MicroRNA-34c Promotes Neuronal Recovery in Rats with Spinal Cord Injury Through the CXCL14/JAK2/STAT3 Axis

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MicroRNA-34c Promotes Neuronal Recovery in Rats with Spinal Cord Injury Through the CXCL14/JAK2/STAT3 Axis

Introduction

Spinal cord injury (SCI) remains a significant global health challenge, leading to severe physical, psychological, and socioeconomic consequences. Secondary complications such as neuroinflammation, apoptosis, and glial scarring further exacerbate the damage, highlighting the urgent need for effective therapeutic strategies. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression in neurological disorders, including SCI. Among these, miR-34c has shown potential in modulating neuropathic pain and neuronal apoptosis. This study investigates the role of miR-34c in SCI recovery, focusing on its interaction with the chemokine CXCL14 and the downstream JAK2/STAT3 signaling pathway.

Methods

Animal Model and Experimental Design

A rat SCI model was established using the modified Allen method. A total of 120 Wistar rats (230–250 g) were divided into six groups: sham, SCI, negative control lentiviral vector (NC-LV), miR-34c-LV, short hairpin negative control (sh-NC), and sh-CXCL14. Post-surgery interventions included intrathecal injections of saline, NC-LV, miR-34c-LV, sh-NC, or sh-CXCL14 vectors.

miRNA Microarray and Target Prediction

Differentially expressed miRNAs in SCI rats were identified using the GeneChip™ miRNA 4.1 Array Strip. Bioinformatics tools (StarBase, miRSearch) predicted CXCL14 as a miR-34c target, validated via dual-luciferase reporter assays in 293T cells.

Molecular and Functional Analyses

  • RT-qPCR: Measured miR-34c and CXCL14 mRNA levels in spinal cord tissues.
  • Western Blot: Assessed CXCL14 protein expression and JAK2/STAT3 phosphorylation.
  • Grip Strength Measurement (GSM): Evaluated motor recovery at multiple time points.
  • TUNEL Assay: Quantified neuronal apoptosis in spinal cord sections.
  • ELISA: Detected levels of thyrotropin-releasing hormone (TRH), prostacyclin (PGI2), and ganglioside (GM).

Statistical Analysis

Data were analyzed using t-tests and ANOVA with Tukey’s post hoc test (SPSS 21.0). Significance was set at P < 0.05.

Results

miR-34c Is Downregulated in SCI Rats

Microarray analysis revealed 189 differentially expressed miRNAs in SCI rats (|Log2FC| > 2). miR-34c exhibited the most significant downregulation (7 days post-SCI: 0.95 ± 0.10 vs. sham 3.78 ± 0.44; P < 0.05). Concurrently, grip strength decreased sharply post-SCI (112.03 ± 10.64 g pre-injury vs. 17.32 ± 1.49 g at 7 days; P < 0.01).

miR-34c Overexpression Enhances Motor Recovery

Lentiviral overexpression of miR-34c (miR-34c-LV) restored grip strength (31.26 ± 2.99 g vs. SCI + NC-LV 11.23 ± 1.08 g; P < 0.01) and reduced neuronal apoptosis (24.59% ± 3.32% vs. 53.61% ± 6.07%; P < 0.01). ELISA confirmed elevated levels of neuroprotective factors: TRH (19.07 ± 1.86 pg/mL vs. 6.94 ± 0.75 pg/mL), PGI2 (37.49 ± 3.52 pg/mL vs. 12.34 ± 1.09 pg/mL), and GM (32.61 ± 3.48 ng/mL vs. 8.69 ± 0.83 ng/mL) (P < 0.01 for all).

miR-34c Targets CXCL14 to Modulate JAK2/STAT3

CXCL14 was identified as a direct miR-34c target via luciferase assays (activity reduced by 64% in CXCL14-WT + miR-34c mimic vs. control; P < 0.01). SCI rats showed upregulated CXCL14 mRNA (7 days: 3.27 ± 0.35 vs. sham 0.91 ± 0.09; P < 0.01) and protein (2.91 ± 0.27 vs. 0.61 ± 0.06; P < 0.01). miR-34c overexpression reversed this trend (CXCL14 protein: 1.29 ± 0.15 vs. SCI + NC-LV 2.87 ± 0.34; P < 0.01).

CXCL14 Silencing Mimics miR-34c Effects

Knockdown of CXCL14 (sh-CXCL14) improved grip strength (29.77 ± 2.75 g vs. sh-NC 12.76 ± 1.13 g; P < 0.01) and reduced apoptosis (26.75% ± 2.84% vs. 55.74% ± 6.24%; P < 0.01). TRH, PGI2, and GM levels increased comparably to miR-34c-LV treatment (P < 0.01).

JAK2/STAT3 Pathway Inactivation

Both miR-34c overexpression and CXCL14 silencing suppressed JAK2/STAT3 phosphorylation. In miR-34c-LV rats, p-JAK2 decreased from 1.32 ± 0.14 to 0.46 ± 0.06 (P < 0.01), and p-STAT3 from 1.19 ± 0.13 to 0.38 ± 0.04 (P < 0.01). Similar reductions occurred with sh-CXCL14 (p-JAK2: 0.49 ± 0.05 vs. 1.36 ± 0.16; p-STAT3: 0.34 ± 0.05 vs. 1.04 ± 0.12; P < 0.01). Total JAK2 and STAT3 levels remained unchanged.

Discussion

miR-34c as a Therapeutic Target in SCI

This study demonstrates that miR-34c downregulation exacerbates SCI by permitting CXCL14 overexpression, which activates the JAK2/STAT3 pathway. miR-34c restoration or CXCL14 silencing attenuated neuronal apoptosis, enhanced neurotrophic factor secretion, and improved motor function. These findings align with prior evidence linking JAK2/STAT3 hyperactivity to neuroinflammation and secondary damage post-SCI.

Mechanistic Insights

The miR-34c/CXCL14 axis directly modulates JAK2/STAT3 signaling. CXCL14, a chemokine with emerging roles in neuroinflammation, likely activates JAK2 via cytokine receptor interactions. Phosphorylated STAT3 then translocates to the nucleus, promoting pro-apoptotic gene transcription. miR-34c interruption of this cascade underscores its neuroprotective potential.

Clinical Implications

Elevated TRH, PGI2, and GM levels following miR-34c intervention highlight its role in neuroregeneration. TRH stabilizes neuronal membranes, PGI2 reduces oxidative stress, and GM supports axon myelination. Combined with JAK2/STAT3 inactivation, these effects create a conducive microenvironment for spinal cord repair.

Conclusion

This study establishes miR-34c as a critical regulator of SCI recovery via CXCL14 suppression and JAK2/STAT3 pathway inactivation. Therapeutic strategies targeting this axis may offer novel avenues for mitigating secondary damage and enhancing functional recovery in SCI patients.

doi.org/10.1097/CM9.0000000000001022

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