LncRNA HCG18 regulates the progression of spinal tuberculosis by modulating the hsa-miR-146a-5p/TGF-β1/SMADs pathway

Abstract

Spinal tuberculosis is the most common extrapulmonary tuberculosis, characterized by intervertebral disc destruction, which seriously affects people's quality of life. Recent studies have suggested that the TGF-β1/SMADs signaling pathway plays an important regulatory role in the process of intervertebral disc destruction caused by spinal tuberculosis. However, the abnormal TGF-β1/SMADs signaling pathway in spinal tuberculosis is not fully understood. Herein, we found for the first time that HCG18 was significantly upregulated in spinal tuberculosis nucleus pulposus clinical samples and confirmed that HCG18 negatively regulates the proliferation and migration ability of nucleus pulposus cells (NPCs). In vitro experiments further suggest that overexpression of HCG18 can significantly promote TGF-β1/SMADs pathway activity and inhibit proliferation, migration, and apoptosis of NPCs, an effect which can be reversed by overexpressing hsa-miR-146a-5p. On the contrary, knocking down HCG18 yields the opposite result. In vivo experiments suggest that knocking down HCG18 can significantly alleviate the destruction of the nucleus pulposus in rats with spinal tuberculosis by inhibiting the activity of the TGF-β1/SMADs pathway. In summary, our research suggests that HCG18 can promote the progression of spinal tuberculosis by alleviating the inhibitory effect of hsa-miR-146a-5p on the TGF-β1/SMADs pathway. This study provides new insights into the occurrence and development of spinal tuberculosis, as well as new strategies for the prevention and treatment of spinal tuberculosis.

Keywords: HCG18; Intervertebral disc destruction; SMADs signaling pathway; Spinal tuberculosis; hsa-miR-146a-5p.

Fig. 1

Extraction of nucleus pulposus cells from tuberculosis infected intervertebral discs. (A) The CT scan shows the nucleus pulposus destruction in sagittal and horizontal planes in ST patient. (B, C) The wound healing assays indicated that tuberculosis infection significantly reduced the migration ability of NPCs (n = 3, * p < 0.05, *** p < 0.001). (D) The CCK-8 assays indicated that tuberculosis infection significantly reduced the proliferation ability of NPCs (n = 3, ** p < 0.01, *** p < 0.001)

Fig. 2

HCG18 inhibits the proliferation and migration ability but promotes apoptosis of NPCs. (A) The expression level of HCG18 significantly increased in clinical nucleus pulposus samples of ST patients (n = 30, *** p < 0.001). (B) The nuclear cytoplasmic separation assays indicated that the expression level of HCG18 in the cytoplasm is significantly higher than that in the nucleus (n = 3, *** p < 0.001). (C) FISH assays indicated that HCG18 is mainly distributed in the cytoplasm of NPCs (n = 3). (D, E) Identification of HCG18 inhibitors and overexpression plasmids (n = 3, *** p < 0.001). (F) Knocking down HCG18 can significantly inhibit apoptosis of NPCs, while overexpression of HCG18 can significantly promote apoptosis of NPCs (n = 3). (G) Knocking down HCG18 significantly promoted the migration capability of NPCs, while overexpression of HCG18 significantly inhibited the migration capability of NPCs (n = 3, *** p < 0.001). (I) Knocking down HCG18 can significantly promote the proliferation capability of NPCs, while overexpression of HCG18 can significantly inhibit the proliferation capability of n NPCs (n = 3, ** p < 0.01, *** p < 0.001)

Fig. 3

hsa-miR-146a-5p is a potential downstream target of HCG18. (A) Venn diagram displays the potential downstream targets of HCG18. (B) The binding sites between HCG18 and hsa-miR-146a-5p. (C) The luciferase reporter gene assays showed that the luciferase activity of 293T cells was significantly reduced when the luciferase reporter gene plasmid containing the wild-type HCG18 sequence was co-transferred with hsa-miR-146a-5p (n = 3, *** p < 0.001). (D) Knocking down HCG18 significantly promoted hsa-miR-146a-5p expression, while overexpression of HCG18 significantly inhibited hsa-miR-146a-5p expression (n = 3, *** p < 0.001). (E) The expression level of hsa-miR-146a-5p was significantly reduced in clinical nucleus pulposus samples of spinal tuberculosis (n = 30, *** p < 0.001). (F) The expression level of hsa-miR-146a-5p was negatively correlated with HCG18 in spinal tuberculosis nucleus pulposus tissue (n = 30, p < 0.0001, R = -0.7130)

Fig. 4

SMAD4 is a potential downstream target of hsa-miR-146a-5p. (A) Venn diagram displays potential downstream targets of hsa-miR-146a-5p. (B) The binding sites between SMAD4 and hsa-miR-146a-5p. (C) The luciferase reporter gene assays showed that the luciferase activity of 293T cells was significantly reduced when the luciferase reporter gene plasmid containing the wild-type SMAD4 3’-UTR sequence was co-transferred with hsa-miR-146a-5p (n = 3, *** p < 0.001). (D) The expression level of SMAD4 significantly increased in clinical nucleus pulposus samples of spinal tuberculosis (n = 30, *** p < 0.001). (E) The expression level of SMAD4 in spinal tuberculosis nucleus pulposus tissue is significantly positively correlated with the expression level of HCG18 (n = 30, p < 0.0001, R = 0.7586). (F) The expression level of SMAD4 in spinal tuberculosis nucleus pulposus tissue is significantly negatively correlated with the expression level of hsa-miR-146a-5p (n = 30, p < 0.0001, R = -0.6858)

Fig. 5

Overexpression of HCG18 can activate the TGF-β1/SMADs pathway activity by inhibiting hsa-miR-146a-5p expression. (A) The expression level of hsa-miR-146a-5p significantly decreased after transfection with HCG18 overexpression plasmid, but could be remedied by co-transfection with hsa-miR-146a-5p mimics (n = 3, *** p < 0.001). (B) The expression level of SMAD4 mRNA significantly increased after transfection with HCG18 overexpression plasmid, but could be remedied by co-transfection with hsa-miR-146a-5p mimics (n = 3, *** p < 0.001). (C) Overexpression of HCG18 can significantly increase the protein expression levels of phospho-SMAD2, phospho-SMAD3, SMAD4, and TGF-β1, and decrease the expression levels of SMAD7, an effect which can be remedied by co-transfection of hsa-miR-146a-5p mimics (n = 3). (D) Overexpression of HCG18 significantly increase apoptosis of NPCs, an effect which can be remedied by co-transfection of hsa-miR-146a-5p mimics (n = 3). Wound healing (E, F) and CCK-8 (G) assays suggest that overexpression of HCG18 can significantly increase the migration and proliferation of NPCs, an effect which can be remedied by co-transfection of hsa-miR-146a-5p mimics (n = 3, *** p < 0.001)

Fig. 6

Inhibition of HCG18 can inhibit TGF-β 1/SMADs pathway activity by promoting hsa-miR-146a-5p expression. (A) The expression level of hsa-miR-146a-5p decreased after HCG18 knocking down, but could be reversed by co-transfection with hsa-miR-146a-5p inhibitor (n = 3, *** p < 0.001). (B) The expression level of SMAD4 mRNA significantly increased after HCG18 knockdown, but can be reversed by co-transfecting hsa-miR-146a-5p inhibitor (n = 3, *** p < 0.001). (C) Knocking down HCG18 can significantly reduce the protein expression levels of phospho-SMAD2, phospho-SMAD3, SMAD4, and TGF - β 1, and increase expression levels of SMAD7, an effect that can be remedied by co transfection of hsa-miR-146a-5p inhibitor(n = 3). (D) Knocking down HCG18 can significantly reduce apoptosis of NPCs, an effect that can be remedied by co transfection of hsa-miR-146a-5p inhibitor (n = 3). Wound healing (E, F) and CCK-8 (G) assays suggest that knocking down HCG18 can significantly increase migration and proliferation of NPCs, an effect which can be remedied by co-transfection of hsa-miR-146a-5p inhibitor (n = 3, *** p < 0.001)

Fig. 7

Knocking down HCG18 in vivo can significantly alleviate nucleus pulposus destruction in rats with spinal tuberculosis. (A) Knocking down HCG18 in vivo significant decresed the expression of HCG18 and SMAD4, but increase the expression of hsa-miR-146a-5p expression in the nucleus pulposus tissue of ST rats (n = 6, *** p < 0.001). (B) Knocking down HCG18 in vivo can significantly decrease the protein expression levels of phospho-SMAD2, phospho-SMAD3, SMAD4, and TGF-β1, and increase expression levels of SMAD7 in nucleus pulposus samples from ST rats (n = 6). (C) The HE and Safranin O staining showed that nucleus pulposus destruction is significant in rats with spinal tuberculosis, but could be improved by injection of si-HCG18 (n = 6)

Fig. 8

HCG18 can promote the progression of spinal tuberculosis nucleus pulposus destruction by alleviating the inhibitory effect of hsa-miR-146a-5p on the TGF-β1/SMADs pathway