restore vision [3]. Studies have confirmed that inhibiting the apoptosis of Müller cells under high glucose conditions can increase the ERG amplitude and restore part of the retinal function and visual function of DR rats [5]. Therefore, we speculate that the development of DR can be inhibited by promoting Müller cell proliferation and protecting the function of Müller cells.

The Hippo signaling pathway plays an important role in cell growth, proliferation, apoptosis, and stem cell regeneration, and is a highly conserved signal transduction pathway [6]. The core of the Hippo pathway is a kinase cascade, and activated LATS1 phosphorylate and inactivate YAP, the main downstream effector of the Hippo pathway [7–9]. LATS1 is a serine/threonine kinase and has been shown to inhibit cell proliferation in vitro via its kinase activity [10–12]. YAP is the main downstream effector of the Hippo pathway, which can directly control cell cycle progression and induce cell proliferation [13]. Studies have found that YAP is specifically expressed in Müller cells of the adult retina [14]. The Hippo signaling pathway may be a mechanism of endogenous regeneration [15, 16]. Therefore, we speculated that regulating the Hippo pathway could promote the proliferation of Müller cells and achieve the goal of delaying the development of DR.

With the attention paid to the therapeutic role of MSCs-EXOs, there is more and more evidence to prove the effectiveness of MSCs-EXOs in the treatment of DR. MSCs-EXOs can transport RNA, cytokines and proteins from MSCs to the target cells to exert biological activity [17]. It has been pointed out that MSCs-EXOs from different sources can promote the axon growth of retinal ganglion cells, thus having a protective effect on nerve cells [18]. In the study of MSCs-EXOs to protect nerve cells, researchers found that MSCs-EXOs functions through the miRNA and neurotrophic factors it carries, and MSCs-EXOs contains more than hundreds of different miRNAs [19]. MSCs-EXOs inhibits photoreceptor cell apoptosis and maintains normal retinal structure, and further proteomic analysis showed that MSCs-EXOs contains anti-inflammatory, neuroprotective, and anti-apoptotic proteins [20].

Based on the evidence, we speculated that MSCs-EXOs could promote cell proliferation in Müller cells via inhibiting the Hippo pathway. Our study aimed to explore the role and potential mechanisms of MSCs-EXOs on Müller cells in the early stage of DR and offer new therapeutic targets for the treatment of DR.

Materials and methods

Isolation and identification of MSCs-EXOs

Isolation and identification of MSCs-EXOs were performed as previously described [21]. Briefly, human umbilical cord mesenchymal stem cells (hUC-MSCs) were obtained from Shandong Qilu Stem Cell Engineering Co. LTD. The extracted conditioned media (CM) was centrifuged at 300 g for 5 min, 2000 g for 10 min, and 10,000 g for 30 min at 4℃. Exosomes were pelleted using a Hitachi CP80WX ultracentrifuge (Hitachi High Technologies, Tokyo, Japan) at 4℃ for 70 min at 100,000 g. After centrifugation the supernatant was collected, and the pellet was resuspended in phosphate-buffered saline (PBS) (Solarbio Technology, Beijing, China). A second round of ultracentrifugation was performed, after which and the pellet was resuspended in PBS and filtered using a 0.22-µm film. The concentration and morphology of the collected exosomes were evaluated using a Hitachi HT-7700 transmission electron microscope (TEM) (Hitachi High Technologies, Japan). The particle size and content of the collected exosomes were confirmed using the Nanosight ns 300 (Malvern, UK) and the nanoparticle tracking analysis (NTA) software. The surface markers CD9, CD81, Alix and Calnexin of MSCs-EXOs were analyzed by Western blot. The concentration of MSCs-EXOs were measured using a BCA assay reagent (Solarbio, Beijing, China).

Cell culture and treatment

Rat retinal Müller cell line (rMC-1) used in this study was purchased from BNCC (Henan, China) and identified using antibodies against glial acidic fibrillary protein (GFAP) (1:200, Proteintech) and glutamine synthetase (GS) (1:200, Proteintech). The rMC-1 were routinely cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (Liji Biotech, China), and 1% antibiotics (Gibco, USA) in a humidified atmosphere of 37 °C and 5% CO2. The rMC-1 were cultured in DMEM medium (5.5mM glucose, normal glucose) until reaching 80% confluency. Subsequently, the cells were maintained in serum-free medium for 24 h and then switched to a medium containing 30mM glucose (high glucose, HG) for 24 h. In the treatment group, the rMC-1 were incubated in HG mixed with MSCs-EXOs at a concentration of 12.5 µg/mL for the same period.

EdU assay

The 5-ethynyl-20-deoxyuridine (EdU) incorporation assay was performed using an EdU assay kit with Alexa Fluor 594 (Beyotime, Biotechnology). The rMC-1 were seeded into 6-well plates at 4 × 10⁴ cells/well for 24 h. Then 10µM EdU reagent was added to each well and continued incubation for 2 h at 37 °C. At the indicated times, the cells were fixed with 4% paraformaldehyde for 15 min. After the cells were washed three times with 3% bovine serum albumin (BSA), 0.3% Triton X-100 was used to block the cells for 10 min. After that, the cells were incubated with the prepared click reaction solution (prepared according to the manufacturer’s instructions) for 30 min at room temperature in the dark. After washing three times with 3% BSA, the cells were stained with 0.1% Hoechst 33,342 for 10 min in the dark for nuclear detection. Finally, the cells were observed under a fluorescence microscope (20×, ZEISS, Germany), and the percentage of EdU-positive cells was calculated.

Western blot analysis

Cultured rMC-1 were lysed in RIPA buffer containing protease inhibitor (Beyotime, China) on ice for 30 min. The resulting lysates were then subjected to centrifugation at a speed of 15,000 rpm for 15 min at 4℃. Protein concentrations were measured using a BCA protein assay reagent (Solarbio, Beijing, China). Subsequently, the samples were loaded onto a 4–12% SDS‒PAGE gel (with a sample amount of 20 µg) and transferred onto immunoblot NC membranes (Roche, pore size 0.22 μm) using the Trans-Blot Turbo Transfer System (Bio-Rad) and then blocked in QuickBlock™ Blocking Buffer for Western Blot (Beyotime, China) at room temperature for 20 min. Finally, the membranes were incubated with the primary antibodies, diluted in a primary antibody dilution buffer (Beyotime, China) at 4℃ overnight. Primary antibodies included β-actin (1:20000, Proteintech), EGFR (1:2000, Abcam), LATS1 (1:1000, Abcam), p-YAP (1:2000, Abcam), YAP (1:2000, Proteintech), CYCLIN D1 (1:2000, Proteintech), CD9 (1:1000, Abcam), CD81 (1:1000, CST), Alix (1:1000, Abcam) and Calnexin (1:1000, Proteintech). After rinsing three times with TBS-T, the membranes were incubated with the fluorescent secondary antibody (1:5000; LI-COR) for 1 h at room temperature. After washing, the bands were detected using Dylight 680 and Dylight 800 (Bio-Rad, USA). The intensity of the bands was analyzed by Image J and normalized to β-actin.

Immunofluorescence staining

The rMC-1 were seeded in 96-well plates (2 × 10³ cells/well) and cultured overnight. At the indicated times, the cells were washed and fixed with 4% paraformaldehyde for 20 min. After washing three times with PBS, 0.3% Triton X-100 and 3%BSA was used to block the cells for 1 h, after which the cells were incubated with the primary antibody overnight at 4℃. Then, the secondary antibody (1:500) was added, and the cells were incubated for 2 h at room temperature with Alexa Fluor^® 488-conjugated streptavidin in the dark. Finally, the cells were stained with DAPI for nuclear detection and observed under a fluorescence microscope (20×, ZEISS, Germany). All images were acquired under identical exposure and gain settings. Nuclei counterstained with DAPI were used to demarcate individual cells. For clustered cells, normalized fluorescence intensity to DAPI to account for clustering. The fluorescent signals were analyzed by Image J.

RNA extraction and quantitative Real-Time PCR (qRT-PCR)

RNA was extracted using a SteadyPure Rapid RNA Extraction Kit (Accurate Biology, Cat# AG21023). Reverse transcription was carried out using a cDNA Synthesis Kit (Vazyme, Nanjing, China) to synthesize cDNA with 1000ng. The cDNA was diluted to 5ng/µl for subsequent qRT-PCR. qRT-PCR was conducted in 96-well plates using a LightCycler 96@Real-Time PCR System (Roche, Germany), and each sample was analyzed in triplicate. Relative mRNA levels were quantified via normalization to the level of β-actin, which was detected using commercially available predesigned primers, which were purchased from Tsingke Biotechnology (Beijing, China). Each reaction (10µL of volume) contained 0.4µL of primer (10µM), 2µL of cDNA, 5µL of SYBR Green Master Mix (2×) (Cat. Q711-02, Vazyme, Nanjing, China) and 2.6µL of ddH2O. The reaction was conducted by 45 cycles of 95℃ for 10s, 60℃ for 15s and 72℃ for 15s (single cycle). The relative mRNA expression of the target genes was acquired using the 2^–ΔΔCt method, with β-actin serving as a normalization gene. The relative expression of miR-21a-5p was normalized by U6.

The primers used in the study were listed as follows (written 5′–3′):

YAP (F): TTCGGCAGGCAATACGGAAT.

YAP (R): TCATCCCGGGAGAAGACACT.

LATS1 (F): TTGACTGACTTTGGCTTGTGC.

LATS1 (R): GCTATCTTGCCGTGGGTGA.

EGFR (F): ATGCTGATAGCCGCCCAAAG.

EGFR (R): CCTCCATCAGGGCTCGGTAA.

AQP4 (F): TTCAAAGGCGTCTGGACTCA.

AQP4 (R): AGCAGAGGGAGATGAGGACC.

β-actin (F): GGAGATTACTGCCCTGGCTCCTA.

β-actin (R): GACTCATCGTACTCCTGCTTGCTG.

U6 (F): GGAACGATACAGAGAAGATTAGC.

U6 (R): TGGAACGCTTCACGAATTTGCG.

miR-21a-5p (F): GCGCGTAGCTTATCAGACTGA.

miR-21a-5p (R): AGTGCAGGGTCCGAGGTATT.

Cell transfection

The small interfering RNA of LATS1 (LATS1 siRNA), the LATS1-wt, the LATS1-mut plasmids, the miR-21a-5p mimics and the miR-21a-5p inhibitor as well as them negative controls (miR-21a-5p mimics-NC and miR-21a-5p inhibitor-NC) were purchased from Tsingke Biotechnology (Beijing, China). The concentration of siRNA, miR-21a-5p mimics and inhibitor both were used 50nM. For in vitro transfection, cells were transfected with the vectors and plasmids using Lipofectamine™ 3000 (Invitrogen, CA, USA). After 24–48 h, cells were used for follow-up experiments following the detection of transfection efficiency by qRT-PCR or Western blot.

Dual-luciferase reporter assay

StarBase (https://starbase.sysu.edu.cn/). was used to predict the binding sites. Cells were co-transfected with LATS1-wt or LATS1-mut plasmids and miR-21a-5p mimics or mimics NC by Lipofectamine™ 3000 (Invitrogen, CA, USA). The transfected cells were lysed in lysis buffer for 10 min. Then, the resulting lysates were then subjected to centrifugation at a speed of 12,000 rpm for 5 min. After centrifugation the supernatant was collected, the supernatant was used to examine luciferase activity. The Luciferase activity was examined using a dual-luciferase reporter assay system (Promega, WI, USA).

MiRNA sequencing

An Illumina HiSeq was used to identify the miRNAs found in MSC-derived exosomes (Ribo Bio, Guangzhou, China). The exosome total RNA was extracted and quantified using a NanoDrop ND-100. The 5′ and 3′ ends of total RNA were then ligated with short RNA adapters. The PCR-amplified fragments were purified from the PAGE gel and the completed cDNA libraries were measured using an Agilent 2100 Bioanalyzer after cDNA syntheses and amplification. Cluster generation was performed on an Illumina cBot, and sequencing was carried out using an Illumina HiSeq 2000 according to the manufacturer’s instructions.

Cell viability

Cell Counting Kit-8 (CCK-8, Antgene, China) was utilized to determine cell viability. The rMC-1 were planted into a 96-well plate (2 × 10³ cells/well) and cultured with a mixture of different concentrations of MSCS-EXOS (0, 12.5, 25, 50 µg/mL) and high glucose for 24 h. Then the cells were incubated with 10µL of CCK8 solution in each well at 37 C for 2 h. A microplate reader (BioTek, USA) was used to measure the optical density (OD) at 450 nm.

Statistical analysis

All the data are presented as the means ± standard deviations (SD). GraphPad Prism 9.0 software was used to perform the statistical analysis. We employed Student’s t test for the analysis of differences between two groups and one-way ANOVA for the analysis of differences among three or more groups. Subsequently, in the case of a statistical significance defined as a p-value of 0.05 or less, a post-hoc analysis of Tukey’s Honest Significant Difference (HSD) test was used to calculate the level of significance between each pair of groups. P- value < 0.05 was defined as statistical significance.

Results

High glucose inhibited cell proliferation in rMC-1

MSCs-EXOs were characterized as previously described [21]. As per the Minimum information for studies of extracellular vesicles (MISEV) guidelines, we added a exosomal negative marker Calnexin (Fig. 1A). Western blot analysis suggested that Calnexin was expressed in the cytosol, while not expressed in exosomes; the exosomal positive markers CD9, CD81 and Alix were expressed in exosomes. The rMC-1 were identified with GFAP and GS protein immunofluorescence. The result showed that the rMC-1 were strongly GFAP and GS positive (Fig. 1B). EdU assay revealed that exposure of rMC-1 to high glucose conditions for 24 h could significantly reduce their proliferative capacity (Fig. 1C). Furthermore, we confirmed the inhibition of cell proliferation by high glucose through Western blot analysis of proliferation-related proteins EGFR, YAP, and cycle-associated protein CYCLIN D1, which revealed decreased expression levels (Fig. 1D-E). Then, we detected the mRNA expression of Yap. High glucose could lead to decreased levels of Yap mRNA (Fig. 1F). These results suggested that high glucose could inhibit cell proliferation in rMC-1.

Fig. 1.

HG reduced the proliferation ability of rMC-1. (A) The protein levels of CD9, CD81, Alix and Calnexin in cell lysate and exosomes were evaluated using western blot. (B) The rMC-1 were identified with GFAP and GS protein immunofluorescence. GFAP, green; GS, green; DAPI, blue. Scale bar, 100 μm. (C) EdU assay was performed to detect cell proliferation and the EdU positive cells were quantitated. DAPI, blue; EdU, red. Scale bar, 50 μm, n = 6. (D) Western blot assays were performed to detect the protein levels of EGFR, YAP and CYCLIN D1. (E) The band grey value was quantified. The intensity of the bands was normalized to β-actin and then to control = 1. n = 3. (F) qRT-PCR was performed to detect the levels of Yap mRNA, with β-actin serving as a normalization gene. n = 3. The data were presented as the means ± SD. *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001 and ns P > 0.05. NC, normal glucose; HG, high glucose. The significant difference was evaluated by the Student’s t test

MSCs-EXOs improved the HG-induced decrease in cell proliferation capacity in rMC-1

We treated rMC-1 with HG medium for 24 h to mimic the diabetic environment in vivo and treated the cells with a mixture of high glucose and MSCs-EXOs to explore the therapeutic role and potential mechanisms. The CCK-8 assay was performed to detect the cell viability after MSCs-EXOs treatment in different MSCs-EXOs concentrations (0, 3.125, 6.25, 12.5, 25, and 50 µg/mL) (Fig. 2A). When the concentration of MSCs-EXOs was 12.5 µg/ml, the cell viability was enhanced. We subsequently concluded that the optimal concentration of MSC-EXOs was 12.5 µg/ml for 24 h. Furthermore, EdU assay results showed that the proliferative capacity of rMC-1 was significantly less in the HG-treated cells than in the NG-treated cells. The HG-induced relative ratio of EdU positive cells was significantly increased in the presence of MSCs-EXOs, demonstrating that MSCs-EXOs could improve the HG-induced reduction in proliferation capacity of rMC-1 (Fig. 2B–C).

Fig. 2.

MSCs-EXOs improved the HG-induced decrease in proliferation capacity of rMC-1. The cells were treated with a mixture of high glucose and different MSCs-EXOs concentrations (0, 3.125, 6.25, 12.5, 25, and 50 µg/mL) for 24 h. (A) Cell viability was detected by a CCK-8 assay, n = 3. (B–C) EdU assay was performed to detect cell proliferation and the EdU positive cells were quantitated. DAPI, blue; EdU, red. Scale bar, 50 μm, n = 3. The data were presented as the means ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001 and ns P > 0.05. NC, normal glucose; HG, high glucose; HG + EXOs, high glucose + MSCs-EXOs (12.5 µg/ml). The significant difference was evaluated by the one-way ANOVA followed by the Tukey’s post-hoc test

MSCs-EXOs protected HG-induced the function of rMC-1

During DR pathogenesis, high glucose could lead to Müller cells’ dysfunction, such as decreased cell viability and expression of functional proteins on cells. The CCK8 assay results revealed that cell viability was lower in the HG-treated cells than in the NG-treated cells. The HG-induced decrease in cell viability was significantly alleviated in the presence of MSCs-EXOs, demonstrating that MSCs-EXOs could improve the HG-induced decline in cell viability of rMC-1(Fig. 3A).

Fig. 3.

MSCs-EXOs protected rMC-1 against HG conditions. Changes in the expression of GS and AQP4 in cells treated with HG or MSCs-EXOs were assessed by immunostaining. AQP4, green; GS, green; DAPI, blue. Scale bar, 50 μm. (A) Cell viability was detected by a CCK-8 assay, n = 3. (B-E) Immunofluorescence was performed to detect the expression of GS and AQP4, and fluorescence intensity was quantified, n = 6. (F) qRT-PCR was performed to detect the levels of Aqp4 mRNA, with β-actin serving as a normalization gene. n = 3. The data were expressed as the means ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001 and ns P > 0.05. NC, normal glucose; HG, high glucose; HG + EXOs, high glucose + MSCs-EXOs (12.5 µg/ml). The significant difference was evaluated by the one-way ANOVA followed by the Tukey’s post-hoc test

Furthermore, Müller cells regulate the excitability of neurons, protecting neurons from excitotoxicity by taking in and recycling neurotransmitters like gamma-Aminobutyric acid (GABA) and glutamine [22–24]. Glutamine synthetase (GS) is the basis for the transport of glutamate. Müller cells also help to maintain water-ion balance. Studies have demonstrated that aquaporin AQP4 on Müller cells plays a crucial role in diabetes-induced retinal neurovascular dysfunction, and increasing AQP4 expression can alleviate diabetes-induced neurovascular dysfunction [25]. Immunofluorescence confirmed the protective effect of MSCs-EXOs on high glucose-treated rMC-1 functional proteins (Fig. 3B–E). The synthesis of GS and AQP4 was significantly suppressed in the HG group, which led to rMC-1 dysfunction. However, MSCs-EXOs alleviated GS and AQP4 protein loss under high glucose conditions. qRT-PCR was performed to detect the levels of Aqp4 mRNA and the results were consistent with the changes in protein expression (Fig. 3F). These results demonstrated that MSCs-EXOs could protect function in rMC-1. The upregulation of GS and AQP4 suggests that MSCs-EXOs treatment not only mitigates proliferation but also restores key metabolic and homeostatic functions of Müller cells.

MSCs-EXOs promoted the proliferation of rMC-1 by inhibiting the Hippo pathway to decrease the expression of LATS1 and to increase the expression of cell proliferation-related proteins and cell cycle-related proteins

In the Hippo pathway, LATS1 has been shown to inhibit cell proliferation in vitro via its kinase activity and YAP can induce cell proliferation [10–13]. When the Hippo pathway is inhibited, the expression of LATS1 decreases and the expression of YAP increases, promoting YAP to translocate into the nucleus to induce cell proliferation. YAP is necessary to maintain levels of Cyclin D1 expression in Müller cells, and in NMDA(n-methyl-d-aspartate)-treated mice, inhibition of YAP decreased the expression of CYCLIN D1, thereby directly inhibiting proliferation [15]. EGFR signaling pathway is a key pathway to induce Müller cells to re-enter cell-cycle [26, 27]. Studies have shown that deletion of EGFR reduces cell proliferation [15]. In our study, the expression of LATS1 was significantly promoted and the expression of cell proliferation-related proteins YAP, EGFR and cell cycle-related protein CYCLIN D1 was significantly inhibited by HG treatment, resulting in the suppression of rMC-1 proliferation. But compared with those in the HG-treated group, the expression of LATS1 and p-YAP, was significantly less and the expression of YAP, EGFR and CYCLIN D1 was significantly higher in the MSCs-EXOs group (Fig. 3A-F). Then, we detected the mRNA expression levels of Lats1, Yap and Egfr. The results were consistent with the changes in protein expression (Fig. 3G). These results demonstrated that MSCs-EXOs could protect rMC-1 from high glucose-induced damage by inhibiting the Hippo pathway to promote cell proliferation (Fig. 4).

Fig. 4.

MSCs-EXOs inhibited the Hippo pathway to decrease the expression of LATS1 and increase the expression of cell proliferation-related proteins and cell cycle-related proteins. (A) Western blot assays were performed to detect the protein levels of LATS1, EGFR, p-YAP, YAP and CYCLIN D1. (B-F) The band grey value was quantified. The intensity of the bands was normalized to β-actin and then to control = 1. n = 3. (G) qRT-PCR was performed to detect the levels of Lats1, Egfr and Yap mRNA, with β-actin serving as a normalization gene. n = 3. The data were presented as the means ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001 and ns P > 0.05. NC, normal glucose; HG, high glucose; HG + EXOs, high glucose + MSCs-EXOs (12.5 µg/ml). The significant difference was evaluated by the one-way ANOVA followed by the Tukey’s post-hoc test

LATS1 knockdown promoted HG-induced cell proliferation and YAP, EGFR and Cyclin D1 expression in rMC-1

To further explore the mechanism by which the Hippo pathway regulates cell proliferation, we successfully constructed the LATS1 knockdown rMC-1 cells (Fig. 5A). And cells were divided into four groups: NG group, HG group, HG + LATS1 SiRNA group and HG + LATS1 SiRNA-NC group. EdU assay results showed that the proliferative capacity of rMC-1 was significantly higher in the HG + LATS1 SiRNA group than in the HG group. Compared with the HG + LATS1 SiRNA-NC group and the HG group, there were no significant changes in the relative ratio of EdU positive cells. Our study demonstrated that LATS1 knockdown could improve the HG-induced decrease in proliferation capacity of rMC-1 (Fig. 5B-C). In addition, we measured cell viability post-transfection. The result of CCK-8 assay showed that the transfection is not significant toxicity (Fig. 5D).

Fig. 5.

LATS1 knockdown suppressed the expressions of LATS1 and improved the HG-induced decrease in proliferation capacity of rMC-1. Cells were transfected with SiRNA-NC or LATS1 SiRNA in high glucose conditions. (A) Western blot was performed to detect LATS1 level in rMC-1cells. (B) EdU assay was performed to detect cell proliferation. DAPI, blue; EdU, red. Scale bar, 50 μm. (C) The EdU positive cells were quantitated, n = 3. (D) Cell viability after transfection was detected by a CCK-8 assay. n = 3. The data were presented as the means ± SD of at least three independent experiments, n = 3. *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001 and ns P > 0.05. NC, normal glucose; HG, high glucose; HG + LATS1 SiRNA, high glucose + LATS1 SiRNA; HG + LATS1 SiRNA-NC, high glucose + LATS1 SiRNA-NC. The significant difference was evaluated by the one-way ANOVA followed by the Tukey’s post-hoc test

Furthermore, the expression of LATS1 and p-YAP was inhibited significantly and the expression of YAP, EGFR and CYCLIN D1 was promoted by HG + LATS1 SiRNA treatment. (Fig. 6A-F). Then, we detected the mRNA expression levels of Lats1, Yap and Egfr. The results were consistent with the changes in protein expression (Fig. 6G). The transfection efficiency was calculated to be approximately 43% based on the mRNA expression levels of Lats1 measured by qRT-PCR after transfection with small interfering RNA. These results demonstrated that LATS1 knockdown could decrease LATS1 and p-YAP expression and increase YAP, EGFR and CYCLIN D1 expression in rMC-1.

Fig. 6.

LATS1 knockdown increased the expression of cell proliferation-related proteins and cell cycle-related proteins. (A) Western blot assays were performed to detect the protein levels of LATS1, EGFR, p-YAP, YAP and CYCLIN D1. (B-F) The band grey value was quantified. The intensity of the bands was normalized to β-actin and then to control = 1. n = 3. (G) qRT-PCR was performed to detect the levels of Lats1, Egfr and Yap mRNA, with β-actin serving as a normalization gene. n = 3. The data were presented as the means ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001 and ns P > 0.05. NC, normal glucose; HG, high glucose; HG + LATS1 SiRNA, high glucose + LATS1 SiRNA; HG + LATS1 SiRNA-NC, high glucose + LATS1 SiRNA-NC. The significant difference was evaluated by the one-way ANOVA followed by the Tukey’s post-hoc test

MSCs-EXOs containing miR-21a-5p inhibited the expressions of LATS1 and regulated the expression of YAP through regulation of LATS1

In order to explore the specific mechanism of MSCs-EXOs’ regulation of the Hippo pathway, we performed bioinformatics sequencing analysis of MSCs-EXOs. Our study used miRNA sequencing to examine the miRNA expression profile in MSCs-EXOs. The data showed that 554 miRNAs were detected within the MSCs-EXOs, with 249 new miRNAs being discovered and published for the first time in our study. We screened MiRNAs with read counts greater than 10,000 and found that the abundance of miR-21a-5p in MSCs-EXOs was the highest (Fig. 7A). In addition, bioinformatics software was employed to predict the potential binding site between miR-21a-5p and LATS1 (Fig. 7B). Dual-luciferase reporter assay was performed to determine their relationship, and the results demonstrated that miR-21a-5p could directly bind to LATS1 and inhibit its expression (Fig. 7C). These results suggested that LATS1 was the downstream target of miR-21a-5p.

Fig. 7.

MSCs-EXOs containing miR-21a-5p regulated the expression of LATS1. (A) We screened MiRNAs with a number greater than 10,000 in MSCs-EXOs. (B) Bioinformatics software was applied to predict the binding site between miR-21a-5p and LATS1. (C) Dual-luciferase reporter assay was employed to verify the binding relationship between miR-21a-5p and LATS1. n = 3. (D) Western blot assays were performed to detect the protein levels of LATS1. (E) The band grey value was quantified. The intensity of the bands was normalized to actin and then to control = 1. n = 3. (F-G) qRT-PCR was performed to detect the levels of miR-21a-5p (with U6 serving as a normalization gene) and Lats1 mRNA (with β-actin serving as a normalization gene). n = 3. The data were expressed as the means ± SD. All data were obtained from at least three replicate experiments. **P < 0.01,***P < 0.001 and ****P < 0.0001 and ns P > 0.05. HG + EXOs, high glucose + MSCs-EXOs (12.5 µg/ml). The significant difference was evaluated by the one-way ANOVA followed by the Tukey’s post-hoc test

In order to explore the direct effect of miR-21a-5p in rMC-1 cells, we transfected miR-21a-5p inhibitor into the MSCs-EXOs group to suppress miR-21a-5p expression. The results of Western blot displayed that the expression of LATS1 was significantly increased in the miR-21a-5p inhibitor transfection group(Fig. 7D-E). The results of qRT-PCR displayed that miR-21a-5p expression was markedly decreased in rMC-1 cells with miR-21a-5p inhibitor transfection(Fig. 7F). The transfection efficiency was calculated to be approximately 88% based on the expression levels of miR-21a-5p after transfection with miR-21a-5p inhibitor. Then, we detected the mRNA expression level of Lats1. The results were consistent with the changes in protein expression(Fig. 7G). Our results of the third part displayed that the protein level of LATS1 was markedly decreased following MSCs-EXOs treatment; while miR-21a-5p inhibition increased the expression of LATS1 and could reverse the effect of MSCs-EXOs treatment. Our results of the fifth part displayed that the protein level of YAP was significantly increased and p-YAP was significantly decreased by LATS1 knockdown, suggesting that YAP was inhibited by LATS1. All the above results demonstrated that MSCs-EXOs containing miR-21a-5p increased the expression of YAP via targeting LATS1, thereby promoting cell proliferation in rMC-1.

4༎Discussion

There is currently a lack of effective therapies or drugs for the treatment at the early stage of DR. Early treatment of DR is mainly to control systemic blood glucose, but this therapy cannot reverse the progression of DR. Therefore, it’s urgent to find new strategies for DR treatment. This study proved that MSCs-EXOs could improve DR, and revealed that miR-21a-5p carried by MSCs-EXOs could promote cell proliferation and protect function in rMC-1 by targeting LATS1.

Current available therapies for DR have limited efficacy, and the pathogenesis and new therapeutic strategies of DR remain to be explored. The retina is the organ with the highest energy consumption per unit of tissue in the human body, and Müller cells directly supply nutrients to retinal neurons, which are the basis of retinal metabolism [28]. Müller cells are the most abundant glial population present within the retina. The study found that Müller cells were lost in the retina of patients with early DR [29]. After 10 months of induction of diabetes mellitus with streptozotocin in mice, Müller cells experienced apoptosis of more than 35%, accompanied by a decrease in the thickness of the retinal core, outer nucleus, and ganglion cell layers in DR mice [30]. In addition, Müller cell apoptosis has been shown to lead to retinal neuronal cell death [5], and inhibition of Müller cell apoptosis will restore DR retinal function. Therefore, reducing Müller cell loss in the early stage of DR may help delay the development of DR. In our study, we found that high glucose could inhibit cell proliferation by decreasing the expression of proliferation-related proteins EGFR, YAP, and cycle-associated protein CYCLIN D1 in rMC-1. Promoting Müller cells proliferation to protect the structural and functional integrity of the retina may be a potential therapeutic approach to control DR.

MSCs are present in almost all organs and tissues, with immunomodulatory, self-replication, and multidirectional differentiation potential, and are commonly used in clinical treatment as adult stem cells, but MSCs transplantation treatment has the problems of tumorigenicity, immune rejection, and embolism [31]. The therapeutic effect of MSCs depends on the extracellular substances secreted by the cells [32]. There is evidence that MSCs achieve their role in the treatment of diseases through paracrine activity rather than direct cell replacement [33, 34]. EXOs is the critical component regulating the paracrine activity of MSCs [35, 36]. MSCs-EXOs have stronger efficacy, safety and convenience than MSCs [37]. The main way that MSCs-EXOs play a role is paracrine, which releases these contents into nearby or distant cells to regulate cell functions, thus participating in various physiological and pathological processes, such as cell proliferation, apoptosis and neurodegeneration [38]. MSCs-EXOs mediate a variety of biological functions and have been used in the treatment of eye diseases such as dry eye, corneal injury, DR, uveitis, optic nerve injury, and retinal detachment in recent years [20, 39–41]. A study found that Adipose Mesenchymal Stem Cell-Derived Exosomes promoted wound healing and regeneration of endothelial cells by inducing a shift in the cell cycle and suppressing senescence and autophagy [42]. The function may vary among MSCs derived from different tissue sources. UC-MSCs showed significantly faster and higher proliferation potencies and higher migration potency than bone marrow MSCs (BM-MSCs) and adipose-derived MSCs (AD-MSCs) [43]. In addition, UC-MSCs produce 2.1-fold higher exosome yields than BM-MSCs and appear to be more promising than BM-MSCs in clinical applications [44]. A study found that UC‑MSCs alleviate streptozotocin‑induced DR in rats by regulating angiogenesis and the inflammatory response at the molecular level [45]. Therefore, we ultimately selected UC-MSCs as the experimental material in this study. Our exosome-mediated delivery strategy aligns with cutting-edge approaches in DR treatment. A study found that exosome-loaded YAP inhibitors ameliorated DR vascular leakage, validating EXOs as effective retinal drug carriersl [46]. In addition, the O-GlcNAc-Hippo axis study and Müller cell-YAP research provided precedent for targeting exosomal pathways in DR [46, 47]. Based on the therapeutic effect of MSCs-EXOs, we investigated the role of MSCs-EXOs in rMC-1 cells induced by HG. As previously described, we found that MSCs-EXOs could improve HG-induced proliferation capacity of rMC-1 and protect cell function. But the specific mechanism of MSCs-EXOs promoting the proliferation of rMC-1 remains to be explored.

The Hippo signaling pathway plays an important role in cell proliferation and stem cell regeneration [6]. Although there has been more and more research on the Hippo signaling pathway in recent years, especially in the fields of cancer, neurology, cardiovascular diseases, regeneration [48–53], there has been little research in ocular diseases. Studies found that enhancing YAP activity can enhance the proliferative capacity of Müller cells, in the mouse retina, where Müller cells are not able to proliferate spontaneously, activating YAP can induce Müller cells to exit their quiescence state via the YAP-EGFR axis and reprogramming into highly proliferative cells [15], which is important for promoting regeneration. Studies found that YAP was significantly upregulated and activated in the astrocytes of the optic nerve in experimental autoimmune encephalomyelitis (EAE) mice. Conditional knockout of YAP in astrocytes caused more severe inflammatory infiltration and demyelination in the optic nerve, and damage of retinal ganglion cells (RGCs) in EAE mice [54]. Studies have found that retinal neurodegenerative diseases are caused by abnormal activation of the Hippo signaling pathway, which inhibits the expression of YAP, and low expression of YAP leads to Müller cell atrophy, retinal degeneration, and retinal ganglion cell death [55, 56], which leads to the occurrence of eye diseases such as retinitis pigmentosa, Sveinsson choreoretinal atrophy (SCRA). SCRA has been shown in the mouse model to be caused by a Tyr421His mutation in Transcriptional enhancer activator domain (TEAD), and missense mutations in TEAD reduce its interaction with YAP, resulting in choreoretinal atrophy [57]. As a result, aberrant activation of the Hippo signaling pathway leads to low YAP expression, causing atrophy of the retina, resulting in decreased vision and even vision loss. At present, there is no effective treatment for degenerative diseases, and we hope to find a regulatory protein in the Hippo signaling pathway to delay the progression of retinal neurodegeneration. We found that MSCs-EXOs promoted the proliferation of rMC-1 by inhibiting the Hippo pathway, thus decreasing the expression of LATS1 and p-YAP and increasing the expression of YAP, EGFR and CYCLIN D1. Accumulating evidence positions YAP as a master regulator of both physiological retinal vascular development [58–60] and pathological angiogenesis in DR. In diabetic models, the YAP-TEAD axis drives neovascularization through: direct transcriptional activation of vascular endothelial growth factor (VEGF) [61], and metabolic reprogramming via 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3(PFKFB3)-mediated glycolysis in human umbilical vein endothelial cells (HUVECs) [62]. Concurrently, YAP induces pericyte dysfunction through pericyte-myofibroblast transition (PMT), accelerating subretinal fibrosis [63].

MSCs-EXOs have the potential to be part of a therapeutic strategy for the treatment of early DR, however the specific active ingredient of MSCs-EXOs regulating the Hippo pathway remains unknown. In order to explore the specific mechanism, we performed bioinformatics sequencing analysis of MSCs-EXOs, and found that the content of miR-21a-5p in MSCs-EXOs was high. In addition, we used bioinformatics software to predict the presence of a potential binding site between miR-21a-5p and LATS1, which may be a downstream target of miR-21a-5p. And we found that miR-21a-5p inhibition increased the expression of LATS1 and could reverse the effect of MSCs-EXOs treatment. All the above evidence displayed that MSCs-EXOs containing miR-21a-5p regulated the Hippo pathway via targeting LATS1. To further explore its molecular mechanism, we knocked out LATS1 in rMC-1. The results showed that LATS1 knockdown could increase YAP, EGFR and CYCLIN D1 expression in rMC-1, which was consistent with the results of the MSCs-EXOs therapy group. While prior work established miR-21/LATS1/YAP as an oncogenic driver [64], our study redefined its role in neurovascular repair. Our work revealed some novel aspects. Prior cancer studies aimed to inhibit this axis, but we exploited it for tissue repair in DR. Prior miR-21/LATS1/YAP studies primarily addressed cancer progression [65]. Our work demonstrated therapeutic potential via exosome-based delivery, contrasting with cancer studies emphasizing inhibition [66].

According to our results mentioned above, we believed that MSCs-EXOs played a protective role in the early stage of DR by targeting LATS1 to promote the cell proliferation of rMC-1, and improved the structural and functional integrity of the retina, alleviating the progression of DR. Although there are some important discoveries revealed by our study, further studies are needed to verify them in animal experiments.

Conclusions

In summary, our study demonstrated that MSCs-EXOs containing miR-21a-5p targets and downregulates LATS1 kinase. The inhibition of the Hippo pathway increased the expression of YAP and ultimately enhanced cellular proliferation. Our study clarified the mechanism of MSCs-EXOs-regulated cell proliferation and function protection in rMC-1 and provided novel theoretical insights for the pathogenesis and treatment of DR. This discovery provided the molecular foundation for developing exosome-based targeted therapies for DR.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

DR

Diabetic retinopathy

MSCs-EXOs

Mesenchymal stem cells-derived exosomes

hUC-MSCs

Human umbilical cord mesenchymal stem cells

rMC-1

Rat retinal Müller cell line

RGCs

Retinal ganglion cells

YAP

Yes-associated protein

LATS1

Large tumor suppressor 1

HG

High glucose

qRT-PCR

Quantitative Real-Time polymerase chain reaction

GFAP

Glial acidic fibrillary protein

GS

Glutamine synthetase

BSA

Bovine serum albumin

CCK-8

Cell Counting Kit-8

OD

Optical density

SD

Standard deviation

EAE

Experimental autoimmune encephalomyelitis

SCRA

Sveinsson choreoretinal atrophy

TEAD

Transcriptional enhancer activator domain

BM-MSCs

Bone marrow MSCs

AD-MSCs

Adipose-derived MSCs

VEGF

Vascular endothelial growth factor

PFKFB3

6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3

HUVECs

Human umbilical vein endothelial cells

PMT

Pericyte-myofibroblast transition

Author contributions

Liya Deng: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Ying Wang: Writing – review & editing, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization. All authors read and approved of the final manuscript.

Funding

This research was supported by the Natural Science Foundation of Hunan Province (2023JJ70043). Research Fund Project of Liaoning Provincial District Research Institute (LNI2025J01).

Data availability

The data used in this article is available from the corresponding author upon appropriate request.

Declarations

Ethics approval and consent to participate

The study was approved by the Science and Technology Ethics Committee of Shandong Qilu Stem Cells Engineering Co., Ltd (“Umbilical cord collection and preservation” Approve NO. QLSC 2024-001, approved November 14, 2024). And written informed consent was obtained from all individual participants (or their legal guardians/representatives) included in the study prior to sample collection.

Consent for publication

Not applicable.

Competing interests

The authors declared no potential conflicts of interest for the research, authorship, and publication of this article.