MicroRNA-138 Inhibits Cell Growth, Invasion, and EMT of Non-Small Cell Lung Cancer via SOX4/p53 Feedback Loop

Abstract

Many studies have shown that downregulation of miR-138 occurs in a variety of cancers including non-small cell lung cancer (NSCLC). However, the precise mechanisms of miR-138 in NSCLC have not been well clarified. In this study, we investigated the biological functions and molecular mechanisms of miR-138 in NSCLC cell lines, discussing whether it could turn out to be a therapeutic biomarker of NSCLC in the future. In our study, we found that miR-138 is downregulated in NSCLC tissues and cell lines. Moreover, the low level of miR-138 was associated with increased expression of SOX4 in NSCLC tissues and cell lines. Upregulation of miR-138 significantly inhibited proliferation of NSCLC cells. In addition, invasion and EMT of NSCLC cells were suppressed by overexpression of miR-138. However, downregulation of miR-138 promoted cell growth and metastasis of NSCLC cells. Bioinformatics analysis predicted that SOX4 was a potential target gene of miR-138. Next, luciferase reporter assay confirmed that miR-138 could directly target SOX4. Consistent with the effect of miR-138, downregulation of SOX4 by siRNA inhibited proliferation, invasion, and EMT of NSCLC cells. Overexpression of SOX4 in NSCLC cells partially reversed the effect of miR-138 mimic. In addition, decreased SOX4 expression could increase the level of miR-138 via upregulation of p53. Introduction of miR-138 dramatically inhibited growth, invasion, and EMT of NSCLC cells through a SOX4/p53 feedback loop.

Figure 1

The expression of microRNA-138 (miR-138) in non-small cell lung cancer (NSCLC) tissues and cell lines. (A) Relative miR-138 level analyzed by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) in seven lung cancer cell lines (H522, H1299, H460, A549, PAa, H1975, and Calu-3) and a human bronchial epithelial cell line (HBE) were normalized with U6 small nuclear RNA (snRNA). (B) Relative miR-138 expression levels in NSCLC tissues (n = 10) and their corresponding adjacent normal tissues (n = 10). (C) Relative sex-determining region Y (SRY)-related high-mobility group (HMG)-box 4 (SOX4) level analyzed by qRT-PCR in seven lung cancer cell lines (H522, H1299, H460, A549, PAa, H1975, and Calu-3) and a human bronchial epithelial cell line (HBE) were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (D) Relative SOX4 expression levels in NSCLC tissues (n = 10) and their corresponding adjacent normal tissues (n = 10). All data are presented as mean ± standard error of the mean (SEM), n = 6. *p < 0.05, **p < 0.01, ***p < 0.001 versus HBE or normal tissues.

Figure 2

Effects of miR-138 on cell proliferation in A549 and H1975 cells. A549 and H1975 cells were transfected with miR-138 mimic or inhibitor. (A) The mRNA levels of miR-138 in A549 and H1975 cells were determined by RT-PCR. (B) Cell proliferation of A549 and H1975 cells was assessed by enzyme-linked immunosorbent assay-bromodeoxyuridine (ELISA-BrdU) assay. (C) The protein expression of proliferating cell nuclear antigen (PCNA) by A549 and H1975 cells was determined by Western blot. α-Tubulin was detected as a loading control. All data are presented as mean ± SEM, n = 6. #p < 0.05, ##p < 0.01 versus miR-negative control (NC) or anti-miR-NC.

Figure 3

The effects of miR-138 on invasion in A549 and H1975 cells. The invasion of A549 and H1975 cells transfected with miR-138 mimic or miR-NC (left) and miR-138 inhibitor or anti-miR-NC (right) was assessed by Transwell assay. All data are presented as mean ± SEM, n = 6. #p < 0.05, ##p < 0.01 versus miR-NC or anti-miR-NC.

Figure 4

Overexpression of miR-138 suppressed expressions and secretions of matrix metalloproteinase 2 (MMP-2) and MMP-9. A549 and H1975 cells were transfected with miR-138 mimic or inhibitor. (A) Levels of MMP-2 and MMP-9 were detected in the culture supernatants of cultured A549 and H1299 cells by ELISA assay. (B) The mRNA levels of MMP-2 and MMP-9 in A549 and H1299 cells were examined by RT-PCR. All data are presented as mean ± SEM, n = 6. ##p < 0.01, ###p < 0.001 versus miR-NC or anti-miR-NC.

Figure 5

The effects of miR-138 on the expression of epithelial–mesenchymal transition (EMT)-related molecules in A549 and H1975 cells. (A) The mRNA and protein levels of epithelial (E)-cadherin, neural (N)-cadherin, and vimentin were determined by qRT-PCR and Western blotting in A549 and H1975 cells transfected with miR-138 mimic or miR-NC, respectively. (B) The mRNA and protein levels of E-cadherin, N-cadherin, and vimentin were determined by qRT-PCR and Western blotting in A549 and H1975 cells transfected with miR-138 inhibitor or anti-miR-NC, respectively. α-Tubulin was detected as a loading control. All data are presented as mean ± SEM, n = 6. #p < 0.05, ##p < 0.01, ###p < 0.001 versus miR-NC or anti-miR-NC.

Figure 6

SOX4 was a direct target of miR-138. (A) The mRNA level of SOX4 was determined by qRT-PCR in A549 and H1975 cells transfected with miR-138 mimic or inhibitor, respectively. (B) The protein expression of SOX4 was determined by Western blot in A549 and H1975 cells transfected with miR-138 mimic or inhibitor, respectively. (C) Schematic representation of SOX4 3′-untranslated regions (3′-UTRs) showing putative miRNA target site. (D) The analysis of the relative luciferase activities of SOX4 wild type (WT) and SOX4 mutation (MUT) in NSCLC cells. All data are presented as mean ± SEM, n = 6. ##p < 0.01, ###p < 0.001 versus miR-NC or anti-miR-NC.

Figure 7

SOX4 silencing also could inhibit cell proliferation, invasion, and EMT in NSCLC cells. A549 and H1975 cells were transfected with small interfering RNA SOX4 (si-SOX4) or si-NC. (A) The mRNA and protein levels of SOX4 were determined by qRT-PCR and Western blot, respectively. (B) Cell proliferation was assessed by ELISA-BrdU assay. (C) The invasion of A549 and H1975 cells was assessed by Transwell assay. (D) The expressions of E-cadherin, N-cadherin, and vimentin were determined by qRT-PCR in A549 and H1975 cells, respectively. All data are presented as mean ± SEM, n = 6. #p < 0.05, ##p < 0.01, ###p < 0.001 versus si-NC.

Figure 8

Overexpression of SOX4 partially rescued miR-138-inhibited cell proliferation, invasion, and EMT in NSCLC cells. A549 and H1975 cells were transfected with miR-NC, miR-138 mimic with or without pcDNA-SOX4 vector. (A) The mRNA and protein levels of SOX4 were determined by qRT-PCR and Western blot, respectively. (B) Cell proliferation was assessed by ELISA-BrdU assay. (C) The invasion of A549 and H1975 cells was assessed by Transwell assay. (D) The expressions of E-cadherin, N-cadherin, and vimentin were determined by qRT-PCR in A549 and H1975 cells, respectively. All data are presented as mean ± SEM, n = 6. ##p < 0.01, ###p < 0.001 versus miR-NC; &p < 0.05, &&p < 0.01 versus miR-138 mimic + pcDNA.

Figure 9

A regulatory network linking miR-138, SOX4, and p53. (A) The protein expression of tumor protein 53 (p53) was determined by Western blot. A549 cells were transfected with miR-138 and pcDNA or pcDNA-SOX4 (B) The protein expression of p53 was determined by Western blot. A549 cells were transfected with si-NC or si-SOX4. (C) The level of miR-138 was determined by qRT-PCR in A549 cells. A549 cells were transfected with si-NC or si-SOX4 with or without si-p53. (D) Cell proliferation was assessed by ELISA-BrdU assay. A549 cells were transfected with miR-NC, miR-138 mimic with or without pcDNA-SOX4, si-tumor protein 53 (p53), si-SOX4, and si-SOX4 with si-p53. (E) The invasion of A549 cells was assessed by Transwell assay. (F) The expressions of E-cadherin, N-cadherin, and vimentin were determined by qRT-PCR in A549. All data are presented as mean ± SEM, n = 6. *p < 0.05, **p < 0.01 versus miR-NC; ##p < 0.01, ###p < 0.001 versus si-NC or miR-138 mimic + si-NC; &p < 0.05, &&p < 0.01 versus si-SOX4.