Abstract
Background
Colorectal cancer (CRC) represents a growing global health burden, particularly in developing countries. Mesenchymal stem cells (MSCs) have been reported to suppress tumor progression partly through the release of exosomes that deliver therapeutic molecules. However, the therapeutic potential of engineered MSC-derived exosomal microRNAs (Exo-miRNAs) in CRC remains largely unexplored.
Methods
Plasma exosome miRNA sequencing was performed to identify dysregulated miRNAs in CRC. Gain- and loss-of-function assays were conducted to evaluate the biological effects of candidate miRNAs in CRC cells. Human mesenchymal stem cells (hMSCs) were engineered to overexpress miR-3614-5p via Lipofectamine-mediated transfection, and exosomes enriched with miR-3614-5p were isolated and characterized. The effects of engineered hMSC-derived exosomal miR-3614-5p (Exo-miR-3614-5p) on CRC progression were assessed both in vitro and in vivo. Mechanistic studies were performed to identify downstream targets and signaling pathways.
Results
miR-3614-5p was identified as a therapeutic miRNA that was significantly downregulated in CRC. Engineered hMSCs efficiently loaded miR-3614-5p into secreted exosomes. Treatment with Exo-miR-3614-5p markedly suppressed CRC cell proliferation in vitro and inhibited tumor growth in vivo. Mechanistically, miR-3614-5p directly bound to the 3′ untranslated region (3′UTR) of IL7Rα, resulting in translational repression and subsequent inactivation of the JAK2/STAT3 signaling pathway. In addition, miR-3614-5p exhibited potential diagnostic value for CRC.
Conclusions
This study demonstrates that engineered hMSC-derived exosomal miR-3614-5p effectively inhibits CRC progression primarily through downregulation of IL7Rα and suppression of the JAK2/STAT3 signaling pathway. These findings suggest that Exo-miR-3614-5p represents a promising therapeutic and diagnostic candidate for CRC.
Graphical Abstract
Supplementary Information
The online version contains supplementary material available at 10.1186/s12967-026-07859-y.
Keywords: Colorectal cancer, Exo-miR-3614-5p, Human mesenchymal stem cells, IL7Rα, JAK2/STAT3
Introduction
Colorectal cancer (CRC) is a global health problem, particularly in developing countries where morbidity and mortality rates continue to rise [1]. Although significant advances have been made in the diagnosis, prevention, surveillance, and intervention of CRC, metastatic CRC presents a poor prognosis with a 5-year survival rate of less than 15% [2]. In the early stages, CRC symptoms are often subtle or absent, leading to frequent misdiagnosis or delayed diagnosis, which is partly due to the current diagnostic methods such as colonoscopy or fecal occult blood testing, that are invasive or have limited sensitivity and specificity. Many CRC patients are diagnosed in the middle-advanced stage or have metastasis [3]. Therefore, there is an urgent need to develop non-invasive and highly sensitive biomarkers for detection and effective therapeutic strategies for CRC.
MicroRNAs (miRNAs) are a class of non-coding RNA molecules approximately 22 nucleotides in length, functioning as gene expression regulators of RNA silencing by binding to the 3’ untranslated regions of mRNAs [4]. Recently, increasing evidence highlighted the significant role of miRNAs in the development and progression of CRC [5]. MiR-3614-5p has been reported as a crucial mediator of several cancers through different cellular mechanisms. For example, miR-3614-5p was found to have therapeutic effects for non-small cell lung cancer by targeting PGAM1 [6], breast cancer by targeting TXNRD1 [7], hepatocellular carcinoma by targeting Yin Yang 1 (YY1) [8]. Furthermore, overexpression of miR-3614-5p inhibited CRC progression through targeting the 3’-untranslated regions of Replication factor C 5 (RFC5) [9], and low level of miR-3614-5p was closely associated with the malignant progression of CRC [10].
Mesenchymal stem cells (MSC) and MSC-derived exosomes (MSC-Exos) have attracted significant attention due to their therapeutic potential. Exosomes are a type of extracellular vesicles (EVs) surrounded by a lipid bilayer with a diameter of 30–150 nm [11]. These EVs encapsulate and deliver functional molecules such as miRNAs and proteins to recipient cells, facilitating cell-cell communication [12]. MSC-Exos demonstrated promising potential as substitutes for MSCs, with low immunogenicity, less toxicity, and easy preparation [13]. However, natural MSC-Exos are often limited by insufficient therapeutic cargos. Therefore, engineering exosomes for efficient loading of therapeutic molecules might provide a potential solution.
In this study, we fabricated miR-3614-5p-engineered exosomes (Exo-miR-3614-5p) for the treatment of CRC. Exo-miR-3614-5p or Exo-miR-NC were isolated from human Mesenchymal Stem Cells (hMSCs) transfected with miR-3614-5p mimics or mimic NC, respectively. We verified the potential therapeutic effect of Exo-miR-3614-5p on CRC cell proliferation, migration, and invasion both in vitro and in vivo. Additionally, we elucidated the regulatory mechanism of miR-3614-5p, which is mainly through inhibiting the IL7R/JAK2/STAT3 signaling pathway in CRC.
Methods
Human CRC specimens and plasma specimens
This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Guangxi Medical University Cancer Hospital (No. LW2024136). Written informed consents were obtained from all participants or their guardians before enrollment. Tumor tissues and adjacent non-cancerous tissues were collected from a total of 65 CRC patients, and 84 plasma samples, including 64 patients diagnosed with CRC based on histopathologic evaluation and 20 healthy individuals, were included in this study. None of the patients received any therapy prior to the biopsy or blood collection.
TCGA MiRNA differential expression and survival analysis
miRNA expression data for CRC were obtained from the TCGA-COAD cohort through the CancerMIRNome database. The dataset was processed using the CancerMIRNome analytical pipeline, in which miRNA expression matrices are pre-normalized and batch effects are minimized through TCGA consortium-level data processing. Differential expression analysis was performed by comparing tumor and normal samples, and miRNAs were considered significantly dysregulated based on an adjusted P value < 0.01 and an absolute log2 (fold change)>1. Survival analysis was conducted using the integrated CancerMIRNome workflow based on the TCGA-COAD cohort. Hazard ratios and corresponding statistical significance were calculated within the platform to assess the prognostic relevance of candidate miRNAs. Volcano plots were generated by plotting log2 fold change against −log10 (P value), with dashed lines indicating the predefined thresholds for statistical significance and fold change.
Cell culture
Human CRC cell lines HCT116 and HT29 were obtained from the Chinese Academy of Sciences (Shanghai, China). The normal intestinal epithelial cell line NCM460 was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The hMSC was purchased from Wuhan Pricella Biotechnology Co., Ltd. All cell lines were authenticated by short tandem repeat (STR) profiling prior to use in the study. HCT116 and HT29 cells were cultured in the DMEM medium (Invitrogen, USA), and NCM460 cells were cultured in the RPMI-1640 medium (Invitrogen, USA), and hMSC was cultured in a serum-free medium (TBD, China). All cells were incubated at 37℃ and 5% CO2.
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from exosomes, cells, and tissues using TRIzol reagent (Invitrogen, USA). GAPDH, U6, and Cel-mir-39 were used as control genes, and the relative expression levels of miRNA and mRNA were determined using the 2−ΔΔCt method. Each assay was repeated at least three times. The primer pairs used for RT-qPCR were as follows: GAPDH: forward: 5’-TCGACAGTCAGCCGCATCTT-3’, reverse: 5’-ACCAAATCCGTTGACTCCGAC-3’; U6: forward: 5’-CTCGCTTCGGCAGCACA-3’, reverse: 5’-AACGCTTCACGAATTTGCGT-3’; miR-3614-5p: forward: 5’-CGCCACTTGGATCTGAAGG-3’, reverse: 5’-AGTGCAGGGTCCGAGGTATT-3’; IL7Rα: forward: 5’-TTTTGACCTGAGTGTCGTCTATCG-3’, reverse: 5’-TCCATTTGTTTTCATCCTTTTCCT-3’; siRNA-IL7R: forward: CAUUCAAGCUAGAGAUGAA, reverse: UUCAUCUCUAGCUUGAAUG.
Western blot
Cells were lysed using RIPA lysis buffer (Beyotime, China). Total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking, the membranes were incubated with the primary antibodies: anti-phosphorylated JAK2 (Abcam, ab32101), anti-JAK2 (ABMART, T55287), anti-phosphorylated STAT3 (Cell Signaling Technology, 9131), anti-STAT3 (Cell Signaling Technology, 9139), anti-IL7Rα (Proteintech, 17626-1-AP), anti-CD63 (Abcam, ab134045), anti-CD9 (Abcam, ab236630, RRID), anti-TSG101 (Abcam, ab133586), anti-GAPDH (Cell Signaling Technology, 2118). anti-β actin (Proteintech, 66009-1-Ig). The next day, the membranes were incubated with a Goat anti-Rabbit lgG (H + L) Secondary Antibody (SA5-35571, Invitrogen) or Goat anti-Mouse lgG (H + L) Secondary Antibody (SA5-35521, Invitrogen). Finally, the signals were detected using the Odyssey CLx Infrared Imaging System.
Dual-luciferase reporter assay
The wild-type or mutant 3’ untranslated regions (UTRs) of the IL7R gene were cloned into the XhoI and NotI sites of the psiCHECK2TM-2 vector (C802A, Promega, USA). In addition, cells were co-transfected with the wild-type/mutant IL7Rα luciferase reporter vector and miR-3614-5p mimics/negative control (NC) using Lipofectamine 3000 reagent (L3000015, Invitrogen, USA). Cells were harvested 24 h after transfection, and luciferase activity was measured using the Duo-Lite Luciferase Assay System (DL101-01, Vazyme, China). Results were expressed as the ratio of firefly luciferase activity to Renilla luciferase activity.
Exosome extraction and identification
Human peripheral blood and hMSCs culture supernatant were collected and centrifuged sequentially at 500 ×g for 10 min, 2000 ×g for 10 min, and 10,000 ×g for 30 min. The supernatant was filtered through a 0.22 μm filter and centrifuged at 100,000 ×g for 120 min to obtain pellets. The pellets were washed twice with PBS and subjected to final centrifugation at 100,000×g for 10 min. The exosome was identified by transmission electron microscopy (TEM) for morphological features. Nanoparticle Tracking Analysis (NAT) for particle size, and Western blot for marker protein expression (TSG101, CD63, and CD9).
Library construction and sequencing for MiRNA
The purity and concentration of RNA samples were determined with NanoDrop ND-1000. Then miRNA sequencing libraries were prepared. After the completed libraries were quantified using the Agilent 2100 Bioanalyzer, the DNA fragments within the libraries were denatured with 0.1 M NaOH to produce single-stranded DNA molecules, which were subsequently captured on Illumina flow cells, amplified in situ, and sequenced for 36 cycles on the Illumina HiSeq 2000 platform. Image analysis and base calling were conducted using the Off-Line Basecaller software (version OLB V1.8.0). Subsequently, the 3’ adapter sequences were removed from the clean reads by passing the Solexa CHASTITY quality filter. The 3’-adapter-trimmed-reads ( > = 15nt) were aligned to the Sanger miRBase 19 using Novoalign (version 2.07.11). To characterize the variability of isomiRs, any sequence that matched the miRNA precursors in the mature miRNA region ± 4 nucleotides (with no more than one mismatch) were accepted as mature miRNA isomiRs and were grouped based on the 5-prime (5p) or 3-prime (3p) arm of the precursor hairpin.
Cell counting kit-8 (CCK) assay
For the CCK-8 assay, 3000 cells (HCT116 and HT29 cells) were seeded per well in a 96-well plate. After culturing for 0, 24, 48, and 72 h, the cells were incubated with CCK-8 reagent (A311-01/02, Vazyme, China) in a 37 °C incubator for 2 h. The absorbance at 450 nm was read using a SpectraMax® iD5 reader.
Scratch assay
HCT116 or HT29 cells (2 × 105/well) were seeded in 6-well plates and allowed to grow to 70–80% confluence. Wounds were made in the cell monolayer using a pipette tip, and the cells were cultured in a medium containing 2% FBS. Wound closure was evaluated at different time points (0, 24, 48, and 72 h) using bright field microscopy.
Transwell assay
For migration and invasion assays, treated CRC cells were placed in the upper chamber of a transwell plate (Corning, NY, USA) with or without Matrigel (Corning, NY, USA). Meanwhile, a medium containing 20% FBS was added to the lower chamber. After incubation for 36 h, the cells that migrated or invaded through the membrane were fixed with 4% paraformaldehyde (Beyotime, China) for 15 min and stained with 0.2% crystal violet (Solarbio, China) for 20 min. The number of cells was counted under an inverted microscope.
Argonaute-2 (Ago2) RNA immunoprecipitation
Argonaute-2 (Ago2) RNA immunoprecipitation was performed using the EZMagna RIP Kit (Millipore, USA). Briefly, cells were lysed with RIP lysis buffer and protein A/G magnetic beads were incubated with Ago2 (ab186733, Abcam, USA). The CRC cell lysates were then incubated with bead-Ago2 complex at 4 °C under rotation overnight, and lgG served as a negative control. Total RNA was harvested for analysis of mRNA and miRNA levels by real-time PCR.
RNA pulldown
The probe was chemically synthesized by Sangon Biotech (Shanghai, China). Cells were lysed using a lysis buffer. Biotin-labeled miR-3614-5p probe or biotin-labeled miRNA-NC probe was then added to the cell lysates. Streptavidin-coated magnetic beads were used to capture the RNA complex, which was analyzed by real-time PCR.
In vivo model
Female BALB/c nude mice (5 weeks old) were obtained from SPF (Beijing) Biotechnology Co., Ltd. and housed under specific pathogen-free conditions in the Experimental Animal Center of Guangxi Medical University. All in vivo procedures complied with national and institutional guidelines and were approved by the Institutional Animal Care and Use Committee of Guangxi Medical University.
For the subcutaneous tumor model, viable HT29 cells (1 × 10^6 cells in 100 µL PBS) were injected subcutaneously into the flank of each mouse. Mice were randomly assigned to two experimental groups (n = 6 per group): Exo-miR-NC and Exo-miR-3614-5p. On days 7, 10, 13, and 16 after tumor cell injection, 100 µg of exosomes (1 µg/µL; Exo-miR-NC or Exo-miR-3614-5p) were administered via tail vein injection. Tumor volumes were measured every 3–4 days using calipers and calculated according to the formula: (width^2 × length) / 2, where width and length represent the shortest and longest tumor diameters, respectively. Mice were sacrificed on day 19 after tumor implantation. Tumors were harvested and subjected to histopathological analysis, RT-PCR, and Western blotting. While blinding and formal power calculations were not performed, all data were collected and analyzed in a standardized manner to minimize experimental bias. These details have been included to ensure transparency and reproducibility of the in vivo experiments.
Immunohistochemical
Paraffin sections were placed in a 65 °C oven for 2 h and then sequentially immersed in deparaffinization solutions I, II, and III, followed by anhydrous ethanol I, II, and III. Sections were rinsed with distilled water and transferred to sodium citrate buffer (pH 6.0). Antigen retrieval was performed by microwaving at medium power for 6 min, cooling for 8 min, and microwaving again at medium–low power for 5 min. Sections were incubated in 3% H₂O₂ at room temperature for 30 min and then blocked with 3% BSA to reduce nonspecific binding. Primary antibodies were applied at 4 °C overnight. The next day, sections were incubated with the secondary antibody at room temperature for 1 h. After PBS washing, DAB substrate was added until brownish-yellow staining developed, followed by rinsing with tap water. Sections were counterstained with hematoxylin for 3 min, differentiated, and blued for 1 min before rinsing with running water. Dehydration was performed through 75%, 85%, and 100% ethanol, followed by clearing in xylene. Finally, sections were air-dried slightly and mounted with mounting medium. Primary antibodies included anti-phosphorylated JAK2 (Abcam, ab32101), anti-JAK2 (ABMART, T55287), anti-phosphorylated STAT3 (Cell Signaling Technology, 9131), anti-IL7Rα (Invitrogen, MA5-50739), and anti-Ki-67 (Servicebio, GB121141).
Statistical analysis
Statistical analyses were performed using GraphPad Prism. Unless otherwise noted, data are presented as mean ± standard error of the mean (SEM). An unpaired two-tailed Student’s t-test was used to compare the means of two groups. For comparisons among multiple groups, if ANOVA results rejected the null hypothesis, Tukey’s multiple comparisons test was conducted to assess differences among group means. Categorical variables for clinical samples were analyzed using Fisher’s exact test. A two-sided P value < 0.05 was considered statistically significant (p < 0.05; p < 0.01; p < 0.001; p < 0.0001). Given the limited sample size, these analyses are considered exploratory, and further validation in independent cohorts is warranted.
Results
miR-3614-5p is decreased in CRC plasma exosomes, tissues, and cell lines
To identify differentially expressed miRNAs in plasma exosomes between healthy controls and CRC patients, miRNA sequencing (miRNA-seq) was conducted. Exosomes were extracted from the plasma of primary CRC patients (n = 6) and healthy controls (n = 3), as illustrated in the flowchart in Fig. 1A. Characterization of the isolated exosomes was performed using Transmission Electron Microscopy (TEM), Nanosight tracking analysis (NTA), and Western blot (Fig. S1A-1 C). RNA-seq analysis of exosomal miRNAs revealed 38 differentially expressed miRNAs between CRC patients and healthy controls. Among these, 5 miRNAs were up-regulated and 33 were down-regulated in CRC patients (fold-change > 1, p-value < 0.05) (Fig. 1B and 1C). Additionally, the miRNA differential expression profile of CRC tissues was obtained from the Cancer Genome Atlas (TCGA) database (Fig. 1D). Candidate exosomal miRNAs were identified by intersecting three groups: differentially expressed miRNAs in exosomes, miRNAs in tissues, and miRNAs with hazard ratio (HR) analysis from the TCGA database. Only miR-3614-5p was identified between CRC patients and controls (Fig. 1E), we used it for further study. Analyzing 64 blood samples from CRC patients and 20 blood samples from healthy controls confirmed that CRC patients had significantly lower expression of plasma miR-3614-5p and plasma exo-miR-3614-5p (Fig. 1F). Receiver operating characteristic (ROC) curve analysis revealed that miR-3614-5p exhibited favorable diagnostic performance for CRC in both plasma and exosomes (Fig. S1D). Stratification based on the optimal cut-off value further showed that CRC patients were significantly enriched in the low miR-3614-5p expression group compared with healthy controls, as confirmed by Fisher’s exact test (Fig. S1E). Further, a total of 65 pairs of primary tumor tissues and corresponding adjacent tissues were collected. Consistently, miR-3614-5p expression was lower in cancer tissues compared to adjacent tissues (Fig. 1G). Multivariable analysis revealed that decreased miR-3614-5p expression was significantly associated with distant metastasis (p = 0.004329), clinical stage (p = 0.01762), and tumor size (p = 0.04459) (Table 1). Consistent with these findings, the CRC cell lines HCT116 and HT29 exhibited lower levels of miR-3614-5p compared to the normal epithelial colon cell line NCM460 (Fig. 1H). Data from the TCGA database also showed that miR-3614-5p levels were lower in colorectal adenocarcinoma (COAD) compared to normal tissues, and low miR-3614-5p expression was associated with poor overall survival in CRC patients (Fig. 1I and J). These findings suggested that miR-3614-5p may act as a therapeutic miRNA for CRC.
Fig. 1.
miR-3614-5p is decreased in CRC plasma exosomes, tissues, and cell lines. (A) The flowchart of identifying differentially expressed miRNAs in plasma exosomes between the healthy control and CRC patients. (B and C) The volcano plots a heatmap showing the differentially expressed exosomal miRNAs between CRC patients and healthy controls. (D) The differentially expressed miRNAs in CRC tissues from TCGA databases. (E) Venn diagram showed the overlapping miRNAs among miRNA-seq of exosomes, differentially expressed miRNAs in CRC tissues from the TCGA database, and prognostic factor from the TCGA database. (F) RT-qPCR was performed to detect the expression of miR-3614-5p in the plasm or plasm exosomes of CRC patients and healthy controls. (G and H) RT-qPCR was performed to detect the expression of miR-3614-5p in CRC tissues or CRC cell lines and adjacent tissues or normal cell line. (I) The TCGA database shows the expression profile of miR-3614-5p in colorectal adenocarcinoma (COAD). (J) The Kaplan-Meier survival curve showed a correlation between miR-3614-5p expression and overall survival in CRC patients
Table 1.
Correlation between miR-3614-5p expression and clinicopathological parameters of CRC
| Clinicopathological parameters | N = 65 | 3614 expression | P value | |
|---|---|---|---|---|
| Low expression | High expression | |||
| n = 32 | n = 33 | |||
| Gender | ||||
| Male | 37 | 20 | 17 | 0.3712 |
| Female | 28 | 12 | 16 | |
| Age | ||||
| <60 | 29 | 11 | 18 | 0.1019 |
| ≥ 60 | 36 | 21 | 15 | |
| Lymph node metastasis | ||||
| Absent | 34 | 13 | 21 | 0.06331 |
| Present | 31 | 19 | 12 | |
| Distant metastasis | ||||
| Absent | 52 | 21 | 31 | 0.004329 |
| Present | 13 | 11 | 2 | |
| Clinical stage | ||||
| I+II | 30 | 10 | 20 | 0.01762 |
| III+IV | 35 | 22 | 13 | |
| T stage | ||||
| T1-2 | 17 | 6 | 11 | 0.1811 |
| T3-4 | 48 | 26 | 22 | |
| Nerve Invasion | ||||
| Absent | 38 | 17 | 21 | 0.6858 |
| Present | 24 | 12 | 12 | |
| Tumor size | ||||
| ≤ 5 cm | 22 | 7 | 15 | 0.04459 |
| > 5 cm | 43 | 25 | 18 | |
| CEA level | ||||
| < 5 µg/mL | 43 | 20 | 23 | 0.5398 |
| ≥ 5 µg/mL | 22 | 12 | 10 | |
miR-3614-5p suppresses proliferation, migration, and invasion properties of CRC cells in vitro
We conducted gain- and loss-of-function experiments to investigate the potential role of miR-3614-5p in CRC. Using miR-3614-5p mimics, we upregulated miR-3614-5p expression in both HCT116 and HT29 cells, as confirmed by RT-qPCR (S2A). CCK-8, wound healing and transwell assays were employed to assess the effect of miR-3614-5p on the malignant characteristics of CRC cells. The results demonstrated that upregulation of miR-3614-5p inhibited CRC cell proliferation, invasion, and migration (S2B-S2D). Although miR-3614-5p exhibits low expression levels in both HT29 and HCT116 cells, miR-3614-5p inhibitors were used to decrease miR-3614-5p expression in HCT116 and HT29 cells by more than 50%, as verified by RT-qPCR (S2E). The knockdown of miR-3614-5p resulted in enhanced CRC cell proliferation, invasion, and migration (S2F-S2H). These findings indicate that miR-3614-5p functions as a tumor suppressor gene, suppressing the proliferation, invasion, and migration of CRC cells in vitro.
Exo-miR-3614-5p from hMSC inhibits CRC cell proliferation, migration and invasion
hMSC was identified by flow cytometry using a group of specific antibodies, positive for CD90 (99.3%) and CD73 (98.8%) and negative for CD45 (0.5%), CD14 (1.7%) and CD34 (1.3%) (S3). Firstly, we assessed the uptake of hMSC-derived exosomes by CRC cells. hMSCs were transfected with an mCherry (red)-labeled lysosomal-associated membrane protein 3 (CD63, an exosome membrane protein) plasmid and then co-cultured with HCT116 cells for 12, 24, and 48 h (Fig. 2A). Confocal Microscopy revealed a time-dependent increase in the number of labeled exosomes within HCT116 cells (Fig. 2B). Next, HCT116 and HT29 cells were co-cultured with hMSCs transfected with either miR-3614-5p mimics or mimic NC. CCK8, wound healing, and transwell assays demonstrated that HCT116 and HT29 cells co-cultured with miR-3614-5p mimic-transfected hMSCs exhibited reduced proliferation, migration, and invasion compared to the control group. Notably, the inhibitory effects of co-culture with hMSCs on CRC cell proliferation, migration, and invasion were significantly diminished by GW4869 (10 µM), an inhibitor of exosome biogenesis (Fig. 2C, 2D and 2E).
Fig. 2.
The inhibitory effects of hMSCs co-culture on CRC cell proliferation, migration, and invasion. (A) Schematic diagram of hMSC and CRC cells co-culture. (B) Representative fluorescence microscopy images showing the time-dependent uptake of hMSC-derived exosomes by HCT116 cells, Scale bars = 20 μm. (C, D and E) CCK8, wound healing, and transwell assays were used to detect the effects of blocking exosome release on the proliferation, migration, and invasion of HCT116 cells induced by co-culture with hMSCs. Scale bars = 500 μm (D) and 100 μm (E)
Subsequently, Exo-miR-3614-5p and Exo-miR-NC were isolated from the engineered hMSCs transfected with either miR-3614-5p mimics or mimic NC (Fig. 3A). TEM, NTA and Western blot analyses were conducted to characterize the morphology, size distribution, and biomarkers of Exo-miR-3614-5p and Exo-miR-NC, respectively. TEM and NTA showed a similar size or mean diameter among Exo-miR-3614-5p and Exo-miR-NC (Fig. 3B and C). Exosome markers including TSG101, CD36, and CD9, were highly expressed in both Exo-miR-3614-5p and Exo-miR-NC (Fig. 3D). RT-qPCR verified the higher miR-3614-5p level in hMSCs transfected with miR-3614-5p mimics and corresponding exosomes (Fig. 3E and F). CCK8, wound healing, and transwell assays demonstrated that Exo-miR-3614-5p significantly suppressed CRC cell proliferation, migration, and invasion relative to the control group (Fig. 3G, 3H and 3I).
Fig. 3.
hMSC-derived Exo-miR-3614-5p and its inhibitory effect in CRC. (A) Schematic diagram of hMSC derived exosomes for CRC treatment. (B-D) Exosomes were identified by Transmission Electron Microscopy (TEM), Scale bars = 100 μm, Nanosight tracking analysis (NTA), and Western blot. (E and F) qRT-PCR detects the expression of miR-3614-5p in exosomes derived from hMSCs transfected with miR-3614-5p mimics or mimic NC. (G-I) CCK8, wound healing, and transwell assays were used to evaluate the effects of hMSC derived exosomal miR-3614-5p on CRC cell proliferation, migration, and invasion. Scale bars = 500 μm (H) and 100 μm (I)
IL7Rα is identified as a target gene of miR-3614-5p in CRC
To investigate the molecular mechanisms underlying the tumor-suppressive role of miR-3614-5p in CRC, HCT116 cells were transfected with miR-3614-5p mimics or mimic NC and subjected to RNA sequencing analysis. Differential expression analysis revealed that, compared with the control group, 31 genes were upregulated and 96 genes were downregulated in miR-3614-5p–overexpressing cells (fold change > 1 or < -1, p < 0.05) (Fig. 4A). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that these differentially expressed genes were primarily enriched in the JAK/STAT signaling pathway, TNFα signaling via NF-κB, and inflammatory response pathways (Fig. 4B). To identify direct targets of miR-3614-5p relevant to these pathways, we integrated the downregulated genes from RNA-seq with miRNA target predictions from the miRWalk database and pathway enrichment results. Through this integrative analysis, Interleukin-7 receptor alpha (IL7Rα) emerged as the only overlapping candidate target (Fig. 4C). The direct interaction between miR-3614-5p and IL7Rα mRNA was further validated by RNA pull-down assays in both HCT116 and HT29 cells (Fig. 4D and E). In addition, Argonaute 2 (AGO2) RNA immunoprecipitation (RIP) assays confirmed the enrichment of IL7Rα mRNA in AGO2-containing RNA-induced silencing complexes following miR-3614-5p overexpression (Fig. 4F and G). Bioinformatic analysis identified two putative miR-3614-5p binding sites within the 3′ untranslated region (3′UTR) of IL7Rα. Accordingly, wild-type and mutant IL7Rα 3′UTR luciferase reporter constructs were generated (Fig. 4H). Dual-luciferase reporter assays demonstrated that miR-3614-5p significantly suppressed the luciferase activity of the wild-type IL7Rα 3′UTR, but not the mutant construct, in both 293T and HCT116 cells (Fig. 4I). Consistent with these findings, miR-3614-5p overexpression markedly reduced IL7Rα expression at both the mRNA and protein levels, as confirmed by RT-qPCR, Western blotting, and flow cytometry analyses in HCT116 and HT29 cells (Fig. 4J, 4K, 4L, and 4M). Notably, miR-3614-5p overexpression also led to a reduction in CD132 (the common γ chain of the IL-7 receptor complex) (Fig. 4K), suggesting that miR-3614-5p may additionally modulate components of the IL-7 receptor signaling complex. Given the KEGG enrichment of TNFα/NF-κB–related pathways, we further examined representative inflammatory signaling molecules. In HCT116 cells, miR-3614-5p overexpression increased p-p65 levels, had no significant effect on TNF-α expression, and reduced IL-6 expression. In contrast, in HT29 cells, miR-3614-5p overexpression decreased p-p65 levels, increased TNF-α expression, and similarly reduced IL-6 expression (Fig. S4A). These results indicate that miR-3614-5p may modulate components of the NF-κB/TNF signaling pathway in a cell line–dependent manner.
Fig. 4.
IL7Rα as a target gene of miR-3614-5p in CRC. (A) The volcano plot showed that there were 127 differentially expressed genes including 31 upregulated and 96 downregulated genes. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed miR-3614-5p down-regulated genes enriched in JAK/STAT3, TNFα, and inflammatory response pathways. (C) Venn diagram showed the overlapping genes among RNA-seq data, miR-3614-5p target genes, and genes enriched in JAK/STAT3, TNFα, and inflammatory response pathways. (D) Schematic diagram of the RNA pull-down assay. (E) The RNA pulldown results showed the enrichment of miR-3614-5p and IL7Rα mRNA by the miR-3614-5p probe. (F) Schematic diagram of the Ago2 RIP assay. (G) The Ago2-RIP results showed the effect the enrichment of IL7Rα mRNA in Ago2-containing miRNPs. (H) Schematic diagram of the binding sites between IL7R mRNA and miR-3614-5p. (I) Dual-luciferase reporter assay was performed to evaluate the effect of miR-3614-5p mimics or inhibitors transfection on the luciferase activity of IL7Rα wild type (Wt) and IL7Rα mutant type (Mut) in 293T and HCT116 cells. (J) RT-qPCR detected the effects of upregulation of miR-3614-5p expression on IL7Rα expression in HCT116 and HT29 cells. (K) Western blot analysis showing reduced IL7Rα and CD132 protein levels following miR-3614-5p overexpression. (L-M) Representative flow cytometry plots (L) and quantification (M) of IL7Rα-positive and IL7Rα-negative cells in HCT116 and HT29 cells, showing decreased IL7Rα expression after miR-3614-5p overexpression.*p < 0.05
IL7Rα is involved in miR-3614-5p-mediated inhibition of CRC cell proliferation, migration, and invasion in vitro
IL7R is a heterodimer consisting of IL7Rα (CD127) and the CD132. Binding of IL7R and Interleukin-7 (IL-7) causes activation of the downstream signaling pathways to promote tumor progression in breast cancer, lung cancer, and prostate cancer [14–16]. IL-7 exhibited a significant increase in CRC patients compared to controls, and this elevation in CRC patients correlates with poor prognosis [17]. However, the functions of IL7Rα in CRC remain unclear. The efficacy of small interfering RNA (siRNA)-mediated IL7Rα knockdown was validated using qPCR and Western blot (Fig. S4B). CCK8, wound-healing, and transwell assay experiments showed that the knockdown of IL7Rα mitigated cell proliferation, migration, and invasion capability in CRC (Fig. 5A, 5B and 5C). Rescue assays revealed that IL7Rα knockdown reversed the promoting effect of miR-3614-5p inhibition on proliferation, migration, and invasion (Fig. 5D, 5E and 5F). We transfected CRC cells with a plasmid encoding IL7Rα, verified by qPCR and Western blot (Fig. S4C). As expected, overexpression of IL7Rα reversed the decreased proliferation, migration, and invasion induced by miR-3614-5p mimics (Fig. 5G and5H).These results suggested that IL7Rα was involved in the effect of miR-3614-5p-induced CRC cell proliferation, migration, and invasion.
Fig. 5.
IL7Rα is involved in miR-3614-5p-mediated inhibition of CRC progression in vitro. (A-C) CCK8, wound healing, and transwell assays were used to detect the effects of siRNA-mediated knockdown of IL7Rα on the proliferation, migration, and invasion of CRC cells, respectively. Scale bars = 500 μm (B) and 100 μm (C). (D-F) CCK8, wound healing, and transwell assays were performed to evaluate the effect of siRNA-IL7Rα on the increased proliferation, migration, and invasion of CRC cells induced by miR-3614-5p inhibitors. Scale bars = 500 μm (E) and 100 μm (F). (G-H) CCK8 and transwell assays were performed to detect the effect of IL7Rα upregulation on the decreased proliferation, migration, and invasion of CRC cells induced by miR-3614-5p mimics. Scale bars = 100 μm (H). *p < 0.05
JAK2/STAT3 pathway acts as an IL7R downstream signaling pathway involved in miR-3614-5p mediated CRC suppression
IL7Rα forms a functional receptor complex with IL-7 that predominantly activates the JAK/STAT signaling pathway [14, 15]. To determine whether this pathway mediates the effects of miR-3614-5p/IL7Rα signaling in CRC, we examined the phosphorylation status of JAK2 and STAT3. Overexpression of miR-3614-5p markedly reduced p-JAK2 and p-STAT3 levels in IL-7–stimulated HCT116 and HT29 cells (Fig. 6A). In contrast, IL7Rα overexpression increased p-JAK2 and p-STAT3 levels (Fig. 6B), whereas IL7Rα knockdown decreased them (Fig. S4D). Importantly, inhibition of the JAK2/STAT3 pathway using AG490 (10 µM; MedChemExpress, Cat. No. HY-12000) abrogated the pro-proliferative, pro-migratory, and pro-invasive effects induced by miR-3614-5p inhibition (Fig. 6C, 6D, and 6E).
Fig. 6.
JAK2/STAT3 pathway acts as IL7R downstream involved in miR-3614-5p mediated CRC suppression. (A) Western blot was used to assess the expression levels of p-JAK2 and p-STAT3 in CRC cells with or without miR-3614-5p mimics or corresponding control. (B) Western blot was used to assess the expression levels of p-JAK2 and p-STAT3 in CRC cells with IL7Rα plasmid or corresponding control. (C-E) CCK8, wound healing, and transwell assays were performed to evaluate the effect of AG490 on the proliferation, migration, and invasion of CRC cells mediated by IL7Rα and miR-3614-5p. Scale bars = 100 μm (D) and 100 μm (E). *p < 0.05
Exo-miR-3614-5p inhibits tumor growth in vivo
Early studies have demonstrated that murine IL-7 exerts biological effects comparable to human IL-7 and is capable of activating IL-7 signaling pathways in human lymphocytes [18]. Consistent with this finding, HCT116 cells were stimulated with recombinant mouse IL-7 at a concentration of 10 ng/mL for 24 h, which resulted in increased phosphorylation of JAK2 and STAT3 (Fig. S4E), indicating activation of the IL-7R/JAK/STAT3 pathway. We next assessed the in vivo therapeutic potential of Exo-miR-3614-5p using a subcutaneous xenograft tumor model established in nude mice with HT29 cells (Fig. 7A). On days 7, 10, 13, and 16 after tumor implantation, nude mice received tail-vein injections of Exo-miR-3614-5p or Exo-miR-NC. As expected, Exo-miR-3614-5p significantly reduced xenograft tumor growth compared with the Exo-miR-NC group (Fig. 7B, 7C, and 7D). The tumors shown in Fig. 7C were harvested on Day 19 after tumor implantation. Tumors from the Exo-miR-3614-5p group exhibited higher miR-3614-5p levels and lower IL7Rα expression than those from the Exo-miR-NC group (Fig. 7E and F). In addition, western blotting and IHC staining demonstrated that IL7Rα, p-JAK2, p-STAT3, and Ki-67 were markedly downregulated in the Exo-miR-3614-5p group (Fig. 7G, 7H, 7I, 7J and 7K). These results indicate that engineered hMSC-derived Exo-miR-3614-5p effectively suppresses CRC tumor growth in vivo.
Fig. 7.
Exo-miR-3614-5p inhibits tumor growth in vivo. (A) Transplanted tumors models were established by subcutaneously inoculating HCT116 cells into tumor-bearing nude mice, which were then divided into two groups upon tumor formation. The groups included: (1) Exo-miR-NC control; (2) Exo-miR-3614-5p, n = 6. (B-C) Transplanted tumor volume and tumors in nude mice treated with tail vein injection of Exo-miR-3614-5p, n = 6. (D-E) qRT-PCR detection of the miR-3614-5p and IL7Rα in tumor tissues. (F) Western blot detection of the IL7Rα, p-JAK2, JAK2, p-STAT3, STAT3, and Ki67 protein expression in tumor tissues. (G) Immunohistochemical staining of the IL7Rα, p-JAK2, p-STAT3, and Ki67 protein expression in tumor tissues. Scale bars = 100 μm (unenlarged picture) and 20 μm (enlarge Image)
Discussion
CRC remains a major global health burden, with advanced-stage patients exhibiting poor five-year survival despite recent improvements in diagnostics and therapeutics [19, 20]. Accumulating evidence has identified miRNAs as crucial regulators of gene expression and promising candidates for both biomarkers and targeted therapy in CRC [21, 22]. However, the clinical development of miRNA-based therapies is still at an early stage, primarily due to challenges in identifying miRNAs with robust tumor-suppressive functions and effective delivery systems. In this study, we systematically analyzed miRNA expression in plasma, exosomes, and tumor tissues, and identified miR-3614-5p as a significantly downregulated miRNA in CRC with favorable prognostic value. While miR-3614-5p has been previously implicated in tumor suppression across various cancers, its role and therapeutic potential in CRC remained largely unexplored [6–8]. Our findings suggest that miR-3614-5p may function as a novel tumor-suppressive miRNA in CRC, offering opportunities for therapeutic intervention.
To address the critical challenge of miRNA delivery, we employed engineered MSCs as biofactories to generate Exo-miR-3614-5p. Compared with conventional synthetic transfection approaches such as direct lipofection, MSC-derived exosomes offer several intrinsic advantages for nucleic acid delivery, including superior biocompatibility with low immunogenicity and cytotoxicity, enhanced stability of encapsulated cargo, intrinsic cellular uptake mechanisms, and an improved ability to traverse biological barriers-features that are particularly important for in vivo applications [23–29]. In addition, exosomes exhibit favorable biodistribution profiles and reduced nonspecific accumulation relative to synthetic transfection reagents, further supporting their suitability as therapeutic delivery vehicles. Importantly, to assess the loading efficiency of miR-3614-5p into MSC-derived exosomes, we quantified its levels in both MSCs and their secreted exosomes following mimic transfection. Approximately 50% of the transfected miRNA was successfully packaged into exosomes, confirming their potential as robust delivery vehicles (data not shown). Although native MSC-Exos generally display limited intrinsic anti-tumor activity, our engineered Exo-miR-3614-5p exhibited markedly enhanced therapeutic efficacy. The modified exosomes efficiently delivered miR-3614-5p into CRC cells, resulting in inhibition of tumor cell proliferation, migration, and invasion both in vitro and in vivo. Collectively, these findings underscore the promise of engineered MSC-derived exosomes as an effective miRNA delivery platform for CRC therapy.
Mechanistically, our study revealed that miR-3614-5p exerts its anti-tumor effects at least in part by directly targeting IL7Rα, leading to inactivation of the JAK2/STAT3 signaling pathway, a well-established oncogenic axis in CRC [30]. Downregulation of IL7Rα significantly impaired CRC cell proliferation and metastasis, and its suppression was required for miR-3614-5p-mediated tumor inhibition. Notably, CRC cells have been reported to endogenously produce and secrete IL-7 [31], suggesting an autocrine or paracrine activation loop that may sustain IL7R signaling. This endogenous IL-7 production could explain why miR-3614-5p effectively suppresses the IL7R/JAK2/STAT3 axis even in the absence of exogenous IL-7, as downregulation of IL7Rα by miR-3614-5p would disrupt both autocrine signaling and downstream oncogenic pathways. Such intrinsic IL-7 expression may contribute to the aggressive phenotype of CRC and highlight the therapeutic relevance of targeting the IL7R axis.
Previous cross-species studies have shown that murine IL-7 can functionally engage the human IL-7 receptor and activate downstream signaling in human lymphocytes [18]. Consistent with this, our data demonstrate that mouse-derived IL-7 induces JAK2 and STAT3 phosphorylation in human CRC cells, validating the biological relevance of targeting the IL-7R/JAK/STAT3 axis in xenograft models.
It should be noted that IL7Rα exists in both soluble and membrane-bound forms [32], and our study focused on overall suppression without dissecting their distinct functional roles. More importantly, while JAK2/STAT3 inactivation is a key downstream mechanism, miR-3614-5p is likely to exert multi-target regulatory effects. Bioinformatics analysis suggests additional targets such as PIK3R1, EZH2, and MCL1, which are involved in critical oncogenic pathways including epithelial–mesenchymal transition (EMT), cell cycle control, apoptosis resistance, and immune evasion. Moreover, miR-3614-5p has been shown to suppress tumorigenesis in other malignancies via distinct mechanisms, such as modulating TGF-β signaling in non-small cell lung carcinoma [33]. Collectively, these observations highlight the complexity and pleiotropic nature of miR-3614-5p in tumor biology.
Despite these findings, several limitations of the present study should be acknowledged. First, the diagnostic analysis was performed in a relatively small cohort comprising 64 CRC patients and 20 control samples, which limits the statistical power and generalizability of the conclusions. Moreover, the absence of an independent validation cohort precludes definitive claims regarding the clinical utility of miR-3614-5p as a diagnostic biomarker; therefore, its diagnostic relevance should be considered exploratory at this stage. Future studies with larger, multi-center cohorts and independent validation sets will be required to confirm its robustness and translational potential. In addition, the functional experiments were conducted using a limited number of CRC cell lines harboring specific genetic alterations. Given the pronounced molecular heterogeneity of colorectal cancer, particularly with respect to KRAS and BRAF mutation status, these models may not fully capture the complexity of CRC biology. Furthermore, the in vivo experiments were performed with a relatively small number of mice per group, and blinding and formal power calculations were not conducted, which may limit the reproducibility and robustness of the reported tumor inhibition results. Finally, although our data demonstrate that miR-3614-5p exerts tumor-suppressive effects at least in part through inhibition of the IL7R/JAK2/STAT3 pathway, miR-3614-5p is likely to regulate multiple downstream targets and signaling networks. Further studies are needed to define its broader targets and regulatory mechanisms in CRC.
Conclusion
In conclusion, this study demonstrates that miR-3614-5p is a potent tumor suppressor in CRC and validates the therapeutic potential of MSC-derived exosomal miRNA delivery. While the JAK2/STAT3 axis represents a critical pathway regulated by miR-3614-5p, further studies are needed to comprehensively map its downstream targets and regulatory networks. A deeper understanding of these pathways will aid in advancing miR-3614-5p as a miRNA-based therapeutic for CRC and potentially other malignancies. Taken together, our data reveal that miR-3614-5p plays a suppressive role in CRC progression at least partially through inhibition of the JAK2/STAT3 pathway. Nonetheless, further investigation is warranted to fully delineate its downstream regulatory network and to identify additional direct targets. A comprehensive understanding of the miR-3614-5p-centered regulatory landscape may provide new opportunities for developing miRNA-based therapeutics in colorectal and other cancers.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
The authors thank the TCGA and miRWalk databases for the available data.
Author contributions
YML drafted the manuscript and carried out the molecular genetic studies. BYZ and LYY conducted animal experiments and part of vitro experiments. LSR and Yuyang Liu performed the statistical analysis. JLX, WJL and XYX participated in vitro experiments. NSF provided experimental guidance. HJL conducted part of the animal experiments. LXX conducted the figures layout and article checking. ZLT participated in the research design and proofread the manuscript. All authors read and approved the final manuscript.
Funding
The research was supported by grants from the Guangxi Science and Technology Base and Talent Special Project (Grant No. GUI KE AD22035047), the Key R&D Program of Scientific Research and Technology Development Project of Guangxi (Grant No. GUI KE AB23026078), the Joint Project on Regional High-Incidence Diseases Research of Guangxi Natural Science Foundation under Grant (Grant No. 2023GXNSFBA026202), the Guangxi Medical and health key discipline construction project, the Key R&D Program of Scientific Research and Technology Development Project of Guangxi (Grant No. 2022AB11046), the Key R&D Program of Scientific Research and Technical Development Project of Qingxiu District, Nanning, Guangxi (Grant No. 2021015), and Guangxi Medical and Health Key Discipline Construction Proiect.
Data availability
The data used to support the findings of this study are available from the corresponding author upon request.
Declarations
Ethics approval and consent to participate
The study was approved by the Ethics Committee of the Guangxi Medical University Affiliated Tumor Hospital.
Consent for publication
All the authors agree to the content of the paper and consent for publication.
Competing interests
The authors declare that there are no conflicts of interest.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Mengling Ye, Yingzhen Bian and Yanyan Lin contributed equally to this work.
Contributor Information
Xinxing Lei, Email: lei.xinxing@szhospital.com.
Litu Zhang, Email: zhanglitu@sr.gxmu.edu.cn.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The data used to support the findings of this study are available from the corresponding author upon request.