Knockdown of circ_0001883 may inhibit epithelial-mesenchymal transition in laryngeal squamous cell carcinoma via the miR-125-5p/PI3K/AKT axis

Abstract

Laryngeal squamous cell carcinoma (LSCC) is a malignant tumor with increasing incidence and poor prognosis. Circular RNAs (circRNAs) are known to modulate tumorigenesis and cancer development that may function through microRNAs (miRs). The aim of the present study was to investigate the functional roles of circ_0001883 in LSCC and the underlying molecular mechanism. The expression of circ_0001883 was upregulated and measured using reverse transcription-quantitative PCR (RT-qPCR) and RNase R. miR-125b-5p expression was downregulated in LSCC tissues and cells as determined using RT-qPCR. Subsequently, knockdown of circ_0001883 inhibited LSCC cell migration, invasion and epithelial-mesenchymal transition (EMT), which were tested by wound healing assays, Transwell assays and western blotting, respectively. Bioinformatics analysis predicted that circ_0001883 was a sponge of miR-125b-5p, which was verified using a dual-luciferase reporter assay. Knockdown of circ_0001883 played a functional role by sponging miR-125b-5p. Additionally, circ_0001883 and miR-125b-5p influenced phosphorylation of PI3K and AKT, detected via western blotting. In an in vivo study, knockdown of circ_0001883 reduced tumor volume and weight in mice, along with enhanced miR-125b-5p and E-cadherin expression levels, and decreased N-cadherin, phosphorylated (p)-PI3K/PI3K and p-AKT/AKT ratios. In conclusion, knockdown of circ_0001883 inhibited cell migration, invasion and EMT of LSCC by sponging miR-125b-5p. This is hypothesized to be via the PI3K/AKT signaling pathway, which suggested that circ_0001883 has potential for LSCC therapy.

Keywords: circ_0001883; epithelial-mesenchymal transition; invasion; laryngeal squamous cell carcinoma; microRNA-125b-5p; migration.

Figure 1

Expression of circ_0001883 and miR-125b-5p expression is increased and decreased, respectively, in LSCC tissues and cells. (A) circ_0001883 expression in 33 pairs of LSCC tissues and corresponding normal tissues was measured using RT-qPCR. (B) Expression of circ_0001883 was measured using RT-qPCR in human bronchial epithelial cell line 16HBE and LSCC cell lines (AMC-HN-8 and Tu686). circ_0001883 and its matching linear mRNA expression were tested in (C) AMC-HN-8 and (D) Tu686 cells, which were treated with RNase R. (E) miR-125b-5p level was detected in tumor and matched non-tumor tissues (n=33) by RT-qPCR. (F) Expression level of miR-125b-5p was detected using RT-qPCR in 16HBE, AMC-HN-8 and Tu686 cell lines. ^*P<0.05. circ, circular; miR, microRNA; LSCC, laryngeal squamous cell carcinoma; RT-qPCR, reverse transcription-quantitative PCR.

Figure 2

circ_0001883 depletion inhibits the migration, invasion and EMT in LSCC cells. (A) Transfection efficiency of AMC-HN-8 and Tu686 cells measured using RT-qPCR. Cell migration capability was tested and wound closure rate was quantified in (B) AMC-HN-8 and (C) Tu686 cells after transfection with si-circ_0001883#3 at 0 and 24 h. Scale bar, 100 µm. Cell invasion was detected using Transwell assays after transfection of si-circ_0001883#3 for 24 h in (D) AMC-HN-8 and (E) Tu686 cells, and the number of invaded cells was quantified. Scale bar, 50 µm. (F) Expression levels of EMT-related markers MMP3, E-cadherin and N-cadherin were detected using western blotting. GAPDH was used as the internal control in LSCC cells. ^*P<0.05 vs. si-NC. circ, circular; EMT, epithelial-mesenchymal transition; LSCC, laryngeal squamous cell carcinoma; RT-qPCR, reverse transcription-quantitative PCR; si-, small interfering; NC, negative control.

Figure 3

circ_0001883 acts as a sponge for miR-125b-5p. (A) miR-125b-5p was predicted using the StarBase v2.0 online database to bind with circ_0001883-WT but not with circ_0001883-MUT. (B) Transfection efficiency of miR-125b-5p mimics in AMC-HN-8 and Tu686 cells. Dual-luciferase reporter assay was performed to test the targeted relationship by co-transfection with circ_0001883-WT or circ_0001883-MUT together with miR-NC or miR-125b-5p in (C) AMC-HN-8 and (D) Tu686 cells. (E) Spearman’s rank correlation analysis was performed to assess the relationship between circ_0001883 and miR-125b-5p expression in LSCC tissues. (F) Expression of miR-125b-5p was tested via reverse transcription-quantitative PCR after transfection of si-circ_0001883#3. ^*P<0.05 vs. miR-NC or si-NC. WT, wild-type; MUT, mutant; miR, microRNA; LSCC, laryngeal squamous cell carcinoma; NC, negative control.

Figure 4

Anti-miR-125b-5p rescues the effects of si-circ_0001883 on migration, invasion and epithelial-mesenchymal transition. Transfection efficiency was tested using reverse transcription-quantitative PCR after (A) transfection of anti-miR-125b-5p, and (B) co-transfection of si-circ_0001883#3 and anti-miR-125b-5p. (C) AMC-HN-8 and Tu686 cell migration capability was tested using a wound healing assay, and (D) wound closure rate was quantified. Scale bar, 100 µm. (E) Invasion of AMC-HN-8 and Tu686 cells was detected using Transwell assays and (F) the number of invading cells was quantified. Scale bar, 50 µm. (G) MMP3, E-cadherin and N-cadherin levels were measured using western blotting and normalized to GAPDH. ^*P<0.05. miR, microRNA; circ, circular; si-, small interfering; NC, negative control.

Figure 5

Knockdown of circ_0001883 suppresses PI3K/AKT pathway by sponging miR-125b-5p. In (A) AMC-HN-8 and (B) Tu686 cells, the protein expression of p-PI3K, PI3K, p-AKT and AKT was detected using western blotting. GAPDH was used for normalization. ^*P<0.05. circ, circular; miR, microRNA; p-, phosphorylated; si-, small interfering; NC, negative control.

Figure 6

Downregulated circ_0001883 inhibits tumor epithelial-mesenchymal transition through the miR-125b-5p/PI3K/AKT axis in vivo. (A) From 7 days post-transfection, tumor volume was measured every 4 days until 27 days via testing tumor length and width. (B) Tumor in mice was dissected, images were captured and tumors were weighed. (C) circ_0001883 and (D) miR-125b-5p expression levels were measured using reverse transcription-quantitative PCR in xenograft tumors. (E) E-cadherin and (F) N-cadherin levels were detected by immunohistochemistry in mice tumors. Scale bar, 50 µm. (G) Protein expression of p-PI3K, PI3K, p-AKT, and AKT was detected using western blotting and quantified through normalization of GAPDH. ^*P<0.05 vs. sh-NC or as indicated. circ, circular; miR, microRNA; p-, phosphorylated; sh-, short hairpin; NC, negative control.