MicroRNA-191 targets CCAAT/enhanced binding protein β and functions as an oncogenic molecule in human non-small cell lung carcinoma cells

Abstract

The aberrant expression of microRNAs (miRs) may be involved in tumor growth and progression in human non-small cell lung carcinoma (NSCLC). The present study aimed to investigate the potential roles of miR-191 in NSCLC. Western blotting and reverse transcription-quantitative polymerase chain reaction were performed to assess protein and/or mRNA levels. Scratch wound healing and transwell assays were performed to determine the NSCLC cell migration and invasion. A luciferase demonstrated that CCAAT/enhanced binding protein β (C/EBPβ) was a target of miR-191. Previously, miR-191 has been reported to act as an oncogenic player in multiple human cancers. C/EBPβ has been identified as a target gene of miR-191; however, the roles and underlying mechanisms of miR-191 associated with the regulation of tumor invasion in NSCLC remain unknown. In the present study, it was demonstrated that miR-191 expression levels were higher in human NSCLC tumors compared with in normal adjacent tissue and elevated miR-191 expression levels were closely associated with tumor node metastasis stage in patients with NSCLC. Furthermore, transfection with miR-191 mimic inhibited C/EBPβ expression at the mRNA and protein levels and promoted A549 cell migration and invasion. C/EBPβ was reported to be the direct target gene of miR-191 using a dual luciferase reporter assay. Finally, C/EBPβ siRNA can mimic the effects of miR-191. These findings indicated that miR-191 may function as an oncogene in NSCLC, at least partially due to its negative regulatory on C/EBPβ.

Keywords: CCAAT/enhanced binding protein β; metastasis; microRNA-191; negative regulation; non-small cell lung carcinoma.

Figure 1.

miR-191 and C/EBPβ expression in human NSCLC tissues. (A) RT-qPCR analysis of miR-191 expression levels in NSCLC clinical tissues relative to adjacent normal control samples. Data were normalized with the expression levels of U6 control. (B) RT-qPCR analysis of C/EBPβ expression levels in NSCLC clinical tissues relative to adjacent controls. Data were normalized with the expression levels of GAPDH control. Results are expressed relative to the value of normal controls that were assigned a value of 1. Data are presented as the mean ± standard deviation. *P<0.01. miR, microRNA; C/EBPβ, CCAAT/enhanced binding protein β; NSCLC, non-small cell lung carcinoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2.

Transfection efficiency of miR-191 mimic and miR-191 inhibitor in A549 cells. A549 cells were transfected with miR-191 mimic, miR-191 inhibitor, miR-191 mimic NC or miR-191 inhibitor NC. (A) Expression levels of miR-191 in miR-191 mimic or miR-191 mimic NC transfected cells were detected at 72 h post-transfection using RT-qPCR. (B) Expression levels of miR-191 in miR-191 inhibitor or miR-191 inhibitor NC transfected cells were detected at 72 h post-transfection using RT-qPCR. Data are presented as the mean + standard deviation. **P<0.01 vs. NC mimic or NC inhibitor. miR, microRNA; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 3.

C/EBPβ siRNA decreased the mRNA and protein expression of C/EBPβ. (A) The mRNA expression level of C/EBPβ was significantly decreased following treatment with siRNA1 and siRNA2. (B) siRNAs significantly downregulated the protein level of C/EBPβ. Results were compared to the value of siRNA-NC assigned a value of 1. (C) Quantification analysis of B. Data are presented as the mean + standard deviation. **P<0.01 vs. siRNA NC. C/EBPβ, CCAAT/enhanced binding protein β; siRNA, small interfering RNA; NC, negative control.

Figure 4.

miR-191 enhanced the motility of A549 cells. Scratch-wound assay was performed to assess the effects of miR-191 on the migration rate of A549. (A) Effect of miR-191 mimic on the migration ability of A549 cells. (B) Effect of miR-191 inhibitor on the migration ability of A549 cells. Data are presented as the mean + standard deviation. **P<0.01 vs. NC mimic or NC inhibitor. miR, microRNA; NC, negative control.

Figure 5.

miR-191 promoted Transwell invasion of A549 cells. (A) Effect of miR-191 mimic on the invasion ability of A549 cells. (B) Effect of miR-191 inhibitor on the invasion ability of A549 cells (magnification, ×200). Data are presented as the mean + standard deviation. **P<0.01 vs. NC mimic or NC inhibitor. miR, microRNA; NC, negative control.

Figure 6.

miR-191 suppressed mRNA and protein level of C/EBPβ. (A) A549 cells were transfected with miR-191 or NC. Relative mRNA and protein expression levels of C/EBPβ were analyzed using RT-qPCR or WB. (B) A549 cells were transfected with miR-191 inhibitor or NC. Relative mRNA and protein expression levels of C/EBPβ were analyzed using RT-qPCR or WB. Data are presented as the mean + standard deviation of three independent experiments. **P<0.01 vs. NC mimic or NC inhibitor. miR, microRNA; C/EBPβ, CCAAT/enhanced binding protein β; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; WB, western blotting.

Figure 7.

C/EBPβ is the direct target of miR-191. (A) Sequence alignment indicates the relative position of the miR-191 binding site in the 3′-UTR of C/EBPβ and the mutated nucleotides are indicated. The sequences were used to construct luciferase reporter plasmids. (B) 293T cells were transfected with plasmids C/EBPβ 3′-UTR wt or mut, along with miR-191 mimic or NC. Dual luciferase assay was conducted at 24 h later. Luciferase activities were normalized to Renilla and presented relative to miR control (arbitrarily set at 1). Results were compared to the value of NC mimics assigned a value of 1. Data are presented as the mean + standard deviation of three independent experiments. **P<0.01 vs. NC mimic. C/EBPβ, CCAAT/enhanced binding protein β; miR, microRNA; UTR, untranslated region; wt, wild-type; mut, mutant; NC, negative control.

Figure 8.

Effect of C/EBPβ on the migration and invasion of A549 cells. (A) Scratch-wound assay was performed to assess the effects of C/EBPβ siRNA on the migration rate A549 cells. (B) Transwell assay was performed to assess the effects of C/EBPβ siRNA on the invasion ability of A549 cells (magnification, ×200). The results were compared with C/EBPβ siRNA NC. Data are presented as the mean + standard deviation of three independent experiments. **P<0.01 C/EBPβ siRNA NC. C/EBPβ, CCAAT/enhanced binding protein β; siRNA, small interfering RNA; NC, negative control.