MicroRNA-374b-5p suppresses osteosarcoma progression via the PDPK1-mediated AKT pathway

Introduction

Osteosarcoma (OS), a common malignant bone tumour, mainly threatens the health of children and adolescents (1,2). With the improvements of surgical technique and the optimization of chemotherapy, the five-year survival rate of patients with localized OS has increased to 70%. However, the survival rate of OS patients with metastasis or relapse is still below 20%, which has not changed over the past 40 years (3-5). Thus, exploring the potential molecular mechanisms of OS and developing more potent treatment strategies are imperative.

MicroRNAs (miRNAs) are small noncoding RNAs that consist of approximately 19-24 nucleotides (6). The major biological functions of miRNAs are involved in their target messenger RNAs (mRNAs) translation inhibition or degradation that accomplished via binding to the 3′ untranslated regions (3′-UTRs) of these mRNAs (7). Substantial evidence shows that miRNAs function as crucial regulators in various biological processes, including tumour progression (8-10). Recent reports indicate that miR-374b-5p, a 22-nt-long miRNA located on chromosome Xq13.2, is aberrantly expressed in some tumours and functions as a prognostic biomarker or tumour suppressor. For example, miR-374b-5p is poorly expressed in non-small cell lung cancer, but upregulating miR-374b-5p expression can suppress the cancer progression by targeting FOXP1 (11). Another study reported that miR-374b-5p suppresses the proliferation and metastasis of pancreatic cancer (PC) cells through inhibiting KDM5B-induced epithelial-mesenchymal transition and that miR-374b-5p, which is down-regulated in PC, may serve as a novel candidate biomarker for PC prognosis (12). However, the functions of miR-374b-5p in OS progression and its regulatory mechanisms involved remain unclear.

Serine/threonine kinase AKT is a central node of numerous signalling pathways, and hyperactivation of AKT commonly occurs in many human malignancies (13,14). Once phosphorylated, AKT is activated and facilitates the proliferation, invasion, and metastasis but inhibits apoptosis of various tumour cells via phosphorylating multiple downstream substrates (15-18). According to previous studies, aberrant AKT activation is also involved in OS progression and could be targeted for therapy (19,20). In addition, some miRNAs can inhibit AKT phosphorylation through suppressing the positive regulators of AKT in some cancers. For instance, Liu et al. reported that miR-18a-5p exhibited an inhibitory effect on breast cancer progression via targeting HER2 to suppress the PI3K/AKT signalling pathway (21). Moreover, Li et al. reported that the low expression of miR-637 results in an upregulation of HEMGN, thereby activating AKT and accelerating the development of papillary thyroid carcinoma cells (22). Nevertheless, whether miR-374b-5p is involved in regulating the activation of AKT and influencing the progression of OS remains unknown.

In our study, the miR-374b-5p expression was found to be down-regulated in OS, and its overexpression inhibited OS progression both in vitro and in vivo. Mechanistically, miR-374b-5p-overexpressing inhibited the AKT pathway in OS cells. Combined with the bioinformatics analysis and the results of a quantitative real-time polymerase chain reaction (qRT-PCR), PDPK1 was suggested to be a target gene of miR-374b-5p in regulating AKT phosphorylation. Subsequently, a luciferase activity assay confirmed the targeted relationship between miR-374b-5p and PDPK1 in OS cells. Moreover, we found that the PDPK1 expressions were upregulated and that could be suppressed by miR-374b-5p in OS cell lines. Furthermore, the following functional experiments revealed that the antitumour characteristics of PDPK1 knockdown were analogous to those of miR-374b-5p overexpression in OS cells. Specially, PDPK1 overexpression obviously rescued the inhibitory effect of miR-374b-5p on AKT phosphorylation. Collectively, these results demonstrate that targeting the miR-374b-5p/PDPK1/AKT axis might be a valuable molecular strategy for OS therapy. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1827/rc).

Methods

Bioinformatics analysis

The GSE65071 dataset, consisting of 15 normal samples and 20 OS samples, originated from the GEO database (http://www.ncbi.nlm.nih.gov). The miRNA profiling data from the dataset were analysed by GEO2R and visualized utilizing the SangerBox website (http://sangerbox.com) (23). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. A protocol was prepared before the study without registration.

Fluorescence in situ hybridization (FISH) assay

A human OS tissue microarray consisting of 40 tumour samples was provided by Alena Biotechnology Co., Ltd. (Xi’an, China). The tumour samples of the tissue microarray were obtained from 40 different patients with primary OS. The main sites of the tumours were the femur, tibia and humerus, with a small portion located in the scapula and rib. Among the patients, 9 cases are stage T1 (tumours are located within the anatomical compartments) OS and 31 cases are stage T2 (The tumours have broken through the anatomical compartments). The tissue slices were hybridized with miR-374b-5p or PDPK1 probes (Servicebio), and a FISH assay was performed as the previous description (24).

Cell culture

All the cell lines used in this study were obtained from Fuheng Biotechnology Co., Ltd. (Shanghai, China) and were authenticated by short tandem repeat analysis in November 2023. The human osteoblast cell line hFOB1.19 was incubated in a humidified atmosphere containing 5% CO₂ at 33.5 ℃, whereas the OS cell lines were incubated in a standard culture conditions (37 ℃, humidified atmosphere with 5% CO₂). Each cell line was cultured in an appropriate complete medium (DMEM for MG-63 and MNNG/HOS, MEM for 143B, McCoy’s 5A for U-2OS, and DMEM/F12 for hFOB1.19) supplemented with 1% penicillin-streptomycin.

qRT-PCR

Total RNA extraction from different cell lines was carried out using a TRIzol reagent kit following the producer’s protocol. The corresponding microRNAs and mRNAs in the extracted RNAs were reverse transcribed to cDNA, respectively, utilizing a Hairpin-it^TM real-time PCR Kit according to the producer’s protocol (GenePharma; Suzhou, China), and a real-time PCR instrument (QuantStudio 5, Thermo Fisher, Waltham, MA, USA) was used to perform the qRT-PCR. The levels of miRNA and mRNA were normalized to U6 and GAPDH, respectively, then calculated utilizing the 2^−ΔΔct method. The sequences of the primers used in the experiment are shown in Table S1.

Transfection assay

Small interfering RNA (siRNA) transient transfection was performed utilizing jetPRIME (Polyplus, USA) following the manufacturer’s instructions. Human si-PDPK1-RNAs and its negative control were provided by GenePharma (Suzhou, China). The sequences of the siRNAs used to target PDPK1 are shown in Table S2.

Plasmids containing miR-374b-5p or PDPK1 and their negative controls were obtained from GenePharma (Suzhou, China). We used HEK293T cells to package these plasmids into lentivirus particles, and the viral titres were measured. For constructing stable miR-374b-5p-overexpressing or/and PDPK1-overexpressing OS cell lines, target cells were seeded into each well of a 6-well plate and infected with 1×10⁸ lentivirus transducing units and 6 µg/mL polybrene (Sigma, Shanghai). Approximately 72 h later, the infected cells were screened with 2.5 µg/mL puromycin, and the overexpression efficacies were assessed by qRT-PCR and/or western blotting.

Western blotting

With RIPA lysis buffer (Beyotime, Shanghai, China), total proteins were extracted from different target cells, and the concentrations of these proteins were measured with a BCA protein quantitation kit (Beyotime, China) following the producer’s protocol. Subsequently, equal amounts of total protein from each cell line were separated utilizing SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes. After being blocked by 5% skimmed milk for 1 h, these membranes were incubated at 4 ℃ overnight with the following primary antibodies: anti-AKT (1:1,000, CST, MA, USA), anti-p-AKT (1:1,000, CST), anti-β-catenin (1:1,000, Proteintech, Wuhan, China), anti-p-β-catenin (1:1,000, Proteintech), anti-p65 (1:1,000, CST) anti-p-p65 (1:1,000, CST), anti-YAP (1:1,000, CST), anti-p-YAP (1:1,000, CST), anti-PDPK1 (1:1,000, Abcam, Cambridge, UK) and anti-Actin (1:2,000, Abcam). Following that, the membranes were washed with TBST and incubated with an HRP-conjugated secondary antibody (1:5,000; Beyotime, China) at room temperature for 1 h. Finally, the protein immunoreactive bands were visualized with an ECL detection kit (Share-bio, Shanghai, China).

Cell counting and colony formation assays

The capacity for cell proliferation was evaluated utilizing a Cell Counting Kit-8 (CCK-8) assay (Share-bio) following the manufacturer’s protocol. First, 2×10³ treated cells were seeded into each well of a 96-well plate and cultured in a cell incubator. Then, at 0, 1, 2, 3, 4 and 5 days, the cells to be tested were incubated in CCK-8 working solution at 37 ℃ for 2 h, and the optical density (OD) value at a wavelength of 450 nm was detected using a spectrophotometer (BioTer EPOCH, USA).

A colony formation assay was also applied to assess the capacity for cell proliferation. Briefly, each well of a 6-well plate was seeded with 1,000 treated cells and incubated in the appropriate complete medium for 2 weeks. Then, following 30 minutes of fixation with 4% formaldehyde, the cell colonies were stained with 0.1% crystal violet for 20 minutes before being photographed with an iPhone and counted by ImageJ software.

Migration and invasion assays

For the migration and invasion assays, 300 µL of serum-free medium containing 2.0×10⁴ treated cells was added to each upper Transwell chamber (24-well inserts, 8-µm pore size; Corning), and 500 µL of complete medium was added to the the lower chamber. For the invasion assay, the membranes in the Transwell chambers were precoated with diluted Matrigel (BD Biosciences, Bedford, MA). Following incubation for 24 h (for the migration assay) or 48 h (for the invasion assay), the cells on the membranes were fixed with 4% formaldehyde and stained with 0.1% crystal violet. Finally, the cells that migrated or invaded across the membranes were photographed utilizing an inverted microscope (Olympus) and counted via ImageJ software.

Flow cytometry

Apoptosis was assessed utilizing a 488-Annexin V and PI Apoptosis Kit (Share-bio) following the producer’s protocol. Briefly, following incubation for 48 h, the transfected cells were collected, and approximately 1×10⁶ cells per sample were resuspended in 500 µL of binding buffer with 488-Annexin V and PI. The cells were subsequently incubated at room temperature in the dark for 15 minutes, and then the apoptotic cells in each sample were counted utilizing a BD Biosciences flow cytometer and analysed using a FlowJo software.

Luciferase activity assays

Possible miR-374b-5p binding sites in the sequence of PDPK1 3′-UTR were predicted by TargetScan (version 8.0; www.targetscan.org). Wild-type (WT) PDPK1 3′-UTR and mutant (MUT) PDPK1 3′-UTR were synthesized and inserted into the pmirGLO vector to generate the corresponding luciferase reporter plasmids. The plasmids were subsequently transfected into the miR-374b-5p-overexpressing or the negative control OS cells by jetPRIME (Polyplus, USA). Following transfection for 48 h, each group cells were collected and the luciferase activities were detected utilizing a dual-luciferase reporter assay system (Promega).

Mouse xenograft model

All animal experiments were performed under a project license (No. IACUC25-0347) granted by the Laboratory Animals Ethics Committee of Nanjing Medical University, in compliance with national guidelines for the care and use of animals. All the BALB/C nude mice used in the experiments were obtained from the Animal Center of Nanjing Medical University at the age of 4 weeks. A total of fifteen nude mice were randomly divided into three groups and fed in a specific pathogen-free animal laboratory. At the age of 5 weeks, each mouse was subcutaneously injected with 1.5×10⁶ treated OS cells. Subsequently, every 5 days, each tumour size was measured with a calliper and the volume was calculated following the formula: length × width² × 0.5. On Day 20, all the mice were euthanized and their tumours were isolated. Finally, the tumours were photographed and fixed with 4% formaldehyde for further histological assay.

Immunofluorescence (IF) staining

The IF assay was carried out as the previous description (25). Cell viability in xenograft tumour tissues was detected by IF staining with a specific antibody against Ki-67 (Servicebio) at a 1:200 dilution. Then a fluorescence microscope (Carl Zeiss) was used to obtain images.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay

Apoptosis in the xenograft tumour tissues was assessed using a TUNEL kit (Servicebio), and the TUNEL assay was carried out in accordance with the previous description (26).

Database analysis

Potential target genes of miR-374b-5p were predicted by StarBase (http://starbase.sysu.edu.cn) and TargetScan (https://www.targetscan.org). The data about the AKT pathway related genes and the data from the above two databases were subsequently applied to construct a Venn diagram utilizing an online website (https://bioinformatics.psb.ugent.be/webtools/Venn/).

Statistical analysis

Statistical analysis were carried out via GraphPad Prism (version 9.5.1 for Windows) and SPSS 26.0 (IBM, USA) software. Comparisons between two groups were conducted utilizing two-tailed Student’s t tests, and comparisons among multiple groups were performed utilizing One-Way ANOVA test (GraphPad Prism). The correlation between the expression of miR-374b-5p and PDPK1 in the human OS tissue microarray was analysed using the chi-square test (SPSS 26.0). All the statistical results are presented as the mean ± standard deviation (SD). A value of P<0.05 was regarded as statistically significant. All the experiments were repeated at least three times.

Results

MiR-374b-5p is markedly down-regulated in OS

The data on the miRNA expression profiles from the GSE65071 dataset were analysed using GEO2R and downloaded from the GEO database. The differentially expressed miRNAs between normal and OS samples in the data were visualized with a volcano plot and heatmap (Figure 1A,1B) using the SangerBox website (logFC > 1, adjusted P value <0.05). The top 30 upregulated and downregulated miRNAs in the GSE65071 dataset were presented in Table S3. Relative to that in normal samples, the expressions of miR-374b-5p were markedly down-regulated in OS samples (Figure 1C). For further confirming the above result, the miR-374b-5p expression levels in an OS tissue microarray (n=40) were detected via FISH with a specific miR-374b-5p probe (Figure 1D). The results revealed that the miR-374b-5p levels in OS tissues were negatively correlated with OS pathological stage (Figure 1E). Additionally, the miR-374b-5p levels in hFOB1.19 cell line and OS cell lines (U-2OS, 143B, MNNG-HOS, and MG63) were detected by qRT-PCR. As displayed in Figure 1F, relative to that in hFOB1.19 cell line, the miR-374b-5p expressions in OS cell lines were markedly down-regulated, especially in 143B and U-2OS cell lines. Collectively, these findings confirmed the down-regulation of miR-374b-5p in OS tissues and cells, which may be associated with OS progression.

Figure 1 MiR-374b-5p was downregulated in osteosarcoma tissues and cells. (A) Volcano plot showing the differential expression of miRNAs in GEO dataset (GSE65071). (B) Heatmap illustrating the top 30 upregulated and downregulated miRNAs in the GSE65071 dataset acquired from the GEO database. (C) The expression levels of miR-374b-5p in the samples of normal and OS from the GSE65071 dataset. (D) Representative FISH photographs of miR-374b-5p expression patterns in human OS tissues. Scale bars =50 μm. (E) Statistical analysis of FISH results based on the level of miR-374b-5p expression in T1 (n=9) and T2 (n=31) stages in OS tissues. (F) Levels of miR-374b-5p in 143B, U-2OS, MG63, and MNNG-HOS relative to that in the hFOB1.19 cells were confirmed via qRT-PCR. Results are exhibited as mean ± standard deviation. *, P<0.05; ***, P<0.001; ****, P<0.0001. FDR, false discovery rate; FISH, fluorescence in situ hybridization; GEO, Gene Expression Omnibus; NS, no significance; OS, osteosarcoma; qRT-PCR, quantitative real-time polymerase chain reaction.

MiR-374b-5p suppresses OS progression in vitro

For exploring the roles of miR-374b-5p in OS progression, 143B and U-2OS cells were applied to the following experiments. Via transfecting miR-374b-5p plasmids into 143B and U-2OS cells, stable miR-374b-5p-overexpressing cell lines were constructed, and the overexpression efficiencies were determined by qRT-PCR (Figure S1A,S1B). As shown in Figure 2A,2B, the CCK-8 assays revealed that the miR-374b-5p-overexpressing OS cells exhibited a poorer proliferation ability than the control OS cells, and the colony formation assays displayed a same result (Figure 2C,2D). Furthermore, miR-374b-5p overexpression markedly attenuated the cell migration (Figure 2E and Figure S1C) and invasion (Figure 2F and Figure S1D) in OS cells, as measured by Transwell assays. However, the flow cytometer results indicated that apoptosis markedly increased in the miR-374b-5p-overexpressing OS cells (Figure 2G,2H). Collectively, these findings confirmed the antitumour roles of miR-374b-5p in regulating OS cell proliferation, apoptosis, migration, and invasion in vitro.

Figure 2 MiR-374b-5p restrains osteosarcoma progression in vitro. (A,B) Overexpression of miR-374b-5p attenuated the proliferation capability of 143B and U-2OS cells assessed by using CCK-8 assay. (C,D) Overexpression of miR-374b-5p suppressed colony formation capacity of OS cells evaluated by using colony formation assay. (E,F) Effects of miR-374b-5p-overexpressing on migration and invasion abilities of OS cells were assessed by migration and invasion assays. Scale bar =100 μm. (G,H) Apoptosis of OS cells induced by miR-374b-5p-overexpressing was detected by flow cytometry. Results are displayed as mean ± standard deviation. ***, P<0.001. CCK-8, Cell Counting Kit-8; FITC, fluorescein isothiocyanate; OS, osteosarcoma; PI, propidium iodide.

MiR-374b-5p targets PDPK1 and suppresses the AKT pathway in OS cells

Numerous studies have indicated that the AKT pathway, β-catenin pathway, NF-κB pathway, and YAP pathway play crucial roles in the progression of various tumours, including OS (27-30). For exploring whether miR-374b-5p exerts its antitumour effects via suppressing these pathways in OS, western blotting was applied to clarify the relationship between miR-374b-5p and the markers of these pathways in OS cells. The results revealed that miR-374b-5p overexpression markedly reduced the degree of phosphorylated AKT (p-AKT), whereas, the protein levels of p-β-catenin, p-p65, p-YAP and all the total protein levels of these biomarkers were not obviously changed (Figure 3A).

Figure 3 MiR-374b-5p suppresses the AKT pathway via direct targeting PDPK1. (A) Protein levels of total and phosphorylated markers of different pathways in the two groups (Vector and miR-374b-5p). (B) Venn diagram exhibiting the AKT-related target genes of miR-374b-5p which were predicted by the two databases (StarBase and TargetScan). (C,D) The mRNA expression levels of miR-374b-5p’s candidate target genes in negative control or miR-374b-5p-overexpressing OS cells. (E) Protein expression levels of PDPK1 in oe-NC and oe-miR-374b-5p groups. (F) The mRNA levels of PDPK1 in 143B, U-2OS, MG63, and MNNG-HOS cell lines relative to that in the hFOB1.19 cells were measured by qRT-PCR. (G) The sequences of PDPK1 3′UTR contains one predicted miR-374b-5p binding site. (H,I) Dual-luciferase reporter assays in OS cells (143B and U-2OS) which were transfected with the luciferase reporter plasmids, containing PDPK1 3′-UTR WT or MUT, in combination with the miR-374b-5p-overexpressing plasmids or the negative control plasmids (Vector). Results are exhibited as mean ± standard deviation. **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, no significance. OS, osteosarcoma; PDPK1, phosphoinositide-dependent protein kinase 1; qRT-PCR, quantitative real-time polymerase chain reaction.

For illustrating the underlying mechanism of miR-374-5p in modulating the AKT pathway in OS progression, StarBase and TargetScan, two target gene prediction website, were applied to predict the target genes of miR-374-5p, respectively. Via constructing a Venn diagram with the above predicted data and AKT pathway-related gene data using a website (https://bioinformatics.psb.ugent.be/webtools/Venn/), twelve candidate mRNAs (EIF4E, LPAR3, CREB1, ATF2, CCNE2, FGF5, PTEN, VEGFC, PDPK1, GNB2, TGFA, and AKT1) were identified to be associated with the AKT pathway (Figure 3B). Next, we detected the mRNA levels of these twelve candidate target genes in miR-374-5p-overexpressing 143B and U-2OS cells by using qRT-PCR. As displayed in Figure 3C,3D, the mRNA expressions of CREB1, CCNE2, FGF5, PDPK1, LPAR3, ATF2, and GNB2 were inhibited at different levels by miR-374-5p overexpression in 143B or U-2OS cells, but only PDPK1 was equally inhibited in both of the OS cell lines, and the inhibition was more significant. Therefore, we speculated that PDPK1 was most likely the target gene of miR-374b-5p. Furthermore, at the protein level, PDPK1 was also suppressed by miR-374-5p overexpression in 143B and U-2OS cells, as detected using western blotting (Figure 3E). Furthermore, the mRNA levels of PDPK1 in hFOB1.19 and OS cell lines (143B, U-2OS, MNNG-HOS and MG63) were detected using qRT-PCR. As presented in Figure 3F, relative to that in hFOB1.19 cells, the mRNA expressions of PDPK1 were markedly upregulated in OS cell lines, especially in 143B and U-2OS cells. We subsequently used the TargetScan website to predict potential sites in the 3′-UTR of PDPK1 for miR-374b-5p targeting, and the results are presented in Figure 3G. Dual-luciferase reporter assays demonstrated that the miR-374b-5p overexpression obviously decreased the luciferase activity of the WT PDPK1 3′ UTR reporter but not the Mut PDPK1 3′ UTR reporter in 143B and U-2OS cells (Figure 3H,3I). Taken together, these results suggest that miR-374b-5p inhibits the AKT pathway via direct targeting of PDPK1 in OS cells.

PDPK1 promotes OS cell progression via activating the AKT pathway

To further explore the potential functions of PDPK1 in OS, a siRNA-mediated gene silencing assay was applied to knock down PDPK1 in 143B and U-2OS cells, and the silencing efficiencies were detected using by qRT-PCR (Figure S2A,S2B) and western blotting (Figure 4A). Notably, western blot analysis demonstrated that PDPK1 knockdown significantly suppressed the activation of the AKT pathway (Figure 4A).

Figure 4 PDPK1 knockdown suppresses OS progression and the AKT pathway in vitro. (A) Knockdown efficacies of PDPK1 siRNAs in OS cell lines were confirmed using western blotting, and PDPK1-silencing decreased p-AKT but not total AKT. (B-D) PDPK1-silencing markedly inhibited OS cell proliferation as assessed by CCK-8 and colony formation assays. (E,F) PDPK1-silencing suppressed OS cell migration and invasion that were evaluated by Transwell assays. Scale bar =100 μm. (G,H) PDPK1 knockdown facilitated the apoptosis of OS cells. Results are exhibited as mean ± standard deviation. ***, P<0.001. CCK-8, Cell Counting Kit-8; CON, control; FITC, fluorescein isothiocyanate; OS, osteosarcoma; PDPK1, phosphoinositide-dependent protein kinase 1; PI, propidium iodide; si, small interfering.

The following CCK-8 assays indicated that the proliferation ability of OS cells was obviously suppressed by PDPK1 knockdown (Figure 4B,4C), and the colony formation assays exhibited a similar result (Figure 4D). In addition, as the Transwell assays demonstrated (Figure 4E,4F and Figure S2C,S2D), PDPK1 silencing obviously suppressed OS cell migration and invasion. However, the flow cytometry results revealed that PDPK1 knockdown markedly promoted the apoptosis of OS cells (Figure 4G,4H). These findings suggest that PDPK1 functions as an oncogene by activating the AKT pathway to regulate OS progression.

MiR-374b-5p inhibits OS progression through the PDPK1-mediated AKT pathway

To further confirm that miR-374b-5p overexpression inhibits OS progression through the PDPK1-mediated AKT pathway, a series of rescue experiments were performed in 143B and U-2OS cells. First, three different cell lines (negative control, miR-374b-5p stable overexpressing, and both miR-374b-5p and PDPK1 stable overexpressing) were constructed with 143B and U-2OS cell lines. As presented in Figure 5A, the protein expressions of PDPK1 in OS cell lines were obviously suppressed by miR-374b-5p overexpression and were partly reversed by PDPK1 overexpression. Similarly, the influence of miR-374b-5p overexpression on p-AKT was partly rescued due to PDPK1 reintroduction, but the total AKT was not changed obviously (Figure 5A). Subsequently, the results of CCK-8 assays indicated that PDPK1 overexpression partly rescued the suppression of proliferation caused by miR-374b-5p overexpression in OS cells (Figure 5B,5C), and the colony formation assays revealed a same result (Figure 5D). Additionally, as revealed by Transwell assays, the abilities of migration and invasion decreased in miR-374b-5p overexpressing OS cells, whereas PDPK1 overexpression partially reversed the inhibitory effects (Figure 5E,5F and Figure S3A,S3B). Furthermore, the results of flow cytometry demonstrated that PDPK1 overexpression partially inhibited the enhancing function of miR-374b-5p overexpression on OS cell apoptosis (Figure 5G,5H). Collectively, these findings further verified that miR-374b-5p acts as a crucial inhibitor in OS progression via regulating the PDPK1-mediated AKT pathway.

Figure 5 PDPK1 overexpression partly rescues the anti-tumour effects of miR-374b-5p on OS cells. (A) Protein levels of PDPK1, AKT and p-AKT in different groups of OS cells (Vecror, miR-374b-5p overexpression, and both miR-374b-5p and PDPK1 overexpression), detected by western blotting, demonstrated that PDPK1 overexpression reversed the inhibition effects of miR-374b-5p on p-AKT expression. (B-D) CCK-8 and colony formation assays showed that the overexpression of PDPK1 reversed the suppression effects of miR-374b-5p overexpression on OS progression in vitro. (E,F) The abilities of OS cell migration and invasion suppressed by miR-374b-5p overexpression were partly reversed after PDPK1 overexpression. (G,H) Flow cytometry assays exhibited that miR-374b-5p-induced OS cell apoptosis was significantly rescued by PDPK1 overexpression. Results are exhibited as mean ± standard deviation. **, P<0.01; ***, P<0.001. FITC, fluorescein isothiocyanate; OS, osteosarcoma; PDPK1, phosphoinositide-dependent protein kinase 1; PI, propidium iodide.

MiR-374b-5p suppresses OS growth through modulating PDPK1 in vivo

For investigating the roles and mechanisms of miR-374b-5p in modulating OS cell proliferation in vivo, we constructed three different cell lines (negative control, miR-374b-5p stable overexpressing, and both miR-374b-5p and PDPK1 stable overexpressing) with 143B cells. Then, the three cell lines were subcutaneously injected into three different groups of nude mice and a xenograft model of OS was established. As presented in Figure 6A,6B and Figure S4A,S4B, relative to those in the control group, the xenograft tumour sizes and volumes in the miR-374b-5p overexpression group were markedly reduced. Nevertheless, the antitumour effect of miR-374b-5p on tumour growth was partially reversed by PDPK1 overexpression. Subsequently, OS cell viability and apoptosis in these xenograft tumours were assessed via Ki-67 staining and a TUNEL assay, respectively. As presented in Figure 6C and Figure S4C,S4D, miR-374b-5p overexpression resulted in an obvious decrease in proliferation and an increase in apoptosis in OS cells in vivo, whereas reintroduction of PDPK1 partially rescued these tumour-suppression effects. Furthermore, the patterns of miR-374b-5p and PDPK1 expression in xenograft tumours and human OS tissue microarray were established via FISH (Figure 6D,6E), and the expression patterns in OS samples from the tissue microarray revealed a negative correlation between miR-374b-5p and PDPK1 (Figure 6F). In summary, these results further verified that miR-374b-5p inhibited OS cell proliferation by targeting PDPK1 in vivo.

Figure 6 MiR-374b-5p suppresses OS growth via PDPK1/AKT axis in vivo. (A) Photograph of the xenograft tumours from three groups of nude mice (negative control, miR-374b-5p overexpression, and both miR-374b-5p and PDPK1 overexpression) (n=5). Scale bar =1 cm. (B) The growth suppression of xenograft tumours due to miR-374b-5p overexpression was partially reversed by PDPK1 overexpression. Tumour volumes were measured every 5 days. (C) OS cell proliferation and apoptosis in each group of xenograft tumour tissues were assessed via Ki-67 staining and a TUNEL assay. Red dots, as the arrows indicated, are TUNEL-positive cells. (D) Representative FISH images displayed miR-374b-5p and PDPK1 expression patterns in OS samples from the xenograft model. Scale bar =50 μm. (E) Representative FISH images displayed miR-374b-5p and PDPK1 expression patterns in the OS tissue microarrays. Scale bars =50 μm. (F) Chi-squared test statistical results showed a negative correlation between miR-374b-5p and PDPK1 expression patterns in the OS tissue microarrays (n=40, r=−0.570, P<0.001). Results are exhibited as mean ± standard deviation. *, P<0.05; **, P<0.01. FISH, fluorescence in situ hybridization; OS, osteosarcoma; PDPK1, phosphoinositide-dependent protein kinase 1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Discussion

As small noncoding RNAs, miRNAs are able to regulate the expression of their target genes posttranscriptionally, and numerous reports have indicated that abnormally expressed miRNAs act as crucial regulators in a variety of human diseases, including OS progression. For example, miR-378a-5p prevents lung adenocarcinoma cell invasion, migration and proliferation by targeting FGR and suppressing its expression (31). Moreover, miR-490-5p inhibits proliferation and metastasis of OS cells by directly regulating the expression of HDAC2 (32). Notably, several previous studies revealed that miR-374b-5p functions as an important regulator for cell proliferation, apoptosis, metastasis, and chemotherapeutic resistance in multiple tumours (33-36). In this study, bioinformatics analysis of a GEO dataset revealed that the levels of miR-374b-5p in OS tissues observably decreased. Consistently, in vitro experiments further confirmed the downregulation of miR-374b-5p in OS tissues and cells. Furthermore, subsequent functional experiments demonstrated that miR-374b-5p restrained OS cell proliferation but stimulated apoptosis in vitro and in vivo and inhibited OS cell migration and invasion in vitro. Briefly, these findings revealed that miR-374b-5p exhibited tumour suppression characteristics in regulating the progression of OS.

AKT, a phosphoprotein, was identified as an oncogene approximately forty years ago (37). As being phosphorylated at Thr308 or Ser473, AKT can phosphorylate and activate more than one hundred downstream protein substrates and, in turn, influences a series of biological functions, involving cell growth, apoptosis, proliferation, and metastasis, in a large number of tumours (37-39). Therefore, as a crucial signalling pivot, AKP phosphorylation is a prerequisite for its biological function to be exerted. For example, Liu et al. studied out that BAIAP2L2 was highyl expressed in human gastric cancer and functioned as a tumour promoter via phosphorylating AKT (40). Bao et al. suggested that elevated MTFR2 expression in hepatocellular carcinoma activated AKT and in turn promoted metabolic reprogramming during HCC progression (41). In our study, we discovered that miR-374b-5p overexpression led to the downregulation of phosphorylated AKT (p-AKT) rather than total AKT in OS cells. According to previous studies, AKT phosphorylation can promote the proliferation, migration, and invasion but suppress the apoptosis of OS cells through phosphorylating multiple downstream substrates (27,42,43). The data of this study suggest that miR-374b-5p restrains OS progression by suppressing the AKT pathway. Additionally, among the three subtypes of AKT (AKT1, AKT2 and AKT3), only AKT1 was predicted as a target gene of miR-374b-5p. However, miR-374b-5p overexpression did not significantly alter the AKT1 mRNA expression in OS cells, as measured by qRT-PCR. Thus, we confirmed that miR-374b-5p did not inhibit the AKT pathway by directly suppressing the phosphorylation of AKT.

PDPK1, also known as PDK1, phosphorylates AKT at the Thr308 site and plays an important role in modulating the progression of several cancers (38,44). For example, Jiang et al. reported that PDK1 interacts with SDC2 to phosphorylate AKT, thereby promoting the development of gastric cancer (45). Yang et al. reported that POU2F2 increased aerobic glycolysis via activating AKT during glioblastoma progression, which was dependent on the intervention with PDPK1 (46). Specifically, a previous study revealed that PDPK1 may be a potential biomarker for OS diagnosis (47). Nevertheless, the roles of PDPK1 in OS progression and its potential molecular mechanism are still need to be further studied. In the present study, as a key upstream regulator of AKT, PDPK1 was predicted to be a target gene of miR-374b-5p through bioinformatics analysis, and a dual-luciferase reporter assay confirmed this interaction. Moreover, as validated by qRT-PCR, the PDPK1 was higher expressed in OS cells relative to that in hFOB1.19 cells, and silencing PDPK1 markedly restrained the proliferation, migration, and invasion but increased the apoptosis of OS cells. Additionally, PDPK1 overexpression rescued the tumour inhibition function of miR-374b-5p in vitro, and these results were further verified in a xenograft model of OS in vivo. Furthermore, upregulating the expression of PDPK1 reversed the inhibitory effect of miR-374b-5p on AKT phosphorylation. In brief, miR-374b-5p suppressed the OS progression via directly targeting PDPK1 to inhibit the AKT pathway.

There are some limitations in our study. Firstly, we only explored the activation changes of four signalling pathways following the miR-374b-5p overexpression in OS cells, whether other pathways are affected by miR-374b-5p still requires further investigation. Secondly, in in vitro and in vivo function rescue experiments, PDPK1 only partially restored the tumour suppressor functions of miR-374b-5p in OS. Therefore, whether miR-374b-5p has other target genes and exerts a synergistic tumour suppressor effect by regulating them still needs further exploration. Furthermore, an animal model of tumour metastasis has not been established due to the urgency of time.

Conclusions

The present study revealed the low expression of miR-374b-5p in OS and the inhibitory role of miR-374b-5p in OS progression both in vitro and in vivo. Mechanistically, miR-374b-5p directly targeted and down-regulated PDPK1, which functions as an oncogene in OS progression, to suppress AKT phosphorylation. These results suggest that the miR-374b-5p/PDPK1/AKT axis might be a potential treatment target for OS.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1827/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1827/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1827/prf

Funding: This study was financially supported by The High-level Talent Project for Health and Wellness in Changzhou City (No. 2024CZLJ004).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1827/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All animal experiments were performed under a project license (No. IACUC25-0347) granted by the Laboratory Animals Ethics Committee of Nanjing Medical University, in compliance with national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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