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. 2024 Oct 10;14:23717. doi: 10.1038/s41598-024-74385-6

miR-16a-5p antagonizes FGF-2 in ligamentogenic differentiation of MSC: a new therapeutic perspective for tendon regeneration

Jibin Yang 1,#, Huaize Dong 1,#, Jin Yang 1, Hao Yu 1, Gang Zou 1, Jiachen Peng 1,✉
PMCID: PMC11479607  PMID: 39390042

Abstract

With the increasing demand for exercise, the population of patients with ankle sprain to anterior talofibular ligament injury has the characteristics of a large base and high requirements for returning to sports, and how to promote the repair of damaged ligaments from a microscopic perspective is an urgent problem to be solved. In many studies, human amniotic mesenchymal stem cells have strong differentiation ability, and can be induced to continuously differentiate into ligament cells to achieve the purpose of repairing damaged ligaments. Human amniotic stem cells were extracted and cultured from human amniotic tissues, evaluated by cell identification and other techniques, and evaluated into ligament differentiation by toluidine blue, alizarin red, oil red O staining and detection of ligament cell differentiation, protein detection by Western blot, mRNA level by qPCR, and finally, the targeted binding relationship between miR-16a-5p and mRNA FGF2 was verified by double luciferase reporter assay. The expression of collagen type 1 (COL 1), collagen type 3 (COL3), SCX and MKX was increased by overexpression of mRNA FGF2, respectively, and miR-16a-5p had a targeted effect on FGF2 and regulated the ligamentous differentiation of human amniotic mesenchymal stem cells. We found that the regulatory effect of overexpressed mRNA FGF2 on mesenchymal stem cells could be inhibited by up-regulation of miR-16a-5p, while the knockdown of FGF2 could reverse the regulatory effect of miR-16a-5p inhibition on ligament-forming differentiation of human amniotic mesenchymal stem cells. In this study, we discovered the existence of the miR-16a-5p–FGF2 axis in human amniotic mesenchymal stem cells, and the differentiation of human amniotic mesenchymal stem cells into ligamentous cells can be regulated by regulating various links in this axis.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-74385-6.

Keywords: MiR-16a-5p, HAMSCs, Ligamentous cells, FGF2

Subject terms: Cell biology, Stem cells

Introduction

Ligament injury is one of the most common manifestations of sports injuries, and with the increasing demand for sports, the need to return to sports after injury is also increasing1. Since the discovery of stem cell technology, the technology has been relatively mature, and mesenchymal stem cells have also been widely used in the diagnosis and treatment of many organs and various diseases in the human body. In the field of tendon healing, many scholars have conducted research from various perspectives, ranging from underlying mechanisms to clinical applications. For instance, cocktail-like gradient gelatin/hyaluronic acid biocomposites have been shown to promote tendon-bone healing in cases of fatty infiltration after rotator cuff injuries2. Additionally, studies have indicated that hyaluronic acid can serve as an effective option for promoting healing in rotator cuff injuries3.In the context of anterior cruciate ligament (ACL) injuries, platelet-rich plasma (PRP) has been demonstrated in both basic and clinical research to significantly enhance tendon-bone healing following ACL reconstruction4. Moreover, intermittent negative pressure has been found to promote tendon-bone healing in rabbit ACL grafts5. From the perspective of stem cell-mediated tendon-bone healing, a combination of cartilage fragments and exosomes derived from bone marrow mesenchymal stem cells can promote tendon-bone healing after ACL reconstruction6. Similarly, exosomes derived from infrapatellar fat pad mesenchymal stem cells have also been shown to facilitate tendon-bone healing following ACL reconstruction7. As a common source of mesenchymal stem cells, human amniotic mesenchymal stem cells are easy to extract and store8, and have the potential for extensive research and treatment of diseases9. We consider improving the condition of the ligaments and promoting ligament repair through the action of mesenchymal stem cells, so this study focuses on human amniotic mesenchymal stem cells.

miRNAs are a small non-coding RNA that is very important in the process of regulating proteins, and can play an important role in physiological and pathological processes, and has a wide range of influences10. Li et al.11 found that miRNA has the ability to regulate the migration of human amniotic mesenchymal stem cells. Relevant studies have also shown that miRNAs can act on the chondrogenic differentiation of hAMSCs under upstream regulation12 to achieve the purpose of microscopic treatment of cartilage injury. In the field of ligament cell research, miRNA can interact with ligament cells, in addition to miRNA as a gene to regulate various phenotypes of ligament cells, Kaneda-Ikeda et al.13 found that periodontal ligament cells can regulate osteogenesis through miR-299-5p in mesenchymal stem cells. As a miRNA, miR-16a-5p plays an important role in arthritis, ligament injury, osteoporosis, and other fields14. However, there are almost no relevant studies on the action of miR-16a-5p on ligament cells, so miR-16a-5p is also the main research object in this project.

FGF2 is a fibroblast growth factor, which participates in the pathophysiological processes of various diseases and plays an important role in human tissue repair. FGF2 acts on downstream signaling pathways to exert specific immunological effects15. In addition, FGF2 also plays an important role in the progression of osteoarthritis16. In the diagnosis and treatment of sports injuries, Zhang et al.17 found that FGF2 is one of the most important factors in promoting tendon bone healing. In recent years, a large number of studies have been carried out on the role of FGF2 through stem cells, and it has been confirmed that FGF2 can promote ligament repair through stem cells18,19. Therefore, this project is an in-depth study of the effect of FGF2 on the ligamentogenic differentiation of mesenchymal stem cells.

At present, there are relatively few studies on the ligamentogenic differentiation of mesenchymal stem cells by miR-16a-5p, and there is still a lack of upstream exploration of the pathway for FGF2 to promote the ligamentogenic differentiation of mesenchymal stem cells, so the study of the role of miR-16a-5p mesenchymal stem cells in ligamentogenic differentiation can make up for this deficiency and play an important role in the diagnosis and treatment of ligament injury。 In this study, we verified our hypothesis by exploring the interaction between miR-16a-5p and FGF2 by (a) miR-16a-5p on the ligamentogenic differentiation of human amniotic mesenchymal stem cells, (b) FGF2 on the ligamentogenic differentiation of human amniotic mesenchymal stem cells, and (c) the interaction between miR-16a-5p and FGF2。.

Materials and methods

Cell culture

In this study, the placenta was collected in the obstetrics department of the Affiliated Hospital of Zunyi Medical University, and the informed consent of each patient was obtained before surgery. We confirm that the experimental protocols were approved by Ethics Committee of the Affiliated Hospital of Zunyi Medical University and all methods were performed in accordance with the relevant guidelines and regulations. Human amniotic mesenchymal stem cells were derived from discarded placenta, primary cells were extracted from the placenta, and the hAMSCs used in this study were isolated from the human placental amniotic membranes of 5 healthy full-term puerperal women by two enzyme mining (Solarbio, China) followed by type II collagenase (Gibco, USA) digestion. After 48 h in primary culture (P0), the morphology of hAMSCs took on a spindle-shaped appearance under an inverted microscope. After several subcultures, the morphology of hAMSCs gradually changed to fusiform. We verified hAMSCs by staining and determined that the cells used in this study were stem cells(Fig. 1A–C). Cells are cultured in complete medium with 10% bovine fetal serum and 1% penicillin streptomycin. The incubation environment was set to 95% air and 5% CO2, and the temperature was set to 37 °C (Table 1).

Fig. 1.

Fig. 1

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After culturing human amniotic mesenchymal stem cells, we performed the following staining: (A) Toluidine Blue Staining (B) Alizarin Red Staining (C) Oil Red O Staining.

Table 1.

Conditions and time points for human amniotic mesenchymal stem cell culture and passaging.

Cell Culture conditions Time points
Primary Cells(P0) T75 Flask, 95% Air and 5% CO2, 37 °C Day1
First-Passage Cells(P1) T25 Flask, 95% Air and 5% CO2, 37 °C Day7
Second-Passage Cells(P2) T25 Flask, 95% Air and 5% CO2, 37 °C Day12
Third-Passage Cells(P3) T25 Flask, 95% Air and 5% CO2, 37 °C Day16
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Cell transfection

In this study, the overexpressed miR-16a-5p (miR-16a-5p mimic group), the inhibited miR-16a-5p (miR-16a-5p inhibiter group), and the down-regulated FGF2 (siFGF2) were all purchased from Shanghai Jikai Gene Chemical Technology Co. Ltd. hASMCs are cultured in 6-well plates at a concentration of 2 × 105 cells per well, and when the cells are approximately 60% grown, the corresponding vector is transfected into hAMSC using reagents, all following the manufacturer’s protocol.

qPCR

HAMSCs from each group are seeded into 6-well plates. RNA was isolated with TRIzol (PrimeScript™ RT Kit, Takara, Japan) and the mRNA was reversed to complementary DNA (cDNA) for RT-qPCR. RT-qPCR was performed using the TaKaRa Ex Taq® Kit (Takara, Japan). GAPDH was used as the housekeeping gene of FGF2 and U6 was used as the internal control of miR-16a-5p, and the relative expression level of the target gene was calculated by 2 − ΔΔCt (ΔCt = Ct target gene − Ct reference gene, and the Ct value indicated the number of cycles when the fluorescence signal in each reaction tube reached the set threshold).

Western blot

Ligament-related proteins, including col3, mkx, scx, were measured in hAMSC. Total protein extracted from hAMSC was determined with a BCA kit (Sigma-Aldrich, MO, USA), followed by 10% SDS-PAGE gel electrophoresis and transfer to a PVDF membrane (Millipore, MA, USA). Membranes are blocked with 5% milk for 2 h, followed by incubation with primary antibodies (including anti-COL3 (1/1000, Abcam, MA, USA), anti-MKX (1/1500, Abcam), anti-SCX (1/800, Abcam), anti-COL1 (1/1000, Abcam), and GADPH (1/3000, Abcam) for 12 h at 4 °C. Subsequently, the immune complex was combined with horseradish peroxidase-labeled immunoglobulin G (IgG; 1/2000, Abcam) for 1 h and visualized using a chemiluminescence kit (Beyotime, Shanghai, China). Finally, the brands were photographed by the IS gel image analysis system and analyzed using Image J.

Dual luciferase test

To verify the targeting relationship between miR-16a-5p and FGF2, we validated it by a dual luciferase reporter assay. (1) Preparation of target cells: 293T cells were seeded in an appropriate proportion (70%) and cultured in 6-well plates for about 16 h. (2) Plasmid co-transfection: Before transfection, the cells prepared in advance need to be changed to serum-free medium at 37°CIncubator to be transferred: take 2ug of FGF2 wild-type plasmid and mutant overexpression plasmid and miR-16a-5p plasmid respectively, and add serum-free medium to tube 1 to 200ul room temperature for 5 min, at the same time, take 8ul transfection reagent and add 192ul serum-free medium to tube 2 and incubate at room temperature for 5 min; after 5 min, add the transfection reagent in tube 2 dropwise to tube 1 plasmid for 20 min, and finally add the bred liposomes dropwise to the 293T cells to be transposed, and mix well. And 6 h after transfection, the DMEM complete medium was replaced. (3) After 48 h, the transfection efficiency should be observed, and samples should be collected for detection. Subsequently, fluorescence intensity detection was performed: (1) the medium in the cultured cells was removed. (2) Wash the culture cells 3 times with 1× PBS. Remove the cleaning solution. (3) Add 1×PLB to 500ul into a 6-well plate in the recommended volume. (4) Passive lysis: gently shake the culture plate at room temperature for 15 min, and transfer the lysate to the test tube. (5) 20ul of the lysed sample was added to a white opaque microplate plate, and 100 µl LARI was added to the lysate/well to detect luciferase; Add 100 µl of Stop&Glo reagent to detect the activity of Renilla luciferase; (6) Finally, the results are analyzed.

Prediction and statistical analysis of gene targeting relationship

Regarding the biological binding of miR-16a-5p to FGF2, we made predictions using the online website starbase (http://starbase.sysu.edu.cn/) and the results were statistically analyzed using GraphPad Prism 8.0 (MacKiev software). The normal distribution (Shapiro-Wilk) and variance uniformity test were performed on multiple sets of data, and those who met the criteria were expressed in the form of “x ± sd”. One-way ANOVA and paired T-test were used to compare two independent samples between and within groups. Each experiment is performed independently three times (n = 3). p < 0.05 on both sides is considered statistically significant.

Results

Overexpression of mRNA FGF2 can promote the differentiation of human amniotic mesenchymal stem cells into ligamentous cells

In our study on the role of FGF2 in the differentiation of hAMSCs into ligament cells, we conducted experiments with different groups, including a control group (blank group), an FGF2 overexpression group, and an FGF2 silencing group. After conducting qPCR and Western Blot (WB) analyses, we observed that the mRNA levels related to ligament differentiation were significantly higher in the FGF2 overexpression group compared to the blank group (Fig. 2B). Conversely, the expression of proteins related to ligament differentiation was significantly lower in the FGF2 silencing group compared to the blank group (Fig. 2C,D).

Fig. 2.

Fig. 2

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After FGF2 transfection into hAMSC, (A) the expression of FGF2-related protein increased by WB technology, indicating that we successfully transfected FGF2 gene into hAMSC to promote its ligament-forming differentiation; (B) Validation of overexpression and low expression levels of FGF2 in hAMSCs by detecting the relevant mRNA; (C,D)Validation of overexpression and low expression levels of FGF2 in hAMSCs by detecting the relative proteins level; **p < 0.01.

First, we explored the role of FGF2 on mesenchymal stem cells. In addition, we verified by WB that after FGF2 overexpression in hAMSC(Fig. 2A), the contents of COL1, MKX and SCX related indexes related to ligament-forming differentiation were significantly higher in the FGF2 overexpression group than in the no-load group. In order to further improve the reliability, we first detected the COL1, MKX, and SCX proteins in the no-load group and the FGF2 overexpression group by western blot technology, and found that the ligament-forming differentiation-related proteins in the FGF2 overexpression group were more than those in the no-load group (Fig. 2C). The detection of ligament-related proteins in the noload group and FGF2 silencing components in blot showed that the noload group was higher than that in the FGF2 silencing group (Fig. 2D).

miR-16a-5p inhibits mRNA FGF2 in promoting ligamentogenic differentiation of human amniotic mesenchymal stem cells

In the study examining the role of miR-16a-5p in regulating FGF2-mediated hAMSC differentiation into ligament cells, we established the following experimental groups: a control group (empty vector), a miR-16a-5p overexpression group (miR-16a-5p mimic group), a miR-16a-5p inhibition group (miR-16a-5p inhibitor group), a miR-16a-5p overexpression + FGF2 overexpression group (miR-16a-5p mimic + FGF2 group), and a miR-16a-5p inhibition + FGF2 knockdown group (miR-16a-5p inhibitor + siFGF2 group).After conducting qPCR and Western Blot (WB) analyses, we found that the expression of mRNA related to ligament differentiation was significantly elevated in the miR-16a-5p mimic group compared to the control group. This mRNA expression was even higher in the miR-16a-5p inhibitor group compared to the miR-16a-5p mimic group. Additionally, the expression of mRNA related to ligament differentiation was higher in the miR-16a-5p mimic + FGF2 group compared to the miR-16a-5p mimic group, indicating that FGF2 still has the potential to promote mesenchymal stem cell differentiation into ligament cells despite the inhibition by miR-16a-5p. Conversely, in the miR-16a-5p inhibitor + siFGF2 group, the mRNA expression related to ligament differentiation was lower than that in the miR-16a-5p inhibitor group, further confirming the role of FGF2 in promoting the differentiation of mesenchymal stem cells into ligament cells (Fig. 3B–D).

Fig. 3.

Fig. 3

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miR-16a-5p was transfected into hAMSC, and the virus transfection was verified by PCR (A), and the mRNA related to ligament differentiation was detected by PCR (B,C,D) when miR-16a-5p was overexpressed, which was lower than that of the noload group, compared with miR-16a-5p In the inhibitor group, when miR-16a-5p was inhibited, the ligament-forming differentiation index was significantly increased, and when miR-16a-5p was overexpressed in the hAMSC group transfected with FGF2, the ligament-forming differentiation indexes were reduced compared with the no-load group When FGF2 was silenced (E,F), it was found that the ligament-forming differentiation index was lower than that of the miR-16a-5p inhibitor group. **p < 0.01.

In order to verify whether miR-16a-5p was successfully transfected into human amniotic mesenchymal stem cells, we detected the content of miR-16a-5p in human amniotic mesenchymal stem cells by PCR after transfection, and found that the content of miR-16a-5p in the mimic group was significantly higher than that in the no-load group, and miR-16a-5p The content of miR-16a-5p in the inhibitor group was significantly lower than that in the no-load group (Fig. 3A), indicating that miR-16a-5p-related genes were successfully transfected into human amniotic mesenchymal stem cells, and in the subsequent experiments, we co-transfected miR-16a-5p and FGF2 into Control Group (empty vector), miR-16a-5p Overexpression Group (miR-16a-5p mimic group), miR-16a-5p Inhibition Group (miR-16a-5p inhibitor group), miR-16a-5p Overexpression + FGF2 Overexpression Group (miR-16a-5p mimic + FGF2 group), miR-16a-5p Inhibition + FGF2 Knockdown Group (miR-16a-5p inhibitor + siFGF2 group)(Fig. 3B–D), we analyzed by PCR technology that the content of mRNA related to ligament-forming differentiation was lower than that of the no-load group when miR-16a-5p was overexpressed, but compared with miR-16a-5p In the inhibitor group, when miR-16a-5p was inhibited, the ligament-forming differentiation indexes (COL1, COL3, MKX) were significantly increased, and we found that miR-16a-5p inhibited the ligament-forming differentiation of hAMSC, and in the miR-16a-5p mimic + FGF2 group, the ligament-forming related differentiation indexes were higher than those of hAMSC + miR-16a-5p. The mimic group increased, which further indicated that FGF2 positively promoted the ligamentogenic differentiation of human amniotic mesenchymal stem cells, while in the verification of the relevant indicators in the miR-16a-5p inhibitor + shFGF2 group, we found that the ligament-forming differentiation related indexes were higher than those of miR-16a-5p alone In addition, we analyzed the effects of co-transfection into FGF2 and silencing FGF2 after overexpression of miR-16a-5p in mesenchymal stem cells by western blot technology (Fig. 3E,F), and finally found that COL1 in the miR-16a-5p + FGF2 group The expression of COL3 (ligament-forming differentiation-related protein) was lower than that of the no-load group, and the expression of related protein in miR-16a-5p + shFGF2 group was lower than that of miR-16a-5p + FGF2, indicating that miR-16a-5p inhibited the differentiation of human amniotic mesenchymal stem cells into ligamentous cells. As for how miR-16a-5p acts on the regulation of FGF2, it is necessary to further verify whether miR-16a-5p has a direct effect on FGF2.

Targeting relationship between miR-16a-5p and mRNA FGF2

We first conducted predictions using authoritative websites to confirm that there are binding sites between miR-16a-5p and FGF2. By using the StarBase online platform, we selected the miRNA-mRNA binding option, with miR-16a-5p as the miRNA and FGF2 as the mRNA. Upon searching, the binding sites between miR-16a-5p and FGF2 were identified.(Fig. 4A)In order to make the results more convincing, we performed a double luciferase reporter assay (Fig. 4B) between miR-16a-5p and FGF2, and the results showed that the content of miR-16a-5p transfected into the FGF2-3’UTR group was significantly lower than that of the FGF2-3’UTR mut group, which directly indicates that miR-16a-5p has a direct biological binding effect with FGF2. It also fully demonstrates that miR-16a-5p can inhibit the ligament-forming differentiation of human amniotic mesenchymal stem cells by inhibiting the phenotype of FGF2 to promote the differentiation of human amniotic mesenchymal stem cells into ligamentous cells.

Fig. 4.

Fig. 4

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(A) The targeting relationship between miR-16a-5p and FGF2 was analyzed online by starbase online website and its binding effect was found, and (B) the targeting relationship between miR-16a-5p and FGF2 was further confirmed by double luciferase reporter assay.

Discussion

With the continuous improvement in living standards, there has been a corresponding increase in people’s demand for exercise. This often leads to various forms of sports injuries, and the pain and the need to return to exercise after an injury are major concerns for many. This study investigates the regulatory factors involved in the differentiation of human amniotic mesenchymal stem cells (hAMSCs) into ligament cells, providing a detailed understanding of the mechanisms of ligament repair at a microscopic level. This research aims to guide the diagnosis and treatment of sports-related anterior talofibular ligament injuries and to lay the groundwork for further studies on tendon-bone healing.In our clinical practice, we have observed a high incidence of anterior talofibular ligament injuries following ankle sprains, with many patients not giving it sufficient attention. Current clinical methods for ligament repair primarily include conservative treatment and surgical repair. Conservative treatment avoids invasive procedures but can result in joint instability and limited range of motion, which may lead to habitual injuries. Surgical repair, while avoiding issues like joint instability, carries risks of damage to blood vessels, nerves, and potential postoperative infections, which deter many patients.This study proposes a new approach to ligament repair, especially applicable to cases where there is no complete rupture of the ligament. By using materials or exosomes loaded with agents or drugs that promote ligament repair through targeted injection, this method can also avoid the trauma associated with surgery. Compared to current clinical methods, this approach promises faster recovery, higher efficiency, and significantly reduced costs.

In our preliminary research, we established that FGF2 promotes tendon-bone healing by enhancing the differentiation of mesenchymal stem cells into ligament cells. However, the upstream regulatory mechanisms of FGF2 have not been explored. Therefore, we identified miRNAs targeting FGF2 through online databases and confirmed the binding relationship using dual-luciferase assays. Based on this, we grouped different treatments to test indicators related to ligament differentiation. We ultimately determined that FGF2 is regulated by miR-16a-5p, which inhibits the differentiation of hAMSCs into ligament cells through competitive binding. This finding guides our subsequent clinical practice, where inhibiting miR-16a-5p during the use of hAMSCs for ligament repair can enhance the repair process.

After past studies found that the differentiation of mesenchymal stem cells is regulated, extensive research has been carried out in the field of medical research on the modularity of their differentiation20, and by transforming extracellular regulatory factors, mesenchymal stem cells can differentiate into various cells under controlled conditions to achieve the purpose of repairing tissues and treating diseases21,22. In the field of liver cancer research, Liu et al.23 finally confirmed that human amniotic mesenchymal stem cells can exhibit significant anti-tumor effects both in vitro and in vivo, thus providing new ideas and methods for the clinical treatment of liver cancer. In addition, in the field of nerve regeneration technology, Li et al24. performed cell transplantation by exposing rat injured nerves to a normal saline environment containing human amniotic mesenchymal stem cells, and finally found that human amniotic mesenchymal stem cells have the effect of promoting nerve injury repair. In other studies, mesenchymal stem cells continue to differentiate into ligament cells to achieve the effect of treating diseases and repairing ligaments in the microscopic field25. Based on the above research, through the in-depth exploration of the process of human amniotic mesenchymal stem cells to ligament cells, we can better think about and solve the problem of post-ligament repair from a microscopic perspective.

With the deepening of research, the regulation of downstream mRNA by miRNA has been further and thoroughly, and we have found that miRNA can play a role in the ligament-forming differentiation of mesenchymal stem cells through the regulation of mRNA26. In terms of regulating mesenchymal stem cells, relevant studies have shown that microRNAs play a role in regulating their osteogenic differentiation27 and chondrogenic differentiation28. At present, there are many studies on the promotion of microRNA in cell ossification, and many other studies have shown that microRNA has a regulatory effect on the osteogenic differentiation of ligament cells29,30, but there are few studies on ligamentogenic differentiation. Therefore, it is of great significance to explore and verify the role of microRNA in the ligament-forming differentiation of mesenchymal stem cells. As a very important intermediate link in this study, miR-16a-5p was initially found to play a role in the diagnosis and treatment of osteoporosis in menopausal women31. miR-16a-5p is relatively blank in the field of ligament injury repair and mesenchymal stem cells, and through the continuous verification of this study, it was found that it has inhibitory effect on the ligamentogenic differentiation of human amniotic mesenchymal stem cells (Fig. 3B, C, D), We can promote the ligament-forming differentiation of human amniotic mesenchymal stem cells by inhibiting miR-16a-5p, so as to achieve the effect of repairing damaged ligaments from the microscopic field. In order to explore how miR-16a-5p inhibits the ligament-forming differentiation of human amniotic mesenchymal stem cells, we conducted further in-depth studies.

FGF2, known as fibroblast growth factor 2, is the most terminal factor in this study and has the effect of promoting the differentiation of hAMCS ligaments (Fig. 2B–D), and as an mRNA, it can play a variety of roles, including promoting tumor growth32, cell migration33, and osteoarthritis34. And there are many bright spots in the field of treatment of sports injuries. Among them, the promotion of stem cell differentiation is undoubtedly the most valuable, according to relevant studies have shown that FGF2 can promote the differentiation of mesenchymal stem cells into cardiomyocytes to repair myocardial damage35, in addition, FGF2 can be regulated by upstream micro RNA in the process of osteogenic differentiation of mesenchymal stem cells36. FGF2 is regulated by many factors, Hu et al.37 found that microRNA 205 can regulate drug resistance in breast cancer through the regulation of FGF2, and miR-16a-1 can promote angiogenesis and improve nerve repair in animals after stroke by upregulating FGF2, achieving a prognostic effect of Ref.38. Other studies have shown that miR-203 can reduce the formation of skin keloids by targeting FGF2 inhibition39; As for how FGF2 regulates diseases, many scholars have shown that FGF2 can regulate the change of disease phenotype by targeting various signal transduction pathways, and Ma et al.40 found that miRNA-21-3p targets FGF2 by acting on AKT/mTOR The pathway inhibits autophagy in bovine ovarian granulosa cells, and miR-653-5p has been shown to inhibit the viability and migration of fibroblast-like synovial cells by targeting FGF2 and Wnt/β-catenin pathway inactivation41. In summary, FGF2 as a growth factor can play many roles in the diseases of many fibrous connective tissues in the body, and has strong research value in research, and in terms of the study of FGF2 in ligament repair, it has been confirmed that FGF2 is an important regulator of tendon bone healing and cartilage repair36, we can basically conclude that FGF2 has the effect of promoting the differentiation of human amniotic mesenchymal stem cells into ligamentous cells (Fig. 5). The promotion of ligamentogenic differentiation of human amniotic mesenchymal stem cells by FGF2 is the cornerstone of this study, and the exploration of the upstream of FGF2 is based on this process. At present, Zhang et al.42 confirmed that FGF2 can promote tendon bone healing through in vivo studies, which is consistent with our findings.

Fig. 5.

Fig. 5

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When miR-16a-5p is overexpressed in human amniotic mesenchymal cells, it will have a binding inhibition effect on FGF2 and delay the differentiation of stem cells into ligaments, thereby inhibiting ligament repair.

In our current study, we investigated whether miR-16a-5p inhibited the differentiation of human amniotic mesenchymal stem cells into ligamentous cells through FGF2. When miR-16a-5p was overexpressed in human amniotic mesenchymal stem cells, the related indexes of ligament-forming differentiation decreased, and when the content of FGF2 increased on this basis, COL1 and other related indexes showed an upward trend (Fig. 2B,C). When miR-16a-5p was inhibited, COL1 and other related indexes also increased, and under the same circumstances, after FGF2 silencing, COL1 and other indexes decreased compared with the previous ones, which indicated that FGF2 silence could reverse the effect of miR-16a-5p inhibition on human amniotic mesenchymal stem cells (Fig. 3B–D), and we could conclude that miR-16a-5p inhibited the ligamentogenic differentiation of human amniotic mesenchymal stem cells. This effect is based on the fact that miR-16a-5p abolishes the role of FGF2 in promoting the differentiation of human amniotic mesenchymal stem cells into ligamentous cells by competitive binding to FGF2 (Fig. 5). This conclusion is also in line with our hypothesis.

Finally, we concluded through this study: FGF2 promotes the differentiation of human amniotic mesenchymal stem cells (hAMSCs) into ligament cells. miR-16a-5p inhibits this process by directly binding to FGF2, thereby suppressing the ability of FGF2 to promote the differentiation of hAMSCs into ligament cells, reveals two genes and one pathway that regulate the ligamentous differentiation of hAMCS, provides a solid and reliable basis for subsequent in-depth research, and through the regulation of miR-16a-5p, it can promote the downstream pathway to promote the differentiation of human amniotic mesenchymal stem cells into ligament cells, so as to repair the damaged ligamentto achieve the effect of clinical cure.

In addition, this study revealed the role of miR-16a-5p in the field of ligamentogenic differentiation of human amniotic mesenchymal stem cells, which laid a foundation for subsequent research, and in-depth understanding of the role of miRNA in regulating downstream mRNA in ligament differentiation of hAMSCs has important clinical significance for ligament injury treatment and tissue engineering. These regulatory networks may provide a theoretical basis for the development of new therapeutic strategies and applications in regenerative medicine. Future studies can further explore the potential application value of miR-16a-p-FGF2 network in clinical treatment. The main limitations and shortcomings of this study are that it primarily focuses on in vitro cell experiments, mainly involving single-cell assays, and lacks in vivo validation through animal experiments. Future research should integrate in vivo studies to enhance credibility. For example, specialized materials could be used to deliver miR-16a-5p and FGF2 to the ankle joints of mice, and the repair of ligament damage in different treatment groups could be observed.In addition, future research will continue to explore the signaling pathways through which FGF2 regulates the differentiation of hAMSCs into ligament cells. It is also important to actively investigate the upstream regulatory factors of miR-16a-5p. Understanding these upstream factors could provide more reliable evidence for the regulation of mesenchymal stem cell differentiation into ligament cells and identify additional targets for ligament repair. This approach aims to improve the chances of successful ligament repair and advance the treatment of ligament injuries from a microscopic perspective.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (135.8KB, pdf)
Supplementary Material 2 (2.6MB, pdf)

Author contributions

Jibin Yang and Huaize Dong are responsible for conceptualization and writing. Gang Zou is responsible for procedures. Hao Yu and Jin Yang conduct an investigation of software. Validation in Jiachen Peng responsible for project management. The written edition of the manuscript has been read and approved by both authors.

Funding

This research was funded by Qian ke he Foundation-ZK[2021]General 390 and Zun shi Ke he HZ word (2023) No. 236.

Data availability

The datasets generated and analysed during the current study are not publicly available due to the current data is being used for subsequent project applications but are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests. All authors disclosed no relevant relationships. The pictures in this article were processed by image J、prism and Figdraw.

Footnotes

Publisher’s note

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

These authors contributed equally: Jibin Yang and Huaize Dong.

References

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Associated Data

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

Supplementary Materials

Supplementary Material 1 (135.8KB, pdf)
Supplementary Material 2 (2.6MB, pdf)

Data Availability Statement

The datasets generated and analysed during the current study are not publicly available due to the current data is being used for subsequent project applications but are available from the corresponding author on reasonable request.

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