let-7c-5p promotes fracture healing by downregulating CDK8

Abstract

Background

Previous studies have shown that let-7c-5p is significantly expressed at a lower level in the serum of fracture patients, suggesting that it may play a role in bone repair. Therefore, this study aims to explore how let-7c-5p regulates osteogenic differentiation in tibial fractures.

Methods

A total of 80 patients with tibial fractures and 83 healthy individuals were included in this study. The level of let-7c-5p in the serum were detected by RT-qPCR. Osteoblast cell line (MC3T3-E1) were cultured in vitro to induce osteogenic differentiation. RT-qPCR was used to detect the expression of let-7c-5p and osteogenic differentiation markers. The activity of alkaline phosphatase (ALP) was determined using an ALP assay kit. Dual-luciferase reporter gene assay and RNA immunoprecipitation were used to verify the targeting relationship between let-7c-5p and CDK8.

Results

In the early stage of tibial fractures, the level of let-7c-5p in the patient’s serum was significantly lower than that of the control group, and as the healing processes progressed, its level gradually increased. In osteogenic induction, let-7c-5p, the activity of ALP, and the levels of osteogenic markers all increase. Increasing the level of let-7c-5p significantly enhanced the expression of osteogenic markers, while inhibiting its expression would weaken this effect. let-7c-5p directly targeted and negatively regulated the expression of cyclin-dependent kinase 8 (CDK8). Overexpression of CDK8 could reverse the osteogenic effect mediated by let-7c-5p.

Conclusion

During the healing process of tibial fractures, let-7c-5p promotes osteogenic differentiation by inhibiting CDK8, thereby accelerating fracture healing.

Background

Fracture is a common type of injury, and such injuries often result in functional impairment of the limbs, imposing a significant burden on society and the economy [1–3]. tibial fractures present unique anatomical features, which poses numerous challenges in clinical treatment [4, 5]. The current treatment methods mainly include surgical fixation, drug therapy and physical therapy [6–9]. Although these approaches offer various options for fractures, there are still some patients still experience delayed healing or non-healing. Furthermore, the early assessment of clinical parameters is of great significance for predicting the outcome of healing [10]. Therefore, exploring the regulatory mechanisms and potential targets related to fracture healing remains of utmost significance.

MicroRNAs (miRNAs) are crucial regulatory factors that can precisely regulate bone formation, bone resorption, and inflammatory responses, and play an important role in bone-related diseases [11–14]. miRNAs participate in fracture healing by regulating osteogenic differentiation, angiogenesis, and the remodeling of extracellular matrix [15–18]. RNA sequencing studies revealed that miR-let-7c-5p is significantly downregulated in fracture patients, indicating that it may play a crucial role in bone injury repair [19]. Furthermore, the research has found that let-7c-5p seems to regulate bone regeneration [20, 21]. Additionally, let-7c-5p alleviates inflammatory responses in osteoarthritis by inhibiting the secretion of pro-inflammatory cytokines in synovial fibroblasts [22]. These studies suggest that let-7c-5p may be involved in the process of fracture healing.

Cyclin-dependent kinase 8 (CDK8) is a member of the cyclin-dependent kinase family. It plays a role in various diseases including cancer, neurodevelopmental disorders, and inflammatory conditions [23–27]. In bone-related diseases, CDK8 promotes the generation of osteoclasts through the STAT1-RANKL axis and accelerates the process of bone resorption [28]. Meanwhile, CDK8 directly inhibits osteogenic differentiation, thereby preventing the formation of the bone cortex [29]. During the healing process of ischemic fractures, CDK8 inhibits the differentiation of mesenchymal cells, promotes fibrosis, hinders the formation of cartilage and the deposition of minerals [30].

Based on this research background, we designed this study to explore how let-7c-5p modulates osteogenic differentiation during fracture and to analyze its targeted relationship with CDK8. Our goal is to identify the therapeutic targets that can facilitate the treatment of fractures.

Materials and methods

Clinical sample collection

This study included 80 patients with tibial fractures who were treated at Zhangzhou Zhengxing Hospital from January 2022 to June 2023, as well as 83 healthy individuals. The inclusion criteria for the tibial fracture were: (1) individuals aged 18 years and above with mature bone development; (2) diagnosed with tibial fractures through imaging examinations; (3) was admitted to the hospital within 3 days after the injury. Exclusion criteria: (1) patients with a history of fractures or orthopedic surgeries; (2) severe organ dysfunction or malignant tumors; (3) systemic autoimmune diseases; (4) having diseases that affect bone metabolism (such as osteoporosis, abnormal parathyroid function). The healthy individuals had no major organic or metabolic diseases.

All the enrolled subjects had their venous blood samples collected in a fasting state. Samples were collected on the 3rd day after the injury and on the 7th, 14th, 21st, and 28th days after receiving standardized fixation surgery. After leaving the blood sample at room temperature and allowing it to coagulate, the upper layer of serum was separated. Subsequently, the serum was aliquoted and stored in ultra-low temperature refrigerator.

This study strictly adhered to the ethical guidelines stipulated in ‌the Declaration of Helsinki‌ and has been approved by the ethics committee of Zhangzhou Zhengxing Hospital(approval number: 2022026) Research Ethics Committee. All participants signed written informed consent forms.

Cell culture

The mouse osteogenic precursor cells (MC3T3-E1) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Cat# SCSP-5218). The cells were routinely cultured in α-MEM basal medium (Cat# 11900-024, Gibco, USA) containing 10% FBS (Cat# A5669201, Gibco, USA) and 1% penicillin-streptomycin (Cat# P1400, Solarbio, China). The cells were maintained in a humidified incubator at 37 °C with 5% CO₂. The fresh medium was replaced every 3 days.

Osteogenic induction

MC3T3-E1 were seeded in 6-well plate at a density of 1 × 10⁵ cells/well. Once the cells reach 80% confluence, discard the original culture medium and slowly add the osteogenic induction culture medium along the wall of the well [31]. The cells were continuously induced for 14 days, with the osteogenic induction medium being replaced every 3 days. The samples were collected at the beginning of induction (day 0), on the 7th day of induction, and on the 14th day of induction.

RT-qPCR

On the 0th day, the 7th day, and the 14th day of osteogenic induction, 1 × 10⁶ cells were collected from each experimental group to assess the expression levels of RNA. Total RNA was extracted from serum and MC3T3-E1 cells using TRIzol reagent (Cat#15596026, Thermo Fisher Scientific, USA). Reverse transcription of CDK8, Runt-related transcription factor 2 (Runx2), Osteocalcin (Ocn), Bone Sialoprotein (BSP), Sp7 Transcription Factor 7 (Sp7) and Collagen Type I alpha 1 (COLIA1) were performed using the PrimScript RT reagent kit (Cat# RR037, Takara, Japan), while let-7c-5p was reverse transcribed using the Mir-X miRNA first-strand synthesis kit (Cat# 638315, Takara, Japan ). Subsequently, qPCR was conducted using TB Green Premix Ex Taq II (Cat# RR430, Takara, Japan ) and Mir-X miRNA RT-PCR SYBR Kit (Cat# 638314, Takara, Japan ). GAPDH and U6 were used as endogenous controls for mRNA and miRNA. Finally, target gene was quantified using the 2^−ΔΔCt method. We set up 5 biological replicates and 3 technical replicates for RT-qPCR.

Measurement of alkaline phosphatase (ALP) activity

The activity of ALP was detected using the kit (Cat# P0321S, Beyotime, China). After 48 h of transfection, cells from each group were collected and counted using the CountStar cell counter (Countstar, China). Then, 1 × 10⁵ cells were taken from each group and lysed with 1% Triton X-100 at 4 °C for 30 min. The supernatant was mixed with the p-nitrophenyl phosphate (pNPP) substrate and incubated in the dark at 37 °C for 15 min. The absorbance was measured at 405 nm using a Microplate Reader. The ALP activity was standardized as the absorbance value corresponding to every 1 × 10⁵ cells. We set up 5 biological replicates and 3 technical replicates for this experiment.

Cell transfection

The MC3T3-E1 cells in the logarithmic growth phase were seeded in 24-well plate at a density of 4 × 10⁴ cells per/well. When the cell confluence reached 70–80%, transfection was performed using Lipofectamine 2000 transfection reagent (Cat# 11668019, Thermo Fisher Scientific, USA). The let-7c-5p mimic, let-7c-5p inhibitor, negative controls (mimic NC, inhibitor NC), as well as pcDNA3.1-CDK8 (pc-CDK8) and pcDNA3.1-control (pc-NC) were all synthesized by Gene Pharma Co., Ltd (China).

Dual-luciferase reporter assay

For the dual-luciferase reporter gene experiment, the cells were seeded at a density of 4 × 10⁴ cells per/well in a 24-well plate and cultured for 24 h until the cell confluence reached the optimal transfection state. The 3’UTR fragment of CDK8 containing the binding site and its mutants were directionally cloned into the pmirGLO dual-reporter vector (Cat# E1330, Promega, USA). Wild-type (CDK8-WT) and mutant (CDK8-MUT) recombinant plasmids were constructed. let-7c-5p mimic, mimic NC, let-7c-5p inhibitor, or inhibitor NC were co-transfected with the above vectors into the cells. After 48 h of transfection, the relative luciferase activity was evaluated using the dual-luciferase reporter assay system (Cat# E1960, Promega, USA). We set up 5 biological replicates and 3 technical replicates for this experiment.

RNA immunoprecipitation

First, 1 × 10⁷ cells were resuspended in RIP lysis buffer (Cat#220 − 188, Millipore, USA). Then, the supernatant was incubated with anti-Ago (Cat# TS-10 × 10ML-U, Millipore, USA) or anti-IgG control antibodies (Cat# MABE-253, Millipore, USA) at 4 °C overnight. After washing the complex with the RIP cleaning solution, RNA was extracted from the immunoprecipitated material. RT-qPCR was used to detect the levels of let-7c-5p and CDK8. We set up 5 biological replicates and 3 technical replicates for this experiment.

Western blot

1 × 10⁶ cells were collected from each treatment group, and total proteins were extracted using 100µL RIPA lysis buffer. Protein concentration was determined using the BCA Protein Assay Kit (Cat#23225, Thermo Fisher Scientific, USA). 20 µg of protein were separated by SDS-PAGE gel electrophoresis and then transferred to PVDF membranes by using wet transfer. The membranes were blocked with 5% skim milk for 1 h. Subsequently, the membranes were incubated with anti-CDK8 primary antibody (1:3000, Cat# FNab01563, FineTest, China) and anti-GAPDH antibody (1:5000, Cat# FNab03345, FineTest, China). After washing with TBST, the membranes were incubated with the secondary antibodies (1:2000, Cat# FNSA-0106 FineTest, China) at room temperature for 1 h. Finally, the membranes were developed using ECL chemiluminescent substrate and the band intensity was quantified using ImageJ software. We set up 5 biological replicates and 3 technical replicates for this experiment.

Statistical analysis

Data analysis was conducted using GraphPad Prism and SPSS. All experiments were set up with 5 biological replicates and 3 technical replicates. The results were presented as mean ± standard deviation (Mean ± SD). Differences between two samples were analyzed using a two-tailed Student’s t-test, whereas comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) with Tukey’s post-hoc test. The threshold for statistical significance was P < 0.05.

Results

The clinical baseline characteristics of the participants

The baseline characteristics of the research subjects are detailed in Table 1. Statistical analysis revealed that there were no statistically significant differences in baseline characteristics such as age, BMI, gender, smoking status, and drinking habits between the healthy control group and the tibial fracture group (P > 0.05). This indicates that the two groups of research subjects were comparable.

Table 1.

Comparison of the baseline data of study objects

Parameters	Health (n = 83)	Tibial fracture (n = 80)	P value
Age (year)	43.82 ± 16.45	45.71 ± 12.92	0.416
BMI (kg/m2)	22.29 ± 3.94	22.83 ± 2.63	0.308
Gender (female/male)			0.481
Female	43	37
male	40	43
Smoking			0.603
Yes	48	43
No	35	37
Alcohol intake			0.791
Yes	46	46
No	37	34

BMI, body mass index

The level of let-7c-5p in the serum of patients with tibial fractures

RT-qPCR was used to analyze the level of let-7c-5p in the serum of the subjects. Compared with healthy controls, the expression of let-7c-5p was significantly downregulated in patients with tibial fractures (Fig. 1A). Additionally, during the process of fracture healing, the expression level of let-7c-5p gradually increases (Fig. 1B). These results suggest that let-7c-5p may play an important regulatory role in fracture healing.

Fig. 1.

The level of let-7c-5p in the serum of patients with tibial fractures. A The serum level of let-7c-5p in the subjects. B The level of let-7c-5p in patients with fractures gradually increased during the healing process. Health, n = 83; tibial fractures, n = 80. The comparison between groups was conducted using t-test or one-way ANOVA followed by Tukey’s post-hoc test

The expression pattern of let-7c-5p during osteogenic differentiation

During the process of osteogenic induction, the activity of ALP and the mRNA levels of osteogenic differentiation markers (OCN, BSP, RUNX2, Sp7, and COLIA1) showed time-dependent upregulation (Fig. 2A-C). Meanwhile, the expression trend of let-7c-5p followed a similar trend to the osteogenic differentiation markers and significantly increased with the induction time (Fig. 2D).

Fig. 2.

The expression pattern of let-7c-5p and osteogenic differentiation markers during osteogenic differentiation. A. The activity of ALP. B-C. The mRNA levels of osteogenic differentiation markers (OCN, BSP, RUNX2, Sp7, and COLIA1). D. The expression levels of let-7c-5p increases with the duration of osteogenic differentiation. n = 5. The comparison between groups was conducted using t-test or one-way ANOVA followed by Tukey’s post-hoc test

The effect of let-7c-5p on the differentiation of osteogenic precursor cells

We investigated the effect of let-7c-5p on osteogenic differentiation by regulating its expression in vitro. The experimental results demonstrated that the let-7c-5p mimic significantly increased the expression level of let-7c-5p (Fig. 3A), and markedly elevated the activity of ALP and the expression levels of osteogenic differentiation markers (Fig. 3B-D). Conversely, The let-7c-5p inhibitor significantly inhibited the expression of let-7c-5p (Fig. 3E), and simultaneously reduced the activity of ALP and the expression of osteogenic differentiation markers (Fig. 3F-H).

Fig. 3.

The effect of let-7c-5p on osteogenic differentiation. A The level of let-7c-5p after transfection with let-7c-5p mimic. B The effect of let-7c-5p mimic on the activity of ALP. C-D. The influence of let-7c-5p mimics on the levels of OCN, BSP, RUNX2, Sp7, and COLIA1. E. The level of let-7c-5p after transfection with let-7c-5p inhibitor. F. The effect of let-7c-5p inhibitors on the activity of ALP. G-H. The effect of let-7c-5p inhibitors on the levels of OCN, BSP, RUNX2, Sp7, and COLIA1. n = 5. The comparison between groups was conducted using t-test or one-way ANOVA followed by Tukey’s post-hoc test

CDK8 is the target of let-7c-5p

Through the TargetScan database, it was found that let-7c-5p and CDK8 have conserved binding sites (Fig. 4A). The luciferase reporter gene assay indicated that following transfection with let-7c-5p mimics, luciferase activity was markedly reduced in the CDK8-WT group, while there was no significant change in the activity of the CDK8-MUT group (Fig. 4B). The RIP results showed that compared with the anti-IgG group, the enrichment levels of let-7c-5p and CDK8 in the anti-Ago2 group were significantly increased (Fig. 4C). The protein and mRNA expression of CDK8 during osteogenic induction was detected. The results showed that the expression level of CDK8 decreased over time (Fig. 4D-E). After transfection with let-7c-5p mimics, CDK8 was significantly downregulated; while after transfection with let-7c-5p inhibitors, CDK8 was significantly upregulated (Fig. 4F-G).

Fig. 4.

CDK8 is the target of let-7c-5p. A The binding site predicted by TargetScan. B The dual-luciferase reporter gene assay verified the targeting relationship between CDK8 and let-7c-5p. C The RNA immunoprecipitation assay determined that CDK8 interacts with let-7c-5p. D The protein level of CDK8 during osteogenic induction. E The mRNA level of CDK8 during osteogenic induction. F-G. The influence of overexpression or inhibition of let-7c-5p on the expression level of CDK8. n = 5. The comparison between groups was conducted using t-test or one-way ANOVA followed by Tukey’s post-hoc test

CDK8 reverses the effect of let-7c-5p on osteogenic differentiation

To clarify the influence of the let-7c-5p/CDK8 axis on osteogenic differentiation, we conducted functional restoration verification through co-transfection experiments. The results of RT-qPCR and Western blot results showed that the let-7c-5p mimic significantly inhibited the expression of CDK8 compared to the control group. When the pc-CDK8 overexpression vector was co-transfected with the cells, the expression level of CDK8 significantly increased (Fig. 5A-B). The overexpression of CDK8 successfully counteracted all the osteogenic-promoting effects mediated by the let-7c-5p mimics (Fig. 5C-H).

Fig. 5.

CDK8 reverses the effect of let-7c-5p on osteogenic differentiation. A-B. The effect of co-transfection of let-7c-5p mimics and pc-CDK8 on the levels of CDK8 protein and mRNA. C. The activity of ALP. D. The mRNA level of OCN. E. The mRNA level of BSP. F. The mRNA level of RUNX2. G. The mRNA level of Sp7. H. The mRNA level of COLIA1. n = 5. The comparison between groups was conducted using t-test or one-way ANOVA followed by Tukey’s post-hoc test

Discussion

Tibial fractures not only cause direct bone damage, but also comes with serious complications, such as malunion, osteomyelitis and deep vein thrombosis [32, 33]. These complications can result in long-term limitations in limb function, increase the risk of amputation, and even cause permanent disability. This study first elucidates the crucial role of the let-7c-5p/CDK8 signaling axis in regulating the healing process of tibial fractures.

miRNA plays a crucial role in fracture healing by acting as a molecular switch that targets and regulates genes related to osteogenic differentiation, signaling pathways, and inflammatory responses. For instance, miR-7-5p promotes fracture healing by inhibiting LRP4 [34]. miR-1224-5p acts on ADCY2 to inhibit osteoclast differentiation and promote osteoblast differentiation [35]. We confirmed that the level of let-7c-5p in the serum of patients with tibial fractures was significantly decreased at the early stage of fracture, but gradually increased with healing time. This is consistent with the previous research findings [19]. Additionally, in vitro experiments showed that overexpression of let-7c-5p significantly enhanced the activity of ALP and the expression level of OCN, BSP RUNX2, Sp7, and COLIA1. These results confirmed the direct promoting effect of let-7c-5p on osteogenic differentiation.

The level of let-7c-5p in the serum showed a significant decline at the early stage of fracture and gradually increased during the fracture healing process. This indicates that it may play a crucial regulatory role in the fracture repair process. We speculate that the early decrease in its expression may have important biological significance. In the early stage of fracture, a strong inflammatory response is crucial for eliminating necrotic tissue. And let-7c-5p has been proven to be able to inhibit the production of pro-inflammatory factors [22]. Therefore, this reduction might be aimed at avoiding the premature suppression of the inflammatory response, thereby laying the foundation for the subsequent repair process. As the healing process progresses from the inflammatory stage to the repair stage, let-7c-5p expression increases, and it promotes osteogenic differentiation by targeting the inhibition of CDK8. Therefore, through the expression pattern of first decreasing and then increasing, let-7c-5p is able to coordinate the balance between the inflammatory response and bone tissue regeneration.

CDK8 has been found to exert a negative regulatory effect in bone metabolism and bone repair processes. For instance, CDK8 regulates the transcription process of SASP and subsequently secretes pro-inflammatory factors, thereby exacerbating the symptoms of osteoarthritis [36]. Additionally, CDK8 also exacerbates osteoporosis by inhibiting osteogenic differentiation [37]. Our experiments have shown that CDK8 is a downstream target gene of let-7c-5p, and let-7c-5p can negatively regulate the expression of CDK8. During osteogenic differentiation, the level of CDK8 is downregulated, and the let-7c-5p mimic substance can effectively inhibit CDK8. The functional recovery experiment further demonstrated that the overexpression of CDK8 could reverse the promoting effect of let-7c-5p on osteogenic differentiation. This demonstrates that CDK8 is a key downstream target of let-7c-5p.

In summary, this study systematically elucidated the molecular mechanism by which let-7c-5p promotes fracture healing. This not only confirms the central role of let-7c-5p as a miRNA that promotes healing and reveals its value as a potential therapeutic target. The research results provide a new strategy for precise treatment of fracture healing. It may be possible to improve the treatment effect of tibial fractures by regulating the let-7c-5p-CDK8 network.

Limitations

This study mainly elucidates the role of the let-7c-5p/CDK8 axis in the process of osteogenic differentiation through quantitative experimental data (such as quantitative detection of osteogenic markers and the activity of ALP). However, this study failed to provide some images that could directly reflect the changes in cells, such as the staining images of ALP or the immunofluorescence localization of CDK8 in cells. These are the limitations of this research. In future research, we will integrate various biological techniques (such as immunohistochemistry or immunofluorescence) to conduct a more systematic analysis of the transformation of cell phenotypes during osteogenesis and the distribution of key regulatory factors, thereby further enhancing the integrity of the evidence chain.

Author contributions

W L, JX Z, BC and QL conceived and designed the experiments. X C, LL and HB Y performed the experiments. W L, JX Z, BC, QL, LL and HB Y contributed sample collection and statistical analysis. BC and QL wrote the manuscript. W L, JX Z, X C and HB Y revised it critically for important intellectual content. All authors read and approved the final manuscript.

Funding

No funding was received to assist with the preparation of this work.

Data availability

Corresponding authors may provide data and materials.

Declarations

Ethics approval and consent to participate

The study protocol was approved by The Ethics Committee of Zhangzhou Zhengxing Hospital (approval number: 2022026)and followed the principles outlined in the Declaration of Helsinki. In addition, informed consent has been obtained from the participants involved.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Conflict of interest

The authors declare that they have no competing interests.