Low expression of miR-192 in NSCLC and its tumor suppressor functions in metastasis via targeting ZEB2

Zhang Yunxia; Dong Hongying

doi:10.1515/biol-2016-0039

Article Open Access

Low expression of miR-192 in NSCLC and its tumor suppressor functions in metastasis via targeting ZEB2

Zhang Yunxia and Dong Hongying

Published/Copyright: November 6, 2016

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From the journal Open Life Sciences Volume 11 Issue 1

Abstract

Objectives

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) accounting for more than 80% of all lung cancer cases. The aim of this study was to investigate the function and underlying mechanism of microRNA-192 (miR-192) in metastasis of NSCLC cells.

Methods

Real-time PCR was applied to quantify the expression of miR-192 in NSCLC tissues and cell lines, matched with their corresponding controls. The biological roles of miR-192 were studied in NSCLC cells using the wound healing and trans well invasion assays. Real-time PCR and western blot were used to evaluate the regulation of ZEB2 by miR- 192.

Results

MiR-192 was expressed significantly lower in NSCLC tissues/cells when compared with controls. Ectopic expression of miR-192 strongly inhibited cell migration and invasion in NSCLC A549 cells. Further investigation revealed ZEB2, an EMT regulator, was one of the downstream targets regulated by miR-192.

Conclusion

These results suggested that miR-192 inhibits the metastasis of NSCLC cells by targeting ZEB2, and thus is an important tumor suppressor.

Keywords: NSCLC; miR-192; metastasis; tumor suppressor; ZEB2

1 Introduction

Lung cancer (LC) is the malignant tumor with the highest rates of morbidity and mortality worldwide, and incidences are ever-increasing due to poor air quality. Non-small cell lung cancer (NSCLC) accounts for over 80% of all LC cases [1]. The outcome of standard surgery and chemotherapy remains disappointing [2] and the post-surgery prognosis for NSCLC patients remains poor: with a 5-year survival rate of only 11% [3]. Due to a lack of precise early diagnostic options and the limitations of early-stage specific clinical indicators, most NSCLC patients are diagnosed at advanced stages, which makes treatment more difficult [4]. Tumor invasion and metastasis are complex processes that lead to the growth of the tumor before detachment from the primary site and metastasis within other organs. Metastasis is the major cause of morbidity and death in NSCLC [5]. Understanding the molecular basis underlying NSCLC progression will therefore hopefully be beneficial in improving both the prognosis and treatment of NSCLC patients.

Recent findings have established that the pathology of NSCLC are not only influenced by protein-coding genes, but that non-coding RNA (ncRNA) also exert important effects on tumorigenesis [6]. MicroRNA (miRNA) is a class of endogenous small ncRNA of 21-25 nt in length and is widely expressed in eukaryotes. miRNA negatively regulate target gene expressions by binding to 3’-UTRs of targeted mRNAs, and lead to mRNA degradation or translational suppression [7, 8]. Recently, it has become more accepted that miRNAs are involved in the regulation of multiple pathological processes that contribute to tumorigenesis and metastasis of LC, including cancer cell differentiation, proliferation, apoptosis, and invasion [9]. Furthermore, it has been reported that the abnormal expression of many miRNAs play roles as oncogenic or tumor suppressing factors in NSCLC [9]. On such miRNA, miR-192, is known to be associated with oncogenesis of gastric, colorectum and prostate cancer [10-12]. Additionally, a previous report has shown that miR-192 inhibits cell proliferation and induces cell apoptosis in LC cells [13]. Currently however, it is unknown whether miR-192 has any effects on metastasis of NSCLC.

In the present study, we first examined the expression levels of miR-192 in NSCLC tissues/cell lines. Functional assays including wound healing and transwell invasion were then performed to evaluate the effects of miR-192 in metastasis of A549 cells. Our study revealed that ZEB2 was negatively regulated by miR-192 and demonstrated that by suppressing tumor metastasis processes, the miR-192/ ZEB2 axis provides a valuable target for NSCLC therapy.

2 Methods

2.1 Patients and tissues

NSCLC tissue and non-cancerous normal lung tissue from patients were obtained from the Human Tissue Bank of the 3rd Peoples’ Hospital of Hangzhou City. Tissue samples were collected from a total of 40 patients from January 2015 to June 2016. All patients were diagnosed with histologically confirmed non-small cell lung cancer. This study was approved by the institutional ethics committee of the 3rd Peoples’ Hospital of Hangzhou City.

2.2 Mimics and transfections

The miR-192 mimics and negative controls (NC) were from Riobio. The cells were seeded in 6-well plates and grown to 50% confluence prior to transfection. The NC or mimics were transfected with Lipofectamine2000 (Life Technologies) according to the manufacturer’s instructions.

2.3 Real-time PCR

Total RNA was prepared from cells using TRIzol (Invitrogen) in accordance with manufacturer’s instructions. For qRT-PCR, 2 μg of total RNA was reverse-transcribed with M-MLV reverse transcriptase (Promega) using a random primer. MiR-192 expression was determined by Taqman MicroRNA Assays (Applied Biosystems). U6 was used as an endogenous reference gene.

2.4 Western blot

Total cell extracts were prepared from cells using RIPA buffer (Beyotime). The proteins were then resolved on 8% SDS-PAGE and transferred to NC membranes. Membranes were blocked with 5% skim milk in PBST and incubated with ZEB2 or GAPDH antibodies (Santa Cruz) followed by incubation with HRP-conjugated secondary antibodies.

2.5 Wound healing assay

Cell migration was determined by wound healing assay. Briefly, when cells reached 90% confluence they were starved for 24 h by incubation in serum-free medium before being scratched with a 1 mL pipette tip and cultured in media containing 10% FBS. Recovery from the disturbance was then observed for a further 24 h.

2.6 Cell invasion assays

Cell invasion assays were performed using Cell Invasion Assay kit (Millipore) and were carried out according to the manufacturer’s instructions. Briefly, cells were starved for 24 h before being plated in an 8 μm pole size chamber coated with a thin layer of polymerized collagen. The chamber was then incubated in a well containing 10% FBS medium for 48 h. The cells on the bottom of the chamber were then stained and photographed and subsequently measured according to the manufacturer’s instructions.

2.7 Statistical analysis

Statistical analyses were performed using SAS v8 software. The results are presented as mean ± SD. The results were analyzed using the Paired or Grouped Student’s t-test. P < 0.05 was considered statistically significant.

3 Results

3.1 The expression of miR-192 was significantly decreased in NSCLC tissues and cell lines

Expression of miR-192 has already been shown to be reduced in LC tissues compared with the corresponding adjacent non-cancerous lung tissues by Feng et al. [13]. To further validate this, we collected 40 samples of NSCLC tissue and five cell lines, matched with non-cancerous normal lung tissue and normal human fetal lung fibroblast cell line MRC-5, respectively. RNA was isolated and miR-192 was quantified using specific primers. As shown in Figure 1A, the levels of miR-192 were significantly decreased in NSCLC tissues (n = 40, P < 0.05) compared to the matched normal lung tissue. This was consistent with Feng et al’s previous data [13]. Also, when analyzing the expression of miR-192 in several NSCLC cell lines, we observed that miR-192 was downregulated in these cell lines compared to MRC-5 (Figure 1B). These results confirmed the downregulation of miR-192 in NSCLC, and provided a solid basis to further explore its role in NSCLC progression.

Figure 1

Expression of miR-192 in human NSCLC tissues and cell lines determined by real-time PCR. (A) Expression of miR-192 in NSCLC tissues compared with non-cancerous normal lung tissues (N = 40, P < 0.05). (B) Expression of miR-192 in NSCLC cell lines compared with normal human fetal lung fibroblast cell line. Experiments were performed at least three times (*: P < 0.05, **: P < 0.01, ***: P < 0.001).

3.2 Ectopic expression of miR-192 inhibited the migration and invasion of A549 cells

To investigate the role of miR-192 in metastasis of NSCLC cells, A549 cells were selected since these expressed the lowest levels of intrinsic miR-192 (Figure 1B). Considering that miR-192 may reduce tumor metastasis of human hepatocellular carcinoma and prostate cancer [12, 14], we hypothesized that miR-192 might also inhibit NSCLC cell migration and invasion. A549 cells were transfected with the miR-192 mimic to overexpress miR-192 with the efficiency of transfection confirmed by real-time PCR (Figure 2A). As expected, overexpressing miR-192 clearly reduced A549 cell migration and invasion (Figure 2B and 2C). Our results suggest that miR-192 also negatively affects NSCLC metastasis.

3.3 ZEB2 was identified to be negatively regulated by miR-192

We further explored possible mechanisms of the antimetastasis effect of miR-192 in NSCLC cells. By analysing previous reports of miR-192 in cancer, we found that interestingly, two papers mentioned the repression of ZEB2 by miR-192 in different cancers [15, 16] ZEB2 is a master transcriptional factor involved in EMT, one of the important determinants of metastasis. In our experiments, we validated that miR-192 could indeed also repress the expression of ZEB2 at both mRNA and protein levels (Figure 3A and 3B). These data suggest that the anti-metastasis role of miR-192 in A549 cells could also be mediated by ZEB2 inhibition.

Figure 2

miR-192 inhibited the migration and invasion of A549 cells. (A) miR-192 expression level in A549 cells transfected with NC or miR-192 mimics determined by real-time PCR (**: P < 0.01). (B) Wound-healing assay using A549 cells transfected with NC or miR-192 mimics. The right panel shows the quantitative results from three independent experiments (**: P < 0.01). (C) Cell invasion assay using A549 cells transfected with NC or miR-192 mimics. A representative experiment out of three independent experiments was given (*: P < 0.05).

Figure 3

ZEB2 may be negatively regulated by miR-192. (A) mRNA levels of ZEB2 in A549 cells transfected with NC of miR-192 mimics determined by real-time PCR (**: P < 0.01). (B) Protein levels of ZEB2 in A549 cells transfected with NC of miR-192 mimics determined by western blot. GAPDH was used as loading control.

4 Discussion

Metastasis is a complex and multi-step process. However, our current knowledge of molecular mechanisms governing the metastatic process of NSCLC still remains very limited. miRNAs are post-transcriptional regulators of gene expression that have been demonstrated to function as oncogenes or tumor suppressors depending on their target genes [17]. Many miRNAs have also been shown to affect metastasis in different cancers. In this study we show that miR-192, a miRNA previously known to inhibit cell proliferation and induce apoptosis in LC cells [13], could also exert anti-metastasis effects on NSCLC cells. Furthermore, this anti-metastasis role might be mediated by ZEB2, a master EMT regulator. Our study suggests that miR-192 could be an important determinant of NSCLC development and progression.

miR-192 was first cloned by Lagos-Quintana et al. [18] and later confirmed by Lim et al. [19]. The miR-192 gene is located on human chromosome 11 and is transcribed as a cluster with miR-194. Expression of miR-192 has been reported to be upregulated or downregulated in certain cancer types [10-12] and its biological effects on these cancers have been partially identified. miR-192 has been shown to inhibit the liver metastasis of colon cancer through targeting Bcl-2, Zeb2, and VEGFA [15], to inhibit cell proliferation and induce apoptosis in LC [13], to suppress tumor progression in renal cell carcinoma [20] and, to be a biomarker of distant metastasis in gastric cancer [11]. However, whether it impairs the metastasis of NSCLC cells remains as a puzzle.

Our study clearly reveals that miR-192 is an antimetastasis factor in A549 cells. Its significantly decreased expression largely contributed to NSCLC progression by promoting the continued metastasis of the disease. We also highlighted that miR-192 targeting of ZEB2 appears to be an important pathway in inhibiting metastasis, although much work still needs to done to better support our conclusion as the specific targeting of ZEB2 by miR-192 is currently lacking robust evidence. We did not perform luciferase reporter assays here since we did not observe a consensus binding motif of miR-192 in its 3’-UTR, and additionally, the potential targets of miR-192 provided in TargetScan (http://www.targetscan.org/) did not include ZEB2. Moreover, it is currently unknown whether other mechanisms are also involved in the inhibition of ZEB2 in A549 cells. The negative regulation of ZEB2, especially by miR-192, is an area which clearly still requires further research.

In summary, our findings confirm the downregulation of miR-192 in NSCLC. Notably, we identify for the first time that miR-192/ZEB2 is an important pathway that governs NSCLC metastasis. Our observations provide new insights into miR-192 as an important tumor suppressor and may be of critical importance to the future of NSCLC therapy.

Competing interests

The authors declare that they have no competing interests.

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Received: 2016-8-18

Accepted: 2016-9-26

Published Online: 2016-11-6

Published in Print: 2016-1-1

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