microRNA-548l is involved in the migration and invasion of non-small cell lung cancer by targeting the AKT1 signaling pathway

Abstract

Purpose

microRNAs (miRNAs) have been documented playing a critical role in cancer development and progression. In this study, we investigated the role of miR-548l in non-small cell lung cancer (NSCLC) migration and invasion.

Methods

microRNAs microarray analysis was used to detect the differentially expressed miRNAs between various metastatic levels of NSCLC cells and further confirmed by real-time PCR analysis. To facilitate the delineation of the role of selected miR-548l in NSCLC pathology, we detected its expression in 22 NSCLC tissues. Proliferation, apoptosis, invasion and metastasis effects of the miRNA were evaluated using MTT, flow cytometry, wound healing and invasion assay following transfection with mimics and inhibitors. Luciferase assay and Western blot analysis were performed to assess miR-548l binding to AKT1 gene. AKT1 expression in the clinical tissues was evaluated using immunohistochemical staining.

Results

The results showed a negative relationship between miR-548l expression and lymph node metastasis of NSCLC. Functional assays showed that over-expression of miR-548l suppressed NSCLC cell migration and invasion. Luciferase assays confirmed that miR-548l could directly bind to the 3′ untranslated region of AKT1. Further data showed that the over-expression of AKT1 could rescue the effects of miR-548l in NSCLC cells, and the miR-548l expression was inversely correlated with AKT1 expression in NSCLC tissues. These results indicated that AKT1 was involved in miR-548l-induced suppression of NSCLC cell migration and invasion.

Conclusion

These results suggested that miR-548l may play a causal role through AKT1 in NSCLC invasion and metastasis.

Background

Lung cancer is one of the most frequently occurring malignant cancers. Among lung cancers, 80 % are classified as non-small cell lung cancer (NSCLC) (Ferlay et al. 2010). Despite recent advances in clinical and experimental oncology, the prognosis of lung cancer is still unfavorable, with a 5-year overall survival rate of approximately 11 %. Many studies have found that the leading causes of death in lung cancer are invasion and metastasis (Verdecchia et al. 2007). Thus, a detailed understanding of the mechanisms underlying NSCLC invasion and metastasis is essential for improving the diagnosis, prevention and treatment of this disease.

microRNAs (miRNAs) are endogenous, ~22 nucleotide-long, non-coding RNAs. It represses gene expression through interaction with 3′ untranslated regions (3′-UTRs) of mRNAs (Lewis et al. 2005). miRNAs are predicted to target over 50 % of all human protein-coding genes, enabling them to have numerous regulatory roles in many physiological and developmental processes, including development, differentiation, apoptosis and proliferation, through imperfect pairing with target mRNAs of protein-coding genes and the transcriptional or post-transcriptional regulation of their expression (Du et al. 2009; Zhang et al. 2014). Increasing evidence shows that aberrantly expressed miRNAs may act as oncogenes or tumor-suppressor genes in lung cancers (Chen et al. 2013; Zhang et al. 2011). In recent years, much research has been intensely focused on the study of the function of altered miRNA expression in lung cancer. miRNA expression signatures seem to hold great promise in lung cancer characterization and could be potential diagnostic and prognostic markers for lung cancer diagnosis and treatment (Rothschild 2013).

In this study, we investigated the potential role of miR-548l in lung cancer cell metastasis. We assessed the expression level of miR-548l in human lung cancer specimens and lung cancer cell lines, and examined its effects on cell growth, migration, invasion and expression of metastasis-related genes. Furthermore, we explored the target of miR-548l in lung cancer cells and the underlining mechanism of its function. This will provide better understanding of lung cancer metastasis.

Methods

Cell lines and cell culture

95C and 95D cells are the sublines of human lung giant cell carcinoma maternal cells. The paired cells have the same genetic background and varied metastatic capacity. 95D cells had the higher metastatic capacity (Sun et al. 2012). The cells were obtained from the Shanghai Institute of Cell Biology (Shanghai, China) and maintained according to vendor’s recommendations. The A549 (adenocarcinoma) cell line was propagated in DMEM (Gibco), and the medium was supplemented with 10 % fetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin. Cells were cultured at 37 °C in 5 % CO₂.

Samples

Between January and December 2012, fresh samples from lung cancer and corresponding normal adjacent tissue (NAT, 0.5 cm from the cancer tissue) were obtained from patients at the Second Xiangya Hospital of Central South University with informed consent (and the approval of the hospital’s Ethical Review Committee). None of the 22 patients in the study received any chemotherapy or radiation therapy before surgery. Clinicopathological information was available, and two pathologists independently determined the diagnoses and histological grades, based on World Health Organization guidelines.

Plasmids

The pcDNA3.1-myr-AKT1 vector plasmids were kindly provided by Dr. Q Deng (Cancer Research Institute, Central South University, Changsha, China). The empty construct pcDNA3.1 was transfected as a control.

miRNA microarray

Total RNA was extracted from the cells using Trizol reagent (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions. miRNAs were isolated using PEG and labeled by an RNA joining enzyme. Subsequently, the miRNAs were hybridized on an miRNA Microarray (CapitalBio V4.0, Beijing, China) and scanned. The LuxScan 3.0 image analysis software was used for analysis, and the original image signals were diverted into digital signals. Finally, the differentially expressed miRNAs were screened and identified though comparisons between the two chips with Significance Analysis of Microarrays (SAM, version 2.1).

Real-time RT-PCR

Real-time RT-PCR was performed using miRNA-specific primers to analyze the miRNA expression. Reverse transcription PCR was performed using the Reverse Transcription System (Promega, Madison, WI). Real-time PCR was performed with iTaqTM SYBR Green Supermix with ROX (Bio-Rad) using an ABI 7500 instrument (Applied Biosystems, Foster City, CA). The primers for miRNA and U6 (control) were from the EzOmicsTM miRNA qPCR detection primer set (Biomics Biotechnologies, Nantong, China).

Transfection

Cells were seeded into six-well plates and allowed to settle overnight until they were 70–90 % confluent. Then, 95D and A549 cells were transfected with pcDNA6.2-GW/EmGFP-miR-548 l or pcDNA6.2-GW/EmGFP-miR-NC (Bioladder, Shanghai, China), and 95C cells were transfected with Anti-miR-548l or Anti-miR-NC (Exiqon, Vedbaek, Denmark) using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions.

Cell proliferation assay

Cells were seeded in 96-well plates at 1,500 cells/well, 24 h after transfection. The MTT assay was used to determine relative cell growth every 24 h. Next, 20 μl of 5 mg/ml MTT (Sigma, USA) was added to the media for 4 h incubation at 37 °C. Following the removal of the culture medium, the remaining crystals were dissolved in 150 μl DMSO (Sigma, USA), and the absorbance at 490 nm was measured by using a microtiter plate reader (Bio-Rad, CA).

Flow cytometry analysis

The transfected cells were washed with phosphate-buffered saline, harvested by treatment with trypsin–EDTA, re-suspended in ice-cold phosphate-buffered saline (pH 7.4) and examined immediately using a FACSort flow cytometer (Becton–Dickinson, Franklin Lakes, NJ).

Wound healing/cell migration assay

After transfection, the cells were grown into six-well culture plates and cultured until 90 % confluent. A P200 pipette tip was used to make a vertical wound, and the cells were washed several times with appropriate culture medium to remove cell debris. The wound healing was observed and photographed in three randomly selected microscopic fields for each condition and time point.

Cell invasion assay

The cell invasion assay was conducted using BD Biocoat Matrigel 24-well invasion chambers, with filters coated with Matrigel on the upper surface (BD Biosciences, Bedford, MA) with 8.0 μm pores. After transfection, the cells were trypsinized and resuspended in serum-free medium and seeded at 1 × 10⁵ cells in 0.2 ml medium to the upper chamber. Next, 0.5 ml 10 % FBS medium was added to the lower chamber, and after incubation at 37 °C with 5 % CO₂ for 24 h, the cells were stained with 0.1 % crystal violet solution. The cells and Matrigel on the top surface of the filter were carefully removed with a cotton swab. The invasive cells attached to the bottom surface of the filter were quantified under a light microscope (×200). The data are presented as the average number of cells from randomly chosen fields. Each treatment condition was assayed using triplicate filters, and all filters were counted in five areas.

Western blot analysis

Cells were collected and lysed. Proteins were separated by 8 % SDS-PAGE, transferred to a tank-based nitrocellulose membrane and blocked for 1 h by TBS containing 5 % nonfat milk. Anti-AKT1 (2967, Cell Signaling, Danvers, MA), anti-mTOR (2983, Cell Signaling, Danvers, MA), anti-MMP-9 (sc-21733, Santa Cruz, CA), anti-MMP-2 (sc-13595, Santa Cruz, CA), ERK (sc-93, Santa Cruz, CA) and tubulin (SC-69969, Santa Cruz, CA) were used according to the manufacturer’s instructions. The ECL nonradioactive detection system was utilized to detect the antibody–protein complexes by photographing with the ChemiDoc XRS system (Bio-Rad, USA).

Dual-luciferase 3′UTR-reporter assay

The plasmid pWT-AKT1-UTR, which contained AKT1 3′UTR and pMUT-AKT1-UTR, comprising mutant AKT1 3′UTR with a fluorescent reporter, was constructed (Ribobio, Guangzhou, China). Cells were transiently co-transfected for 48 h with reporter plasmids (200 ng) and 100 nM of miR-548l or anti-miR-548l and were then harvested in reporter lysis buffer. Both firefly luciferase and Renilla luciferase activities were measured using the Dual-Luciferase assay kit (Promega, Madison, WI), according to manufacturer’s instructions. The luciferase activity normalized against the protein concentration was expressed as a ratio of firefly luciferase to Renilla luciferase units.

Immunohistochemistry

Immunohistochemistry (IHC) was performed using a Histomouse SP Broad Spectrum DAB kit (Invitrogen–Zymed, Carlsbad, CA). Paraffin sections were immunostained using a streptavidin peroxidase procedure after microwave antigen retrieval. The signal was detected using a diaminobenzidine solution. In each case, two arbitrary separate microscopic fields (200×) containing >200 tumor cells were evaluated. After quantification of both the immunoreactive cells and total number of tumor cells, the average percentages of immunoreactive cells were calculated.

Statistical analysis

Data were shown as the mean ± standard deviation (SD). All statistical analyses were performed using the SPSS15.0 software. Differences/correlations between groups were compared using the Pearson’s chi-square test for qualitative variables and the Student’s t test for continuous variables. p value <0.05 was considered statistically significant.

Results

Differential miRNAs between 95D and 95C cells

To identify the miRNAs potentially involved in NSCLC invasion and metastasis, we examined the change in miRNA expressions between the 95D and 95C lines using a miRNA microarray. The data showed that a variety of miRNAs exhibited significantly different expression levels between the two cells (Fig. 1). In particular, 15 miRNAs were up-regulated, and one miRNA was down-regulated in 95D cells versus 95C cells. Of these, miR-548l displayed the most significantly different expression, with 9.14 times the expression in a 95C cell than in a 95D cell. To validate the miRNA microarray data, we performed real-time RT-PCR to analyze the expression levels of 16 miRNAs in two cells. The results showed that 15 of the 16 miRNAs tested with real-time RT-PCR gave results consistent with the microarray data. Considering the exceptions were has-miR-487b, a concordance rate of 93.7 % was found. Among these miRNAs, the miR-548l was focused on as it is one of the most evidently altered miRNAs.

Fig. 1.

Changes of the miRNA expression between 95D and 95C cells. CapitalBio Human miRNA array, which contained 988 probes based on Sanger miRBase release 12.0, was used to scan miRNAs that were differentially expressed between 95D and 95C cells. These were the 16 miRNAs which exhibited significantly differential expression and real-time RT-PCR validated the expression of these miRNAs of the 95D cells compared with that of 95C cells

miR-548l was associated with lymph node metastasis in NSCLC

To further confirm the previous observation, we used real-time RT-PCR to investigate miR-548l expression in fresh tissues from the primary tumors of 22 NSCLC patients. These included 17 tumors with lymph node metastasis (pN1, 2 stages) and five NSCLC patients without lymph node metastasis (pN0 stage). The miR-548l level in each tumor sample was normalized to match a normal colorectal tissue sample from the same patient. The results indicated that the miR-548l expression level was visibly higher in adjacent non-tumor tissues than that in NSCLC (p < 0.01). This observation demonstrated that miR-548l was down-regulated in NSCLC (Fig. 2a). Furthermore, associations between miR-548l expression and lymph node metastasis were observed to be statistically significant (p < 0.05, two-sided Fisher’s exact test) (Fig. 2b). No correlation was observed between miR-548l expression and gender, age, differentiation, Histology type, maximum tumor diameter and pathological type (Table 1).

Fig. 2.

The real-time RT-PCR analysis of miR-548l in NSCLC tissues and adjacent non-tumor tissues. Relative expression of miR-548l in 22 paired NSCLC and adjacent non-tumor lung tissues was measured by real-time RT-PCR. a Statistical significance between the NSCLC and adjacent non-tumor lung tissues. b Statistical significance between the non-lymph node metastasis tumor and lymph node metastasis tumor tissues. *p < 0.05 compared with the control, **p < 0.01 compared with the control

Table 1.

Clinicopathologic characteristics of the 22 cases with NSCLC and miR-548l expression level in NSCLC tissues

Clinicopathological characteristics	n	miR-548 l		p value*
		Low expression	High expression
Gender				>0.05
Male	17	13 (76.5 %)	4 (23.5 %)
Female	5	4 (80 %)	1 (20 %)
Age (years)				>0.05
≤65	10	8 (80 %)	2 (20 %)
>65	12	9 (75 %)	3 (25 %)
Differentiation				>0.05
Well-moderate	4	3 (75 %)	1 (25 %)
Poor	18	14 (77.8 %)	4 (22.2 %)
Histology type				>0.05
Adenocarcinoma	12	9 (75 %)	3 (25 %)
Squamous cancer	10	8 (80 %)	2 (20 %)
Maximum tumor diameter				>0.05
≤5 cm	15	11 (78.6 %)	4 (21.4 %)
>5 cm	7	6 (85.7 %)	1 (14.3 %)
T grade				>0.05
T1	13	10 (76.9)	3 (23.1 %)
T2–3	9	7 (77.8)	2 (22.2 %)
N grade				<0.05
N0	5	1 (20 %)	4 (80 %)
N1–2	17	16 (94 %)	1 (6 %)

* Two-sided Fisher’s exact test

miR-548l suppressed the proliferation of NSCLC cells but did not influence cell apoptosis

To examine the effect of miR-548l on cell proliferation and apoptosis, the cells after transfection and the miR-548l expression levels were assessed by real-time RT-PCR (Fig. 3a). The results of MTT and flow cytometry showed that the down-regulation of miR-548l, as a result of transfection by miR-548l inhibitor, was able to promote the growth of 95C cells (Fig. 3b, p < 0.05), although cell apoptosis was not different after the transfection (Fig. 3e). Meanwhile, the up-regulation of miR-548l, as a result of transfection by pcDNA6.2-GW/EmGFP-miR-548l, was able to restrain the growth of 95D and A549 cells (Fig. 3c, d, p < 0.05), and cell apoptosis rates did not differ (Fig. 3f, g). The results showed that miR-548l was able to restrain the growth of lung tumor cells, even though the influence of miR-548l on the cell apoptosis in NSCLC was not obvious.

Fig. 3.

Effect of miR-548l on cell proliferation and apoptosis of NSCLC cells. 24 h after cells were transfected with anti-miR-548l or miR-548l mimics, real-time RT-PCR assay was performed to detect the expression of miR-548l, MTT analysis of growth and flow cytometry analysis of apoptosis. a The expression of miR-548l in cells after transfection; b, e the proliferation and apoptosis of 95C cell after transfection with the anti-miR-548l; c, f the proliferation and apoptosis of 95D cell after transfection with miR-548l mimics; d, g the proliferation and apoptosis of A549 cell after transfection with miR-548l mimics. Data are presented as the means of three repeated experiments. *p < 0.05 compared with the control. **p < 0.01 compared with the control

miR-548l inhibited the migration and invasion of NSCLC cells

To examine the effect of miR-548l on the migration and invasion of NSCLC cells, we conducted a cell migration (wound healing) assay and Transwell assays. Compared with 95C cells, the capacities for wound healing (Fig. 4a) and invasion (Fig. 4b) in 95D and A549 cells were higher (p < 0.05). We observed a significant increase in the capacities for wound healing and invasion in 95C cells after transfection with anti-miR-548l, compared with control cells (p < 0.05). Meanwhile, we also found a significant decrease in the capacities for wound healing and invasion in 95D and A549 cells after transfection with miR-548l, compared with control cells (p < 0.05). The results demonstrated that the over-expression of miR-548l inhibits the migration and invasion ability of NSCLC cells in vitro.

Fig. 4.

Effect of miR-548l on cell migration and invasion of NSCLC cells. 24 h after cells were transfected with anti-miR-548l or miR-548l mimics, wound healing and Transwell assays were performed to analyze of migration and invasion. a Wound healing assay to evaluate the effect of miR-548l expression on cell migration capacity, with healing determined at the indicated times; b invasion assays of 95C cells transfected with anti-miR-548l, 95D and A549 cells transfected with a miR-548l mimics. Values give the average number of invasive cells per field from three independent experiments ± SE. *p < 0.05 compared with the control

AKT1 is the target gene of miR-548l

To find a potential target gene of miR-548l that might contribute to its metastatic suppressor functions, we searched for such genes using the bioinformatics algorithms TargetScans (http://targetscan.org), miRanda (http://www.microrna.org/microrna/home.do) and DIANA microTest (www.microrna.org/microrna). The three algorithms identified multiple target genes, including AKT1 and ERK, which were associated with cancer metastasis (Lee et al. 2011).

Western blot analysis and the luciferase reporter assay helped us to determine whether AKT1 and/or ERK was regulated by miR-548l. The Western blot analysis showed that the expression of AKT1 in 95C cells was lower than in 95D cells (Fig. 5a). AKT1 expression was down-regulated in 95D cells after transfection with miR-548l but up-regulated in 95C cells after transfection with anti-miR-548l. ERK1/2 had no difference in any of the above groups. Previous studies have shown that the AKT1-mTOR pathway was reported to play crucial roles in cancer invasion and metastasis (Kim et al. 2011) and to crosstalk with MMPs (Dilly et al. 2013), which are critically involved in the processes of tumor invasion and metastasis. Western blot analysis showed that the expressions of AKT1, mTOR, MMP-2 and MMP-9 in 95C cells were lower than in 95D cells. Further, AKT1, mTOR, MMP-9 and MMP-2 expressions were down-regulated in 95D cells after transfection with miR-548l but up-regulated in 95C cells after transfection with anti-miR-548l (Fig. 5a).

Fig. 5.

miR-548l targeted the AKT1. a 24 h after cells were transfected with anti-miR-548l or miR-548l mimics, Western blot analysis of AKT1, ERK1/2, mTOR, MMP-2 and MMP-9; b the targeting site for miR-548l was identified in AKT1 3′-UTRs (nt 2179 to nt 2204, NM_005163); c dual-luciferase reporter assays were performed to test the interactions of miR-548l and the targeting sequences in the AKT1 mRNA using constructs containing the predicted targeting sequences and the corresponding mutants cloned into the 3′-UTR of the reporter gene. Luciferase reporter vectors co-transfected into cells with miR-548l or anti-miR-548l, respectively. Luciferase activity was determined 48 h after transfection. Values are the mean ± SE of three replicates. *p < 0.05 compared with the control

Finally, we constructed a luciferase reporter assay to verify that AKT1 was the direct target of miR-548l. The results showed that, among 95C cells that were co-transfected with AKT1-WT and anti-miR-548l, the relative luciferase activity of the cells was significantly higher than those of the control groups (p < 0.05). Among 95D cells that were co-transfected with AKT1-WT and miR-548l, the relative luciferase activity of the cells was significantly lower than that of the control group (p < 0.05). However, in cells co-transfected with AKT1-MUT, no difference was found between the relative luciferase activity of either 95D or 95C cells. The above observations show that miR-548l can bind to the putative binding sites for miR-548l in AKT1 mRNA 3′UTR; therefore, it is able to decrease AKT1 expression.

Over-expression of AKT1 could rescue the effects of miR-548l in NSCLC cells

The expression levels of AKT1 protein were determined, 24 h after pcDNA/AKT1 or pcDNA/control vector was transfected into A549/miR-548l cells. As shown in Fig. 6a, pcDNA/AKT1 could rescue the decreased protein expression in A549/miR-548l cells. Also, pcDNA/AKT1 could partially reverse growth and invasion inhibition of A549/miR-548l cells (Fig. 6b, c). These results suggested that over-expression of AKT1 could rescue the effects of miR-548l on phenotypes of NSCLC cells.

Fig. 6.

DNA vector-mediated AKT1 over-expression could rescue the effects of miR-548 l mimics on malignant phenotypes of A549 cells. a Western Blot analysis of AKT1 protein expression in A549/miR-NC, A549/miR-548l or A549/miR-548l co-transfected with pcDNA/control or pcDNA/AKT1. b MTT analysis of growth in A549/miR-NC, A549/miR-548l or A549/miR-548l co-transfected with pcDNA/control or pcDNA/AKT1. c Analysis of invasion in A549/miR-NC, A549/miR-548l or A549/miR-548l co-transfected with pcDNA/control or pcDNA/AKT1. Values are the mean ± SE of three replicates. *p < 0.05 compared with the control

miR-548l expression was inversely correlated with AKT1 expression in NSCLC tissues

As down-expressed miR-548l was related to the invasion and metastasis of NSCLC and targeted AKT1 by binding to its 3′ UTR, we next determined whether AKT1 expression was negatively associated with miR-548l levels in primary NSCLC patient tissues. Analysis of AKT1 expression level in 22 NSCLC tissues by immunohistochemical staining revealed that associations between AKT1 expression and lymph node metastasis were observed to be statistically significant (p < 0.05, two-sided Fisher’s exact test) (Fig. 7a, b). Furthermore, Spearman’s correlation test revealed a negative correlation between miR-548l relative expression and AKT1 expression (r = –0.423, p = 0.017) (Fig. 7c). Thus, these data further support that down-regulation of miR-548l was inversely correlated with up-regulation of AKT1 in NSCLC tissues. In addition, no correlation was observed between AKT1 expression and gender, age, differentiation, histology type, maximum tumor diameter and pathological type (data not shown).

Fig. 7.

AKT1 expression in NSCLC tissues was inversely correlated with miR-548l expression. a In a tissue sample from case 1, that showed low immunohistochemistry staining of AKT1, whereas in case 7, that showed highly immunohistochemistry staining of AKT1; b relative expression levels of AKT1 protein was detected in N0 lymph node metastasis (n = 5) and N1, 2 lymph node metastasis (n = 17) tissues. c A statistically significant inverse correlation between miR-548l and AKT1 protein levels in 22 cases of NSCLC tissues (Spearman’s correlation analysis, r = − 0.423; p = 0.017). Corresponding p values analyzed by a t test or Spearman correlation test are indicated

Discussion

Numerous miRNAs, many of which play essential roles in the regulation of invasion and metastasis in NSCLC, have been found to be decreased in NSCLC. For instance, decreased miR-148a has been found to be associated with lymph node metastasis and poor clinical outcomes, acting as a suppressor of tumor metastasis in NSCLC by target DNA (cytosine-5)-methyltransferase 1 (DNMT1) (Chen et al. 2013). MiR-149 was down-regulated in NSCLC and inhibited the epithelial-to-mesenchymal transition process of A549 cells by targeting FOXM1 (Ke et al. 2013). MiR-29c suppressed lung cancer cell adhesion to the extracellular matrix and metastasis by targeting MMP-2 (Wang et al. 2013a). microRNA let-7a inhibited the proliferation and invasion of the non-small cell lung cancer cell line 95D by regulating K-ras and HMGA2 gene expression (Wang et al. 2013c)

In this study, the data indicated that miR-548l was involved in the migration and invasion of NSCLC cells, and the expression of miR-548l appeared to be correlated with advanced lymph node metastasis. Furthermore, the over-expression of miR-548l remarkably inhibited the proliferation, migration and invasion of NSCLC cells. Using a luciferase activity assay and Western blotting, we found that AKT1 was a direct target of miR-548l. Further data showed that the over-expression of AKT1 could rescue the effects of miR-548l in NSCLC cells, and the miR-548l expression was inversely correlated with AKT1 expression in NSCLC tissues. AKT is the key kinase of the comprehensive phosphoinositide 3-kinase (PI3K)/AKT/mTOR signal pathway, which increases glucose utilization, promotes cell growth and regulates cell motility (Franke 2008; Lee et al. 2011). The upstream activators, downstream targets and negative regulators of this signaling pathway, which are often aberrantly expressed in lung cancers, have been reported to be the targets of miRNAs (Bueno et al. 2008; Lin et al. 2010; Wang et al. 2013b).

The PI3K-AKT1-mTOR pathway has been reported to play crucial roles in cancer invasion and metastasis, as well as in crosstalk with the matrix metalloproteinases (MMPs) (Karrasch et al. 2011; Kim et al. 2012; Lima et al. 2012; Mihai et al. 2012). MMPs are critically involved in the processes of tumor cell invasion and metastasis. MMPs, including MMP-2 and MMP-9, act as key enzymes in the degradation of the extracellular matrix (Mott and Werb 2004), and high tissue levels of MMP-2 and MMP-9 have been associated with tumor growth and invasion (Cawston and Wilson 2006). In this study, the data showed that MMP-2 and MMP-9 are likely to be important regulators of this phenomenon. Taken together, impact of miR-548l on cell invasion and proliferation is likely to be regulated via the target protein AKT1, which then down-regulates the expression of mTOR, MMP-2 and MMP-9.

Conclusion

In conclusion, we demonstrated that the expression of miR-548l significantly impacted cell proliferation, migration and invasion in NSCLC cells. miR-548l is down-regulated in NSCLC, which has been associated with lymph node metastasis. These biologic effects may be due to the modulated expressions of AKT1, mTOR and MMP- 2/9.