LOXL1-AS1 contributes to the proliferation and migration of laryngocarcinoma cells through miR-589-5p/TRAF6 axis

Abstract

Background: LOXL1-AS1 is a long non-coding RNA (lncRNA) that plays crucial roles in various cancers. However, the functional role of LOXL1-AS1 in laryngocarcinoma remains unclear. Thus we planned to probe into the function and underlying mechanism of LOXL1-AS1 in laryngocarcinoma.

Methods: Gene expression was evaluated in laryngocarcinoma cells using RT-qPCR. The ability of cell proliferation and migration was assessed by CCK8, colony formation, wound healing and transwell assays. The interaction among LOXL1-AS1, miR-589-5p and TRAF6 was detected by Ago2-RIP, RNA pull down and luciferase reporter assays.

Results: LOXL1-AS1 was overexpressed in laryngocarcinoma cells. Silencing of LOXL1-AS1 suppressed cell proliferation, migration and EMT in laryngocarcinoma. Moreover, miR-589-5p, the downstream of LOXL1-AS1, directly targeted TRAF6 in laryngocarcinoma. Importantly, LOXL1-AS1 augmented TRAF6 expression in laryngocarcinoma cells by sequestering miR-589-5p. Besides, miR-589-5p worked as a tumor-inhibitor while TRAF6 functioned as a tumor-facilitator in laryngocarcinoma. Of note, rescue experiments both in vitro and in vivo validated that LOXL1-AS1 aggravated the malignancy in laryngocarcinoma by targeting miR-589-5p/TRAF6 pathway.

Conclusions: LOXL1-AS1 promotes the proliferation and migration of laryngocarcinoma cells through absorbing miR-589-5p to upregulate TRAF6 expression.

Keywords: LOXL1-AS1; Laryngocarcinoma; TRAF6; miR-589-5p.

Fig. 1

LOXL1-AS1 aggravates laryngocarcinoma cell proliferation, migration and EMT. a RT-qPCR analysis detected the overexpressed LOXL1-AS1in laryngocarcinoma cell lines (SNU-899, SNU-46, AMC-HN-8, Tu-177 and M4E) compared to normal pharyngal epithelial cell line (NP69). b The knockdown efficiency of LOXL1-AS1 was examined using RT-qPCR after transfecting three shRNAs targeting LOXL1-AS1 into Tu-177 and M4E cells. c, d CCK8 and colony formation assays detected the hindered cell proliferation in response to LOXL1-AS1 silencing. e Wound healing assay demonstrated the suppression on migration ability induced by LOXL1-AS1 silencing in Tu-177 and M4E cells. Relative wound width was calculated using the relative value of wound widths at 24 h to that at 0 h. f Transwell assay determined the hinder migration of Tu-177 and M4E cells with LOXL1-AS1 inhibition. G. IF examined the elevated expression of E-cadherin and the reduced level of N-cadherin in Tu-177 and M4E cells under LOXL1-AS1 inhibition. The standard deviation of control groups in a and b was calculated by 2^−ΔΔCt method as follow: ΔCt (Control) = ΔCt (target, Control) – ΔCt (reference, Control); mean ΔCt (Control) = {ΔCt−1 (Control) + ΔCt−2 (Control) + ΔCt−3 (Control)}/3; ΔΔCt (Control) = ΔCt (Control) − mean ΔCt (Control). *p < 0.05, **p < 0.01

Fig. 2

LOXL1-AS1 sponges miR-589-5p in laryngocarcinoma cells. a, b Nuclear separation and FISH experiments confirmed the main cytoplasmic location of LOXL1-AS1 in both Tu-177 and M4E cells. c RNA pull down assay detected that the high enrichment of miR-589-5p but not miR-423-5p in Bio-LOXL1-AS1 groups. d Ago2-RIP experiments illustrated the strong enrichment of LOXL1-AS1 and miR-589-5p in anti-Ago2 groups. e A binding site of miR-589-5p to LOXL1-AS1 was presented by using starBase. f Luciferase reporter assays examined the declined luciferase activity of pmirGLO/LOXL1-AS1-WT but not pmirGLO or pmirGLO/LOXL1-AS1-Mut under the overexpression of miR-589-5p. The standard deviation of control groups in c, d and f was calculated by 2^−ΔΔCt method as follow: ΔCt (Control) = ΔCt (target, Control) – ΔCt (reference, Control); mean ΔCt (Control) = {ΔCt−1 (Control) + ΔCt−2 (Control) + ΔCt−3 (Control)}/3; ΔΔCt (Control) = ΔCt (Control)−mean ΔCt (Control). **p < 0.01

Fig. 3

MiR-589-5p restrains cell proliferation, migration and EMT in laryngocarcinoma. a, b CCK8 and colony formation assays examined the repressive impact of overexpressed miR-589-5p on the proliferation of Tu-177 and M4E cells. c, d. Wound healing and transwell assays manifested the hampered capacity of migration in Tu-177 and M4E cells in face of up-regulation of miR-589-5p. Relative wound width in c was calculated using the relative value of wound widths at 24 h to that at 0 h. e IF assessed the augmented expression of E-cadherin and declined level of N-cadherin in Tu-177 and M4E cells with up-regulated miR-589-5p. The standard deviation of control groups in Figure was calculated by 2^−ΔΔCt method as follow: ΔCt (Control) = ΔCt (target, Control) – ΔCt (reference, Control); mean ΔCt (Control) = {ΔCt−1 (Control) + ΔCt−2 (Control) + ΔCt−3 (Control)}/3; ΔΔCt (Control) = ΔCt (Control)−mean ΔCt (Control). **p < 0.01

Fig. 4

MiR-589-5p directly targets TRAF6 in laryngocarcinoma cells. a RIP experiments demonstrated the obvious enrichment of TRAF6 but not SORT1, ABL2 or NPTN in Bio-miR-589-5p groups. b Ago2-RIP assays examined that the apparent enrichment of LOXL1-AS1, miR-589-5p and TRAF6 in anti-Ago2 groups. c RNA pull down assay disclosed the high enrichment of LOXL1-AS1 and TRAF6 in Bio-miR-589-5p-WT groups. d Luciferase reporter assays detected the luciferase activity of pmirGLO/3′UTR-WT/Mut in Tu-177 and M4E cells co-transfected with miR-NC, miR-589-5p mimics, or miR-589-5p mimics + LOXL1-AS1. **p < 0.01

Fig. 5

LOXL1-AS1 drives laryngocarcinoma cell proliferation and migration through miR-589-5p/TRAF6 axis. Rescue experiments were conducted in Tu-177 cells transfected with sh-NC, sh/LOXL1-AS1#1, sh/LOXL1-AS1#1 + inhibitor and sh/LOXL1-AS1#1 + inhibitor + sh/TRAF6. a, b CCK8 and colony formation assays detected the changes on the proliferation of Tu-177 cells in indicated groups. c, d Wound healing and transwell assays detected the migration ability of indicated Tu-177 cells. Relative wound width in c was calculated using the relative value of wound widths at 24 h to that at 0 h. **p < 0.01