Mechanism underlying long non‑coding RNA ILF3‑AS1‑mediated inhibition of cervical cancer cell proliferation, invasion and migration, and promotion of apoptosis

Abstract

Long non‑coding RNA ILF3 divergent transcript (ILF3‑AS1) displays a tumor‑suppressing effect. StarBase predicted that the potential target microRNA (miR) of ILF3‑AS1 was miR‑454‑3p; therefore, the present study investigated the effect of ILF3‑AS1 and its target miR‑454‑3p on cervical cancer (CC). Gene Expression Profiling Interactive Analysis was used to predict the expression of ILF3‑AS1 in CC and the overall survival rate of patients. The present study demonstrated that ILF3‑AS1 expression was significantly downregulated in human CC tissues and cells compared with adjacent tissues (ANTs) and normal cervical epithelial cells (NCEs), respectively. Patients with CC with high ILF3‑AS1 expression displayed higher survival rates compared with patients with low ILF3‑AS1 expression. Cell viability, apoptosis, migration and invasion were detected by performing Cell Counting Kit‑8, flow cytometry, wound healing and Transwell assays, respectively. Compared with the negative control (NC) group, ILF3‑AS1 overexpression significantly inhibited CC cell viability and migration, but significantly increased CC cell apoptosis. Moreover, ILF3‑AS1 overexpression significantly upregulated E‑Cadherin expression levels, but significantly downregulated N‑Cadherin and snail family transcriptional repressor 1 expression levels compared with the NC group. miR‑454‑3p expression was negatively correlated with ILF3‑AS1, and highly expressed in CC tissues and cells compared with ANTs and NCEs, respectively. PTEN, which was predicted and verified as the target gene for miR‑454‑3p, was significantly downregulated in CC tissues and cells compared with ANTs and NCEs, respectively. ILF3‑AS1 expression was positively correlated with PTEN expression, and ILF3‑AS1 overexpression partially reversed the inhibitory effect of miR‑454‑3p on PTEN expression. In conclusion, the present study indicated that ILF3‑AS1 inhibited CC cell proliferation and migration, and promoted CC cell apoptosis by inhibiting epithelial‑mesenchymal transition, and ILF3‑AS1 overexpression partially reversed the inhibitory effect of miR‑454‑3p on PTEN expression.

Keywords: PTEN; cervical cancer; lncRNA ILF3 divergent transcript; microRNA‑454‑3p.

Figure 1.

Gene Expression Profiling Interactive Analysis database analysis of ILF3-AS1 expression levels in tumor tissues. Red dots represent the transcripts levels of ILF3-AS1 in tumor tissues. Green dots represent the transcripts levels of ILF3-AS1 in healthy tissues. Green text indicates the tumours with the lowest ILF3-AS1 expression. Red text indicates the tumours with the highest ILF3-AS1 expression. ILF3-AS1, ILF3 divergent transcript; T, tumor; N, healthy.

Figure 2.

ILF3-AS1 expression is downregulated in CC tissues. (A) GEPIA database analysis demonstrated that ILF3-AS1 expression was downregulated in CC tissues compared with healthy tissues. The red box represents CC tissues and the gray box represents healthy tissues. (B) Low ILF3-AS1 expression levels were associated with the stage of CC. (C) GEPIA database analysis of overall survival in patients with CC. (D) Reverse transcription-quantitative PCR was performed to measure ILF3-AS1 expression levels in CC tissues (stage I/II=15; stage III/IV=17) and ANTs (n=32). All experiments were repeated three times. *P<0.05 and ***P<0.001 vs. healthy tissues or ANTs; ^{^^^}P<0.001 vs. stage I/II. ILF3-AS1, ILF3 divergent transcript; CC, cervical cancer; GEPIA, Gene Expression Profiling Interactive Analysis; ANT, adjacent tissue; lncRNA, long non-coding RNA; CESC, cervical and endocervical cancer; T, tumor; N, healthy; TPM, transcripts per million.

Figure 3.

ILF3-AS1 inhibits CC cell proliferation and promotes CC cell apoptosis. (A) ILF3-AS1 expression levels in NCEs and CC cells were detected via RT-qPCR. Transfection efficiency of ILF3-AS1 in (B) SiHa and (C) HeLa cells. (D) SiHa and (E) HeLa cell viability were assessed by performing Cell Counting Kit-8 assays. (F) SiHa and (G) HeLa cell proliferation were assessed by conducting cell colony formation assays (magnification, ×1). (H) SiHa and (I) HeLa cell apoptosis was assessed via flow cytometry. All experiments were repeated three times. ***P<0.001 vs. NCEs; ^{^}P<0.05 and ^{^^^}P<0.001 vs. NC. ILF3-AS1, ILF3 divergent transcript; CC, cervical cancer; NCE, normal cervical epithelial cell line; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; lncRNA, long non-coding RNA; OD, optical density.

Figure 4.

ILF3-AS1 inhibits CC metastasis by inhibiting EMT. (A) SiHa and (B) HeLa cell migration were assessed by performing the wound healing assay. (C) SiHa and (D) HeLa cell invasion were assessed by conducting the Transwell invasion assay. Expression levels of EMT-associated proteins in (E) SiHa and (F) HeLa cells were determined via western blotting. All experiments were repeated three times. ^{^^^}P<0.001 vs. NC. ILF3-AS1, ILF3 divergent transcript; CC, cervical cancer; EMT, epithelial-mesenchymal transition; NCE, normal cervical epithelial cell line; NC, negative control; E-cad, E-cadherin; N-cad, N-cadherin; Snail, snail family transcriptional repressor 1.

Figure 5.

miR-454-3p expression is upregulated in CC cells and is negatively correlated with ILF3-AS1. (A) StarBase predicted that miR-454-3p was the targeted miRNA of ILF3-AS1. The sequences of ILF3-AS1 WT, ILF3-AS1 MUT and miR-454-3p. Dual-luciferase reporter assays were performed to verify the relationship between ILF3-AS1 and miR-454-3p in (B) SiHa and (C) HeLa cells. (D) RT-qPCR was performed to measure miR-454-3p expression levels in ANTs and CC tissues. Pearson's correlation coefficient was used to analyze the correlation between miR-454-3p and ILF3-AS1 expression in (E) ANTs, (F) stage I/II CC tissues and (G) stage III/IV CC tissues. (H) RT-qPCR was performed to measure miR-454-3p expression levels in NCEs and CC cells. Transfection efficiency of miR-454-3p mimic in (I) SiHa and (J) HeLa cells. All experiments were repeated three times. ***P<0.001 vs. mimic control; ^{^^^}P<0.001 vs. ANT; ^###P<0.001 vs. stage I/II; ^&&&P<0.001 vs. NCEs; ⁺⁺⁺P<0.001 vs. blank. miR/miRNA, microRNA; CC, cervical cancer; ILF3-AS1, ILF3 divergent transcript; WT, wild-type; MUT, mutant; RT-qPCR, reverse transcription-quantitative PCR; ANT, adjacent tissue; NCE, normal cervical epithelial cell line.

Figure 6.

PTEN is a target gene for miR-454-3p and is correlated with miR-454-3p and ILF3-AS1 expression. (A) TargetScan was used to predict the target gene of miR-454-3p. Dual-luciferase reporter assays were performed to verify the relationship between PTEN and miR-454-3p in (B) SiHa and (C) HeLa cells. (D) RT-qPCR was performed to measure miR-454-3p expression levels in ANTs and CC tissues. Pearson's correlation coefficient was used to analyze the correlation between miR-454-3p and PTEN expression in (E) ANT, (F) stage I/II CC tissues and (G) stage III/IV CC tissues. Pearson's correlation coefficient was used to analyze the correlation between PTEN and ILF3-AS1 in (H) ANTs, (I) stage I/II CC tissues and (J) stage III/IV CC tissues. RT-qPCR was performed to measure (K) miR-454-3p expression levels in SiHa cells following transfection, (L) PTEN expression levels in NCEs and CC cells, and PTEN expression levels in (M) SiHa and (N) HeLa cells following transfection. All experiments were repeated three times. ***P<0.001 vs. mimic control; ^{^^^}P<0.001 vs. ANT; ^###P<0.001 vs. stage I/II; ^&&&P<0.001 vs. NCEs; ^∆∆P<0.01; ^∆∆∆P<0.001 vs. control; ^§§§P<0.001 vs. ILF3-AS1; ⁺⁺⁺P<0.001 vs. miR-454-3p mimic. miR, microRNA; ILF3-AS1, ILF3 divergent transcript; RT-qPCR, reverse transcription-quantitative PCR; ANT, adjacent tissue; CC, cervical cancer; NCE, normal cervical epithelial cell line; WT, wild-type; MUT, mutant; UTR, untranslated region.