lincRNA‑p21 inhibits the progression of non‑small cell lung cancer via targeting miR‑17‑5p

Abstract

Non‑small‑cell lung cancer (NSCLC) is well established as one of the major subtypes of human lung cancer. NSCLC is characterized by a high incidence rate and poor patient prognosis. Previous studies have identified that long intergenic non-coding RNA (lincRNA) serves a key role in the development of tumor and malignant metastasis. However, the majority of the underlying mechanisms for lincRNA deregulation in various diseases, including cancer and diabetes, have not been completely elucidated. In the present study, the deregulation of lincRNA‑p21 in NSCLC tumor tissues in comparison to adjacent healthy tissues was examined using reverse transcription‑quantitative polymerase chain reaction. Furthermore, the effect of lincRNA‑p21 overexpression and knockdown on different NSCLC cell lines was further investigated in vitro. The association between lincRNA‑p21 expression and microRNA (miR)‑17‑5p level in NSCLC tumor cells was also investigated to clarify the underlying mechanism. The influence of miR‑17‑5p on different NSCLC cell lines A549 and PC9 were also examined in vitro using miR‑17‑5p mimics and inhibitors. Bioinformatics and luciferase assays were conducted to verify the direct binding sites on lincRNA‑p21 for miR‑17‑5p. The results demonstrated that there was a significant low‑expression of lincRNA‑p21 in NSCLC tumor tissues, and lincRNA‑p21 effectively inhibited the progression of lung cancer cells by suppressing cell proliferation and migration and promoting cell apoptosis. An evident negative association between lincRNA‑p21 and miR‑17‑5p expression was observed, and the inhibitory effect of overexpressed lincRNA‑p21 on lung cancer cells was counteracted by miR‑17‑5p. Bioinformatics and luciferase reporter analysis results confirmed that miR‑17‑5p is a direct target for lincRNA‑p21. The present study provides evidence for lincRNA‑p21 to inhibit the progression of NSCLC via direct targeting of a miR‑17‑5p associated signaling pathway.

Figure 1.

lincRNA-p21 expression in normal and NSCLC tumor tissues. lincRNA-p21 expression levels in 40 pairs of lung cancer tissue samples and adjacent tissue samples were detected by reverse transcription-quantitative polymerase chain reaction. NSCLC, non-small cell lung carcinoma; lincRNA, long intergenic non-coding RNA.

Figure 2.

lincRNA-p21 expression in NSCLC cell lines. (A) The expression of lincRNA-p21 in A549 and PC9 cells were detected by RT-qPCR. The lincRNA-p21 expression in A549 cells was set as 1, and the lincRNA-p21 expression in PC9 cells was normalized to that of A549 cells. (B) A549 cells were seeded into 24-well plates and then transfected with empty vector (vector) or lincRNA-p21 overexpression plasmid (lincRNA-p21). The relative lincRNA-p21 expression in overexpression plasmid group was normalized to the empty vector group. (C) PC9 cells were seeded into 24-well plates and then transfected with scrambled siRNA (NC) or lincRNA-p21 siRNA (si-lincRNA-p21). The lincRNA-p21 expression in lincRNA-p21 siRNA group was normalized to the scrambled siRNA group. **P<0.01. RT-qPCR, reverse transcription quantitative polymerase chain reaction; NSCLC, non-small cell lung carcinoma; lincRNA, long intergenic non-coding RNA; siRNA/si, small interfering RNA; NC, negative control.

Figure 3.

Effects of lincRNA-p21 on lung cancer cell proliferation, apoptosis and migration. (A) A549 and PC9 cells were transfected with lincRNA-p21 overexpression plasmid or lincRNA-p21 siRNA for 72 h, and the cell proliferation was detected using a Cell Counting Kit-8 assay. (B) The cells were transfected with lincRNA-p21 overexpression plasmid or lincRNA-p21 siRNA for 72 h, and cell apoptosis was detected by flow cytometric analysis. (C) The transfected cells were subjected to Transwell analysis for 24 h (magnification ×200). *P<0.05 and **P<0.01 vs. vector group; ^#P<0.05 and ^##P<0.01 vs. NC group. NSCLC, non-small cell lung carcinoma; lincRNA, long intergenic non-coding RNA; siRNA/si, small interfering RNA; NC, negative control; PI, propidium iodide.

Figure 3.

Effects of lincRNA-p21 on lung cancer cell proliferation, apoptosis and migration. (A) A549 and PC9 cells were transfected with lincRNA-p21 overexpression plasmid or lincRNA-p21 siRNA for 72 h, and the cell proliferation was detected using a Cell Counting Kit-8 assay. (B) The cells were transfected with lincRNA-p21 overexpression plasmid or lincRNA-p21 siRNA for 72 h, and cell apoptosis was detected by flow cytometric analysis. (C) The transfected cells were subjected to Transwell analysis for 24 h (magnification ×200). *P<0.05 and **P<0.01 vs. vector group; ^#P<0.05 and ^##P<0.01 vs. NC group. NSCLC, non-small cell lung carcinoma; lincRNA, long intergenic non-coding RNA; siRNA/si, small interfering RNA; NC, negative control; PI, propidium iodide.

Figure 4.

lincRNA-p21 regulates the expression of miR-17-5p in lung cancer cells. (A) miR-17-5p expression levels in different cell groups following transfection with lincRNA-p21 overexpression plasmid or lincRNA-p21 siRNA for 24 h. (B) miR-17-5p expression levels in different cell groups following treatment with miR-17-5p mimics or inhibitor for 24 h. (C) Western blot analysis for Bcl-2 and MMP9 protein expression levels in different cell groups following treatments with miR-17-5p mimics or inhibitor for 24 h. **P<0.01 vs. vector group. ^#P<0.05 and ^##P<0.01 vs. NC group, ^&&P<0.01 vs. lincRNA-p21+miR-17-NC, ^@@P<0.01 vs. si-lincRNA-p21+miR-17-NC. lincRNA, long intergenic non-coding RNA; siRNA/si, small interfering RNA; NC, negative control; miR, microRNA; Bcl-2, B-cell lymphoma-2; MMP9, matrix metalloproteinase.

Figure 5.

Effects of miR-17-5p on lung cancer cell proliferation, apoptosis and migration. (A) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 72 h, and cell proliferation was detected using a Cell Counting Kit-8 assay. (B) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 72 h, and the cells apoptosis were detected by flow cytometric analysis. (C) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 24 h, and the cells were subjected to Transwell analysis (magnification, ×200). *P<0.05 and **P<0.01 vs. NC group. lincRNA, long intergenic non-coding RNA; siRNA/si, small interfering RNA; NC, negative control; miR, microRNA; PI, propidium iodide; OD, optical density.

Figure 5.

Effects of miR-17-5p on lung cancer cell proliferation, apoptosis and migration. (A) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 72 h, and cell proliferation was detected using a Cell Counting Kit-8 assay. (B) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 72 h, and the cells apoptosis were detected by flow cytometric analysis. (C) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 24 h, and the cells were subjected to Transwell analysis (magnification, ×200). *P<0.05 and **P<0.01 vs. NC group. lincRNA, long intergenic non-coding RNA; siRNA/si, small interfering RNA; NC, negative control; miR, microRNA; PI, propidium iodide; OD, optical density.

Figure 6.

miR-17-5p is a direct gene target of lincRNA-p21. (A) Bioinformatics results for the direct binding sites of lincRNA-p21 and miR-17-5p. (B) Cells in different groups were transfected with luciferase reporters, internal control and indicated RNAs. At 72 h following transfection, luciferase activities were monitored using a luciferase reporter assay system. **P<0.01 (lincRNA-p21 vs. lincRNA-p21+miR-17-5p mimics). (C) lincRNA-p21 overexpression plasmid- or lincRNA-p21 siRNA-transfected lung cancer cells were treated miR-17-5p mimics or inhibitor for 72 h, and the protein SMOC1 expression was detected by western blot analysis. **P<0.01 vs. vector group; ^##P<0.01 vs. lincRNA-p21 group; ^&&P<0.01 vs. NC group; ^@@P<0.01 vs. si-lincRNA-p21 group. lincRNA, long intergenic non-coding RNA; siRNA/si, small interfering RNA; NC, negative control; miR, microRNA; SMOC1, SPARC-related modular calcium binding 1.

Figure 7.

SMOC1 is a direct gene target of miR-17-5p. (A) Bioinformatics results for the direct binding sites position 127–133 on SMOC1 for miR-17-5p. (B) Luciferase reporter analysis for A549 and PC9 cell groups with wild or mutant type SMOC1 3′UTR, and miR-NC or miR-17. (C) Relative SMOC1 expression between 40 pairs of lung cancer tissue samples and adjacent normal tissues. (D) The expression level of SMOC1 demonstrated by representative immunohistochemical staining of lung cancer tissues and adjacent normal tissues. (magnification, ×200) *P<0.01 vs. 3′UTR wt+miR-NC. SMOC1, SPARC-related modular calcium binding 1; UTR, untranslated region; lincRNA, long intergenic non-coding RNA; siRNA/si, small interfering RNA; NC, negative control; miR, microRNA.

Figure 8.

Inhibitory effect of lincRNA-p21 on lung tumor growth in vivo. (A) Tumor volumes of different groups were monitored over 4 weeks. **P<0.01 vs. vector group. (B) Tumor tissues from vector control, lincRNA-p21 treated, lincRNA-p21 and miR-17-5p treated groups were isolated and (C) weighed. The relative levels of (D) lincRNA-p21 and (E) miR-17 in tumors isolated from the vector, lincRNA-p21 and lincRNA-p21+miR-17 mimics groups, as demonstrated by RT-qPCR. **P<0.01 vs. vector control group, ^##P<0.01 vs. lincRNA-p21 group. (F) The SMOC1 protein expression in tumors isolated from the vector, lincRNA-p21 and lincRNA-p21+miR-17 mimics groups, as demonstrated by RT-qPCR. *P<0.05 vs. vector control group, ^#P<0.05 vs. lincRNA-p21 group. RT-qPCR, reverse transcription quantitative polymerase chain reaction; lincRNA, long intergenic non-coding RNA; siRNA/si, small interfering RNA; NC, negative control; miR, microRNA; SMOC1, SPARC-related modular calcium binding 1.