Downregulated microRNA-140-5p expression regulates apoptosis, migration and invasion of lung cancer cells by targeting zinc finger protein 800.

Despite advances in the diagnosis and treatment in recent years, lung cancer is still one of the primary causes of cancer-associated morbidity and mortality in globally. Abnormally expressed microRNAs (miRNAs/miRs) in tumor tissues serve vital roles in the pathological mechanism of tumors and have become prospective biomarkers for cancer diagnosis. The present study aimed to investigate the effects of the miR-140-5p/zinc finger protein 800 (ZNF800) axis in lung carcinoma, and determine its potential underlying molecular mechanisms. The degree of cell proliferation was assessed via the MTT assay, while the migratory and invasive abilities of lung cancer cells were determined via the Transwell and Matrigel assays. The expression levels of miR-140-5p and ZNF800 were detected via reverse transcription-quantitative PCR and western blot analyses. The results demonstrated that miR-140-5p expression was notably higher in normal human bronchial epithelial cells compared with the respective lung cancer cell lines, H292, PC-9, CL1-5 and H460. Furthermore, miR-140-5p expression increased in the lung cancer cells compared with the control cells following transfection with miR-140-5p mimic. Overexpressing miR-140-5p significantly suppressed the proliferative, invasive and migratory abilities of H460 and PC-9 cells, and stimulated cell apoptosis by upregulating the expression of cleaved-caspase-3. Notably, these effects were reversed following transfection with miR-140-5p inhibitor. miR-140-5p was predicted as a negative regulator of ZNF800, and ZNF800 knockdown significantly suppressed the proliferative and metastatic abilities of lung adenocarcinoma (LUAD) cells, which was comparable to the effects of miR-140-5p mimic. Taken together, these results suggest that miR-140-5p may block the malignant phenotype of LUAD by negatively regulating ZNF800 expression. Thus, the miR-140-5p/ZNF800 axis may be used as an alternative therapeutic target for lung carcinoma in general, and LUAD in particular. Copyright: © Zhuo et al.

Introduction

Lung carcinoma constitutes a fatal tumor affecting the lungs (1,2). The spread of lung carcinoma cells is the causative process driving cancer metastasis (3). The majority of cancers derived from the lungs are classified as primary lung neoplasms (1,2). Lung carcinoma remains one of the most common causes of mortality worldwide (4). According to estimates from the World Health Organisation (WHO), ~1.7 million people died of lung cancer worldwide in 2018, accounting for 20% of all cancer-associated deaths (5,6). Despite advancements in the diagnosis and treatment, lung carcinoma remains a principal element that contributes to high morbidity and mortality in global malignancies (6,7). Recently, an assortment of therapeutics has been developed, including the latest anti-programmed cell death protein 1 (PD1) drugs (8). However, according to reports, the 5-year survival rate for 75% of patients with lung carcinoma is <15% (4,9,10). Thus, it remains critical to understand the pathological processes of lung carcinoma and identify novel treatment targets.

MicroRNAs (miRNAs/miRs) are miniscule non-coding RNA molecules, which consist of ~22 nucleotides and are expressed in animals, plants and specific viruses (11,12). For example, miR-155 and miR-168 can be found in plants and animals (11), and viral miR-H6 is essential for the in vivo replication of herpes simplex virus 2 (HSV-2) (13). miRNAs play diverse roles as RNA silencers and post-transcriptional modulators of gene expression (14–16). The majority of miRNAs are intracellular, such as miR-25, miR-302c and miR-181a; however, a minority known as circulating miRNAs originate from the extracellular compartments of several biological fluids, such as exosomal miR-9, miR-211 and miR-133b (17,18). Cancer cells can generate an extensive number of exosomal miRNAs compared with normal cells, thus rendering miRNAs as prospective biomarkers in cancer diagnosis (19–21). Abnormal miRNA expression has been reported in different types of tumor tissues such as lung, bladder and cervical cancer, and it is well-known that miRNAs play notable roles in tumor development (16,22,23). A recent study demonstrated that miR-140-5p is implicated in different types of human and animal cancers, including colorectal cancer (24), glioma (25), non-small cell lung cancer (26,27) and hypopharyngeal squamous cell carcinoma (28), through different molecular mechanisms. miR-140-5p is downregulated in several types of cancer and impairs the proliferation and metastasis of cancer cells by regulating the vascular endothelial growth factor A (VEGFA) gene or regulating VEGFA/matrix metalloproteinase 2 signaling (24,25,29). In addition, miR-140-5p inhibits invasion and migration of FaDu cells by targeting A disintegrin and metalloproteinase 10 in the Notch1 signaling pathway (28). miR-140-5p also inhibits gastric cancer by regulating the WNT1-mediated WNT/β-catenin signaling pathway (30).

Although miR-140-5p has been extensively reported to play an important inhibitory role in several types of cancer cells (31–33), considering the complex molecular mechanisms associated with tumor pathogenesis and the diversity of miR-140-5p functions, the impact of miR-140-5p in different types of cancer requires further investigation, particularly in lung cancer. Zinc finger protein 800 (ZNF800) is a member of the 1 Ensembl protein family, which contains eight splice variants and 141 orthologues that interact with a single phenotype (34). Currently, the research and clinical data associated with ZNF800 are very limited, and its function and molecular mechanism in lung cancer remain unknown.

In the process of predicting the target gene of miR-140-5p via bioinformatics analysis, the results of the present study demonstrated that there are two 7-base binding sites between miR-140-5p and the 3′-untranslated region (UTR) of ZNF800 mRNA, which suggests that the miR-140-5p/ZNF800 pathway is a potential regulatory axis involved in different pathological conditions, such as lung cancer. To further elucidate this hypothesis, the present study performed an association analysis based on information from the publicly available LinkedOmics database (http://www.linkedomics.org/admin.php). However, no significant association between miR-140 and ZNF800 was observed, based on The Cancer Genome Atlas (TCGA)-lung adenocarcinoma (LUAD) and TCGA-lung squamous cell carcinoma (LUSC) datasets. In addition, bioinformatics analysis on gene expression demonstrated opposite trends in the expression levels of miR-140 and ZNF800 in the TCGA-LUAD dataset downloaded from TCGA database (https://www.cancer.gov/tcga).

Thus, the present study aimed to investigate the regulatory association between miR-140-5p and ZNF800. The functions of these two factors in lung cancer cells were assessed to investigate their effects on apoptosis, migration and invasion, and determine whether the miR-140-5p/ZNF800 pathway can be used as an alternative therapeutic candidate for lung carcinoma.

Materials and methods

Lung cancer cell lines

Based on the results of the bioinformatics analysis, four LUAD cell lines commonly used in cancer research (H292, CL1-5, PC-9 and H460) and the normal human bronchial epithelial (NHBE) control cells were selected for the experiments. The purpose of selecting multiple cell lines was to determine whether the expression levels of miR-140-5p and ZNF800 were similar in different lung cancer cell lines compared with NHBE cells. Among these cell lines, H460 is pathological and established from the pleural fluid of patients with large cell lung carcinoma (35), whereas PC-9 is an epidermal growth factor receptor (EGFR)-positive mutant, with 19 mutations in the exon of EGFR (36). In the present study, the pathological cell line (H460) and the EGFR mutant cell line (PC-9) were selected as the representative lung cancer cell lines for downstream overexpression and silencing experiments. Both cell lines were selected independent of their association with miR-140-5P expression to accurately assess the potential role of the miR-140-5p/ZNF800 axis in mutant and non-mutant lung cancer cells. All cell lines were purchased from the American Type Culture Collection and cultured in DMEM supplemented with 10% fetal bovine serum (FBS, both purchased from Gibco; Thermo Fisher Scientific, Inc.). The culture condition was maintained in 5% CO2 and 95% humidified air at 37°C.

miRNA and RNA interference

H460 and PC-9 cells were seeded into 24-well plates at a density of 2×104 cells/well and cultured with either 50 nM hsa-miR-140-5p mimic or negative-control mimics, or 100 nM hsa-miR-140-5p inhibitor or negative-control inhibitor (all purchased from Thermo Fisher Scientific, Inc.) using Dharma FECT 4 transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. In total, 50 nM Human ZNF800 small interfering (si)RNA Oligo (5′-GTCAAGCAGTACTCCTGAACA-3′) or control siRNA (5′-GCCATAGCACAACTCGATAGT-3′) (both purchased from Advanced Biomatrix) were transfected into cells using the X-tremeGENE siRNA Transfection kit (cat. no. 04476093001; Roche Molecular Diagnostics) at 37°C for 48 h, according to the manufacturer's instructions. The subsequent experiments were performed 48 h after transfection.

Cell proliferation assay

Cell proliferation of H460 and PC-9 cell lines was assessed using the MTT assay kit (cat. no. 11465007001; Sigma-Aldrich; Merck KGaA). Briefly, cells were seeded into 96-well plates at a density of 1×104 cells/well and cultured in DMEM for 72 h at 37°C until they reached 80–90% confluence. Cells were subsequently washed twice with phosphate-buffered saline (PBS), prior to incubation with 5 mg/ml MTT reagent for 4 h at 37°C. Following the MTT incubation, the purple formazan crystals were dissolved using 150 µl dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) and proliferation was subsequently analyzed at a wavelength of 490 nm. All experiments were performed in triplicate.

Apoptosis analysis

Following transfection for 48 h, the apoptosis of lung cancer cells (H460 and PC-9) was detected by annexin V-APC/7-AAD (cat. no. KGA1023-1026; Nanjing KeyGen Biotech Co., Ltd.) double staining via flow cytometry. Briefly, cells were digested using 0.25% trypsin (without EDTA) and collected by centrifugation at 300 × g for 5 min at 4°C. The harvested cells were washed twice with PBS and resuspended in 500 µl binding buffer. Cells were subsequently stained with 5 µl Annexin V-APC and 5 µl 7-AAD at room temperature for 15 min in the dark. Apoptotic cells were subsequently analyzed using a flow cytometer (FC500; Beckman Coulter, Inc.) with FlowJo software (version 10.0; FlowJo LLC; BD Bioscience).

Cell migration and invasion assays

Cell migration and invasion of H460 and PC-9 cell lines were detected in vitro via the Boyden Chamber Assays (Essen Bioscience). For cell migration, 3×104 cells were seeded onto a fibronectin-coated polycarbonate membrane in a Boyden Chamber (Essen Bioscience), in 100 µl DMEM (cat. no. 11965092; Thermo Fisher Scientific, Inc.) without FBS. For the invasion assay, Transwell membranes (Corning Life Sciences, Inc.) were precoated with 24 µg/µl Matrigel (R&D Systems, Inc.) for 8 h at 37°C in 5% CO2. A total of 500 µl DMEM supplemented with 10% FBS was plated in the lower chamber as a chemoattractant. Following incubation for 6 h at 37°C in 5% CO2, cells were rinsed with PBS twice and cells in the upper chamber were gently removed using a cotton swab. Migratory cells in the lower chamber were fixed with 100% methanol for 10 min and stained with a 0.1% crystal violet solution for 30 min at room temperature. Then, migratory cells were counted in five predetermined fields using a light microscope (magnification, ×200). All experiments were performed in triplicate.

Reverse transcription-quantitative (RT-q) PCR

Total RNA was extracted from lung cancer cells (H292, CL1-5, PC-9 and H460) and NHBE cells using TRIzol® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. For miR-140-5p, cDNA was synthesized using the PrimeScript RT Enzyme Mix kit (cat. no. DRR037A; Takara Biotechnology Co., Ltd.), according to the manufacturer's instructions. SYBR-Green PCR master mix (cat. no. 4367660; Thermo Fisher Scientific, Inc.) in a ViiA 7 Real-Time PCR system (cat. no. 4453545; Thermo Fisher Scientific, Inc.) was used for qPCR. The following primer sequences were used for qPCR: miR-140-5p forward, 5′-GAGTGTCAGTGGTTTTACCCT-3′ and reverse, 5′-GCAGGGTCCGAGGTATTC-3′; ZNF800 forward, 5′-GGACGAAGAGGGGTCTACCT-3′ and reverse, 5′-CTTCTTTCAACAGCCACGCC-3′; U6 forward, 5′-GGTCGGGCAGGAAAGAGGGC-3′ and reverse, 5′-GCTAATCTTCTCTGTATCGTTCC-3′; and GAPDH forward, 5′-AGACACCATGGGGAAGGTGAA-3′ and reverse, 5′-ATTGCTGATGATCTTGAGGCTG-3′. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 10 min, followed by 45 cycles of amplification (95°C for 15 sec, 58°C for 30 sec for miR-140-5p or 60°C for 15 sec for ZNF800, and 72°C for 10 sec), and a final extension step of 72°C for 5 min. Relative miR-140-5p and ZNF800 expression levels were quantified using the 2−ΔΔCq method (37) and normalized to the internal reference genes U6 small nuclear RNA and GAPDH, respectively. All experiments were performed in triplicate.

Western blotting

Western blotting was performed using the Bio-Rad general protocol (38). Cell lysates were prepared from lung cancer cells (H292, CL1-5, PC-9 and H460) and NHBE cells and total protein were subsequently extracted using (1X) radioimmunoprecipitation assay (RIPA) buffer (cat. no. 2114-100; BioVision, Inc.) and protein concentration was quantified using the Bradford protein assay kit (cat. no. C503031; Bio-Rad Inc.). Protein lysates were purified by SDS-PAGE (15 µg protein/gel lane) on 10–12% gels and subsequently transferred onto polyvinylidene fluoride membranes (EMD Millipore). The membranes were blocked with 5% skim milk for 1 h at 25°C and then incubated with primary antibodies against ZNF800 (1:500; cat. no. PAB21502; Abnova, Inc.), pro-caspase-3 (1:1,000; cat. no. GTX74264; GeneTex, Inc.), cleaved caspase-3 (1:1,000; cat. no. GTX01201; GeneTex, Inc.) and β-actin (1:5,000; cat. no. GTX109639; GeneTex, Inc.) overnight at 4°C. Following the primary incubation, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibodies (cat. no. GTX213110-01; 1:1,000; GeneTex, Inc.) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system (Amersham; Cytiva) and detected using Multi Gauge software (version 2.0; Fujifilm, Inc.).

Bioinformatics analysis of gene expression from TCGA database

Clinical data of miR-140 and ZNF800 from the LUAD and LUSC datasets were downloaded from TCGA database using the ‘TCGAbiolinks’ package in R (version 3.6.3; Lucent Technologies, Inc.). Information from each dataset was divided into two groups (normal tissues and cancer tissues), according to the sampling tissue type.

Prediction of ZNF800 as a target of miR-140-5p

The regulatory association between miR-140-5p and the 3′-UTR of ZNF800 mRNA in H460 and PC-9 cells was predicted in silico using the TargetScan database (release 7.2; http://www.targetscan.org/vert_72/) and validated via the dual-luciferase reporter assay.

Dual-luciferase reporter assay

H460 and PC-9 cells were transfected with 40 ng of wild-type (WT) pGL3-ZNF800 plasmid or pGL3-ZNF800 3′-UTR mutant plasmid (MUT). The pGL3 luciferase reporter plasmid was purchased from Promega Corporation and co-transfected with pRL-TK Renilla luciferase plasmid (40 ng; Promega Corporation), miR-140-5p mimic (5′-CAGUGGUUUUACCCUAUGGUAG-3′; GenePharma, Inc.) or control mimic (5′-UUCUCCGAACGUGUCACGUTT-3′; GenePharma, Inc.) using Lipofectamine® 2000 Transfection Reagent kit (cat. no. 11668030; Thermo Fisher Scientific, Inc.). The pGL3 basic vector (Promega Corporation) was used to construct the pGL3-ZNF800-WT and pGL3-ZNF800 3′-UTR-MUT vectors. The mutation in the 3′-UTR of ZNF800 was generated using the Q5 Site-Directed Mutagenesis kit (cat. no. E0554S; New England Biolabs, Inc.). Following incubation for 24 h at 37°C, luciferase activities were detected using a Dual-Glo™ Luciferase Assay System (Promega Corporation) and normalized by using the ratio of firefly to Renilla luciferase activity, according to the manufacturer's protocol.

Statistical analysis

Statistical analysis was performed using GraphPad Prism v6.01 (GraphPad Software, Inc.). Data are presented as the mean ± standard deviation. One-or two-way analysis of variance, followed by Bonferroni's post hoc analysis were performed to compare differences between multiple groups. A paired Student's t-test was used to compare differences between two groups. An unpaired Student's t-test with Welch's correction was used to compare the differences in the expression levels of miR-140 and ZNF800 between the normal and cancer tissue samples. Each experiment was performed and analyzed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-140-5p is downregulated in lung cancer cells

The clinical data of miR-140 from TCGA database was analyzed and the results demonstrated that miR-140 expression in the LUAD and LUSC datasets were significantly downregulated in cancer tissues compared with normal tissues (P<0.01; Fig. 1A). RT-qPCR analysis was performed to detect miR-140-5p expression in the different lung cancer cells. The results demonstrated that miR-140-5p expression was significantly higher in NHBE cells compared with the H292, PC-9, CL1-5 and H460 cancer cell lines (P<0.01; Fig. 1B), which was consistent with the findings obtained from the public database.

Figure 1.

miR-140-5p regulates the proliferation and apoptosis of human lung cancer cells. (A) miR-140 expression in normal tissues and cancer tissues from the LUAD and LUSC datasets downloaded from The Cancer Genome Atlas database. (B) Reverse transcription-quantitative PCR analysis was performed to detect miR-140-5p expression in lung cancer cell lines (H292, PC-9, CL1-5 and H460) and NHBE cells. The transfection efficiency of overexpressing or silencing miR-140-5p expression in (C) H460 and (D) PC-9 cells. The effect of overexpressing or silencing miR-140-5p expression on the proliferation of (E) H460 and (F) PC-9 cells. The effect of overexpressing or silencing miR-140-5p expression on the apoptotic rate of (G) H460 and (H) PC-9 cells. The effect of overexpressing or silencing miR-140-5p expression on apoptosis-related proteins of (I) H460 and (J) PC-9 cells. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. **P<0.01 vs. NHBE cells, Ctrl mimic or Ctrl inhibitor. miR, microRNA; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; NHBE, normal human bronchial epithelial; Ctrl, control.

miR-140-5p regulates proliferation, apoptosis and metastasis of human lung cancer cells

Following transfection with miR-140-5p-mimic, miR-140-5p expression was significantly upregulated in H460 and PC-9 cells (P<0.01; Fig. 1C and D). Conversely, transfection with miR-140-5p-inhibitor significantly downregulated miR-140-5p expression in H460 and PC-9 cells compared with the respective controls (P<0.01; Fig. 1C and D).

The results of the MTT assay demonstrated that H460 and PC-9 cell proliferation significantly declined following overexpression of miR-140-5p (P<0.01; Fig. 1E and F). Conversely, the proliferation of both cell lines significantly improved following miR-140-5p knockdown (P<0.01; Fig. 1E and F). As presented in Fig. 1G and H, overexpression of miR-140-5p significantly increased the apoptotic rates of H460 and PC-9 cells compared with the control mimic group, whereas silencing miR-140-5p expression significantly decreased the apoptotic rates of both cell lines (P<0.01).

Western blot analysis demonstrated that there was no significant difference in pro-caspase 3 expression between the different groups (P>0.05; Fig. 1I and J). However, cleaved-caspase 3 protein expression was significantly higher in the miR-140-5p mimic group compared with the control mimic group (P<0.01), the effects of which were reversed following miR-140-5p knockdown (P<0.01; Fig. 1I and J). Taken together, these results suggest that overexpressing miR-140-5p increases programmed cell death of lung cancer cells.

H460 and PC-9 cell migration significantly decreased following overexpression of miR-140-5p compared with the control mimic group (P<0.01), the effects of which were reversed following miR-140-5p knockdown (P<0.01; Fig. 2A and B). Comparable findings were observed in the cell invasion assay (P<0.01; Fig. 2C and D). Collectively, these results suggest that miR-140-5p regulates proliferation, apoptosis and metastasis of human lung cancer cells.

Figure 2.

miR-140-5p regulates migration and invasion of human lung cancer cells. The effect of overexpressing or silencing miR-140-5p expression on the migratory ability of (A) H460 and (B) PC-9 cells. The effect of overexpressing or silencing miR-140-5p expression on the invasive ability of (C) H460 and (D) PC-9 cells. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. **P<0.01. miR, microRNA; Ctrl, control.

miR-140-5p regulates ZNF800 expression in human lung cancer cells

To further clarify the molecular mechanism by which miR-140-5p affects lung cancer cells, the regulatory association between ZNF800 and miR-140-5p was assessed. As presented in Fig. 3A, the results of the LUAD dataset demonstrated that ZNF800 expression was significantly higher in cancer tissues compared with normal tissues (P<0.05); however, the opposite was observed in the LUSC dataset (P<0.01), suggesting that there may be a negative regulatory association between miR-140 and ZNF800 in lung cancer cells of the LUAD category. Similarly, ZNF800 mRNA expression was significantly lower in NHBE cells compared with the LUAD cell lines (H292, PC-9, CL1-5 and H460; P<0.01; Fig. 3B). Western blot analysis demonstrated that ZNF800 protein expression was significantly higher in the LUAD cell lines compared with normal cells (P<0.01; Fig. 3C).

Figure 3.

Regulatory role of miR-140-5p on ZNF800 expression in human lung cancer cells. (A) ZNF800 expression in normal tissues and cancer tissues from the LUAD and LUSC datasets downloaded from The Cancer Genome Atlas database. (B) RT-qPCR and (C) western blot analyses were performed to detect ZNF800 mRNA and protein expression levels in lung cancer cell lines (H292, PC-9, CL1-5 and H460) and NHBE cells, respectively. (D) TargetScan software was used to predict the binding site between miR-140-5p and ZNF800. The dual-luciferase reporter assay verified the negative regulatory association between miR-140-5p and ZNF800 in (E) H460 and (F) PC-9 cells. Western blot analysis was performed to assess the effect of overexpressing or silencing miR-140-5p expression on ZNF800 protein expression in (G) H460 and (H) PC-9 cells. RT-qPCR analysis was performed to assess the effect of overexpressing or silencing miR-140-5p expression on ZNF800 mRNA expression in (I) H460 and (J) PC-9 cells. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. *P<0.05; **P<0.01 vs. NHBE cells, Ctrl mimic or Ctrl inhibitor. miR, microRNA; ZNF800, zinc finger protein 800; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; RT-qPCR, reverse transcription-quantitative PCR; NHBE, normal human bronchial epithelial; Ctrl, control; UTR, untranslated region; WT, wild-type; MUT, mutant.

TargetScan software analysis demonstrated that miR-140-5p shares a binding site in the ZNF800 mRNA 3′-UTR sequence (Fig. 3D). The results indicated that cotransfection with miR-140-5p mimic significantly decreased the luciferase activity of the lung cancer cells (H460 and PC-9) in the WT group (P<0.01; Fig. 3E and F). However, there was no change in luciferase activity following cotransfection with miR-140-5p in the MUT group (P>0.05; Fig. 3E and F).

Western blot analysis demonstrated that overexpression of miR-140-5p decreased ZNF800 protein expression in both H460 and PC-9 cells, the effects of which were reversed following transfection with miR-140-5p inhibitor, suggesting that miR-140-5p is a negative regulator for ZNF800 (Fig. 3G and H). Notably, overexpression or silencing of miR-140-5p had no effect on ZNF800 mRNA expression (Fig. 3I and J). Taken together, these results suggest that ZNF800 is a direct post-transcriptional target for miR-140-5p in LUAD.

ZNF800 siRNA induces effects similar to those of miR-140-5p overexpression

Transfection with ZNF800-siRNA was performed to suppress ZNF800 expression in H460 and PC-9 cells. The results demonstrated that both ZNF800 mRNA and protein expression levels were significantly downregulated following transfection with the siRNA vector compared with the control (ctrl) siRNA (P<0.01; Fig. 4A-D). The results of the MTT assay demonstrated that miR-140-5p overexpression and ZNF800 siRNA had similar adverse effects on the proliferative ability of H460 and PC-9 cells (Fig. 4E and F). In addition, both ZNF800 siRNA and miR-140-5p overexpression induced apoptosis of H460 and PC-9 cells (Fig. 4G and H), and significantly increased cleaved-caspase 3 protein expression (P<0.01; Fig. 4I and J).

Figure 4.

ZNF800 siRNA induces effects similar to those of miR-140-5p overexpression. Reverse transcription-quantitative PCR analysis was performed to verify the transfection efficiency of ZNF800 silencing in (A) H460 and (B) PC-9 cells. Western blot analysis was performed to verify the transfection efficiency of ZNF800 silencing in (C) H460 and (D) PC-9 cells. The effect of ZNF800 silencing on the proliferation of (E) H460 and (F) PC-9 cells. The effect of ZNF800 silencing on the apoptotic rate of (G) H460 and (H) PC-9 cells. The effect of ZNF800 silencing on apoptosis-related proteins of (I) H460 and (J) PC-9 cells. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. **P<0.01 vs. Ctrl siRNA or Ctrl mimic. ZNF800, zinc finger protein 800; si, small interfering; miR, microRNA; Ctrl, control.

The effects of ZNF800 silencing and miR-140-5p overexpression on cell migration and invasion were assessed. The results demonstrated that these metastatic features weakened in both H460 and PC-9 cells (Fig. 5). Collectively, these results suggest that compared with ZNF800 siRNA, miR-140-5p partly exerts antitumor activity in lung cancer cells by targeting the 3′-UTR of ZNF800 mRNA.

Figure 5.

ZNF800 silencing affects the migration and invasion of human lung cancer cells. The effect of ZNF800 silencing on the migratory ability of (A) H460 and (B) PC-9 cells. The effect of ZNF800 silencing on the invasive ability of (C) H460 and (D) PC-9 cells. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. **P<0.01. ZNF800, zinc finger protein 800; Ctrl, control; miR, microRNA; si, small interfering.

Discussion

A number of miRNAs play significant roles in the pathophysiology of different types of cancer, including lung cancer (39,40). Based on previous studies, let-7, miR-126, miR-598, miR-148a/b and miR-7 are known to be dysregulated in lung cancer (41–44). To the best of our knowledge, the present study was the first to demonstrate that miR-140-5p exhibits anticancer functions in lung cancer by regulating ZNF800. Taken together, the results of the present study suggest that the miR-140-5p/ZNF800 axis is a candidate therapeutic target for lung cancer treatment.

Previous studies have demonstrated that miR-140-5p is associated with the pathophysiology of several neoplasms, including breast and stomach cancers (29,31). The results of the present study demonstrated that miR-140-5p expression was downregulated in lung cancer cell lines (H292, PC-9, CL1-5 and H460), which is consistent with findings from previous clinical and experimental studies (13,45,46). For example, Tang et al (13) and Flamini et al (46) demonstrated that miR-140-3p and miR-140-5p expression levels are significantly lower in lung cancer tissues compared with adjacent normal tissues. In addition, Huang et al (45) assessed the expression of the miR-140-5p sister strand, miR-140-3p, in 52 squamous cell lung cancer tissues and 22 adjacent normal tissues from clinical samples, and reported that miR-140-3p expression is significantly higher in adjacent normal tissues compared with cancer tissues. A previous study demonstrated that miR-140-5p expression decreases in malignant melanoma, which exerts anticancer functions by modulating the expression of its target gene, YES proto-oncogene 1 (31). Li and He (26) reported that miR-140-5p hinders the cell proliferation and metastasis of non-small cell lung cancer, and in 2018, Yang et al (27) confirmed these findings.

Consistent with previous findings (26,27,46), the results of the present study demonstrated that miR-140-5p inhibited cell proliferation and induced apoptosis of lung cancer cells by upregulating the expression level of cleaved-caspase-3. In addition, miR-140-5p hindered the migration and invasion of lung carcinoma cells. Mechanistic experiments were performed to verify that ZNF800 is the direct target of miR-140-5p. The results suggest that the miR-140-5p/ZNF800 pathway may be an effective regulatory pathway for studying the molecular mechanisms associated with lung cancer. Previous studies have reported that miR-140-5p inhibits the development of cancer through various means. For example, miR-140-5p prevents the cancer cell invasion by regulating VEGFA expression in non-small cell lung cancer (NSCLC) or monocyte to macrophage differentiation-associated protein in hypopharyngeal squamous cell carcinoma (HSCC) (26,27). However, the molecular function and mechanism of ZNF800 in lung cancer remain unknown.

The results of the present study demonstrated that ZNF800 expression was increased in lung carcinoma cell lines compared with NHBE cells, and that miR-140-5p may act as an effective negative regulator of ZNF800. The results verified that miR-140-5p hinders the cancer phenotype of lung carcinoma cells by negatively regulating ZNF800, which contributes to the current understanding on the pathological mechanisms involved in the proliferation, migration and invasion of cancer cells. Notably, the results of the present study demonstrated that transfection with ZNF800 siRNA exerted effects similar to miR-140-5p overexpression. Taken together, these results suggest that miR-140-5p, which negatively regulates ZNF800, inhibits the proliferation and metastasis of lung cancer cells, and thus may be used as a therapeutic target for tumor therapy.

The present study is not without limitations. First, the effects of miR-140-5p inhibitor were only assessed in lung carcinoma cells rather than NHBE cells. Studying the role of miR-140-5p/ZNF800 axis in normal cells would be helpful to analyze the role of this axis under normal physiological conditions, and whether disruption causes adverse reactions or even other diseases. Furthermore, additional cell lines, particularly LUSC-related cell lines, need to be assessed in prospective studies to accurately conclude the effect of the miR-140-5p/ZNF800 axis in lung cancer. In addition, both animal and clinical studies are required to validate the results of the present in vitro studies.

In conclusion, the results of the present study demonstrated that miR-140-5p inhibited the proliferation, invasion and migration of LUAD cells by inhibiting ZNF800 protein expression. Taken together, these results suggest that the miR-140-5p/ZNF800 axis is a novel potential pharmacological target for drug development against lung cancer in general, and LUAD in particular.