Upregulation of Spinal miR-155-5p Contributes to Mechanical Hyperalgesia by Promoting Inflammatory Activation of Microglia in Bone Cancer Pain Rats

Abstract

Bone cancer pain (BCP) seriously deteriorates the life quality of patients, but its underlying mechanism is still unclear. Spinal microRNAs might contribute to the development of BCP and the role of microglial activation is controversial. In this study, we established a BCP model by injecting Walker 256 breast carcinoma cells into the tibial intramedullary cavity of rats and significant hyperalgesia was observed in the BCP rats. The lumbar spinal cords were harvested to perform RNA sequencing (RNA-seq), and 31 differentially expressed miRNAs (26 upregulated and 5 downregulated) were identified in the BCP rats. Among them, miR-155-5p was significantly upregulated in the BCP rats. Spinal microglial activation was observed during BCP development. miR-155-5p could be expressed in spinal microglia and was significantly upregulated in microglia treated with lipopolysaccharide (LPS) in vitro. Serum/glucocorticoid regulated kinase family member 3 (Sgk3) was predicted to be the possible downstream target of miR-155-5p and this was confirmed using a dual-luciferase reporter assay in vitro. The inhibition of miR-155-5p restored Sgk3-expression-attenuated microglial activation and alleviated hyperalgesia in the BCP rats. In conclusion, spinal miR-155-5p/Sgk3/microglial activation might play an important role in BCP pathogenesis.

Keywords: Sgk3; bone cancer pain; mechanical hyperalgesia; miR-155-5p; microglia.

Figure 1

Establishment of a bone cancer pain (BCP) model and transcriptome sequencing (RNA-seq) of miRNA. (A) Mechanical hyperalgesia was induced in bone cancer pain rats six days after Walker 256 carcinoma cell inoculation and was maintained to the end of the experiment. n = 5, *** p < 0.001 vs. the sham-operated group. (B) Bone destruction was found in the BCP rats using H&E staining of tibia sections, the black arrow indicates the discontinuity of cortical bone (scale bar: 500 μm). (C) Sham-operated and bone cancer pain samples (marked by different colors) were separated in the principal component analysis (PCA) of the general gene expression value. B1, B2, and B3 represent biological replicates of the BCP group, while S1, S2, and S3 represent biological replicates of the sham-operated group. (D) After neglecting unannotated and extremely lowly expressed miRNAs, 31 differentially expressed miRNAs (26 upregulated miRNAs and 5 downregulated miRNAs), which were filtered using the threshold of |log2FC|> 0.8 and an adjusted p < 0.05, were listed and clustered in a heatmap. (E) Differentially expressed miRNAs (marked using different colors) are shown in the scatter plot. (F) RT-qPCR of miR-155-5p verified its gradually increased expression in the BCP rats. n = 5, * p < 0.05, ** p < 0.01 vs. the sham-operated group.

Figure 2

Fluorescence in situ hybridization (FISH) of miR-155-5p (green) showed its co-localization with Iba1 (red, a marker of microglia) and outside of the nucleus (marked with DAPI, blue) (scale bar: 10 μm) in the lumbar spinal cord of BCP rats.

Figure 3

Activation of microglia in the lumbar spinal cord of bone cancer pain rats. (A,B) Immunofluorescence of Iba1 showed the morphology and number of activated microglia (scale bar: 200 μm). (C–E) RT-qPCR analysis of inflammatory factors showed the surge of Tnf, Il1b, and Il6 in bone cancer pain. n = 5, * p < 0.05, *** p < 0.001 vs. the sham-operated group.

Figure 4

Knockdown of miR-155-5p alleviated the inflammatory storm in microglia caused by the LPS treatment. (A) The core sequence of the recombinant lentivirus-mediated miR-155-5p sponge was designed to be partially complementary to that of miR-155-5p. (B) The lentivirus-mediated miR-155-5p sponge knocked down the expression of miR-155-5p in LPS-treated microglia. n = 3, * p < 0.05, LPS + NC vs. Con + NC; * p < 0.05, LPS + sponge vs. LPS + NC. (C–E) The miR-155-5p sponge alleviated the surge of Tnf, Il1b, and Il6 induced by LPS. n = 3, *** p < 0.001, LPS + NC vs. Con + NC; *** p < 0.001, LPS + sponge vs. LPS + NC.

Figure 5

miR-155-5p suppressed the Sgk3 expression in vitro. (A) The probable pairing sequences of miR-155-5p and the 3′ untranslated region (UTR) of Sgk3 (seed region shown in grey boxes). (B) The structure of pMIR-REPORTTM Luciferase system and sequences of wild type and mutated 3′ UTR of Sgk3. (C) Dual-luciferase activity reporter assay showed that miR-155-5p mimics decreased the luciferase activity in 293T cells transfected with Sgk3-WT, but not in the MUT vector. n = 3, *** p < 0.001, Sgk3-WT + miR-155-5p vs. Sgk3-WT + NC; *** p < 0.001, Sgk3-WT + miR-155-5p vs. Sgk3-MUT + miR-155-5p. (D,E) Knockdown of miR-155-5p using a miR-155-5p sponge restored the expression of Sgk3 suppressed by LPS treatment assayed using RT-qPCR (D) and Western blot (E). n = 3, * p < 0.05, ** p < 0.01, LPS + NC vs. Con + NC; ** p < 0.01, *** p < 0.001, LPS + sponge vs. LPS + NC.

Figure 6

Intrathecal administration of a miR-155-5p sponge knocked down miR-155-5p expression, restored Sgk3 expression, and attenuated mechanical hyperalgesia in the bone cancer pain rats. (A) The mechanical hyperalgesia was attenuated in the bone cancer pain rats after the miR-155-5p sponge administration. n = 5, *** p < 0.001 vs. the sham + NC group; n = 5, # p < 0.05, ## p < 0.01 vs. the BCP + NC group. (B) The lentivirus-mediated miR-155-5p sponge knocked down the expression of miR-155-5p in vivo. n = 5, ** p < 0.01 vs. the sham + NC group; * p < 0.05 vs. the BCP + NC group. (C,D) Administration of the miR-155-5p sponge restored the expression of Sgk3 suppressed in bone cancer pain according to an assay using RT-qPCR (C) and Western blot (D). n = 5, * p < 0.05 vs. the sham + NC group; * p < 0.05, ** p < 0.01 vs. the BCP + NC group.

Figure 7

Intrathecal administration of a miR-155-5p sponge alleviated microglial activation in the bone cancer pain rats. (A–C) Immunofluorescence of Iba1 showed the morphology and number of activated microglia in different treatments (scale bar: 200 μm). (D–F) The administration of a miR-155-5p sponge alleviated the inflammatory factors (i.e., Tnf, Il1b, and Il6) that were increased in the bone cancer pain condition. n = 5, *** p < 0.001 vs. the sham + NC group; * p < 0.05 vs. the BCP + NC group.