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Upregulation of miR‑128 inhibits neuronal cell apoptosis following spinal cord injury via FasL downregulation by repressing ULK1

Liu, Ruixuan, Peng, Zhibin, Zhang, Yubo, Li, Rui, Wang, Yansong·2021·Molecular Medicine Reports
DOI 10.3892/mmr.2021.12306PMC PMC8335739
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Figure 1. Rats with SCI exhibit increased neuronal apoptosis, intense inflammatory response and lowly expressed miR-128. (A) Locomotor functions of rats evaluated by BBB score. (B) Hematoxylin and eosin staining of morphological changes of neuronal cells (magnification, ×200). (C) The survival of neurons in spinal cord of rats determined by Nissl staining. (D) The number of nestin-positive cells detected using immunofluorescence. (E) The expression of inflammatory factors (IL-1β, IL-6 and TNF-α) in rat serum determined by ELISA. (F) Apoptosis of spinal cord cells detected using TUNEL. (G) Protein expression of apoptosis-related factors measured by western blot analysis. (H) The expression of miR-128 assessed by reverse transcription-quantitative PCR. An unpaired t-test was performed for comparisons of data between two groups. BBB score was used for comparison of data at different time points. Repeated-measures analysis of ANOVA was performed, followed by Bonferroni post-hoc test. *P<0.05 vs. normal. SCI, spinal cord injury; miR, microRNA; BBB, Basso, Beattie and Bresnahan; ANOVA, analysis of variance.
Figure 1. Rats with SCI exhibit increased neuronal apoptosis, intense inflammatory response and lowly expressed miR-128. (A) Locomotor functions of rats evaluated by BBB score. (B) Hematoxylin and eosin staining of morphological changes of neuronal cells (magnification, ×200). (C) The survival of neurons in spinal cord of rats determined by Nissl staining. (D) The number of nestin-positive cells detected using immunofluorescence. (E) The expression of inflammatory factors (IL-1β, IL-6 and TNF-α) in rat serum determined by ELISA. (F) Apoptosis of spinal cord cells detected using TUNEL. (G) Protein expression of apoptosis-related factors measured by western blot analysis. (H) The expression of miR-128 assessed by reverse transcription-quantitative PCR. An unpaired t-test was performed for comparisons of data between two groups. BBB score was used for comparison of data at different time points. Repeated-measures analysis of ANOVA was performed, followed by Bonferroni post-hoc test. *P<0.05 vs. normal. SCI, spinal cord injury; miR, microRNA; BBB, Basso, Beattie and Bresnahan; ANOVA, analysis of variance.CC BY-NC-ND · PubMed Central
Figure 2. Upregulation of miR-128 can alleviate SCI in rats. Vectors containing LV-control, LV-miR-128, or LV-sponge miR-128 were injected into rats with SCI (n=10/group). (A) miR-128 expression was assessed by reverse transcription-quantitative PCR. *P<0.05 vs. SCI rats injected with LV-control. (B) Locomotor functions of rats indicated by BBB score. (C) Hematoxylin and eosin staining of morphological changes of neuronal cells (magnification, ×200). (D) The survival of neuronal cells in rat spinal cord determined by Nissl staining. (E) The number of nestin-positive cells evaluated by immunofluorescence. (F) The levels of inflammatory factors in rat serum detected by ELISA. (G) Cell apoptosis was assessed by TUNEL staining. (H) The expression of apoptosis-related factor protein in rat spinal cord determined by western blot analysis. An unpaired t-test was performed for comparisons of data between two groups. ANOVA was performed for comparisons of data among multiple groups, followed by Tukey's post-hoc test. BBB score was used as the comparisons of different time points using repeated-measures analysis of ANOVA, followed by Bonferroni post-hoc test. *P<0.05 vs. SCI rats infected with LV-control. miR, microRNA; SCI, spinal cord injury; LV, Lentiviral vector; ctrl, control; BBB, Basso, Beattie and Bresnahan; ANOVA, analysis of variance.
Figure 2. Upregulation of miR-128 can alleviate SCI in rats. Vectors containing LV-control, LV-miR-128, or LV-sponge miR-128 were injected into rats with SCI (n=10/group). (A) miR-128 expression was assessed by reverse transcription-quantitative PCR. *P<0.05 vs. SCI rats injected with LV-control. (B) Locomotor functions of rats indicated by BBB score. (C) Hematoxylin and eosin staining of morphological changes of neuronal cells (magnification, ×200). (D) The survival of neuronal cells in rat spinal cord determined by Nissl staining. (E) The number of nestin-positive cells evaluated by immunofluorescence. (F) The levels of inflammatory factors in rat serum detected by ELISA. (G) Cell apoptosis was assessed by TUNEL staining. (H) The expression of apoptosis-related factor protein in rat spinal cord determined by western blot analysis. An unpaired t-test was performed for comparisons of data between two groups. ANOVA was performed for comparisons of data among multiple groups, followed by Tukey's post-hoc test. BBB score was used as the comparisons of different time points using repeated-measures analysis of ANOVA, followed by Bonferroni post-hoc test. *P<0.05 vs. SCI rats infected with LV-control. miR, microRNA; SCI, spinal cord injury; LV, Lentiviral vector; ctrl, control; BBB, Basso, Beattie and Bresnahan; ANOVA, analysis of variance.CC BY-NC-ND · PubMed Central
Figure 3. miR-128 inhibits the expression of ULK1 and knockdown of ULK1 attenuates SCI in rats. Vectors containing LV-control, LV-miR-128, or LV-sponge miR-128 were constructed and injected into rats with SCI in A-G, while vectors containing sh-NC or sh-ULK1 were injected into rats with SCI in H-P (n=10/group). (A) Venn diagram of the target genes of miR-128 predicted using RNAInter, miRDB, TargetScan and miRWalk. (B) The mRNA expression of ULK1 in normal rats and rats with SCI detected using RT-qPCR. *P<0.05 vs. normal rats. (C) Protein expression of ULK1 detected by western blot analysis. *P<0.05 vs. normal rats. (D) The binding site of miR-128 and ULK1 predicted using TargetScanHuman 7.2. (E) The targeting relationship between ULK1 and miR-128 measured by dual-luciferase report assay. *P<0.05 vs. NC mimic. (F) The mRNA expression of ULK1 examined by RT-qPCR. *P<0.05 vs. SCI rats injected with LV-control. (G) The protein expression of ULK1 measured using western blot analysis. *P<0.05 vs. SCI rats infected with LV-control. (H) The mRNA expression of ULK1 measured using RT-qPCR. *P<0.05 vs. Blank or sh-NC. (I) The protein expression of ULK1 expressing sh-NC and sh-ULK1 evaluated by western blot analysis. *P<0.05 vs. Blank or sh-NC. (J) Locomotor functions of rats evaluated by BBB score. (K) H&E staining of morphological changes of neurons (magnification, ×200). (L) Survival of neuronal cells detected by Nissl staining. (M) The number of nestin-positive cells detected by immunofluorescence. (N) The content of inflammatory factors in rat serum determined by ELISA. (O) Apoptosis of rat spinal cord cells determined using TUNEL. (P) The protein expression of apoptosis-related factor in rats with SCI evaluated by western blot analysis. J-P, *P<0.05 vs. SCI rats transfected with sh-NC. An unpaired t-test was performed for comparisons of data between two groups. ANOVA was performed for comparisons of data among multiple groups, followed by Tukey's post-hoc test. BBB score was used as comparisons of data at different time points using repeated-measures analysis of ANOVA, followed by Bonferroni post-hoc test. miR, microRNA; ULK1, Unc-51 like autophagy activating kinase 1; SCI, spinal cord injury; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; LV, Lentiviral vector; ctrl, control; sh, short hairpin; ANOVA, analysis of variance.
Figure 3. miR-128 inhibits the expression of ULK1 and knockdown of ULK1 attenuates SCI in rats. Vectors containing LV-control, LV-miR-128, or LV-sponge miR-128 were constructed and injected into rats with SCI in A-G, while vectors containing sh-NC or sh-ULK1 were injected into rats with SCI in H-P (n=10/group). (A) Venn diagram of the target genes of miR-128 predicted using RNAInter, miRDB, TargetScan and miRWalk. (B) The mRNA expression of ULK1 in normal rats and rats with SCI detected using RT-qPCR. *P<0.05 vs. normal rats. (C) Protein expression of ULK1 detected by western blot analysis. *P<0.05 vs. normal rats. (D) The binding site of miR-128 and ULK1 predicted using TargetScanHuman 7.2. (E) The targeting relationship between ULK1 and miR-128 measured by dual-luciferase report assay. *P<0.05 vs. NC mimic. (F) The mRNA expression of ULK1 examined by RT-qPCR. *P<0.05 vs. SCI rats injected with LV-control. (G) The protein expression of ULK1 measured using western blot analysis. *P<0.05 vs. SCI rats infected with LV-control. (H) The mRNA expression of ULK1 measured using RT-qPCR. *P<0.05 vs. Blank or sh-NC. (I) The protein expression of ULK1 expressing sh-NC and sh-ULK1 evaluated by western blot analysis. *P<0.05 vs. Blank or sh-NC. (J) Locomotor functions of rats evaluated by BBB score. (K) H&E staining of morphological changes of neurons (magnification, ×200). (L) Survival of neuronal cells detected by Nissl staining. (M) The number of nestin-positive cells detected by immunofluorescence. (N) The content of inflammatory factors in rat serum determined by ELISA. (O) Apoptosis of rat spinal cord cells determined using TUNEL. (P) The protein expression of apoptosis-related factor in rats with SCI evaluated by western blot analysis. J-P, *P<0.05 vs. SCI rats transfected with sh-NC. An unpaired t-test was performed for comparisons of data between two groups. ANOVA was performed for comparisons of data among multiple groups, followed by Tukey's post-hoc test. BBB score was used as comparisons of data at different time points using repeated-measures analysis of ANOVA, followed by Bonferroni post-hoc test. miR, microRNA; ULK1, Unc-51 like autophagy activating kinase 1; SCI, spinal cord injury; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; LV, Lentiviral vector; ctrl, control; sh, short hairpin; ANOVA, analysis of variance.CC BY-NC-ND · PubMed Central
Figure 4. Upregulation of miR-128 repressed ULK1 expression and thereby attenuating SCI via the downregulation of FasL. The neuronal cells were transfected with inhibitor NC and sh-NC, miR-128 inhibitor and sh-NC or miR-128 inhibitor and sh-ULK1. (A) The morphology of spinal cord neurons observed under an inverted microscope (magnification, ×200). (B) Neuronal cell nestin-positive expression determined by immunofluorescence (magnification, ×200). (C) The expression of miR-128 and ULK1 detected using reverse transcription-quantitative PCR. (D) The protein expression of ULK1 measured by western blot analysis. (E) Cell proliferation assessed by MTT assay. (F) Cell apoptosis determined by flow cytometry. (G) The protein expression of apoptosis-related factors determined by western blot analysis. (H) The enrichment of ULK1 and FasL determined by ChIP assay. (I) The regulatory relationship of ULK1 on FasL assessed using dual-luciferase reporter assay. *P<0.05 vs. cells co-transfected with inhibitor NC and sh-NC or #P<0.05 vs. cells co-transfected with miR-128 inhibitor and sh-NC, or cells co-transfected with si-NC. An unpaired t-test was performed for comparisons of data between two groups. ANOVA was conducted for comparison of data among multiple groups, followed by Tukey's post-hoc test. miR, microRNA; ULK1, Unc-51 like autophagy activating kinase 1; SCI, spinal cord injury; FasL, Fas ligand; NC, negative control; sh, short hairpin; WT, wild type; MUT, mutated; ChIP, Chromatin immunoprecipitation; IgG, immunoglobulin G.
Figure 4. Upregulation of miR-128 repressed ULK1 expression and thereby attenuating SCI via the downregulation of FasL. The neuronal cells were transfected with inhibitor NC and sh-NC, miR-128 inhibitor and sh-NC or miR-128 inhibitor and sh-ULK1. (A) The morphology of spinal cord neurons observed under an inverted microscope (magnification, ×200). (B) Neuronal cell nestin-positive expression determined by immunofluorescence (magnification, ×200). (C) The expression of miR-128 and ULK1 detected using reverse transcription-quantitative PCR. (D) The protein expression of ULK1 measured by western blot analysis. (E) Cell proliferation assessed by MTT assay. (F) Cell apoptosis determined by flow cytometry. (G) The protein expression of apoptosis-related factors determined by western blot analysis. (H) The enrichment of ULK1 and FasL determined by ChIP assay. (I) The regulatory relationship of ULK1 on FasL assessed using dual-luciferase reporter assay. *P<0.05 vs. cells co-transfected with inhibitor NC and sh-NC or #P<0.05 vs. cells co-transfected with miR-128 inhibitor and sh-NC, or cells co-transfected with si-NC. An unpaired t-test was performed for comparisons of data between two groups. ANOVA was conducted for comparison of data among multiple groups, followed by Tukey's post-hoc test. miR, microRNA; ULK1, Unc-51 like autophagy activating kinase 1; SCI, spinal cord injury; FasL, Fas ligand; NC, negative control; sh, short hairpin; WT, wild type; MUT, mutated; ChIP, Chromatin immunoprecipitation; IgG, immunoglobulin G.CC BY-NC-ND · PubMed Central
Figure 5. Molecular mechanism of miR-128/ULK1/FasL axis in protecting spinal cord injury. The upregulation of miR-128 could suppress ULK1 to inhibit the apoptosis of neuronal cells, thereby alleviating SCI via the downregulation of FasL. miR, microRNA; ULK1, Unc-51 like autophagy activating kinase 1; FasL, Fas ligand.
Figure 5. Molecular mechanism of miR-128/ULK1/FasL axis in protecting spinal cord injury. The upregulation of miR-128 could suppress ULK1 to inhibit the apoptosis of neuronal cells, thereby alleviating SCI via the downregulation of FasL. miR, microRNA; ULK1, Unc-51 like autophagy activating kinase 1; FasL, Fas ligand.CC BY-NC-ND · PubMed Central

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Citations

Cites

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Metadata

Authors
Liu, R., Peng, Z., Zhang, Y., Li, R., & Wang, Y.
Affiliations
  • Department of Orthopaedics, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China
  • Department of Orthopaedics, Binzhou Medical University Hospital, Binzhou, Shandong 256603, P.R. China
Venue
Molecular Medicine Reports
Publisher
Spandidos Publications
Published
2021-07-20
Volume
24
Issue
3
ISSN
  • 1791-2997
  • 1791-3004
DOI
10.3892/mmr.2021.12306
PMC
PMC8335739
Cited by
9
References
1

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