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. 2025 Aug 11;18(3-4):297–309. doi: 10.1007/s12195-025-00856-z

Enhancing Pluripotency and Adipogenic Differentiation in Human Adipose Tissue-Derived Stem Cells Through miR-302/367 Cluster Overexpression

Hossein Faghih 1,#, Maryam Khani 1,2,#, Mehdi Shamsara 3, Hossein Taghizadeh 1, Arash Javeri 1,✉, Masoumeh Fakhr Taha 1,✉
PMCID: PMC12436253  PMID: 40963718

Abstract

Purpose

Human adipose tissue-derived stem cells (hADSCs) have emerged as a promising source of cells for tissue engineering and regenerative medicine. However, their differentiation potential is restricted and requires enhancements. This study explores the reprogramming of hADSCs through exogenous induction of the miR-302/367 cluster.

Methods

Human ADSCs were transfected with the mock or miR-302/367 cluster-expressing vectors. One week after transfection, expression levels of several pluripotency-related genes, epithelial-to-mesenchymal (EMT) markers, and mechanistic target of rapamycin kinase (mTOR) signaling factors were assessed by qPCR and western blot. Additionally, the influence of miR-302/367 cluster overexpression on the proliferation and adipogenic differentiation of the ADSCs was evaluated.

Results

One week after transfection, the expression of several pluripotency-related genes and epithelial markers was significantly upregulated, while mesenchymal markers were downregulated in the miR-302/367-transfected cells compared with the mock group. Additionally, the levels of several mTOR signaling factors were reduced in the miR-302/367-transfected ADSCs. Flow cytometry analysis showed a decrease in the abundance of ADSCs in the S phase and an increase in the population of cells in the G1 phase of the cell cycle. Moreover, the adipogenic differentiation of the miR-302/367-transfected cells was significantly enhanced.

Conclusion

The overexpression of the miR-302/367 cluster directed the ADSCs towards a more pluripotent state and promoted their adipogenic potential. However, miR-302/367 overexpression diminished the proliferative capacity of hADSCs, which warrants a comprehensive investigation. Further evaluations are needed to fully elucidate the differentiation potential and regenerative capacity of the ADSCs reprogrammed by the miR-302/367 cluster before any clinical application.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12195-025-00856-z.

Keywords: miR-302/367, ADSC, Reprogramming, Pluripotency, mTOR signaling pathway, Proliferation

Introduction

Human adipose tissue-derived stem cells (hADSCs) represent a valuable source of mesenchymal stem cells (MSCs), with significant potential applications in the field of tissue engineering and regenerative medicine. The importance of these cells is attributed to their easy isolation, high proliferation rate, multipotency, and secretion of numerous anti-apoptotic, anti-fibrotic, pro-angiogenic, anti-inflammatory, immunomodulatory, and trophic factors [1–5]. However, the differentiation potential of ADSCs is more limited than that of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and is further influenced by several factors, such as the age, gender, and metabolic index of the donors. Specifically, ADSCs obtained from individuals who are old, have a high body mass index (BMI), suffer from diabetes mellitus, or have experienced radiotherapy and hormone therapy exhibit compromised proliferation and differentiation capabilities compared to those from normal subjects [6, 7]. To improve the regenerative potential of ADSCs, it is crucial to boost their ability to proliferate, differentiate, and secrete regeneration-promoting factors through various strategies, such as employing miRNAs. Among the miRNAs that show promise in this regard, the pluripotency-associated miRNAs, like miR-302/367 cluster, stand out for their ability to reprogram somatic cells towards a more pluripotent state.

The miR-302/367 cluster is located in an intron of the La-related protein 7 (LARP7) gene in the 4q25 region of human chromosome 4. This highly conserved cluster is specific to vertebrates and contains 5 members, including miR-302a, miR-302b, miR-302c, miR-302d, and miR-367. The seeding sequence of miR-367 is slightly different from the other members, but they all regulate common mRNA targets [8].

The miR-302/367 cluster was initially identified as a miRNA specifically expressed in undifferentiated cells, ESCs, and malignant embryonic carcinoma cells and implicated in the maintenance of stem cell pluripotency [9]. Subsequent studies showed that the miR-302/367 cluster is the most significantly and specifically expressed miRNA cluster in human ESCs and iPSCs and plays significant roles in maintaining pluripotency, cell proliferation, differentiation, and reprogramming [10, 11]. According to the results of several studies, the miR-302/367 cluster enhances the reprogramming of somatic cells to iPSCs in the presence of pluripotency transcription factors or other ESC-associated miRNAs [12–17]. However, the ability of this individual cluster to generate fully reprogrammed iPSCs is still a matter of controversy; while several studies have successfully produced fully reprogrammed iPSCs from somatic cells using miR-302 cluster alone [18–21], others have failed to achieve similar results with different cell types or protocols [13, 15, 17].

In the present study, we evaluated the capability of the miR-302/367 cluster to induce pluripotency in hADSCs and to enhance the adipogenic differentiation capacity of these cells. Our findings have significant implications for the use of hADSCs in regenerative medicine and the development of new cell-based therapies.

Materials and Methods

Culture of Human ADSCs

Human ADSCs were previously isolated by our research team from adipose tissue samples harvested by abdominoplasty surgery from 40 to 50-year-old healthy women [22–24]. All samples were collected with informed consent from the donors. The experiments were conducted in accordance with the Declaration of Helsinki and were approved by the Bioethics Committee of the National Institute of Genetic Engineering and Biotechnology (Ethics code: IR.NIGEB.EC.1400.2.26.C).

The cells were cultured in growth medium containing Dulbecco’s Modified Eagle’s Medium (DMEM), 20% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Gibco, Thermo Fisher Scientific). Culture medium was changed every two days, and the cells were passaged after reaching 80–90% confluency.

Transfection of hADSCs with a Plasmid Expressing the miR-302/367 Cluster

For miR-302/367 cluster overexpression, the third-passaged hADSCs were transfected with a miR‐302bcad/367 expressing Lentivector (TDH101PA‐GP; System Biosciences, SBI, Palo Alto, CA) or the same lentivector lacking the miR‐302/367 cluster sequence, as the mock group (Supplementary Fig 1). This lentivector was used solely in plasmid form, without any lentiviral packaging system, and thus no viral particles were produced. As such, the vector functioned as a non-integrating expression system. The transfection was performed by electroporation using a Neon Transfection System (Invitrogen, Thermo Fisher Scientific, USA), according to the manufacturer’s instructions (1400 volts, 10 milliseconds, and 3 pulses). For selection of the transfected cells, 1 µg/ml puromycin was added to the culture medium from day 2 after transfection.

The miRNA and Gene Expression Analysis

Two days after transfection, transfected cells were harvested by trypsinization and total RNAs, including small RNAs, were extracted using the RNX-Plus RNA Isolation Solution (Sinaclon, Tehran, Iran), based on the acid guanidinium thiocyanate–phenol–chloroform method. Equal amounts of RNAs from the mock and miR‐302/367 transfected groups were reverse‐transcribed into cDNA using BON‐miR miRNA 1st‐Strand cDNA Synthesis Kit (Stem Cell Technology Co., Tehran, Iran), based on the poly‐adenylation method. Then, expression of the miR-302/367 members was assessed by quantitative real-time PCR using RealQ-PCR Master (Ampliqon A/S, Denmark) and specific primers for miR-302a, miR-302b, miR-302c, miR-302d, and miR-367. SNORD47 was used as the internal reference for miRNA quantification.

For analysis of mRNA expression, one week after transfection, total RNAs were extracted from the cells using the High Pure RNA Isolation Kit (Roche), according to the manufacturer’s protocol. Then, cDNAs were synthesized from equal amounts of RNA using RevertAid H Minus Reverse Transcriptase (Thermo Fisher Scientific). Quantitative real-time PCR was performed using RealQ-PCR Master (Ampliqon A/S, Denmark) and specific primers as described in supplementary Table 1. B2M and GAPDH were used as the internal reference controls.

Reactions were run on a Rotor-Gene 6000 (Corbett Research, Australia) real-time PCR instrument. Relative quantification of gene expression was performed between different groups using REST 2009 software (Relative Expression Software Tool, Qiagen GmbH, Hilden, Germany) based on Pair Wise Fixed Reallocation Randomization Test [25]. Four replicates of each sample were included in the qPCR experiments.

Western Blot Analysis

One week after transfection, the cells were collected by trypsinization and homogenized in ice-cold Radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with a protease inhibitor cocktail (Sigma). The homogenized samples were then centrifuged at 13000 g for 15 min at 4 °C. The resulting supernatants were collected, and the protein concentration was measured by the Bradford assay. Next, 50 µg protein from each group was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to Polyvinylidene fluoride (PVDF) membranes, and blocked with 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). After blocking, the membranes were incubated with the primary antibodies against β-actin, Octamer-binding transcription factor 4 (OCT4), NANOG, Vimentin (VIM), transforming growth factor beta receptor 2 (TGFBR2), E-cadherin, Protein kinase B (AKT), mechanistic target of rapamycin kinase (mTOR), phosphorylated AKT (p-AKT), Phosphorylated mTOR (p-mTOR), tuberous sclerosis complex (TSC) and Ras homologue enriched in brain (RHEB), followed by incubation with a mouse anti-rabbit horseradish peroxidase conjugated (IgG-HRP) or mouse IgG kappa binding protein conjugated to Horseradish Peroxidase (m-IgGκ BP-HRP) as secondary antibodies. The antibodies employed in western blot analysis are detailed in Supplementary Table 2. To detect immunoreactive bands, an Electrochemiluminescence (ECL) Detection Kit (CytoMatin Genae, Esfahan, Tehran, Iran) was used. The bands were then exposed to X-ray films and scanned. Densitometry of the bands was performed using Image Studio Lite version 5.2 (LI-COR Biosciences, Lincoln, USA), statistical analysis was carried out by unpaired t-test, and the charts were generated using GraphPad Prism 8 (GraphPad Software Inc., La Jolla, USA).

Cell Cycle Analysis by Flow Cytometry

One week after transfection, the cells were collected using 0.25% trypsin/EDTA and fixed with ice-cold 70% ethanol. The DNA content was stained with a propidium iodide (PI) solution consisting of 0.1% (v/v) Triton X-100, 10 μg/ml PI, and 100 μg/ml DNase-free RNase A (Sigma) in phosphate-buffered saline (PBS) for 30 min at room temperature in the dark. Detection of the cell cycle phases was performed by a BD FACSCalibur™ flow cytometer (BD Biosciences, USA). The obtained data were analyzed, and histogram charts were generated using FlowJo software (TreeStar Inc., Ashland, OR, USA).

Adipogenic Differentiation and Quantification

Adipogenic differentiation potential of hADSCs was examined one week after transfection with the mock and mir-302/367 cluster-expressing vectors. The cells were cultured in adipogenic induction medium consisted of DMEM, 10% FBS, 1 µM dexamethasone, 100 µM indomethacin, 5 µg/ml insulin, and 500 µM isobutylmethylxanthine (IBMX) for a duration of two weeks. Subsequently, Oil Red O staining was used to visualize lipid accumulations within the differentiated cells. To quantify the extent of adipogenesis, unbound dye was washed out, and the stained lipid droplets were eluted using isopropanol for 10 min. The optical density of the resulting supernatant was measured using a microplate reader (BioTek Instrument) at an absorbance wavelength of 500 nm. Data from three biological replicates (5 wells) were analyzed using GraphPad Prism 8 (GraphPad Software Inc).

Results

Overexpression of miR-302/367 Cluster in hADSCs

The third-passage ADSCS were transfected with either a vector expressing the miR-302/367 cluster or a Mock vector. 48 hours after transfection, the expression of GFP protein was observed in a significant proportion of the cells (Fig. 1A–D). The expression levels of miR-302a, miR-302b, miR-302c, miR-302d, and miR-367 in the miR-302/367 cluster-overexpressing cells were significantly higher than the mock group, with fold increases of 262, 1479, 70, 867, and 23, respectively (Fig. 1E).

Fig 1.

Fig 1.

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Transfection of hADSCs with the miR-302/367 cluster-expressing vector (A) and the mock vector (C), and the respective phase contrast images (B and D), 48 h after transfection. (E) QPCR analysis for the members of miR-302/367 cluster in the transfected cells, 48 h after transfection. The expression level of genes in the mock group was assumed to be 1, and their expression levels (fold change) in the miR-302/367-transfected group were compared to that. Significant differences have been indicated by their respective P values (Pair Wise Fixed Reallocation Randomization Test performed by REST 2009 software, n = 4)

Upregulation of Pluripotency-Associated Transcription Factors in hADSCs Transfected with miR-302/367 Cluster

One week after transfection, the expression of OCT4, SOX2, NANOG, REX1 and NODAL genes in the hADSCs transfected with miR-302/367 cluster were upregulated by mean factors of 7 (P = 0.01), 2.08 (P < 0.001), 2.56 (P = 0.006), 2.71 (P = 0.011) and 2.12 (P = 0.018) compared to the mock group, respectively (Fig. 2A). OCT4 and NANOG proteins were also increased in the miR-302/367-transfected ADSCs (Fig. 2B, C and Supplementary Fig. 2). However, the miR-302/367 cluster alone was not able to generate human iPSC-like colonies.

Fig 2.

Fig 2.

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Examining the expression of some pluripotency-associated markers in hADSCs, 1 week after transfection with the mock and miR-302/367 cluster-expressing vectors. (A) QPCR analysis. The expression levels of the genes in the mock group have been assumed to be 1 (as represented by the red line), and their expression levels in the miR-302/367 group were compared to this baseline. Significant differences were indicated by their respective P values (Pair Wise Fixed Reallocation Randomization Test performed by REST 2009 software, n = 4). (B, C) Western blot analysis and densitometry of the bands using Image Studio Lite version 5.2 (LICOR Biosciences, USA). Statistical analysis was performed using an unpaired t-test (n = 3), and the charts were generated with GraphPad Prism 8 (GraphPad Software Inc., La Jolla, USA). *** P < 0.001

Regulation of Epithelial and Mesenchymal Markers in hADSCs Transfected with miR-302/367 Cluster

One week after transfection, the expression levels of SNAI1, SNAI2, VIM, and TGFBR2 genes in the ADSCs transfected with miR-302/367 cluster were downregulated by mean factors of 0.4, 0.43, 0.68, and 0.43 compared to the mock group, respectively, while the expression of CDH1 was mildly upregulated by 1.6-fold. Additionally, Ras homologue gene family member C (RHOC) and CD105 genes were respectively downregulated by mean factors of 0.82 and 0.59 after miR-302/367 cluster overexpression (Fig. 3A). Western blot analysis further confirmed these findings, showing that VIM and TGFBR2 proteins were downregulated, while E-cadherin was upregulated in the miR-302/367 cluster-transfected ADSCs compared to the mock group (Fig. 3B–D and Supplementary Fig 3).

Fig 3.

Fig 3.

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Examining the expression of some epithelial and mesenchymal markers, 1 week after transfection of the hADSCs with the mock and miR-302/367 cluster-expressing vectors. (A) QPCR analysis. The expression levels of the genes in the mock group have been assumed to be 1 (as represented by the red line), and their expression levels in the miR-302/367 group were compared to this baseline. Significant differences were indicated by their respective P values (Pair Wise Fixed Reallocation Randomization Test performed by REST 2009 software, n = 4). (B-D) Western blot analysis and densitometry of the bands using Image Studio Lite version 5.2 (LICOR Biosciences, USA). Statistical analysis was performed using an unpaired t-test (n = 3), and the charts were generated with GraphPad Prism 8 (GraphPad Software Inc., La Jolla, USA). *** (P < 0.001)

Suppression of mTOR Signaling Factors in the ADSCs Transfected with miR-302/367 Cluster

One week after transfection, the expression levels of RAC (Rho family)-alpha serine/threonine-protein kinase (AKT1), MTOR, regulatory-associated protein of mTOR (RPTOR), and independent companion of MTOR complex 2 (RICTOR) genes were significantly downregulated in the ADSCs transfected with miR-302/367 cluster compared to the mock group. The expression levels of these genes were downregulated to approximately 0.6, 0.61, 0.58 and 0.48 of the mock group, respectively (Fig. 4A). Additionally, western blot analysis revealed that the expression of AKT1 and MTOR proteins remained unchanged in the miR-302/367 cluster-transfected ADSCs, while TSC1 was upregulated and the levels of p-AKT1, RHEB and p-mTOR proteins were downregulated significantly compared to the mock group (Figure 4B–G and Supplementary Fig 4).

Fig 4.

Fig 4.

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Examining the expression of some mTOR signaling markers, 1 week after transfection of hADSCs with the mock and miR-302/367 cluster-expressing vectors. (A) QPCR analysis. The expression levels of the genes in the mock group have been assumed to be 1(as represented by the red line), and their expression levels in the miR-302/367 group were compared to this baseline. Significant differences were indicated by their respective P values (Pair Wise Fixed Reallocation Randomization Test performed by REST 2009 software, n = 4). (B-G) Western blot analysis and densitometry of the bands using Image Studio Lite version 5.2 (LICOR Biosciences, USA). Statistical analysis was performed using an unpaired t-test (n = 3), and the charts were generated with GraphPad Prism 8 (GraphPad Software Inc., La Jolla, USA). *** (P < 0.001). This figure has been adapted from our recently published work (in Persian), with additional modifications [26]

Analysis of the Cell Cycle in hADSCs Transfected with miR-302/367 Cluster

Flow cytometry analysis on the PI-stained cells showed the suppressive effect of miR-302/367 on the cell proliferation in hADSCs. The exogenous induction of the miR-302/367 cluster in ADSCs resulted in a significant decrease in the S-phase population, with approximately 22% observed in the mock-transfected cells versus 8% in the miR-302/367-transfected cells. Meanwhile, the G1-phase population increased from approximately 61% in the mock group to about 73% in the miR-302/367 group. No significant change was detected in the G2/M phase (Fig. 5).

Fig 5.

Fig 5.

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Cell cycle analysis by flow cytometry on the PI-stained cells. (A) The distribution of cell cycle in the ADSCs, one week after transfection with the mock and miR-302/367 cluster-expressing vectors. (B) The statistical analysis and corresponding charts were generated using GraphPad Prism 8 (GraphPad Software Inc.). The quantitative data are expressed as mean ± SD (n = 4). The significance of the differences was assessed using one-way analysis of variance (ANOVA), with *** denoting P < 0.001 and n.s. indicating no significant difference

Enhanced Adipogenic Differentiation of hADSCs Transfected with miR-302/367 Cluster

One week after transfection of ADSCs with the mock and miR-302/367 cluster-expressing vectors, adipogenic differentiation of the cells was evaluated. In both the mock and miR-302/367 groups, the formation of fat droplets was observed starting from day three of differentiation and progressively increased in size and quantity. Figures 6A, B illustrate the accumulation of intracellular lipid droplets within the cells after two weeks of differentiation. Quantitative analysis of the Oil Red O staining intensity revealed that overexpression of the miR-302/367 cluster significantly increased lipid accumulation in the differentiated cells (Fig. 6C).

Fig 6.

Fig 6.

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Adipogenic differentiation of hADSCs after transfection with the mock and miR-302/367 cluster-expressing vectors. (A and B) Oil red O staining showed the accumulation of intracellular lipid droplets in two-week differentiated ADSCs of the miR-302/367 and Mock groups, respectively. (C) Quantitative analysis of the lipid accumulation by eluting the Oil Red O dye in isopropanol and measuring the absorbance at 500 nm. The data was analyzed using GraphPad Prism 8 Software (three replicates, 5 wells). The significant difference was indicated by the respective P value.

Discussion

Certain groups of miRNAs, such as the miR-302/367 cluster, are specifically expressed in pluripotent stem cells, and their expression decreases with differentiation and is ultimately silenced in adult tissues [27]. The miR-302/367 cluster has been shown to target several hundred genes, most of which work to initiate or facilitate the differentiation of specific cell types during embryonic development [18]. In this study, we transfected hADSCs with a vector expressing the miR-302/367 cluster and examined the expression of several pluripotency-related genes, mesenchymal-to-epithelial transition (MET) markers, and mTOR signaling mediators. We also evaluated the impact of the miR-302/367 cluster on the proliferation and adipogenic differentiation of hADSCs.

Reprogramming of hADSCs with miR-302/367 Cluster

Overexpression of the miR-302/367 cluster in hADSCs upregulated the expression of OCT4, SOX2, NANOG, NODAL, and REX1 genes, as well as OCT4 and NANOG proteins. OCT4 is a crucial transcription factor during early embryonic development. It works in conjunction with SOX2 and NANOG to form a self-regulatory network that maintains pluripotency and controls the expression of numerous downstream genes [28, 29]. Here we examined the expression of two downstream genes, REX1 and NODAL. REX1 is highly expressed in the embryo’s inner cell mass (ICM), but its expression decreases as the differentiation process begins. NANOG directly regulates the expression of REX1 [30]. NODAL, on the other hand, is a member of the TGF-β superfamily. The activin/Nodal signaling pathway plays a role in maintaining the proliferation of ESCs [31].

Previous studies have demonstrated that the miR-302/376 cluster plays a significant role in regulating the expression of OCT4, SOX2, and NANOG. In turn, these factors are essential for the expression of the miR-302/367 cluster, thereby establishing an interconnected autoregulatory loop in pluripotent cells [32, 33]. To this end, several authors have utilized the miR-302/367 cluster along with pluripotency-related transcription factors or other miRNAs to enhance the efficiency of iPSC production [12–17]. Moreover, several previous studies [18, 34, 35], including some works by our team [36–40], have demonstrated that the miR-302/367 cluster can be used to reprogram human cancer cells through induction of MET and suppression of their proliferative capacity. However, the ability of this cluster alone to reprogram somatic cells to fully reprogrammed iPSCs remains uncertain. In a previous study, the miR-302/367 cluster alone was unable to generate iPSC colonies from human embryonic fibroblasts, umbilical cord blood, and endothelial cells [15]. Conversely, some other studies demonstrated the successful generation of iPSC colonies solely by the miR-302/367 cluster from various human cell types, including skin cancer cells [18], hair follicle cells [19], HEK293 embryonic kidney cells [21], and fibroblasts [20]. In the present study, as well as in our earlier investigation concerning mouse ADSCs [41], the exogenous induction of the miR-302/367 cluster increased the expression of some pluripotency-related transcription factors to a certain extent, but it was not successful in producing fully reprogrammed iPSCs, even after being cultured for 30 days on MEF feeder layer and in human ESC culture medium (unpublished data). These inconsistent results may be attributed to employing various starting cell types and different delivery methods. Furthermore, it is possible that additional factors, such as specific miRNAs or small molecules, are needed to achieve complete reprogramming of hADSCs to iPSCs.

The Impact of miR-302/367 Cluster on the Expression of mTOR Signaling Molecules in hADSCs

Previous studies have identified AKT1 as a direct target of miR-302s [35]. Moreover, in pluripotent stem cells, AKT1 and OCT4 interplay at several levels and form an AKT-OCT4 regulatory subunit [42] which maintains OCT4 expression at a high level [43].

AKT1 is a serine-threonine kinase that is recruited to the cell membrane through binding of its PH domain to PIP3 and is fully activated after phosphorylation by PDK1 (3-phosphoinositide-dependent kinase) and mTOR-sensitive rapamycin complex 2 (mTORC2) [44]. Activated AKT1 phosphorylates several important proteins, such as forkhead box O (FOXO), TSC2, and proline-rich Akt substrate of 40 kDa (PRAS40). The phosphorylation of FOXO increases cell proliferation and survival. On the other hand, TSC1 (hamartin) and TSC2 (tuberin) form an intracellular complex that inhibits the activity of the small G protein RHEB through the GAP activity of TSC2. Phosphorylation of TSC2 impairs its inhibitory effect on RHEB activity [44]. RHEB-GTP directly targets TOR complex 1 through interactions with both the catalytic domains of mTOR and LST8, which activate mTOR kinase activity. Additionally, RHEB regulates mTOR expression through FK506 binding protein 38 (FKBP38), which binds to mTOR and inhibits its activity. AKT also phosphorylates PRAS40 protein, thereby reducing the inhibitory effect of PRAS40 on the mTOR and mTORC1 complex [45]. In our study, the expression of AKT1 and p-AKT decreased following the miR-302/367 cluster overexpression in the ADSCs, which is in agreement with previous studies [43]. We also detected upregulation of TSC1 protein, which is a known negative regulator of mTORC1 signaling, and downregulation of RHEB protein, a positive inducer of mTORC1. These coordinated alterations provide a mechanistic basis for the observed downregulation of p-mTOR despite unchanged total mTOR protein levels.

The mTOR forms part of two distinct complexes called mTORC1 and mTORC2. The mTORC1 complex includes mTOR, RAPTOR, mLST8, and PRAS40 [46]. This complex is sensitive to rapamycin, increases protein synthesis and translation [47] and influences cell proliferation, growth, and autophagy [48]. In our study, in addition to MTOR, RAPTOR expression was downregulated following the miR-302/367 cluster overexpression. Both of these changes can affect cell proliferation and autophagy processes. The mTORC2 complex consists of mTOR, RICTOR, mLST8, and mammalian stress-activated protein kinase-interacting protein (mSIN1). The mTORC2, in conjunction with PDK1, phosphorylates and results in full activation of AKT1 [49–51]. We demonstrated that, in addition to MTOR, the expression of RICTOR was downregulated following the overexpression of the miR-302/367 cluster. This can influence the activity of the mTORC2 complex and reduce the phosphorylation and activation of AKT1.

Altogether, our findings indicated that the overexpression of the miR-302/367 cluster results in functional inhibition of the mTOR signaling. This mechanism may contribute to the observed decrease in cell proliferation [52] and an increase in the adipogenic potential of hADSCs [53].

The Impact of miR-302/367 Cluster on the Expression of Epithelial and Mesenchymal Markers in hADSCs

To assess the epithelial or mesenchymal characteristics of hADSCs following miR-302/367 overexpression, we assessed several well-characterized markers. Snail family transcriptional repressor 1 (SNAI1) and SNAI2 encode SNAIL and SLUG proteins, which are two important transcriptional repressors of epithelial markers, such as CDH1, and are crucial drivers of the EMT process. VIM (vimentin) and TGFBR2 are also associated with mesenchymal phenotype, whereas CDH1 encodes E-cadherin, a transmembrane protein with a significant role in epithelial cell adhesion and maintenance of polarity [54]. We demonstrated that the overexpression of the miR-302/367 cluster in hADSCs downregulated the expression of SNAI1, SNAI2, VIM, and TGFBR2, while CDH1 expression was upregulated. Also, VIM and TGF-β receptor 2 proteins were downregulated, while E-cadherin protein was upregulated in the miR-302/367 cluster-transfected ADSCs compared to the mock group.

We also evaluated the expression of Ras Homolog Family Member C (RHOC) and CD105/Endoglin genes. RHOC is a member of the Rho GTPase family and plays key roles in cytoskeletal dynamics, cell migration, and the EMT process [55]. The downregulation of RHOC corresponds with the suppression of mesenchymal characteristics and loss of migratory phenotype. CD105 (endoglin) is a co-receptor in the TGF-β signaling pathway. It is abundantly expressed in mesenchymal stem cells and modulates their proliferation, migration, and differentiation potential [56]. Downregulation of CD105 usually happens during epithelial-like transitions or differentiation away from the mesenchymal phenotype. Taken together, these findings showed that the overexpression of the miR-302/367 cluster promotes the MET process by suppressing the mesenchymal and enhancing the epithelial characteristics of the ADSCs.

MET plays an essential role during the early stages of somatic cell reprogramming [57]. This process is regulated by various signaling molecules, which are influenced by the miR-302/367 cluster. The members of this cluster directly target TGFBR2 and RHOC transcripts [12, 58]. It also suppresses the bone morphogenetic protein (BMP) inhibitors, such as TOB2, DAZAP2, and SLAIN1. Both of these mechanisms play significant roles in MET and thereby in reprogramming [58]. Additionally, AKT1, which is directly targeted by the miR-302/367 cluster, facilitates the EMT by suppressing the E-cadherin expression and activating the mesenchymal transcription factors. On the other hand, mTORC2 regulates the EMT by changing the phosphorylation status of Protein kinase C (PKC) and affecting the cytoskeletal actin dynamics [59]. AKT1 also induces the NF-κB pathway through activation of IκB kinase (IKK), resulting in upregulation of SNAI1 and SNAI2 expression, which facilitates EMT through inhibition of E-cadherin and PTEN [60]. PTEN inhibits migration and invasion through the αVβ6 integrin signaling pathway [61]. Notch4 represents an additional target of miR-302a [62]. It is well established that the Notch signaling pathway induces the EMT during both embryonic development and tumor progression [63].

Analysis of the Cell Cycle in the ADSCs Transfected with the miR-302/367 Cluster

The overexpression of the miR-302/367 cluster increased the population of hADSCs in the G1 phase while concurrently decreasing the proportion of cells in the S phase of the cell cycle. This indicates a negative effect of the miR-302/367 cluster on the proliferation rate of hADSCs. There are conflicting reports regarding the impact of the miR-302/367 cluster on the cell cycle progression in human cells. This cluster has been shown to diminish tumorigenicity in pluripotent stem cells by augmenting multiple pathways that induce G1 phase arrest. Specifically, miR-302 can simultaneously inhibit the cyclin E-cyclin-dependent kinase 2 (CDK2) and cyclin D-CDK4/6 pathways, thereby blocking over 70% of the G1-to-S phase transition [34]. It also regulates the pluripotency and teratoma formation through an AKT1/OCT4-dependent manner [43]. Moreover, the PI3K/AKT1/mTOR pathway is a major regulator of survival, cell growth, proliferation, and metabolism, and its suppression can lead to G1/S arrest. [64–66]. In our study, AKT1, RPTOR, and RICTOR genes, as well as p-AKT1 and p-mTOR proteins decreased following the miR-302/367 cluster overexpression, which may adversely affect the proliferation rate of hADSCs. Similarly, the miR-302/367 cluster attenuates the proliferation rate of cancer cells by suppressing the G1-phase checkpoint regulators, such as cyclin E-CDK2 and cyclin D-CDK4/6, as well as silencing the cancer stem cell marker BMI-1, which promotes the expression of two tumor suppressor genes p16Ink4a and p14/p19Arf [34, 67]. However, our findings are in contrast to the results obtained by Li and coworkers [43], who observed an enhancement in the proliferation of hADSCs following transfection with miR-302abcd mimics. Additionally, another investigation indicated that the overexpression of miR-302d led to downregulation of cyclin-dependent kinase inhibitor 1A (CDKN1A) expression and thereby promoted the proliferation of hADSCs [68]. Altogether, it seems that the miR-302/367 cluster directly targets different positive and negative cell cycle regulators [43], and that the ultimate impact of miR-302 on proliferation depends on the various pathways it affects, with the presence of miR-367 also playing a crucial role in determining the outcome. It is also plausible that cells undergoing partial reprogramming experience a transitional proliferation pause [69], during which they modify their gene expression and chromatin structure to assume a new cellular identity, such as that of iPSCs. All of the mentioned mechanisms may work together to mediate the anti-proliferative effects we detected in our study.

The Impact of the miR-302/367 Overexpression on Adipogenic Differentiation of hADSCs

The overexpression of the miR-302/367 cluster markedly enhanced the adipogenic differentiation of hADSCs, as evidenced by quantifying the Oil Red O staining degree of lipid accumulations in two-week differentiated hADSCs. To date, there has been no research specifically evaluating the impact of the mir-302/367 cluster on the adipogenic differentiation of MSCs. Nonetheless, a previous study has indicated that the overexpression of miR-302b facilitates adipogenic differentiation in both ADSCs [70] and bovine preadipocytes [71]. Conversely, another investigation indicated that miR-302a inhibits adipogenesis in 3T3-L1 adipocytes by downregulating the peroxisome proliferator-activated receptor γ (PPARγ) [72]. This controversy may arise from the varying initial cell types and the specific members of the miR-302/367 cluster utilized for transfection. In MSCs, adipogenic differentiation is regulated by various signaling pathways that function either as activators, like BMP pathway, or as repressors, such as Wnt, SHH, and TGF-β pathways. All of these pathways converge in the nucleus and exert strong regulatory effects on key factors, like peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT-enhancer-binding protein-α (C/EBP-α) [73]. The miR-302/367 cluster enhances the adipogenic differentiation of MSCs, probably through several interacting mechanisms. Firstly, previous studies indicate that miR-302b enhances preadipocyte differentiation by targeting cell cycle regulators, such as CDK2, which leads to suppressing proliferation and promoting differentiation [71]. Secondly, this cluster downregulates certain effectors within the EphA2/Wnt/β-catenin [37, 74] and TGF-β signaling pathways [39], thereby relieving the inhibitory signals upon adipogenic processes. Thirdly, the activation of pluripotency-related genes and the induction of MET may have driven the hADSCs into an even more plastic and receptive state, making them more responsive to adipogenic signals. Lastly, inhibition of the mTOR signaling pathway may be another driving force behind the enhancement of adipogenesis. In certain contexts, partial depletion of the mTOR activity is associated with increased adipogenesis, likely through alleviation of repression on the adipogenic transcription factors, PPARγ and C/EBPα [53].

Potential Translational Forecast

From a clinical perspective, the miR-302/367 cluster has the potential to reprogram hADSCs towards a more pluripotent state, as well as to induce their adipogenic differentiation. This has significant implications for reconstructive and regenerative medicine. The exogenous induction of the miR-302/367 cluster in hADSCs via a transient, non-integrative method, like nucleofection, offers a safer approach for clinical applications [75]. Reprogramming of hADSCs with this cluster may improve outcomes in the repair of soft tissue defects caused by congenital anomalies, traumas, and tumor resections. This approach is also beneficial for fat tissue engineering, especially where efficient adipogenesis is needed. Additionally, priming hADSCs with the miR-302/367 cluster may be particularly valuable when applying ADSCs from elderly or metabolically weakened donors, and the cells may not function optimally [76]. It is noteworthy that this strategy avoids full reprogramming to iPSCs, thereby reducing the oncogenic risk while improving the regenerative efficacy of ADSCs [77]. These findings may justify the development of future priming strategies for stem cell-based therapies, although additional research studies are necessary, particularly regarding in vivo validation and long-term assessments.

Conclusion

In the present study, the overexpression of the miR-302/367 cluster in hADSCs significantly increased the expression of various pluripotency-associated genes and facilitated the MET process, while downregulating the expression of several mTOR signaling molecules. Furthermore, the miR-302/367 cluster notably enhanced the adipogenic differentiation of hADSCs. These alterations showed the capability of the miR-302/367 cluster to reprogram hADSCs towards a more pluripotent state, which is valuable for their application in reconstructive and regenerative medicine. However, we found that the miR-302/367 cluster suppresses the proliferation of hADSCs, which warrants a comprehensive investigation. Moreover, it is necessary to conduct further assessments to fully elucidate the differentiation potential, as well as the regenerative capability of hADSCs reprogrammed by the miR-302/367 cluster before considering any clinical application.

Supplementary Information

Below is the link to the electronic supplementary material.

12195_2025_856_MOESM1_ESM.pdf (211.9KB, pdf)

Supplementary file1 (PDF 212 KB)—System Biosciences map for TDH101PA‐GP, a miR‐302bcad/367 expressing Lentivector.

12195_2025_856_MOESM2_ESM.pdf (269.5KB, pdf)

Supplementary file2 (PDF 270 KB)—Original, unprocessed images of western blots, related to Fig. 2.

12195_2025_856_MOESM3_ESM.pdf (229.6KB, pdf)

Supplementary file3 (PDF 230 KB)—Original, unprocessed images of western blots, corresponding to Fig. 3.

12195_2025_856_MOESM4_ESM.pdf (322.6KB, pdf)

Supplementary file4 (PDF 323 KB)—Original, unprocessed images of western blots, corresponding to Fig. 4.

12195_2025_856_MOESM5_ESM.pdf (74.1KB, pdf)

Supplementary file5 (PDF 74 KB)—Primers used for RT-PCR and qPCR.

12195_2025_856_MOESM6_ESM.pdf (199.5KB, pdf)

Supplementary file6 (PDF 200 KB)—The list of antibodies used for western blot analysis.

Acknowledgement

This work was supported by the Iran National Science Foundation (INSF; grant number 91059038).

Author Contribution

Hossein Faghih: Investigation, Methodology, Data curation. Maryam Khani: Investigation, Methodology, Data curation. Mehdi Shamsara: Methodology, Writing—review and editing. Hossein Taghizadeh: Investigation. Arash Javeri: Supervision, Methodology, Funding acquisition, Writing—review and editing. Masoumeh Fakhr Taha: Conceptualization, Supervision, Validation, Writing—review and editing.

Funding

Iran National Science Foundation, 91059038, Arash Javeri.

Data Availability

The original, uncropped images of western blots are included as supplementary information. The datasets that support the other findings of this study are available from the corresponding authors upon request.

Declarations

Competing Interests

The authors declare that they have no relevant financial or non-financial interests to disclose.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Hossein Faghih and Maryam Khani have contributed equally to this work.

Contributor Information

Arash Javeri, Email: arashj@nigeb.ac.ir.

Masoumeh Fakhr Taha, Email: mftaha@nigeb.ac.ir.

References

  • 1.Frese, L., P. E. Dijkman, and S. P. Hoerstrup. Adipose tissue-derived stem cells in regenerative medicine. Transfusion Medicine and Hemotherapy. 43(4):268–274, 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Lattanzi, W., M. C. Geloso, N. Saulnier, S. Giannetti, M. A. Puglisi, et al. Neurotrophic features of human adipose tissue-derived stromal cells: in vitro and in vivo studies. J Biomed Biotechnol. 2011:468705, 2011. 10.1155/2011/468705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Rehman, J., D. Traktuev, J. Li, S. Merfeld-Clauss, C. J. Temm-Grove, et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation. 109(10):1292–1298, 2004. 10.1161/01.CIR.0000121425.42966.F1. [DOI] [PubMed] [Google Scholar]
  • 4.Ribeiro, T. B., A. S. Duarte, A. L. Longhini, F. Pradella, A. S. Farias, et al. Neuroprotection and immunomodulation by xenografted human mesenchymal stem cells following spinal cord ventral root avulsion. Sci Rep. 5:16167, 2015. 10.1038/srep16167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Al-Ghadban, S., M. Artiles, and B. A. Bunnell. Adipose Stem Cells in Regenerative Medicine: Looking Forward. Front Bioeng Biotechnol. 9:837464, 2021. 10.3389/fbioe.2021.837464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Zhang, J., Y. Liu, Y. Chen, L. Yuan, H. Liu, et al. Adipose-Derived Stem Cells: Current Applications and Future Directions in the Regeneration of Multiple Tissues. Stem Cells Int. 2020:8810813, 2020. 10.1155/2020/8810813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Varghese, J., M. Griffin, A. Mosahebi, and P. Butler. Systematic review of patient factors affecting adipose stem cell viability and function: implications for regenerative therapy. Stem Cell Res Ther. 8(1):45, 2017. 10.1186/s13287-017-0483-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Lipchina, I., L. Studer, and D. Betel. The expanding role of miR-302–367 in pluripotency and reprogramming. Cell cycle. 11(8):1517–1523, 2012. [DOI] [PubMed] [Google Scholar]
  • 9.Houbaviy, H. B., M. F. Murray, and P. A. Sharp. Embryonic stem cell-specific MicroRNAs. Developmental cell. 5(2):351–358, 2003. [DOI] [PubMed] [Google Scholar]
  • 10.Gao, Z., X. Zhu, and Y. Dou. The miR-302/367 cluster: a comprehensive update on its evolution and functions. Open biology. 5(12):150138, 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Sugawara, T., Y. Kawamoto, T. Kawasaki, A. Umezawa, and H. Akutsu. A single allele of the hsa-miR-302/367 cluster maintains human pluripotent stem cells. Regen Ther. 21:37–45, 2022. 10.1016/j.reth.2022.05.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Subramanyam, D., S. Lamouille, R. L. Judson, J. Y. Liu, N. Bucay, et al. Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells. Nature biotechnology. 29(5):443, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Hu, S., K. D. Wilson, Z. Ghosh, L. Han, Y. Wang, et al. MicroRNA-302 increases reprogramming efficiency via repression of NR2F2. Stem cells. 31(2):259–268, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Liao B, Bao X, Liu L, Feng S, Zovoilis A, et al., MicroRNA cluster 302-367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition. Journal of Biological Chemistry. 2011:jbc. C111. 235960 [DOI] [PMC free article] [PubMed]
  • 15.Lee, M. R., N. Prasain, H. D. Chae, Y. J. Kim, C. Mantel, et al. Epigenetic regulation of NANOG by miR-302 cluster-MBD2 completes induced pluripotent stem cell reprogramming. Stem Cells. 31(4):666–681, 2013. 10.1002/stem.1302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Miyoshi, N., H. Ishii, H. Nagano, N. Haraguchi, D. L. Dewi, et al. Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell stem cell. 8(6):633–638, 2011. [DOI] [PubMed] [Google Scholar]
  • 17.Sokka, J., M. Yoshihara, J. Kvist, L. Laiho, A. Warren, et al. CRISPR activation enables high-fidelity reprogramming into human pluripotent stem cells. Stem Cell Reports. 17(2):413–426, 2022. 10.1016/j.stemcr.2021.12.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lin, S.-L., D. C. Chang, S. Chang-Lin, C.-H. Lin, D. T. Wu, et al. Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. Rna. 14(10):2115–2124, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Lin, S.-L., D. C. Chang, C.-H. Lin, S.-Y. Ying, D. Leu, et al. Regulation of somatic cell reprogramming through inducible mir-302 expression. Nucleic acids research. 39(3):1054–1065, 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Anokye-Danso, F., C. M. Trivedi, D. Juhr, M. Gupta, Z. Cui, et al. Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell stem cell. 8(4):376–388, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Koide, N., K. Yasuda, K. Kadomatsu, and Y. Takei. Establishment and optimal culture conditions of microrna-induced pluripotent stem cells generated from HEK293 cells via transfection of microrna-302s expression vector. Nagoya journal of medical science. 74(1–2):157, 2012. [PMC free article] [PubMed] [Google Scholar]
  • 22.Faghih, H., A. Javeri, H. Amini, and M. F. Taha. Directed differentiation of human adipose tissue-derived stem cells to dopaminergic neurons in low-serum and serum-free conditions. Neurosci Lett. 708:134353, 2019. 10.1016/j.neulet.2019.134353. [DOI] [PubMed] [Google Scholar]
  • 23.Soheilifar MH, Javeri A, Amini H,Taha MF, Generation of dopamine-secreting cells from human adipose tissue-derived stem cells in vitro. Rejuvenation research. 2018 [DOI] [PubMed]
  • 24.Asgharian, R., A. Hashemi, A. Javeri, and M. Fakhr Taha. DNA Methyltransferase Inhibition by RG108 Improves Stemness and Multipotential Differentiation of Human Adipose Tissue-derived Stem Cells. Iran J Biotechnol. 22(2):e3863, 2024. 10.30498/ijb.2024.435096.3863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Pfaffl, M. W., G. W. Horgan, and L. Dempfle. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30(9):e36, 2002. 10.1093/nar/30.9.e36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Khani, M. J. A., A. Bazargani, G. Karimi, and M. F. Taha. Investigating the Effect of miR-302/367 Cluster Overexpression on the Expression of mTOR Signaling Pathway Genes in Human Adipose Tissue- Derived Stem Cells. Research in Medicine. 48(1):28–37, 2024. [Google Scholar]
  • 27.Suh, M. R., Y. Lee, J. Y. Kim, S. K. Kim, S. H. Moon, et al. Human embryonic stem cells express a unique set of microRNAs. Dev Biol. 270(2):488–498, 2004. 10.1016/j.ydbio.2004.02.019. [DOI] [PubMed] [Google Scholar]
  • 28.Jaenisch, R., and R. Young. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell. 132(4):567–582, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Shenghui, H., D. Nakada, and S. J. Morrison. Mechanisms of stem cell self-renewal. Annual Review of Cell and Developmental. 25:377–406, 2009. [DOI] [PubMed] [Google Scholar]
  • 30.Son, M. Y., H. Choi, Y. M. Han, and Y. Sook Cho. Unveiling the critical role of REX1 in the regulation of human stem cell pluripotency. Stem cells. 31(11):2374–2387, 2013. [DOI] [PubMed] [Google Scholar]
  • 31.Pauklin, S., and L. Vallier. Activin/Nodal signalling in stem cells. Development. 142(4):607–619, 2015. [DOI] [PubMed] [Google Scholar]
  • 32.Card, D. A., P. B. Hebbar, L. Li, K. W. Trotter, Y. Komatsu, et al. Oct4/Sox2-regulated miR-302 targets cyclin D1 in human embryonic stem cells. Mol Cell Biol. 28(20):6426–6438, 2008. 10.1128/MCB.00359-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Marson, A., S. S. Levine, M. F. Cole, G. M. Frampton, T. Brambrink, et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell. 134(3):521–533, 2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Lin, S. L., D. C. Chang, S. Y. Ying, D. Leu, and D. T. Wu. MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of the CDK2 and CDK4/6 cell cycle pathways. Cancer Res. 70(22):9473–9482, 2010. 10.1158/0008-5472.CAN-10-2746. [DOI] [PubMed] [Google Scholar]
  • 35.Cai, N., Y. D. Wang, and P. S. Zheng. The microRNA-302-367 cluster suppresses the proliferation of cervical carcinoma cells through the novel target AKT1. RNA. 19(1):85–95, 2013. 10.1261/rna.035295.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Maadi, H., A. Moshtaghian, M. F. Taha, S. J. Mowla, A. Kazeroonian, et al. Multimodal tumor suppression by miR-302 cluster in melanoma and colon cancer. Int J Biochem Cell Biol. 81(Pt A):121–132, 2016. 10.1016/j.biocel.2016.11.004. [DOI] [PubMed] [Google Scholar]
  • 37.Ramezankhani, B., M. F. Taha, and A. Javeri. Vitamin C counteracts miR-302/367-induced reprogramming of human breast cancer cells and restores their invasive and proliferative capacity. J Cell Physiol. 234(3):2672–2682, 2019. 10.1002/jcp.27081. [DOI] [PubMed] [Google Scholar]
  • 38.Hoseinbeyki, M., M. F. Taha, and A. Javeri. miR-16 enhances miR-302/367-induced reprogramming and tumor suppression in breast cancer cells. IUBMB Life. 72(5):1075–1086, 2020. 10.1002/iub.2249. [DOI] [PubMed] [Google Scholar]
  • 39.Ahmadalizadeh Khanehsar, M., M. Hoseinbeyki, M. Fakhr Taha, and A. Javeri. Repression of TGF-beta Signaling in Breast Cancer Cells by miR-302/367 Cluster. Cell J. 21(4):444–450, 2020. 10.22074/cellj.2020.6193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Rezania, M. A., A. Eghtedari, M. F. Taha, A. M. Ardekani, and A. Javeri. A novel role for aspirin in enhancing the reprogramming function of miR-302/367 cluster and breast tumor suppression. J Cell Biochem. 123(6):1077–1090, 2022. 10.1002/jcb.30264. [DOI] [PubMed] [Google Scholar]
  • 41.Taha, M. F., A. Javeri, S. Rohban, and S. J. Mowla. Upregulation of pluripotency markers in adipose tissue-derived stem cells by miR-302 and leukemia inhibitory factor. Biomed Res Int. 2014:941486, 2014. 10.1155/2014/941486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Su Tong, D. S. Wang Yingjie Akt–Oct4 regulatory circuit in pluripotent stem cells. Chin. Sci. Bull. 59:936–943, 2014. [Google Scholar]
  • 43.Li, H. L., J. F. Wei, L. Y. Fan, S. H. Wang, L. Zhu, et al. miR-302 regulates pluripotency, teratoma formation and differentiation in stem cells via an AKT1/OCT4-dependent manner. Cell Death Dis. 7(1):e2078, 2016. 10.1038/cddis.2015.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Chalhoub, N., and S. J. Baker. PTEN and the PI3-kinase pathway in cancer. Annu Rev Pathol. 4:127–150, 2009. 10.1146/annurev.pathol.4.110807.092311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Long, X., Y. Lin, S. Ortiz-Vega, K. Yonezawa, and J. Avruch. Rheb binds and regulates the mTOR kinase. Curr Biol. 15(8):702–713, 2005. 10.1016/j.cub.2005.02.053. [DOI] [PubMed] [Google Scholar]
  • 46.Rosner, M., M. Hanneder, N. Siegel, A. Valli, C. Fuchs, et al. The mTOR pathway and its role in human genetic diseases. Mutat Res. 659(3):284–292, 2008. 10.1016/j.mrrev.2008.06.001. [DOI] [PubMed] [Google Scholar]
  • 47.Brotelle, T., and J. O. Bay. PI3K-AKT-mTOR pathway: Description, therapeutic development, resistance, predictive/prognostic biomarkers and therapeutic applications for cancer. Bull Cancer. 103(1):18–29, 2016. 10.1016/j.bulcan.2015.09.011. [DOI] [PubMed] [Google Scholar]
  • 48.Fantus, D., N. M. Rogers, F. Grahammer, T. B. Huber, and A. W. Thomson. Roles of mTOR complexes in the kidney: implications for renal disease and transplantation. Nat Rev Nephrol. 12(10):587–609, 2016. 10.1038/nrneph.2016.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Guertin, D. A., and D. M. Sabatini. Defining the role of mTOR in cancer. Cancer Cell. 12(1):9–22, 2007. 10.1016/j.ccr.2007.05.008. [DOI] [PubMed] [Google Scholar]
  • 50.Bhaskar, P. T., and N. Hay. The two TORCs and Akt. Dev Cell. 12(4):487–502, 2007. 10.1016/j.devcel.2007.03.020. [DOI] [PubMed] [Google Scholar]
  • 51.Yang, Q., and K. L. Guan. Expanding mTOR signaling. Cell Res. 17(8):666–681, 2007. 10.1038/cr.2007.64. [DOI] [PubMed] [Google Scholar]
  • 52.Romanyuk, N., K. Sintakova, I. Arzhanov, M. Horak, C. Gandhi, et al. mTOR pathway inhibition alters proliferation as well as differentiation of neural stem cells. Front Cell Neurosci. 18:1298182, 2024. 10.3389/fncel.2024.1298182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Yoon, M. S., C. Zhang, Y. Sun, C. J. Schoenherr, and J. Chen. Mechanistic target of rapamycin controls homeostasis of adipogenesis. J Lipid Res. 54(8):2166–2173, 2013. 10.1194/jlr.M037705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Sterneck, E., D. K. Poria, and K. Balamurugan. Slug and E-Cadherin: Stealth Accomplices? Front Mol Biosci. 7:138, 2020. 10.3389/fmolb.2020.00138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Jansen, S., R. Gosens, T. Wieland, and M. Schmidt. Paving the Rho in cancer metastasis: Rho GTPases and beyond. Pharmacol Ther. 183:1–21, 2018. 10.1016/j.pharmthera.2017.09.002. [DOI] [PubMed] [Google Scholar]
  • 56.Aslan, H., Y. Zilberman, L. Kandel, M. Liebergall, R. J. Oskouian, et al. Osteogenic differentiation of noncultured immunoisolated bone marrow-derived CD105+ cells. Stem Cells. 24(7):1728–1737, 2006. 10.1634/stemcells.2005-0546. [DOI] [PubMed] [Google Scholar]
  • 57.Pei, D., X. Shu, A. Gassama-Diagne, and J. P. Thiery. Mesenchymal-epithelial transition in development and reprogramming. Nat Cell Biol. 21(1):44–53, 2019. 10.1038/s41556-018-0195-z. [DOI] [PubMed] [Google Scholar]
  • 58.Kuo, C.-H., J. H. Deng, Q. Deng, and S.-Y. Ying. A novel role of miR-302/367 in reprogramming. Biochemical and biophysical research communications. 417(1):11–16, 2012. [DOI] [PubMed] [Google Scholar]
  • 59.Karimi Roshan, M., A. Soltani, A. Soleimani, K. Rezaie Kahkhaie, A. R. Afshari, et al. Role of AKT and mTOR signaling pathways in the induction of epithelial-mesenchymal transition (EMT) process. Biochimie. 165:229–234, 2019. 10.1016/j.biochi.2019.08.003. [DOI] [PubMed] [Google Scholar]
  • 60.Lin, K., S. Baritaki, L. Militello, G. Malaponte, Y. Bevelacqua, et al. The Role of B-RAF Mutations in Melanoma and the Induction of EMT via Dysregulation of the NF-kappaB/Snail/RKIP/PTEN Circuit. Genes Cancer. 1(5):409–420, 2010. 10.1177/1947601910373795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Fedorova, O., S. Parfenyev, A. Daks, O. Shuvalov, and N. A. Barlev. The Role of PTEN in Epithelial-Mesenchymal Transition. Cancers (Basel). 2022. 10.3390/cancers14153786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Costa, F. F., E. A. Seftor, J. M. Bischof, D. A. Kirschmann, L. Strizzi, et al. Epigenetically reprogramming metastatic tumor cells with an embryonic microenvironment. Epigenomics. 1(2):387–398, 2009. 10.2217/epi.09.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Bui, Q. T., J. H. Im, S. B. Jeong, Y. M. Kim, S. C. Lim, et al. Essential role of Notch4/STAT3 signaling in epithelial-mesenchymal transition of tamoxifen-resistant human breast cancer. Cancer Lett. 390:115–125, 2017. 10.1016/j.canlet.2017.01.014. [DOI] [PubMed] [Google Scholar]
  • 64.Paplomata, E., and R. O’Regan. The PI3K/AKT/mTOR pathway in breast cancer: targets, trials and biomarkers. Ther Adv Med Oncol. 6(4):154–166, 2014. 10.1177/1758834014530023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Ryskalin, L., G. Lazzeri, M. Flaibani, F. Biagioni, S. Gambardella, et al. mTOR-Dependent Cell Proliferation in the Brain. Biomed Res Int. 2017:7082696, 2017. 10.1155/2017/7082696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Peng, Y., Y. Wang, C. Zhou, W. Mei, and C. Zeng. PI3K/Akt/mTOR Pathway and Its Role in Cancer Therapeutics: Are We Making Headway? Front Oncol. 12:819128, 2022. 10.3389/fonc.2022.819128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Lin, S. L., J. S. Chen, and S. Y. Ying. MiR-302-Mediated Somatic Cell Reprogramming and Method for Generating Tumor-Free iPS Cells Using miR-302. Methods Mol Biol. 2115:199–219, 2020. 10.1007/978-1-0716-0290-4_12. [DOI] [PubMed] [Google Scholar]
  • 68.Kim, J. Y., K. K. Shin, A. L. Lee, Y. S. Kim, H. J. Park, et al. MicroRNA-302 induces proliferation and inhibits oxidant-induced cell death in human adipose tissue-derived mesenchymal stem cells. Cell Death Dis. 5(8):e1385, 2014. 10.1038/cddis.2014.344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Rand, T. A., K. Sutou, K. Tanabe, D. Jeong, M. Nomura, et al. MYC Releases Early Reprogrammed Human Cells from Proliferation Pause via Retinoblastoma Protein Inhibition. Cell Rep. 23(2):361–375, 2018. 10.1016/j.celrep.2018.03.057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Jian-feng, W. miR-302 regulates the osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells. Chinese Journal of Tissue Engineering Research. 19:6595–6599, 2015. 10.3969/j.issn.2095-4344.2015.41.007. [Google Scholar]
  • 71.Lei, Z., D. Wei, Y. Ma, L. Tang, S. Wang, et al. miR-302b promotes bovine preadipocyte differentiation and inhibits proliferation by targeting CDK2. Anim Biotechnol. 34(4):1447–1454, 2023. 10.1080/10495398.2022.2029743. [DOI] [PubMed] [Google Scholar]
  • 72.Jeong, B. C., I. H. Kang, and J. T. Koh. MicroRNA-302a inhibits adipogenesis by suppressing peroxisome proliferator-activated receptor gamma expression. FEBS Lett. 588(18):3427–3434, 2014. 10.1016/j.febslet.2014.07.035. [DOI] [PubMed] [Google Scholar]
  • 73.Bae, K. H., W. K. Kim, and S. C. Lee. Involvement of protein tyrosine phosphatases in adipogenesis: new anti-obesity targets? BMB Rep. 45(12):700–706, 2012. 10.5483/bmbrep.2012.45.12.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.Huang, J., Y. He, H. L. McLeod, Y. Xie, D. Xiao, et al. miR-302b inhibits tumorigenesis by targeting EphA2 via Wnt/ beta-catenin/EMT signaling cascade in gastric cancer. BMC Cancer. 17(1):886, 2017. 10.1186/s12885-017-3875-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75.Agostini, F., C. Vicinanza, G. Biolo, P. Spessotto, F. Da Ros, et al. Nucleofection of Adipose Mesenchymal Stem/Stromal Cells: Improved Transfection Efficiency for GMP Grade Applications. Cells. 2021. 10.3390/cells10123412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 76.Holvoet, P. Aging and Metabolic Reprogramming of Adipose-Derived Stem Cells Affect Molecular Mechanisms Related to Cardiovascular Diseases. Cells. 2023. 10.3390/cells12242785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77.Talkhabi, M. Partial reprogramming as a therapeutic approach for heart disease: A state-of-the-art review. J Cell Biochem. 120(9):14247–14261, 2019. 10.1002/jcb.28900. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12195_2025_856_MOESM1_ESM.pdf (211.9KB, pdf)

Supplementary file1 (PDF 212 KB)—System Biosciences map for TDH101PA‐GP, a miR‐302bcad/367 expressing Lentivector.

12195_2025_856_MOESM2_ESM.pdf (269.5KB, pdf)

Supplementary file2 (PDF 270 KB)—Original, unprocessed images of western blots, related to Fig. 2.

12195_2025_856_MOESM3_ESM.pdf (229.6KB, pdf)

Supplementary file3 (PDF 230 KB)—Original, unprocessed images of western blots, corresponding to Fig. 3.

12195_2025_856_MOESM4_ESM.pdf (322.6KB, pdf)

Supplementary file4 (PDF 323 KB)—Original, unprocessed images of western blots, corresponding to Fig. 4.

12195_2025_856_MOESM5_ESM.pdf (74.1KB, pdf)

Supplementary file5 (PDF 74 KB)—Primers used for RT-PCR and qPCR.

12195_2025_856_MOESM6_ESM.pdf (199.5KB, pdf)

Supplementary file6 (PDF 200 KB)—The list of antibodies used for western blot analysis.

Data Availability Statement

The original, uncropped images of western blots are included as supplementary information. The datasets that support the other findings of this study are available from the corresponding authors upon request.

Articles from Cellular and Molecular Bioengineering are provided here courtesy of Springer

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