MicroRNA-1285-5p influences the proliferation and metastasis of non-small-cell lung carcinoma cells via downregulating CDH1 and Smad4

Abstract

Abnormal expression of microRNAs has been reported to regulate gene expression and cancer cell growth, invasion, and migration. Recently, upregulation of hsa-miR-1285 was demonstrated in bronchoalveolar lavage fluid samples from patients with lung cancer and downregulation in plasma level of stage-I lung cancer patients. However, the function and the underlying mechanism of miR-1285 in non-small-cell lung carcinoma have not been elucidated. In this study, we found that miR-1285-5p, the mature form of miR-1285, was significantly upregulated in human non-small-cell lung carcinoma cell lines A549 and SK-MES-1. Additionally, cells transfected with the miR-1285-5p inhibitor LV-anti-miR-1285-5p demonstrated significantly inhibited proliferation and invasion and depressed migration. Further analysis demonstrated that the miR-1285-5p precursor LV-miR-1285-5p attenuated the expression of Smad4 and cadherin-1 (CDH1) but that LV-anti-miR-1285-5p showed opposite results. A luciferase reporter assay confirmed that miR-1285-5p targeted Smad4 and CDH1. Mechanism analyses revealed that silence of Smad4 and CDH1 significantly attenuated the inhibitory effects of LV-anti-miR-1285-5p on non-small-cell lung carcinoma growth and invasion. Taken together, our data suggest that miR-1285-5p functions as a tumor promoter in the development of non-small-cell lung carcinoma by targeting Smad4 and CDH1, indicating a novel therapeutic strategy for non-small-cell lung carcinoma patients.

Keywords

Non-small-cell lung carcinoma microRNA-1285-5p Smad4 cadherin-1 proliferation invasion migration

Introduction

Lung cancer is the leading cause of cancer-associated mortality and causes more deaths than the next three most common tumors combined (breast, prostate, and colon).¹ According to the statistics, 87% of lung cancer cases are non-small-cell lung cancer (NSCLC).² MicroRNAs (miRNAs) are a class of non-coding RNA genes whose final product is a 22-nt functional RNA molecule.^3,4 MiRNAs have been found to target several tumor-related genes by combining with their 3′-untranslated regions (UTRs).^5–8 In recent years, miR-1285 and its family members have been widely studied and have been found to play a regulatory role in the development of various tumors.^9–11 Rehbein et al.¹² found that hsa-miR-1285 was upregulated in bronchoalveolar lavage samples from patients with lung cancer, and Gao et al.¹³ found that miR-1285 was downregulated in plasma level of stage-I lung cancer patients. However, a more in-depth study of miR-1285 in NSCLC tissues and cells lines has not been elucidated.

Abnormal expression of miR-1285 could modulate cancer cell growth and metastasis and regulate its target genes.¹⁴ For example, miR-1285 is downregulated in renal cell carcinoma and restoration of mature miR-1285 markedly inhibited cancer cell proliferation.¹⁰ MiR-1285 significantly inhibits cancer cell proliferation, invasion, and migration following its inhibitor transfecting with carcinoma cells. Additionally, abnormal expression of miR-1285-5p and -3p was also found in some kinds of tumor cells and could play important roles in cancer tumor progression. Prior studies indicated that miR-1285-5p was at a high expression levels and associated with infiltrative growth of follicular variant of papillary thyroid carcinomas.¹¹ Additionally, miR-1285-3p could directly repress JUN oncogene expression in hepatocellular carcinoma (HCC) cells.⁹ MiR-1285-3p was significantly dysregulated in patients with pancreatic cancer.¹⁵ MiR-1285 also regulates transglutaminase 2 (TGM2) expression.^10,16 Cadherin-1 (CDH1; also called E-cadherin) is downregulated in a variety of cancer cells, and loss of CDH1 function contributes to cancer progression.^16,17 Moreover, CDH1, folliculin (FLCN), and Smad4 also have the potential to interact with miR-1285 and other miRNAs.^16,17 It is reported that long-term effects of tobacco carcinogens can promote the migration and invasion of human bronchial epithelial cell line 2 (HBEC-2), but CDH1 showed opposite effects.^16,18 FLCN and Smad4 are currently thought to be tumor-suppressor genes that are regulated by miR-1285,^16,19 which suggest that miR-1285 could influence lung cancer by inhibiting the tumor-suppressor gene.²⁰ However, few studies focused on the target gene and the role of miR-1285-5p in NSCLC cell lines.

This study used NSCLC cell lines from the human lung adenocarcinoma cell line (A549) and the human lung squamous cell carcinoma cell line (SK-MES-1) to study the alterations of miR-1285-5p expression in and its effects on NSCLC. We found that miR-1285-5p was upregulated in NSCLC cell lines and primary NSCLC tissues which suggested that miR-1285-5p may play important roles in lung cancer. Our aim is to provide new insights into the treatment of NSCLC.

Materials and methods

Tissue of samples

The study randomly selected 22 prospectively enrolled patients with histologically confirmed primary NSCLC undergoing curative treatment at the Second Affiliated Hospital of Zhengzhou University from 2009 onward. Detailed demographic and clinicopathological data, including information on smoking history and alcohol consumption, were collected at enrollment by medical interview, along with pathological staging of the tumors (Table 1). Primary NSCLC tissues and adjacent normal colon tissue (NCT) samples (>5 cm from edge of primary lung cancer tissues) were collected by surgical resection or biopsy and snap-frozen in liquid nitrogen in the same pathology laboratory within minutes of collection. All human materials were obtained with informed consent. The Clinical Research Ethics Committee of the Second Affiliated Hospital of Zhengzhou University approved the study.

Table 1.

Clinicopathological variables and the expression of miR-1285-5p in NSCLC patients.

	Cases	MiR-1285-5p expression
		Low	High	High %
Total	22	8	14	63.6
Age (years)
<55	6	2	4	66.7
⩾55	16	6	10	62.5
Gender
Female	5	2	3	60
Male	17	6	11	64.7
Alcohol^a
Yes	14	4	10	71.4
No	6	3	3	50
Cigraette^a
Yes	16	6	10	62.5
No	5	2	3	60.0
Differentiation
Well	8	3	5	62.5
Moderate/poor	14	5	9	64.3
Histology
Squamous	15	5	10	66.7
Adenoma	7	3	4	57.1
T stage
T1–T2	4	2	2	50.0
T3–T4	18	6	12	66.7
N stage
N0	6	2	4	66.7
N+	16	6	10	62.5
Clinical stage
I–II	12	4	8	66.7
III–IV	10	4	6	60.0

Some patients were not in neither of subgroups due to undetermined record.

Cell culture

NSCLC cell lines A549 and SK-MES-1, HBEC-2, and human normal lung cells (HMRC-5) were obtained from American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich Co., St. Louis, MO, USA) and 2 mM l-glutamine under an atmosphere of 95% air and 5% CO₂ at 37°C.

Cell transfection

Recombinant lentivirus vectors carrying the miR-1285-5p precursor (LV-miR-1285-5p) or miR-1285-5p inhibitor (LV-anti-miR-1285-5p) were obtained from GenePharma (Shanghai, China). Lentivirus vectors were transduced into NSCLC cells according to the supplier’s instructions. Cells were cultured in a normal medium for 24 h. Then, they were cultured in a medium containing Polybrene (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Thereafter, cells were transfected with the lentivirus on a 0.5 × 10⁵ plaque-forming unit overnight. The medium was refreshed and incubated overnight without Polybrene. Then, stable clones expressing miR-1285-5p or anti-miR-1285-5p were selected with puromycin dihydrochloride (Santa Cruz Biotechnology). The construction of recombinant lentiviral vectors of siRNA specific for Smad4 (si-Smad4) and CDH1 (si-CDH1) were performed by Sangon Biotechnology Co. Ltd. (Shanghai, China). For mechanism research, cells which were pre-transfected with LV-anti-miR-1285-5p were further transfected with the siRNA of Smad4 or CDH1 for 24 h. Cells transfected with null vectors served as the negative control (NC).

Cell Counting Kit-8 assay

The number of viable cells was determined by performing the Trypan blue dye exclusion test, as previously described.²¹ For assessing proliferation, a CCK-8 assay was performed. Cells were seeded in 96-well plate and cultured for 12, 24, 48, and 72 h. Then, Cell Proliferation Reagent Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) was added to each well, and the cultures were incubated for 1 h. Absorbance of the formazan product was detected at 450 nm with a 96-well spectrophotometric plate reader.

Scratch wound-healing assay

NSCLC cells were seeded in 48-well plates and grown to 80% confluence and cultured by serum-free RPMI 1640 medium for 24 h. Wound areas were made by dragging sterile 200 µL pipettes across the bottoms of the culture plates. The medium was then removed and replaced with a fresh serum-free medium containing either test material or controls. Photographs were captured at 4× magnification using an Olympus IX70 microscope equipped with a digital camera at 0 and 24 h. Scratch width was measured by calculating the distance between both edges of the scratch using ImageJ software.

Transwell invasion assay

Transwell membranes coated with Matrigel (Becton-Dickinson, Franklin Lakes, NJ, USA) were used to assay the invasion of the A549 and SK-MES-1 cells in vitro. Cells were plated at 2 × 10⁴ per well in the upper chamber in serum-free medium, and 20% FBS was added to the medium in the lower chamber. The chambers were then incubated in the culture box for 24 h. After incubation, the cells on the upper surface were scraped off, and the invasive cells attached to the lower surface of the membrane inserts were fixed and stained with Diff-Quik (Sysmex, Kobe, Japan). The invading cells were observed and counted under a microscope in four random fields. All assays were performed in triplicate.

Quantitative real-time polymerase chain reaction

According to the manufacturer’s instructions, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used for extracting total RNA. After a reverse transcription step, SYBR Green Master Mix (Life Technologies, Carlsbad, CA, USA) was used for the quantitative analysis of gene expression. The amplification involved a denaturation step, and amplification and quantification were repeated for 40 cycles. Primer sequences were as follows: p53, forward 5′-CAGCCAAGTCTGTGACTTGCACGTAC-3′ and reverse 5′-CTATGTCGAAAAGTGTTTCTGTCATC-3′; TGM2, forward 5′-CACCATGGGAAACTGTTGTG-3′ and reverse 5′-TCTCCGACAGTCTCAGGTCA-3′; FLCN, forward 5′-TGCAGGTGCTGGTGAAGGTAACCT-3′ and reverse 5′-GGGATTGGGCAAGTCAGATGCTTG-3′; Smad4, forward 5′-AAAGGTGAAGGTGATGTTTGGGTC-3′ and reverse 5′-CTGGAGCTATTCCACCTACTGATCC-3′; CDH1, forward 5′-GGTTATTCCTCCCATCAGCT-3′ and reverse 5′-CTTGGCTGAGGATGGTGTA-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5′-ATTGTTGCCATCAATGACCC-3′ and reverse 5′-AGTAGAGGCAGGGATGATGT-3′. The data of the relative gene expression levels were calculated by the 2^−ΔΔCt method and presented as the fold change of transcripts for genes. GAPDH was used as internal control for normalization in quantitative real-time polymerase chain reaction (qRT-PCR).

Western blot

Cells were lysed, the supernatant was quantitated, and total protein was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Millipore, Temecula, CA, USA). The membranes were blocked by 3% nonfat milk for 1.5 h at room temperature and then incubated at 4°C overnight with primary antibodies (Santa Cruz Biotechnology) against Smad4 (1:600), CDH1 (1:800), and GAPDH (1:2000). Then, the membranes were incubated for 1.5 h at room temperature with horse radish peroxidase (HRP)-conjugated secondary antibody (1:18,000; Santa Cruz Biotechnology). The protein bands were visualized using an enhanced chemiluminescence detection system. Densitometry values were normalized to the levels of GAPDH. Quantitation analysis for WB was performed using Image-Pro Plus 6.0 Software (Media Cybernetics, Inc., Rockville, MD, USA).

Luciferase reporter assay

The putative binding site of miR-1285-5p which was contained in the Smad4 3′-UTR or CDH1 3′-UTR complementary DNA (cDNA) fragment and the Smad4 3′-UTR or CDH1 3′-UTR which had been mutated were both amplified and subcloned into a pGL3-luciferase promoter vector (Promega, Madison, WI, USA). We use six-well plates to seed the human embryonic kidney 293 cells at a density of 1 × 10⁵ cells per well and co-transfected with the pGL3-luciferase promoter vector (containing the original or mutated 3′-UTR of Smad4 or CDH1) and miR-1285-5p for 24 h. Subsequently, cells were harvested and lysed in a reporter lysis buffer, and the relative luciferase activity was determined using a dual-luciferase reporter assay kit (Beyotime, Nantong, China).

Statistics

Data are presented as mean ± standard deviation (SD). Statistical differences were analyzed with SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests.

Results

MiR-1285-5p increased in NSCLC cells lines and tissues

The levels of miR-1285-5p in NSCLC tissues were significantly higher than in NCT group (Figure 1(a)) and the high expression rate of miR-1285-5p was 63.6% in NSCLC tissues from 22 patients with NSCLC (Table 1). As shown in Figure 1(b), in A549 and SK-MES-1 groups, the levels of miR-1285-5p were upregulated compared with HBEC-2 and HMRC-5 groups. These findings imply that miR-1285-5p likely functions as a tumor promoter in NSCLC.

Figure 1.

The expression levels of miR-1285-5p are upregulated in NSCLC tissues and cell lines and the inhibition of miR-1285-5p decreases the cell activity of A549 and SK-MES-1. (a) The expression of miR-1285-5p in NSCLC tissues (***p < 0.001 vs NCT). (b) The expression of miR-1285-5p in A549 and SK-MES-1 cells (^##p < 0.01 vs HBEC-2 or HMRC-5). (c) LV-anti-miR-1285-5p inhibits the expression levels of miR-1285-5p in A549 and SK-MES-1 cells; LV-anti-miR-1285-5p inhibits the absorbance OD (450 nm) of (d) A549 and (e) SK-MES-1 cells. (f) The viable cell rates (%) of A549 and SK-MES-1 cells transfected with LV-anti-miR-1285-5p at 72 h (^&p < 0.05 and ^&&p < 0.01 vs control).

MiR-1285-5p contributed to NSCLC cell proliferation

As shown in Figure 1(c), the level of miR-1285-5p was downregulated in the cells transfected with LV-anti-miR-1285-5p. CCK-8 assay demonstrated that the LV-anti-miR-1285-5p markedly inhibited the viable cell rate of the A549 and SK-MES-1 cells (Figure 1(d)–(f)). Furthermore, LV-anti-miR-1285-5p exhibited nearly a time-dependent decrease in cell proliferation (Figure 1(d) and (e)). Quantitative analysis confirmed that the inhibition of miR-1285-5p significantly inhibited viability of both A549 and SK-MES-1 cells at 72 h (Figure 1(f)).

Inhibition of miR-1285-5p partially abolished cell invasion

We further investigated the role of miR-1285-5p in cell invasion. The A549 and SK-MES-1 cells were transfected with LV-anti-miR-1285-5p, and we found that the invasive potential of A549 and SK-MES-1 cells were both markedly declined, compared with the control (Figure 2(a) and (b)).

Figure 2.

The inhibition of miR-1285-5p using LV-anti-miR-1285-5p decreases the cell invasion and migration of A549 and SK-MES-1. (a) and (b) Cell invasion was tested using Transwell invasion assay. (c) and (d) Cell migration ability was tested using scratch wound-healing assay (^&&p < 0.01 vs control).

Inhibition of miR-1285-5p partially blocked cell migration

The effect of miR-1285-5p on cell migration was further investigated. As shown in Figure 2(c) and (d), inhibition of miR-1285-5p significantly decreased the migration rate of both A549 and SK-MES-1 cells. These data indicate that miR-1285-5p might serve as a significant migration promoter in NSCLC cells.

MiR-1285-5p is a negative regulator of the expression of CDH1 and Smad4

The mRNA levels of p53, CDH1, FLCN, and Smad4 were markedly downregulated in NSCLC tissues, compared with NCT (Figure 3(a)). These levels also decreased in A549 and SK-MES-1 cell lines, compared with HBEC-2 and HMRC-5, but the levels of TGM2 did not change (Figure 3(b)).

Figure 3.

Smad4 and CDH1 are the two of the target genes of miR-1285-5p. The mRNA levels of p53, TGM2, FLCN, Smad4, and CDH1 in (a) NSCLC tissues and (b) cell lines (**p < 0.01 vs NCT; ^##p < 0.01 vs HBEC-2 or HMRC-5). (c) The level changes of miR-1285-5p in LV-miR-1285-5p-transfected A549 and SK-MES-1 cells. (d) and (e) The mRNA level changes of p53, TGM2, FLCN, Smad4, and CDH1 in LV-miR-1285-5p-transfected A549 and SK-MES-1 cells. (f) and (g) The mRNA level changes of p53, TGM2, FLCN, Smad4, and CDH1 in LV-anti-miR-1285-5p-transfected A549 and SK-MES-1 cells (^&&p < 0.01 vs control). (h) The protein expression changes of Smad4 and CDH1 in LV-miR-1285-5p or LV-anti-miR-1285-5p-transfected A549 and SK-MES-1 cells. Interaction between miR-1285-5p and the 3′-UTR of (i) Smad4 or (j) CDH1 was tested using dual-luciferase reporter assay (^$$p < 0.01 vs NC). (k) The upregulation of Smad4 and CDH1 protein expression levels in LV-anti-miR-1285-5p-transfected cells was downregulated by si-Smad4 and si-CDH1, respectively.

We then used the vector carrying LV-miR-1285-5p and LV-anti-miR-1285-5p to transfect NSCLC cells (Figures 1(c) and 3(c)). We found that the expression of Smad4 and CDH1 was downregulated by LV-miR-1285-5p transfection (Figure 3(d) and (e)). Smad4 and CDH1 were remarkably upregulated by LV-anti-miR-1285-5p transfection (Figure 3(f) and (g)). However, levels of p53, FLCN, and TGM2 did not significantly change (Figure 3(d)–(g)).

Smad4 and CDH1 are common target genes of miR-1285-5p

Next, we found that LV-miR-1285-5p downregulated Smad4 and CDH1 protein levels and that LV-anti-miR-1285-5p showed opposite results (Figure 3(h)). To validate whether miR-1285-5p regulated Smad4 or CDH1-3′-UTR through the binding sites, the wild-type and mutant type of Smad4-3′-UTR and CDH1-3′-UTR were constructed and cloned into the downstream of the luciferase reporter genes. The results from the luciferase reporter assay demonstrated that miR-1285-5p decreased the luciferase intensity of wild-type Smad4-3′-UTR and CDH1-3′-UTR, compared with the miR-NC cells. However, there was no change in luciferase activity in the 3′-UTR-mutated groups, indicating that Smad4 or CDH1 were the targets of miR-1285-5p (Figure 3(i) and (j)).

MiR-1285-5p regulated A549 and SK-MES-1 cell proliferation, invasion, and cell migration progression through CDH1 and Smad4

To further confirm the relationship between miR-1285-5p and Smad4 or CDH1 in NSCLC cells, LV-anti-miR-1285-5p-transfected A549 and SK-MES-1 cells were further transfected with si-Smad4 and si-CDH1. WB analysis demonstrated that the increases in Smad4 and CDH1 in LV-anti-miR-1285-5p-transfected cells were downregulated by si-Smad4 and si-CDH1 (Figure 3(k)), respectively. Furthermore, in the presence of si-Smad4 or si-CDH1, the values of the absorbance OD (450 nm), the number of invaded cells, and the relative percentage of wound healed (%) of A549 and SK-MES-1 cells, which had been decreased by LV-anti-miR-1285-5p, were all increased (compared with LV-anti-miR-1285-5p group; Figure 4(a)–(e)). Collectively, these data proved that the inhibition of miR-1285-5p negatively regulated NSCLC cell growth, invasion, and migration by targeting both Smad4 and CDH1.

Figure 4.

MiR-1285-5p regulated A549 and SK-MES-1 cell proliferation, invasion, and cell migration progression through CDH1 and Smad4. (a) and (b) The changes in the cell invasion numbers. (c) The changes in the absorbance OD (450 nm) of (d) A549 and (e) SK-MES-1 cells. (d) and (e) The changes in the cell migration ability (*p < 0.05 and ** p < 0.01).

Discussion

MiR-1285 is presently demonstrated as an important NSCLC cell regulator that modulates Smad4 and CDH1, two anti-oncogenes that have been demonstrated in previous studies.¹⁶ In our study, a high level of miR-1285-5p was demonstrated in A549 and SK-MES-1 cells, compared with HBEC-2 and HMRC-5. Prior studies indicated that miR-1285-5p was at a high expression levels and associated with infiltrative growth of follicular variant of papillary thyroid carcinomas.¹¹ Additionally, the levels of miR-1285-5p in NSCLC tissues were significantly higher than in carcinoma adjacent tissues. Consistent with our results, increased expression of miR-1285 was found in bronchoalveolar lavage samples from patients with lung cancer.¹² But miR-1285 was downregualted in plasma of stage-I laryngeal squamous cell carcinoma (LSCC) patients compared to healthy controls,¹³ suggesting that a differential expression of miR-1285 and miR-1285-5p can be found in different tissues or stages of patients with lung cancer. These results stimulated us to investigate the role of miR-1285 in regulating NSCLC cell growth, migration, and invasion.

Previously, miR-1285 was suggested to be an oncogene in MCF-7, SH-SY5Y, and HepG2 cell lines, and it inhibits the expression of the tumor suppressor p53 by directly targeting its 3′-UTR region.¹⁴ MiR-1285 is also positively implicated in the development and progression of several human cancers.^9,10,22 Interestingly, high level of miR-1285-5p was significantly correlated with the malignant development of NSCLC cells. In our study, the overexpression of miR-1285-5p increased the viable cell rate and LV-anti-miR-1285-5p abolished the invasion and movement which are consistent with the above studies. These results demonstrated that miR-1285-5p also plays a role of oncogene in NSCLC cells.

Target genes of miRNAs have been identified as the mediators that help play outstanding gene-regulatory roles. Smad4 and CDH1, two important tumor-suppressing genes, were determined to be the target of miR-1285-5p in this study. Luciferase assay indicated that miR-1285-5p could bind to the 3′-UTR region of Smad4 and CDH1. Western blot (WB) results found that miR-1285-5p significantly decreased the expression of Smad4 and CDH1 as well. Further studies also demonstrated that effect of miR-1285-5p on cell growth, migration, and invasion was mediated by Smad4 and CDH1. These data are consistent with previous predictions that CDH1 and Smad4 are the predicted target genes of miR-1285.^10,16,20 These studies demonstrate that miR-1285-5p could act as an oncogene regulator and promote NSCLC progress by downregulating Smad4 and CDH1.

CDH1, which could be regulated by many miRNAs, was confirmed to be a key modulator in NSCLC cell migration and invasion. In this study, we found that CDH1 was downregulated in NSCLC cells which were controlled by miR-1285-5p by directly targeting its 3′-UTR region. Accumulating evidence supports that CDH1 was regulated by miRNAs and could negatively influence tumor progression. MiR-124 was found to inhibit cell migration and invasion activity but increased expression of CDH1 and apoptosis of A549 cells.²³ Yu et al.²⁴ found a high level of miR-196b in mesenchymal-like NSCLC cells and lung cancer tissues; miR-196b upregulation stimulates cell invasion by targeting homeobox A9, and homeobox A9 increased CDH1 expression. Further results confirmed that CDH1 is the functional target gene of miR1285-5p, which anti-proliferation, invasion and migration effects of LV-anti-miR-1285-5p was attenuated by CDH1 silence in present study.

Recent studies suggest that miR-1285 has the potential to interact with Smad4 by combining with its 3′-UTR in cancer cells.¹⁶ Smad4 is a member of the Smad super family, which is a well-defined tumor inhibitor in NSCLC, pancreatic cancer, and colon cancer.^20,25 In our study, Smad4 expression was inhibited by miR-1285-5p. Reciprocally, downregulation of Smad4 could attenuate the function of LV-anti-miR-1285-5p in the proliferation, migration, and invasion of A549 and SK-MES-1. Previous studies have demonstrated that Smad4 was downregulated by miR-205, which could modulate NSCLC cell epithelial–mesenchymal transition, migration, and invasion.²⁶ Additionally, it has been demonstrated that miR-224 is upregulated in NSCLC tissues, particularly in resected NSCLC metastasis. Increased miR-224 expression promotes cell migration, invasion, and proliferation by directly targeting the tumor suppressors tumor necrosis factor alpha (TNFα)-induced protein 1 and Smad4.²⁷ These results suggest that CDH1 and Smad4 were directly regulated by many miRNAs which include miR-1285-5p and could have a close relationship in the proliferation and metastasis of NSCLC cells.

Previous studies show that loss of CDH1 and Smad4 leads to synergistic carcinogenic action in NSCLC and other cancer progression.²⁸ Loss of CDH1 could promote the carcinogenic potential of epithelial cells in squamous cells carcinoma.²⁹ Hypoxia was reported to induce epithelial–mesenchymal transition in NSCLC,^30,31 which also inhibits CDH1 expression in A549 and HCC2935 cells.³² Additionally, Haeger et al.²⁰ show that loss of Smad4 both initiates and promotes NSCLC development. Park et al.²⁸ find that loss of CDH1 cooperates with Smad4 loss to promote the development and metastatic progression of gastric adenocarcinomas. Our study showed that CDH1 and Smad4, the two target genes of miR-1285-5p, were remarkably inhibited by miR-1285-5p. Furthermore, CDH1 can be increased by deguelin, which is known to suppress the growth of cancer cells and block the phosphorylation of Smad3 and nuclear translocation of Smad4 in PanC-1 cells.³³ Smad4 plays a crucial role as a tumor-suppressor protein in the nucleus via regulating CDH1 and participating in tumor-infiltrative growth and lymph node metastasis.^34,35 These studies indicate that miR-1285-5p modulates the loss of Smad4 and CDH1 and accelerates NSCLC cell growth and metastasis.

In conclusion, we demonstrated that miR-1285-5p was upregulated in NSCLC tissues and cell lines, and inhibition of miR-1285-5p inhibited NSCLC cell proliferation and invasion as well as cell migration by targeting Smad4 and CDH1. Our findings provide a novel insight into miR-1285-5p as a key carcinogenic gene in progression of NSCLC.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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