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. 2018 Dec 13;234(8):12828–12838. doi: 10.1002/jcp.27939

Ornithine aminotransferase promoted the proliferation and metastasis of non‐small cell lung cancer via upregulation of miR‐21

Yanfeng Liu 1,2,†, Lei Wu 3,†, Kai Li 2, Fengrui Liu 2, Li Wang 2, Dongling Zhang 2, Jing Zhou 4, Xuan Ma 4, Shengyu Wang 4, Shuanying Yang 1,✉
PMCID: PMC13483213  PMID: 30549035

Abstract

The incidence and mortality of lung cancer ranked the first among all types of cancer in China, and non‐small cell lung cancer (NSCLC) is the most common type of lung cancer accounting for 85% of all lung cancers. Given that the survival rate of patients with advanced NSCLC is still poor nowadays, identification of novel therapeutic targets and the development of effective therapies are desired for the treatment of NSCLC in clinics. In this study, we reported the upregulation of ornithine aminotransferase (OAT) in NSCLC cells and clinical tumor samples as well as its association with the advanced TNM stage, metastasis, and poor tumor differentiation of lung cancer. Using different NSCLC cell lines, we demonstrated that OAT promoted the proliferation, invasion, and migration, inhibited the apoptosis, and altered cell cycle of NSCLC cells; besides, the involvement of OAT‐miR‐21‐glycogen synthase kinase‐3β signaling in the functional role of OAT in NSCLC was also revealed. Importantly, in the absence of OAT, the growth and metastasis of tumor lung cancer xenograft was significantly suppressed in the nude mice. Based on our findings, OAT may be a potential novel biomarker for the diagnosis and therapeutic outcome monitoring of NSCLC. Inhibition of OAT may also represent a new therapeutic strategy of NSCLC.

Keywords: GSK‐3β, invasion and migration, miR‐21, non‐small cell lung cancer, ornithine aminotransferase, proliferation

In this study, we reported the upregulation of ornithine aminotransferase (OAT) in non‐small cell lung cancer (NSCLC) cells and clinical tumor samples as well as its association with the advanced TNM stage, metastasis, and poor tumor differentiation of lung cancer. Based on our findings, OAT may be a potential novel biomarker for the diagnosis and therapeutic outcome monitoring of NSCLC. Inhibition of OAT may also represent a new therapeutic strategy of NSCLC.

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1. INTRODUCTION

Based on the statistics from the National Central Cancer Registry (NCCR) of China, incidence and mortality of lung cancer ranked the first among all types of cancer in 2015. The estimated new cases of lung cancer in China were 733,300 in 2015 and 610,200 lung cancer‐related deaths were expected (W. Chen et al., 2016b). Of note, non‐small cell lung cancer (NSCLC), any type of epithelial lung cancer other than small cell lung cancer, is the most common type of lung cancer, accounting for 85% of all lung cancers. NSCLC basically includes squamous cell carcinoma, lung adenocarcinoma, and large cell carcinoma, and each type of NSCLC has different kinds of cancer cells that grow and spread in different ways. The leading risk factor of NSCLC is cigarette smoking, and other risk factors also include exposure to radiation and air pollution, etc (Katanoda et al., 2011; Li et al., 2014). The treatment of NSCLC depends on the cancer stages and individual characteristics of the patients. Nowadays, the standard treatment options of NSCLS include surgery, chemotherapy, radiation therapy, immunotherapy, and targeted therapy, and so forth. NSCLC is less sensitive to chemotherapy and radiation therapy, more than one treatment option is often used for patients with NSCLC for a better outcome (Non‐small Cell Lung Cancer Collaborative, 2000); however, the survival rate of patients with advanced NSCLC is still poor. For Stage III lung cancer, the survival rate of patients is about 13%, and the survival rate is even lower in Stage IV lung cancer patients (Curran et al., 2011). Thus, identification of novel therapeutic targets and the development of effective therapies are desired for the treatment of NSCLC in clinics.

The identification of mutations in NSCLC has led to the development of targeted therapy to improve the survival rates of different subsets of patients, such as the mutations in epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene, anaplastic lymphoma kinase receptor, and human epidermal growth factor receptor 2 (Pao & Girard, 2011). Ornithine aminotransferase (OAT), an enzyme encoded by the OAT gene in humans, is involved in the ultimate formation of proline from the amino acid ornithine (Katagiri et al., 2014). It has been reported that OAT is needed to establish spindle formation at least in rapidly proliferating cancer cells (Wang, Shang, Burgett, Harran, & Wang, 2007). Recently, OAT was reported to be overexpressed in hepatocellular carcinoma and inactivation of OAT using gabaculine was found to significantly suppress the proliferation of liver cancer cell lines including Hep3B and HepA1−6 (Zigmond et al., 2015). As a potentially novel therapeutic target for cancer, OAT is known to be expressed in almost all the human tissues, with relatively low expression in lungs (Ginguay, Cynober, Curis, & Nicolis, 2017; Wang et al., 2013). However, its expression in lung cancer, especially in NSCLC, remains unclear.

Nowadays, substantial evidence have demonstrated the functional roles of microRNAs (miRNAs) in the development and progression of lung cancer, in particular, in the regulation of the proliferation, differentiation, growth, mobility, and apoptosis of lung cancer cells (Inamura & Ishikawa, 2016). Different miRNAs including miR‐205, miR‐196b, and miR‐375 also have the potential to be used as biomarkers for the histological classification of different NSCLCs, especially the squamous cell carcinoma and adenocarcinoma (Hamamoto et al., 2013). In this study, we for the first time examined the expression of OAT in lung cancer cells and tissues and elucidated the functional role of OAT in the progression of lung cancer. Given the importance of miRNAs in NSCLC, we also investigated the possible interactions between OAT and downstream miRNAs as well as the underlying signaling pathways, with an aim to identify potentially novel biomarkers and therapeutic targets for the diagnosis and treatment of NSCLC.

2. MATERIALS AND METHODS

2.1. Cell culture and clinical samples

Normal lung fibroblast MRC‐5 and NSCLC cell lines A549, H226, and H1299 were obtained from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were maintained in EMEM (MRC‐5), F‐12K (A549) or RPMI‐1640 (H226 and H1299) medium supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C.

Eighty pairs of human lung cancer samples and normal adjacent tissues were collected during the surgical resection at the Second Affiliated Hospital of Xi'an Jiaotong University. The tissues were frozen stored in liquid nitrogen immediately after the collection. This study was approved by the Institutional Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University, and the written informed consent from all the patients was collected before sample collection. Both lung cancer samples and normal adjacent tissues were confirmed by pathological examination. The general clinical information of the patients was shown in Table 1.

Table 1.

The relationship between OAT mRNA expression and clinicopathological features in patients with NSCLC

Characteristics Case number, n OAT mRNA expression p value
Low (n = 40) High (n = 40)
Age, y
<60 28 12 16 0.4823
≥60 52 28 24
Gender
Male 44 23 21 0.8224
Female 36 17 19
TNM stage
I‐II 43 27 16 0.0243
III‐IV 37 13 24
Histological type
Adenocarcinoma 31 14 17 0.6466
Squamous carcinoma 49 26 23
Distant metastasis
No 45 30 15 0.0014
Yes 35 10 25
Lymph node metastasis
No 33 22 11 0.0225
Yes 47 18 29
Tumor differentiation
Well/Moderate 30 20 10 0.0368
Poor 50 20 30
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2.2. Plasmids, small interfering RNAs, miRNAs, and cell transfections

The OAT‐overexpressing vector (pcDNA3.1‐OAT) and the control plasmid (pcDNA3.1) were constructed by GenePharma (Shanghai, China). The siRNAs targeting OAT (si‐OAT) and control scrambled siRNAs (si‐NC) were designed and synthesized by Ribobio (Guangzhou, China). The miR‐21 mimics, miR‐21 inhibitors and the respective negative controls (mimics NC and inhibitors NC) were also purchased from Ribobio. The cell transfections with plasmids, small interfering RNAs (siRNAs) or miRNAs was performed by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. At 24 hr after transfection, cells were processed for further experimentation.

2.3. Reverse transcription and quantitative real‐time polymerase chain reaction

Total RNA from cells and tissues was isolated by using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The same amount of total RNA (1 μg) was used to synthesize cDNA by reverse transcription using the PrimeScript RT Master Mix Kit (Takara‐Bio, Dalian, China). Real‐time PCR was performed using an ABI 7900 system (Applied Biosystems, Foster City, CA). Data analysis was carried out using the 2−▵▵Ct method for relative quantification. GAPDH was used as the internal control for OAT mRNA expression, and U6 was used as the internal control for miRNA expression.

2.4. Cell counting Kit‐8 (CCK‐8) assay

CCK‐8 assay was performed to determine cell proliferation. Briefly, transfected cells were seeded into a 96‐well plate, and cell viability was evaluated with CCK‐8 (Beyotime, Beijing, China) at 0, 24, 48, and 72 hr after seeding. Following the Cell Counting Kit (CCK)‐8 assay at 37°C for 1 hr, the absorbance at 450 nm was measured by using a microplate reading (Bio‐Tek, Winooski, VT).

2.5. Cell colony formation assay

Briefly, 0.5% agar in 1.5 ml cell culture medium was added into each well of the 6‐well plate. When the medium was solid, 5,000 cells suspended in 2 ml culture medium with 0.3% agar were seeded on the top of the basal gel. Cells were then incubated at 37°C in a humidified atmosphere with 5% CO2 for 10 days, and the medium was refreshed every 3 days. Afterward, cells were fixed with 4% formaldehyde and stained with 0.1% crystal violet, then rinsed with phosphate buffered saline (PBS). The number of colonies was recorded using microscopy and calculated.

2.6. Cell migration and invasion assay

The in vitro cell migration and invasion assays were carried out using transwell inserts from Corning (Corning, NY). For the transwell migration assay, treated cells were re‐suspended in 100 μl FBS‐free medium at a final density of 1 × 106 cells/mL and seeded into the upper chambers of transwell inserts in a 24‐well plate. The lower chambers were filled with 600 μl medium with 10% FBS. After incubation at 37°C for 36 hr, invaded cells on the lower surface of the transwell membrane were fixed and stained with crystal violet. The images of the cells were recorded using a Nikon microscope equipped with a digital camera, 4–6 observation areas were randomly selected for each group. For the cell invasion assay, the transwell membrane was pre‐coated with 50 μl Matrigel (30 μg/well; BD, San Jose, CA) before seeding the cells, and the following procedures were the same as transwell migration assays.

2.7. Apoptosis by flow cytometry

Annexin V and propidium iodide (PI) double staining were used to determine the level of apoptosis in the lung cancer cells. Cells were seeded into 12‐well plates at a density of 1.0 × 105 cells/mL and allowed to incubate overnight. For apoptosis determination, cells were harvested, washed with PBS and re‐suspended in Annexin‐binding buffer containing Annexin V and PI. After incubation for 15 min at room temperature, the cells were subject to a BD LSRFortessa Cell Analyzer (BD Biosciences, San Jose, CA) using 488 nm excitation and 530&575 nm emission wavelengths. Data were finally analyzed using Flow Jo 7.6.1 software (Tree Star, Inc., Ashland).

2.8. Cell cycle distribution by flow cytometry

Cells were seeded into 6‐well plates at a density of 1.0 × 105 cells/mL and allowed to incubate overnight. Before the cell cycle distribution measurement, cells were harvested, washed with PBS and fixed with 70% ethanol at − 20oC overnight. Next day, after centrifugation at 4,000 rpm for 5 min, the pellet of cells was re‐suspended in staining solution containing PI and RNase A for 15 min. Finally, the samples were analyzed using a BD LSRFortessa Cell Analyzer (BD Biosciences) using 488 nm excitation and 575 nm emission wavelengths. Data were finally analyzed using Flow Jo 7.6.1 software (Tree Star, Inc.).

2.9. Dual luciferase reporter assay

To validate if miR‐21 directly targets the glycogen synthase kinase‐3β (GSK‐3β) 3′‐untranslated region (3′UTR), we performed a firefly luciferase reporter assay. Wild type (WT) and mutated (MUT) putative miR‐21 seed‐matching sites in GSK‐3β 3′UTR were amplified from human cDNA by PCR and inserted into pGL3 vector (Promega, Madison, WI). HEK293T cells were seeded in 24‐well plate and cotransfected with WT or MUT reported plasmid, Renilla luciferase (pRL) plasmids, or miR‐21 mimic, miR‐21 inhibitor or the relative negative controls. After transfection for 48 hr, the cells were harvested and luciferase activity was analyzed with the Dual‐Luciferase Reporter Assay System (Promega).

2.10. Western blot analysis

Equal amount of the proteins (30 μg) were resolved by on a 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio‐Rad, Hercules, CA). After blocking in 5% nonfat milk for 1 hr at room temperature, the PVDF membranes were incubated with primary antibodies of anti‐OAT, anti‐β‐catenin, anti‐cyclin D1, anti‐c‐myc, anti‐GSK‐3β, anti‐N‐cadherin, anti‐vimentin, anti‐E‐cadherin, and anti‐β‐action diluted in 5% bovine serum albumin at 4°C overnight. Afterward, the membranes were incubated with respective horseradish peroxidase‐conjugated secondary antibodies at room temperature for 2 hr. The chemiluminescent signals were developed and detected by the ChemiDoc XRS gel documentation system (Bio‐Rad).

2.11. Tumor xenograft model in nude mice

Male BALB/c nude mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing China). This study was approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Xi'an Jiaotong University. Lentivirus carrying sh‐OAT or sh‐control were designed and packaged by Genechem (Shanghai, China). Stable cell lines were established by infecting lentivirus into H1299 cells and selected by puromycin. Generally, xenograft tumors were established in male BALB/c mice (6‐week old) by injection of sh‐control and sh‐OAT H1299 cells into the posterior flank area of the mice. Food intake and body weight of the mice were monitored during the study. Tumor volume was monitored every week for 5 weeks, and the tumor volume was measured by using the following formula: length × width2/2. At the time of sacrifice, tumors were removed, weighed and stored at − 80°C for further analysis.

For the in vivo lung metastasis assay, cells were injected into the lateral tail vein of male BALB/c mice (6‐week old). The injected mice were euthanized after 5 weeks. The lungs were removed and fixed in 10% formalin, and the number of metastatic nodules in the lung was counted under a dissecting microscope.

2.12. Statistical analysis

Statistical analysis of the data was performed using Prism 5.0 (GraphPad Software, La Jolla, CA). The data were expressed as mean ± standard error of the mean (S.E.M.). Significant differences were analyzed Student's t‐test (for the comparison between two groups) or by one‐way analysis of variance (ANOVA) followed by Dunnett's post hoc test (for the comparison among more than two groups). p < 0.05 indicated statistically significant.

3. RESULTS

3.1. Upregulation of OAT in NSCLC tissues

To study the functional role of OAT in the lung cancer progression, the expression of OAT in human NSCLC tissues and normal adjacent tissues was first examined. As shown in Figure 1a, the relative mRNA expression of OAT in lung cancer samples was significantly higher than that in normal adjacent tissues. The mRNA expression of OAT in the lung cancer tissues was divided into low expression and high expression based on the expression median values. Of note, the upregulated expression of OAT was also associated with the advanced TNM stage, distant metastasis, lymph node metastasis and poor tumor differentiation of lung cancer (Figure 1b–e, Table 1). More important, based on the Kaplan‐Meier survival analysis (Figure 1f), lung cancer patients with high expression of OAT mRNA had a shorter 5‐year survival rate than the patients with low expression of OAT mRNA. These findings indicated that OAT may play a promoting role in lung cancer progression.

Figure 1.

Figure 1

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Upregulation of OAT mRNA in non‐small cell lung cancer tissues. (a) QRT‐PCR determined the expression of OAT mRNA in normal adjacent tissues (n = 80) and lung cancer tissues (n = 80). The expression levels of OAT mRNA were significantly higher in patients with (b) advanced TNM stage, (c) distant metastasis, (d) lymph node metastasis and (e) poor tumor differentiation. (f) Kaplan‐Meier survival analysis of patients with “low expression of OAT mRNA” or “high expression of OAT mRNA.” *p < 0.05 and **p < 0.01 [Color figure can be viewed at wileyonlinelibrary.com]

3.2. Overexpression of OAT promoted the growth and metastasis of A549 cells

The relative mRNA and protein expression of OAT was also examined in normal lung fibroblast MRC‐5 cells and three NSCLC cell lines including A549, H226, and H1299. Consistent with the results in human samples, the OAT mRNA and protein expression in three lung cancer cells were significantly higher than its expression in normal lung fibroblast (Figure 2a,b). To study the functional role of OAT in lung cancer, A549 cells were transfected pcDNA3.1‐OAT to generate OAT‐overexpressing cells. As shown in Figure 2c,d after transfection with pcDNA3.1‐OAT, the mRNA and protein expression of OAT in A549 cells was significantly increased when compared with the pcDNA3.1 control group.

Figure 2.

Figure 2

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In vitro effects of OAT overexpression in A549 cells. (a) QRT‐PCR determined the expression of OAT mRNA in normal lung cell line and lung cancer cell lines. (b) Western blot assay determined the expression of OAT protein in normal lung cell line and lung cancer cell lines. (c and d) A549 cells transfected with pcDNA3.1‐OAT had a higher expression level of OAT mRNA and protein than the control group. Overexpression of OAT promoted cell proliferation (e), increased the number of colonies (f), and invaded cells (g) and migrated cells (h) as determined by CCK‐8 assay, colony formation assay, transwell invasion, and migration assays, respectively. (i) Overexpression of OAT inhibited cell apoptosis. (j) Flow cytometry analysis determined the cell cycle changes in A549 cells transfected with pcDNA3.1 or pcDNA3.1‐OAT. Overexpression of OAT enhanced Wnt/β‐catenin signaling activity (k) and promoted EMT (l) in A549 cells as determined by western blot assay. *p < 0.05 and ***p < 0.001 [Color figure can be viewed at wileyonlinelibrary.com]

Based on the CCK‐8 assay (Figure 2e) and colony formation assay (Figure 2f), overexpression of OAT in A549 cells resulted in a significant increase of both the cell proliferation rate and the number of colonies. Transwell invasion and migration assays (Figure 2g,h) showed that the number of both invaded and migrated A549 cells was also significantly increased after overexpression of OAT, suggesting an increased metastasis capability of the cells by OAT overexpression. Besides, the level of apoptosis was also determined in pcDNA3.1 control and pcDNA3.1‐OAT A549 cells. As shown in Figure 2i, overexpression of OAT reduced the basal level of apoptosis in A549 cells, partially contributing to the increased proliferation rate of the OAT –overexpressing A549 cells.

Given that OAT overexpression increased the proliferation rate of A549 cells, the cell cycle distribution of the cells was also examined. As shown in Figure 2j, when compared with the pcDNA3.1 control group, the G0/G1 populations of OAT‐overexpressing A549 cells were significantly decreased, whereas S populations were increased, respectively. Additionally, overexpression of OAT led to an upregulated protein expression of β‐catenin, cyclin D1, and c‐myc as well as a downregulated protein expression of GSK‐3β (Figure 2k), indicating its stimulation on the Wnt/β‐catenin signaling activity. Given the increased protein expressions of N‐cadherin, vimentin and decreased protein expression of E‐cadherin (Figure 2l), overexpression of OAT also promoted the epithelial–mesenchymal transition (EMT) of OAT‐overexpressing A549 cells. Taken together, these findings demonstrated that overexpression of OAT promoted the proliferation and metastasis of A549 cells.

3.3. Knockdown of OAT inhibited the proliferation and metastasis of H1299 cells

On the other hand, OAT expression was downregulated by siRNA transfection in H1299 cells to further confirm its functional role in the growth and metastasis of lung cancer cells. As shown in Figure 3a,b when compared with the si‐NC control H1299 cells, the mRNA and protein expression of OAT was significantly reduced in si‐OAT transfected H1299 cells, suggesting the successful knockdown of OAT in H1299 cells. After knockdown of OAT, the cell proliferation rate and the number of colonies were reduced significantly as determined by CCK‐8 assay and colony formation assay, respectively (Figure 3c,d). Transwell invasion and migration assays (Figure 3e,f) showed that the number of both invaded and migrated H1299 cells was also significantly decreased after knockdown of OAT, suggesting a suppressed metastasis capability of H1299 cells by OAT knockdown. Besides, after knockdown of OAT, the basal level of apoptosis was increased in H1299 cells (Figure 3g), consistent with the reduced cell proliferation rate caused by si‐OAT interference.

Figure 3.

Figure 3

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In vitro effects of OAT knockdown in H1299 cells. (a and b) H1299 cells transfected with OAT siRNA had a lower expression level of OAT mRNA and protein than the control group. Knockdown of OAT suppressed cell proliferation (c), decreased the number of colonies (d), and invaded cells (e) and migrated cells (f) as determined by CCK‐8 assay, colony formation assay, transwell invasion and migration assays, respectively. (g) Knockdown of OAT promoted cell apoptosis of H1299 cells. (h) Flow cytometry analysis determined the cell cycle changes H1299 cells transfected with si‐NC or si‐OAT. Knockdown of OAT suppressed Wnt/β‐catenin signaling activity (i) and reversed EMT (j) in H1299 cells as determined by western blot assay. *p < 0.05 and ***p < 0.001 [Color figure can be viewed at wileyonlinelibrary.com]

The cell cycle distribution of H1299 cells was also examined after OAT knockdown. As shown in Figure 3h, knockdown of OAT led to an increased G0/G1 population and a decreased S population in H1299 cells, indicating the occurrence of G0/G1 phase cell cycle arrest caused by OAT knockdown. Moreover, after knockdown of OAT in H1299 cells, protein expressions of β‐catenin, cyclin D1, and c‐myc were downregulated and protein expression of GSK‐3β was upregulated (Figure 3i), suggesting the suppression of Wnt/β‐catenin signaling activity by OAT knockdown. The EMT of H1299 cells was also suppressed after OAT knockdown based on the decreased protein expressions of N‐cadherin, vimentin and increased protein expression of E‐cadherin (Figure 3j). These findings in H1299 cells further confirm the functional role of OAT in promoting the growth and metastasis of lung cancer cells.

3.4. OAT promoted the proliferation and metastasis of NSCLC cells via upregulation of miR‐21

To further explore the possible downstream targets of OAT in the regulation of growth and metastasis of lung cancer cells, the relative expression levels of a panel of miRNAs including miR‐21, miR‐200c, miR‐125b, miR‐148b, miR‐365, miR‐124, miR‐32, miR‐146a, and miR‐357 were examined in lung cancer cells after overexpression or knockdown of OAT. Interestingly, as shown in Figure 4a, among all the microRNAs tested, miR‐21 was upregulated by ~4 folds in A549 cells after overexpression of OAT. Accordingly, the level of miR‐21 was significantly downregulated in H1299 cells after knockdown of OAT (Figure 4b), despite the changes of miR‐200c, miR‐365 and miR‐124. Therefore, miR‐21 was selected as a possible downstream target of OAT in lung cancer cells for further experimentation.

Figure 4.

Figure 4

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In vitro effects of miR‐21 in lung cancer cells with OAT overexpression. (a) The expression levels of miRNAs were determined by qRT‐PCR in A549 cells transfected with pcDNA3.1 or pcDNA3.1‐OAT. (b) The expression levels of miRNAs were determined by qRT‐PCR in A549 cells transfected with si‐NC or si‐OAT. (c) MiR‐21 inhibitor transfection suppressed the expression of miR‐21 in A549 cells. (d) miR‐21 inhibitor transfection suppressed the cell proliferation of A549 cells. (e) Cell proliferation, (f) colony formation, (g) cell invasion and (h) cell migration in A549 cells cotransfected with pcDNA3.1 + inhibitor NC, pcDNA3.1‐OAT + inhibitor NC, or pcDNA3.1‐OAT + miR‐21 inhibitor were determined by CCK‐8 assay, colony formation assay, transwell invasion, and migration assay, respectively. *p < 0.05, **p < 0.01, and ***p < 0.001 [Color figure can be viewed at wileyonlinelibrary.com]

An inhibitor of miR‐21 was used to suppress miR‐21 expression with an aim to further examine its role in mediating the functional role of OAT in lung cancer. In the presence of miR‐21 inhibitor, the expression of miR‐21 was significantly downregulated in A549 cells (Figure 4c), suggesting the successful inhibition of miR‐21 by this inhibitor. At 72 hr after transfection with miR‐21 inhibitor, the cell proliferation of A549 cells was significantly suppressed (Figure 4d). More important, as shown in Figure 4e, in the presence of miR‐21 inhibitor, the promoting effect of OAT overexpression on the A549 cells proliferation rate was significantly attenuated, demonstrating that miR‐21 mediated the functional role of OAT in promoting the proliferation of lung cancer cells. In addition, the increased number of colonies, invaded and migrated A549 cells by OAT expression was also attenuated in the presence of miR‐21 inhibitor (Figure 4f–h). These results demonstrated that OAT promoted the growth and metastasis of lung cancer cells via upregulation of miR‐21.

3.5. GSK‐3β was a direct target of miR‐21 in NSCLC

In addition, miR‐21 was found to potentially bind to the 3′UTR of GSK‐3β with putative binding sites indicated in Figure 5a. As shown in Figure 5b, dual‐luciferase reporter assay demonstrated that the luciferase activity of the reporter containing GSK‐3β 3′UTR‐WT, rather than GSK‐3β 3′UTR‐MUT, was significantly decreased by miR‐21 overexpression and increased by the miR‐21 knockdown in HEK293T cells, indicating the direct binding relationship between miR‐21 and GSK‐3β 3′UTR. Overexpression of miR‐21 suppressed the expression of GSK‐3β mRNA in A549 cells, whereas knockdown of miR‐21 increased the expression of GSK‐3β mRNA in A549 cells (Figure 5 c,d). In addition, knockdown of miR‐21 attenuated the effects of OAT overexpression on the levels of Wnt/β‐catenin signaling‐related and EMT‐related proteins (Figure 5e,f).

Figure 5.

Figure 5

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GSK‐3β is a direct target of miR‐21. (a) Putative binding sites between miR‐21 and 3′UTR of GSK‐3β. (b) Luciferase activity of wild‐type (WT) reporter was suppressed by miR‐21 overexpression and increased by the miR‐21 knockdown, and luciferase activity of mutant (MUT) reporter was not affected by miR‐21 overexpression or knockdown. (c) Overexpression of miR‐21 suppressed the expression of GSK‐3β mRNA in A549 cells. (d) Knockdown of miR‐21 increased the expression of GSK‐3β mRNA in A549 cells. The protein levels of (e) Wnt/β‐catenin signaling‐related mediators and (f) EMT markers in A549 cells cotransfected with pcDNA3.1 + inhibitor NC, pcDNA3.1‐OAT + inhibitor NC, or pcDNA3.1‐OAT + miR‐21 inhibitor were determined by western blot assay. (g) Cell proliferation, (e) colony formation, (f) cell invasion and (g) cell migration of OAT‐overexpressing A549 cells in the presence of XAV939 were determined by CCK‐8 assay, colony formation assay, transwell invasion, and migration assay, respectively. *p < 0.05, **p < 0.01, and ***p < 0.001 [Color figure can be viewed at wileyonlinelibrary.com]

Besides, in the presence of XAV939, a Wnt/β‐catenin signaling inhibitor, the promoting effect of OAT overexpression on the A549 cells proliferation rate was significantly attenuated (Figure 5g), suggesting the importance of Wnt/β‐catenin signaling activation in mediating the functional role of OAT in promoting the proliferation of lung cancer cells. As shown in Figure 5h–j, the increased number of colonies, invaded and migrated A549 cells by OAT overexpression were also attenuated in the presence of XAV‐939. Collectively, these results demonstrated that GSK‐3β was a direct target of miR‐21 and activation of Wnt/β‐catenin signaling was also involved in the functional role of OAT in promoting the growth and metastasis of lung cancer cells.

3.6. Knockdown of OAT inhibited the in vivo tumor growth in the nude mice

The lung cancer xenograft model in nude mice derived from control and OAT knockdown H1299 cells was used to further study the in vivo effect of OAT on the tumor growth and metastasis of lung cancer. As shown in Figure 6a, the average volume of tumors in the OAT knockdown group was significantly lower than the volume of tumors in the control group after 21 d. At the time of sacrifice, the tumor weight in OAT knockdown group was also significantly lower than that in control group (Figure 6b). These in vivo results were consistent with the in vitro findings showing that OAT promoted the growth of lung cancer. In addition, knockdown of OAT also reduced the number of lung metastatic nodules in the nude mice as determined by the in vivo lung metastasis assay (Figure 6c). Furthermore, when compared with the tumors from the control group, the relative expressions of both OAT mRNA and protein and the expression of miR‐21 were significantly downregulated in tumors from OAT knockdown group (Figure 6d–f). Consistent with in vitro findings, these findings also demonstrated that in vivo functional role of OAT in promoting the growth and metastasis of lung cancer was also mediated by the upregulation of miR‐21.

Figure 6.

Figure 6

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In vivo effects of OAT knockdown on tumor growth. (a) Knockdown of OAT suppressed the tumor growth in the nude mice as determined by tumor volume measurement. (b) The dissected tumor weight was significantly lower in the sh‐OAT group than that in the sh‐NC group. (c) Knockdown of OAT suppressed the tumor metastasis in the nude mice as determined by lung metastasis assay The expression levels of (d) OAT mRNA and (e) OAT protein were determined by qRT‐PCR and western blot assay, respectively. (f) The expression level of miR‐21 was determined by qRT‐PCR in dissected tumor tissues from the sh‐NC and sh‐OAT group. *p < 0.05, **p < 0.01, and ***p < 0.001 [Color figure can be viewed at wileyonlinelibrary.com]

4. DISCUSSION

The scientific reports on the relationship between OAT and different cancers were relatively rare. As mentioned earlier, OAT has been reported by Zigmond et al. (2015) to be overexpressed in hepatocellular carcinoma, but its functional role in liver cancer was not further investigated. In the current study, we reported the significant upregulation of OAT in NSCLC cells and, more importantly, in clinical lung cancer samples. The high expression of OAT is associated with the advanced stage, metastasis, and differentiation of NSCLC as well as the shorter overall survival rates of the cancer patients. Hence, OAT has the potential to serve as a potential biomarker in the diagnosis and therapeutic outcome monitoring of NSCLC in clinics.

Moreover, our findings demonstrated that OAT promoted the growth and metastasis of NSCLC cells. Accordingly, knockdown of OAT suppressed the proliferation, invasion and migration of NSCLC cell line (H1299) but elevated its basal apoptosis. In this regard, inhibition and inactivation of OAT may represent a novel therapeutic strategy for the treatment of NSCLC. As a matter of fact, Zigmond et al. (2015) reported the inhibition of the proliferation of hepatocellular carcinoma cells by gabaculine, an inhibitor of OAT and gabaculine also suppressed the secretion of Alpha‐fetoprotein (AFP), a biomarker of hepatocellular carcinoma, in liver cancer cells. Besides, the authors also identified a more potent and selective inhibitor of OAT: (1 S,3 S)− 3‐Amino‐4‐(hexafluoropropan‐2‐ylidene) cyclopentane‐1‐carboxylic acid. In liver cancer cells Hep3B and HepG2, this compound significantly suppressed the cell proliferation and AFP secretion. Importantly, in vivo administration of OAT inhibitor significantly suppressed AFP serum levels and tumor growth in hepatocellular carcinoma‐harboring mice (Zigmond et al., 2015). These previous findings demonstrated the potential application of OAT inhibitors in the treatment of hepatocellular carcinoma. Because our study also showed the suppression of NSCLC by OAT knockdown, the anticancer potential of above‐mentioned OAT inhibitors against NSCLC is also worthy of investigation in the future work.

Additionally, upregulation of miR‐21 was also found to be involved in the functional role of OAT in promoting the growth and metastasis of NSCLC cells. MiR‐21 was previously reported to promote the development of NSCLC, partially by downregulation of suppressor of cytokine signaling 1 (SOCS1), suppressor of cytokine signaling 6 (SOCS6) and phosphatase and tensin homolog (PTEN) (Hu et al., 2015; Xue et al., 2016). Our findings on the promoting role of miR‐21 in NSCLC were consistent with this report. Besides, the role of miR‐21 in lung cancer, in particular, its role as a biomarker of therapy response and as a therapeutic target has already been reviewed recently (Markou, Zavridou, & Lianidou, 2016). In addition to SOCS1, SOCS6 and PTEN, the downstream targets of miR‐21 also include programmed cell death protein 4, tropomyosin 1, transforming growth factor‐β, EGFR and reversion‐inducing cysteine‐rich protein with Kazal motifs (Markou et al., 2016; Wang et al., 2015). In NSCLC, plasma miR‐21 level was also reported to be associated with the resistance of cancer cells to platinum‐based chemotherapy, and this kind of resistance in cancer would decrease the therapeutic efficacy of anticancer drugs and lead to chemotherapy failure (Hu et al., 2014; Hu, Li, Gao, & Cho, 2016). Accordingly, downregulation of miR‐21 was able to enhance the sensitivity of A549 cells to anticancer drug cis‐diamminedichloride platinum II both in vitro and in vivo (Xu et al., 2014). In this regard, it would be also interesting to explore the possible relationship between OAT expression and the therapeutic response of NSCLC to chemotherapy, radiation therapy and targeted therapy.

Based on the bioinformatics prediction, GSK‐3β was one of the predicted targets of miR‐21, GSK‐3β was chosen for further investigation in our study due to the fact that GSK‐3β is a pivotal mediator of cancer progression and resistance to therapy (Domoto et al., 2016; Walz et al., 2017). We demonstrated that GSK‐3β was a direct target of miR‐21 in NSCLC and activation of Wnt/β‐catenin signaling was also involved in the functional role of OAT in promoting the growth and metastasis of NSCLC cells. GSK‐3β is a constitutively active protein kinase that acts as a negative regulator in the hormonal control of glucose homeostasis and Wnt/β‐catenin signaling (Llorens‐Martín, Jurado, Hernández, & Avila, 2014). GSK‐3β has been found to be regulated by different miRNAs such as miR‐129 (Chen, Sun, Liu, Zong, & Zhao, 2016a). Our findings not only showed the direct interactions between miR‐21 and GSK‐3β but also enhanced the current understanding of the downstream targets of miR‐21 in NSCLC. Mutations of Wnt/β‐catenin signaling components have been extensively reported in various diseases such as focal dermal hypoplasia, obesity, type II diabetes and different cancers (MacDonald, Tamai, & He, 2009; Wang et al., 2016; Xu et al., 2017). The roles of Wnt/β‐Catenin signaling in the carcinogenesis of lungs have also been reported and reviewed by different groups. In NSCLC, as reported, the mutations of adenomatosis polyposis coli and β‐catenin are uncommon. However, overexpression of Wnt‐1, frizzled‐8, porcupine and transcription factor 4 is common in resected NSCLC and is found to be associated with the poor prognosis of lung cancer (Stewart, 2014). In contrast, other Wnt/β‐Catenin signaling molecules including axis inhibition protein 1 and Dickkopf 3 were found to be downregulated in lung cancer (Rapp, Jaromi, Kvell, Miskei, & Pongracz, 2017). In this regard, the possible regulation of these Wnt/β‐catenin signaling components is also worth further investigation. Nevertheless, given the involvement of Wnt/β‐catenin signaling in the functional role of OAT in NSCLC cells revealed in this study, we provided the scientific community with new evidence to support the important role of Wnt/β‐catenin signaling in the carcinogenesis of lungs. Regulation of Wnt/β‐catenin signaling by different modulators may play an active role in the treatment of NSCLC towards a better therapeutic effect.

Several limitations may be considered in the present study. The present study only selected several lung cancer‐associated miRNAs to examine the effects of OAT on the expression of these miRNAs, and future miRNA array may be performed to further elucidate the role of OAT in modulating miRNA expression. The predicted targets of miR‐21 by using TargetScan are limited to only GSK‐3β, and other predicted targets of miR‐21 may be investigated; in addition, other bioinformatics databases could also be used to determine more novel downstream targets of miR‐21 in the further studies. The present study also lacked the role of miR‐21 as well as GSK‐3β in the lung cancer progression from the clinical perspective, which may be worthy of further investigation.

In summary, this study reported the upregulation of OAT in NSCLC cells and clinical tumor samples as well as the correlation between low OAT expression and increased survival rates of lung cancer patients in clinics. Using different NSCLC cell lines and lung cancer xenograft models in nude mice, we also demonstrated that OAT promoted the growth and metastasis of NSCLC cells and the involvement of OAT‐miR‐21‐GSK‐3β signaling in the functional role of OAT in NSCLC. Based on our findings, OAT may be a potential novel biomarker for the diagnosis and therapeutic outcome monitoring of NSCLC. Inhibition of OAT may also represent a new therapeutic strategy of NSCLC.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

ACKNOWLEDGMENT

This study was supported by Natural Science Basic Research Plan in Shaanxi Province of China (Grant number 2017JM8094), National Natural Science Foundation of China (Grant numbers 81350032 and 81672300).

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