Agomir

The human LSCC cell lines TU177, TU686, TU212, LSC-1 and Hep-2 were obtained from Shanghai Institute of Biological Science Cell Center. Hep-2 cells were cultured in F12K medium (Invitrogen), and the remaining cells were cultured in RPMI 1640 medium (Invitrogen).
Cell transfection was carried out when cells in culture reached 60–80% confluence using Lipofectamine® 2000. Small interfering RNAs (siRNAs) targeting circZNF609#1 (si-circZNF609#1, 5ʹ-GTCAAGTCTGAAAAGCAATGA-3ʹ), circZNF609#2 (5ʹ-TGCCCTAGTACTACCCTGCAT-3ʹ) and circZNF609#3 (5’-TTGACTGCATCGTAGCCAAAC-3’) and negative control (si-NC, 5’-UUCUCCGAACGUGUCACGUTT-3’) were purchased from Shanghai GenePharma Co., Ltd. The miR-134-5p mimetic, agomir and controls were purchased from Guangzhou RiboBio Co., Ltd. The transfection concentrations of oligonucleotides were as follows: si-NC, 40 nM; si- circZNF609, 40 nM; si-NC, 40 nM; si-EGFR, 40 nM; miR-134-5p mimetic, 50 nM; and miRNA control, agomir and miRNA control, 50 nM. Lipofectamine® 2000 Reagent and si-RNAs or miR-mimic were diluted with serum-free DMEM medium, mixed together and incubated at room temperature for 20 min. This solution was subsequently added to LSC-1 and Hep-2 cells for transfection at 37°C for 4–6 h in a humidified incubator containing 5% CO₂.

We used Sprague-Dawley rats aged between 6 and 7 weeks (n = 36 rats) weighing approximately between 225 and 250 g. The animal study protocols were sanctioned by the Animal Ethical Review Board of Zhongnan Hospital of Wuhan University, China. The rats were housed under pathogen-free standard lab conditions at 25℃ with a relative humidity of 50–60% in 12 h dark and light cycle. The rats were provided free access to food and water. The rats were divided into four groups (n = 6/group), namely, the SCI-induced group, sham-operated group, miR-142-3p agomir group and miR-142-3p antagomir group (NC group). The sham-operated rats were subjected to laminectomy at T10 without the weight-drop injury; similarly, the SCI group rats underwent T10 laminectomy by the New York spinal segment impactor, and miR-142-3p antagomir-(NC) and agomir-treated rats were subjected to SCI and were given dose intrathecally (1 µL/h, 20  nmol/mL). The agomir and antagomir were obtained from RiboBio (Guangzhou, China).

Thirty-six female WT C57BL/6 mice aged 6 weeks were equally randomized to 6 groups, of which 5 groups underwent OVX operation, and the remaining group underwent sham operation. Two months after operation, the 5 OVX groups of mice respectively received 10nmol/per mouse of agomir-106b, con-agomir-106b, mut-antagomir-106b, antagomir-106b or 0.2ml PBS through the tail vein on day 1-3 for 3 consecutive weeks. A section of coccygeal vertebrae were get out from all the mice before injection and at 3, 4.5 and 6 weeks after the first injection (Figure 7A). Six weeks after the first injection, mice were euthanized. Bone and serum samples were collected for further experiments. The agomir, con-agomir, mut-antagomir and antagomir were all purchased from RiboBio (RiboBio, Guangzhou, China). All procedures involving the mice were approved by the Animal Management Committeeof the Second Military Medical University.