Objective To investigate the expression of miR-16 in spinal cord and the effects of miR-16 mimics on the pain behavior and the expression of its target gene BDNF during the development and maintenance of bone cancer pain.Methods The bone cancer pain model rats were developed by intra-tibia inoculation of Walker 256 mammary gland cells.Before inoculation and 3,5,7,10,14 d after inoculation,the samples of spinal cord L4～6 lumbar enlargement were collected to detect the expression of miR-16 using real-time PCR.Mice in group BM and group BN were intrathcal injected of miR-16 mimics and its negative control products on 10 d,11 d,12 d after inoculation.Paw withdrawal mechanical threshold (PWMT) was measured using von Frey hair mechanical stimulation.The expression of BDNF was detected using western-blot on 14 d after behavior tests.Results Compared with the base level and the level in group S,the significant decrease of miR-16 expression was observed at 5 d～14dingroupT (5d (0.91±0.04),7 d (0.77±0.01),10 d (0.73±0.03),14d (0.42±0.08)) (all P＜ 0.05).Compared with SH group,PWMT was significantly decreased in BC and BP groups at 5 d～ 14 d (P＜0.05),and in group BM at 5 d～ 10 d(P＜0.05).Compared with BP group,PWMT was significantly higher in BM group at 10～12 d(P＜0.05).Compared with SH group,the expression of BDNF was significantly increased in BN and BP groups((2.78±0.31),(2.34±0.23)) (all P＜0.01).Compared with BP group,the expression of BDNF was significantly decreased in BM group (1.42±0.16) (P＜0.01).Conclusion miR-16 is downregulated in spinal cord of bone cancer pain rats,while intrathcal injection of miR-16 mimics can attenuate the pain behaviors in a rat model of bone cancer pain and decrease the expression of BDNF.miR-16 may modulate bone cancer pain through BDNF.

Objective To explore the mechanism by which zinc-regulated transporters, iron-regulated transporter-likeprotein 4 (ZIP4) regulates glycolysis and its impact on tumor progression in cholangiocarcinoma (CCA), providing a theoretical basis for targeted therapy of CCA. Methods ZIP4 expression in CCA was analyzed using the GEPIA database. Immuno-histochemistry (IHC) was used to detect ZIP4 expression in 20 paired CCA and adjacent non-tumor tissues. Stable ZIP4-overexpressing CCA cell lines (ZIP4-OE) were established. Gene set enrichment analysis was used to screen differentially expressed genes and pathways in ZIP-OE CCA cells. ZIP4, N-myc proto-oncogene protein (MYCN), and histone-lysine N-methyltransferase 2E (KMT2E) were knocked down using small interfering RNAs (siRNAs). The expression of glycolysis-related gene (glucose transporter 1 [Glut1], hexokinase 2 [HK2], and lactate dehydrogenase A [LDHA]) was measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Glycolytic activity was assessed by measuring the extracellular acidification rate (ECAR). Cell proliferation was evaluated using colony formation assays, and cell migration was assessed using Transwell assays. A xenograft mouse model was constructed to examine CCA tumor growth. Protein levels of ZIP4, KMT2E, H3K4me3 (tri-methylation of lysine 4 on histone H3), and MYCN were detected by Western blotting. Results GEPIA database analysis and IHC results confirmed significantly higher ZIP4 expression levels in CCA tissues compared to adjacent non-tumor tissues (P＜0.05). Compared to the control group, the ZIP4-OE group exhibited a significantly increased ECAR, along with significantly enhanced proliferation and migration abilities (P＜0.01). Conversely, knockdown of ZIP4 suppressed CCA cells proliferation and migration. GEPIA analysis indicated that ZIP4 upregulates the transcription of oncogene MYCN, as well as glycolysis-related genes. Knockdown of MYCN abolished the ZIP4 overexpression-induced upregulation of Glut1, HK2, and LDHA gene transcription, reduced glycolysis, and significantly inhibited CCA cell proliferation and migration (P＜0.05). Mechanistic studies demonstrated that ZIP4 increases H3K4me3 level via KMT2E, leading to MYCN transcription. Knockdown of KMT2E in CCA cells suppressed the ZIP4 overexpression-induced enhancement in H3K4me3 modification, resulting in MYCN downregulation and significantly reduced CCA cells proliferation and migration (P＜0.05). Conclusions ZIP4 upregulates H3K4me3 modification through KMT2E, which recruits transcription factors to activate the transcription of MYCN. This subsequently enhances cellular glycolysis and promotes the proliferation and migration of CCA cells.