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miR-155-5p in the spinal cord regulates hypersensitivity in a rat model of bone cancer pain - PubMed

. 2022 Apr:18:17448069221127811.

doi: 10.1177/17448069221127811.

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miR-155-5p in the spinal cord regulates hypersensitivity in a rat model of bone cancer pain

Qiuli He et al. Mol Pain. 2022 Apr.

Abstract

Background: Noncoding microRNAs have emerged as critical players of gene expression in the nervous system, where they contribute to regulating nervous disease. As stated in previous research, the miR-155-5p upregulation happens in the spinal cord at the nociceptive state. It was unclear if miR-155-5p is linked to bone cancer pain (BCP). Herein, we aimed at investigating the miR-155-5p functional regulatory function in BCP process and delineating the underlying mechanism.

Methods: The miRNA-155-5p levels and cellular distribution were determined by RNA sequencing, fluorescent in situ hybridization (FISH), and quantitative real-time PCR (qPCR). Immunoblotting, qPCR, dual-luciferase reporter gene assays, immunofluorescence, recombinant overexpression adeno-associated virus, small interfering RNA, intraspinal administration, and behavioral tests were utilized for exploring the downstream signaling pathway.

Results: The miR-155-5p high expression in spinal neurons contributes to BCP maintenance. The miR-155-5p blockage via the intrathecal injection of miR-155-5p antagomir alleviated the pain behavior; in contrast, upregulating miR-155-5p by agomir induced pain hypersensitivity. The miR-155-5p bounds directly to TCF4 mRNA's 3' UTR. BCP significantly reduced protein expression of TCF4 versus the Sham group. The miR-155-5p inhibition relieved the spinal TCF4 protein's down-expression level, while miR-155-5p upregulation by miR-155-5p agomir intrathecal injection decreased TCF4 protein expression in naïve rats. Additionally, TCF4 overexpression in BCP rats could increase Kv1.1. Moreover, TCF4 knockdown inhibited Kv1.1 expression in BCP rats. Indeed, TCF4 and Kv1.1 were co-expressed in BCP spinal cord neurons.

Conclusion: The study findings stated the miR-155-5p pivotal role in regulating BCP by directly targeting TCF4 in spinal neurons and suggested that miR-155-5p could be a promising target in treating BCP.

Keywords: Kv1.1; bone cancer pain; microRNA-155; spinal cord; transcription factor 4.

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Conflict of interest statement

Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.

BCP establishment model was successfully proved by behavioral tests and histochemical analysis. (a) The flowchart includes the study design to assure that BCP model was established successfully. (b) The left hind paw’s PWT in BCP group started decreasing after 6 days. The sham group’s pain sensitivity did not alter significantly (F(2,27) =349.94, ***p < 0.001; BCP versus Sham group; two-way ANOVA, then the Bonferroni test; n = 10). (c–d) CT scanning and HE staining created after 12 days following the tumor inoculation in sham and BCP groups. Active cancer cell invasion in bone medullary cavity destroyed normal structure. (e–g) Representative CatWalk gait, including print and timing view, and print intensity, in Sham and BCP groups. Tumor invasion significantly declined. (e) Max Contact Area and (f) Max Contact Mean Intensity on days 12 following tumor inoculation treatment. (*p < 0.05, **p < 0.01, unpaired t-test). The detected animals’ number within each column is determined. (LH, left hind, RH, right hind. (h) Representative animal tracks from Sham and BCP group rats in the open field test (***p < 0.001, versus Sham group; unpaired t test; n = 10, Fig. 1(h)).

Figure 2.
Figure 2.

The miR-155-5p upregulation mediates neuropathic pain. (a) This part experiments’ timeline (b) heatmap of 45 microRNAs was significantly changed on day 12 post-operation. (c) The miR-155-5p relative expression level in the spinal dorsal horn at different time points following tumor inoculation treatment (F(3,12) = 73.913,***p< 0.001 vs. the Sham group, one-way ANOVA, n=4). As the mean ± SD, the data are presented. (d) FISH analysis revealed that miR-155-5p was co-expressed with spinal dorsal horn neurons (arrow). Scale bar= 100 μm; (n=4). (e) Immunofluorescence showed miR-155-5p immunoactivities in different groups. (f–g) Effects of suppression and enhancement miR-155-5p on mechanical sensitivity. Intrathecal injection of miR-155-5p agomir (1 nmol each day over five consecutive days) and miR-155-5p antagomir (1 nmol every day for 5 days consecutively) were performed. Compared to controls, miR-155 agomir intrathecal injection significantly decreased PWT in naïve rats. (F(2,27) = 122.16,**p< 0.01, ***p< 0.001, control rats versus rats with agomir, two-way ANOVA, then the Bonferroni test; n = 10). Conversely, miR-155 antagomir intrathecal injection significantly increased PWT compared with BCP +control group (F(2,27) = 14.31,*p<0.05, **p<0.01, ***p<0.001 control rats versus rats with antagomir, two-way ANOVA, then the Bonferroni test; n = 10). (h) Representative animal tracks from rats treated with miR-155 antagomir/control at BCP for 12 days in the open field test. (*p<0.001, unpaired t-test, vs. the corresponding antagomir control group, n = 10).

Figure 3.
Figure 3.

TCF4 is a miR-155-5p target. (a) A potential binding site was predicted between miR-155-5p and the TCF4 mRNA’s 3’UTR. (b) miR-155-5p administration mimics significantly decreased the luciferase activity (**p<0.01 vs. miR-155-5p control group, unpaired t-test, n=3). (c) miR-155-5p regulates TCF4 expression in spinal in bone cancer pain. (**p < 0.01, ***p < 0.001, unpaired t-test.) (d) Western blot analysis revealed the expression of TCF4 dynamic changes in the spinal cord. (F(3,12) = 30.07, **p< 0.01, ***p < 0.001, versus Sham group; n = 4, one-way ANOVA). (e) Immunofluorescence showed immunoactivities of TCF4 in different groups. Scale bar: 100 μm. (f) The TCF4 relative immunofluorescence expression level in the spinal dorsal horn at different time points following the tumor inoculation treatment (F(3,12) = 30.77,**p< 0.01, ***p < 0.001, versus Sham group; n = 4, one-way ANOVA). (g–h) Expression of spinal TCF4 protein in the ipsilateral and contralateral on day 12 after BCP surgery (**p< 0.01, unpaired t-test, compared to the contralateral side).

Figure 4.
Figure 4.

TCF4 expression and localization in BCP. TCF4 (green), NeuN (red), Iba-1 (red), and GFAP (red) representative double-immunofluorescence images suggesting that TCF4 is co-localized with a neuron in the spinal cord. Arrowheads show double-labeled cells. Scale bar: 100 μm.

Figure 5.
Figure 5.

TCF4 regulates the nociceptive behaviors of BCP rats. (a) The experiment schedule. (b) Western blot analysis of TCF4 dynamic changes following a TCF4 siRNA’s specific inhibitor’s intrathecal injection (F(2,9) = 49.37, ***p< 0.001, versus BCP + scramble siRNA group; by one-way ANOVA, then the Bonferroni test; n=4 rat in every group). (c) Mechanical allodynia for rats receiving A TCF4 overexpression AAV was measured with von Frey tests (F(2,27) = 457.41, *p < 0.05, **p < 0.01 versus BCP + AAV control group using one-way ANOVA, then the Bonferroni test; n=10 rat in every group). (d) Schematic showing experimental strategy for AAV injection. AAV transfection was confirmed by the enhanced green fluorescent protein (EGFP) expression in the spinal dorsal horn. Double-immunofluorescence images (e) and Western blots (f) show efficient TCF4 overexpression in the spinal cord. (f) Following the recombinant AAV-TCF4 intraspinal injection, TCF4 expression was significantly upregulated. (F(2,9) = 10.48,*p< 0.05, **p< 0.01 versus BCP + AAV control group; by one-way ANOVA, then the Bonferroni test; n=4 rat in every group). (g–h) Quantification of behavioral parameters in OF after AAV injection. (*p<0.05 unpaired t-test, vs. the corresponding AAV control group, n = 10).

Figure 6.
Figure 6.

Kv1.1 expression in the rats’ spinal cord having bone cancer pain. (a) Kv1.1 expression in the rats’ spinal cord with BCP significantly decreased on day 12. (***p<0.001 unpaired t-test, vs. the Sham group, n = 4). (b) Kv1.1 proteins in the spinal cord after the overexpression of AAV-TCF4 microinjection or control in BCP rats. Representative Western immunoblots and the densitometric analysis statistical summary are shown. Per group (F(2,9) = 73.17,**p<0.01; by one-way ANOVA, then the Bonferroni test; n=4 rat in every group, versus the corresponding AAV control group). (c) Immunofluorescence showed that Kv1.1 was localized to the spinal dorsal horn. Immunostaining revealed that Kv1.1 and TCF4 were colocalized with a neuronal marker. Original magnification: ×200; scale bar:100 μm.

Figure 7.
Figure 7.

Schematic shows that increased miR-155-5p regulated BCP via interaction with TCF4 and the subsequent Kv1.1 pathway in the spinal neuron.

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