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Introduction

Lung cancer is one of the most common cancer types with a high mortality rate globally (1). Non-small cell lung cancer (NSCLC) represents a large proportion of lung cancer cases (2). The 5-year survival rate of patients with lung cancer is only 15%, which is due to metastasis (3). Early stage diagnosis of NSCLC might lead to higher survival rates and patients could be effectively treated by surgery, radiotherapy or chemotherapy sooner (4). Therefore, it is important to gain an improved understanding of NSCLC progression.

Long non-coding RNA (lncRNA) is a form of non-coding RNA, ~200 nucleotides in length, that cannot be translated into protein (5). A number of lncRNAs regulate cancer metastasis and progression. In several studies, Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1; transcribed into the antisense strand of OIP5) functions as an oncogenic lncRNA in several cancer types, including oral squamous cell carcinoma (6), bladder cancer (7) and hemangioma (8). OIP5-AS1 was reported to promote lung cancer progression (9,10). Nonetheless, the underlying molecular mechanisms of OIP5-AS1 function in this phenomenon have not been fully elucidated.

MicroRNAs (miRs/miRNAs) are non-coding RNAs ~22 nucleotides in length (11). miRs regulate gene expression levels via occupying 3′-untranslated (3′-UTR) regions of target genes (12). miRs are involved in a number of cancer types, including NSCLC (13,14). miR-140-5p was demonstrated to inhibit proliferation, invasion and drug resistance of NSCLC via the vascular endothelial growth factor A (VEGFA) and Wnt signaling pathways (15,16).

Histone deacetylase 7 (HDAC7) is a class 2 member of the HDAC family (17). Previous studies have shown that HDACs affect gene transcription through complexing with classical transcription factors, such as STAT3, hypoxia-inducible factor-1α, forkhead box protein (FOX) P3 and FOXA1 (18–20). Caslini et al (21) suggested that HDAC7 was necessary for stem cell-like properties of breast cancer cells. Additionally, HDAC7 deletion in endothelial progenitor cells repressed the neovascularization of NSCLC tumors via regulating the transcription of angiogenic genes, such as VEGF (22). However, whether HDAC7 exerts a role in NSCLC cells remains to be elucidated.

The present study aimed to investigate and resolve the novel molecular mechanism underlying OIP5-AS1-induced metastasis in NSCLC. These findings may help advance the understanding of NSCLC progression and aid the development of novel therapeutic targets for treating NSCLC.

Materials and methods

Cell lines

Human lung cancer cells (A549 and H1299) were obtained from American Type Culture Collection. Lung cancer cells were cultured in DMEM (HyClone; Cytiva) supplemented with 10% FBS (Hyclone; GE Healthcare Life Sciences) and 1% penicillin/streptomycin at 5% CO2 and 37°C. In addition, human dermal lymphatic endothelial cells (HDLECs) were obtained from PromoCell GmbH. HDLECs were cultured in endothelial cell growth medium (PromoCell GmbH) at 5% CO2 and 37°C. After 72 h, the conditioned medium (CM) from A549 and H1299 cells were harvested and filtered. HDLECs (2×105 per well) were plated in 6-well cell culture plates.

Cell transfection

HDAC7 and VEGFA cDNA sequences were cloned and ligated into a pcDNA6 vector (Youbio Inc., http://www.youbio.cn/). In brief, RNAs were extracted from A549 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration and purity of RNA were determined using NanoDrop™ 2000 (Thermo Fisher Scientific, Inc.). cDNA was synthesized from 2 µg RNA using the PrimeScript RT kit (Takara Biotechnology Co., Ltd.) at 42°C for 30 min and 85°C for 5 sec. cDNA samples were diluted with 50 µl RNase-free water. HDAC7 and VEGFA cDNA were cloned and amplified by PCR. Q5 High-Fidelity DNA Polymerase (M0491, New England Biolabs Inc.) was used to amplify DNA. HDAC7, forward: 5′-GGTACCATGCACAGCCCCGGCGCTGATGG-3′ and reverse: 5′-CTCGAGTTAGAGATTCATAGGTTC-3′; VEGFA, forward: 5′-GGTACCATGACGGACAGACAGACAGACAC-3′ and reverse: 5′-CTCGAGTCACCGCCTCGGCTTGTCA-3′. The following thermocycling conditions were used: Initial denaturation at 98°C for 30 sec, followed by 30 cycles of 98°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec with final extension at 72°C for 2 min. The constructed vectors (2 µg), NC mimic (50 µM), NC inhibitor (50 µM), miR-140-5p mimic (50 µM) and miR-140-5p inhibitor (50 µM) were transiently transfected into A549 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc). NC mimic: 5′-GGUUCCAUCGUACACUGUUCA-3′; miR-140-5p 5′-mimic: CUCACAGAGAAGGGCAGUG-3′; NC inhibitor: 5′-CCAUCAGUCCCCAUCGCCA-3′; miR-140-5p inhibitor: 5′-CAGUAGUAGAACGGCG-3′. After 48 h of transfection, the cells were used for subsequent experimentation. Transfection efficiency reached 50–70% through visualizing EGFP positive cells under microscopy. Small interfering (si)-negative control (NC) (20 µM), si-OIP5-AS1 (20 µM), miR-140-5p mimic and inhibitor were all synthesized by Shanghai GenePharma Co., Ltd. The sequences were as follows: si-NC:UACCGACUGGCAAUUCAUG; si-OIP5-AS1:GGCAGUAGAAUCACUUAAA and si-HDAC7:GGGCUGACAAAGAAGAAGU.

Gene cloning bacterial transformation

The cDNAs and pcDNA6 vector were double-digested with restriction enzymes (KpnI and XhoI) at 37°C for 12 h. The digested linear sequences were ligated using T4 ligase at 16°C for 12 h. The ligated vectors were then transformed into DH5α competent bacteria (Sangon Biotech Co., Ltd). A tube containing ligated vectors and competent bacteria were incubated at 42°C for 90 sec and then placed on ice.

Bioinformatic prediction

Starbase version 2.0 online program (developed by Sun Yat-sen University) was to perform prediction of interaction between OIP5-AS1 and miR-140-5p. TargetScan (developed by the Massachusetts Institute of Technology) was also used to predict interaction between miR-140-5p and HDAC7.

Dual-luciferase reporter assay

Wild-type and mutant sequences of OIP5-AS1 and HDAC7 were subcloned into a pGL3 vector (Promega Corporation). HDAC7 and VEGFA cDNA were cloned and amplified by PCR. Following 12 h of A549 cell inoculation at density of 3×104 cells/well (24-well plate), the vectors were transfected into cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, cells were collected. Luciferase activities were determined using a Dual-Luciferase Reporter Assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.

Transwell invasion assay

H1299 and A549 cells (~1×105 cells per well) were resuspended in 250 µl DMEM. Cells were plated in the upper chamber containing Matrigel-coated membranes (precoating for 12 h at 4°C). Approximately 300 µl DMEM supplemented with 50% FBS was plated in the bottom chambers. Following incubation for 48 h, invaded cells were stained with 0.005% crystal violet for 2 h at room temperature and visualized under light microscope (magnification, ×400).

Reverse transcription-quantitative PCR (RT-qPCR)

RNA extraction was performed as aforementioned. Subsequently, 0.5 µl cDNA was used for qPCR in a total volume of 10 µl using the SYBR Green I Master Mix kit (Thermo Fisher Scientific, Inc.) and primers. qPCR was performed on an ABI 7500 instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the two-step qPCR program: Initial denaturation at 94°C for 15 min; followed by 40 cycles of 94°C for 30 sec, 60°C for 30 sec and 70°C for 30 sec. GAPDH served as the internal control for OIP5-AS1, VEGFA and HDAC7, whereas U6 was the internal control for miR-140-5p. Relative gene expression was calculated using 2−ΔΔCq method (23). The following primer pairs were used for the qPCR: OIP5-AS1 forward, 5′-TGCGAAGATGGCGGAGTAAG-3′ and reverse, 5′-TAGTTCCTCTCCTCTGGCCG-3′; miR-140-5p forward, 5′-CAGTGGTTTTACCCTATGGTAG-3′ and reverse, 5′-ACCATAGGGTAAAACCACTGTT-3′; HDAC7 forward, 5′-TGCTCTTGTCCTTGTGGAGA-3′ and reverse, 5′-CCACCACCTCTTCCTAGCAG-3′; VEGFA forward, 5′-GGCCAGCACATAGGAGAGAT-3′ and reverse, 5′-ACGCTCCAGGACTTATACCG-3′; GAPDH forward, 5′-CTGACTTCAACAGCGACACC-3′ and reverse, 5′TGCTGTAGCCAAATTCGTTG-3′; U6 forward, 5′CGCTTCGGCAGCACATATACTA-3′ and reverse, 5′GAATTTGCGTGTCATCCTTGCG-3′.

Lymphatic vessel formation assay

HDLECs were subjected to CM treatment on Growth Factor Reduced Matrigel. Matrigel was diluted with PBS buffer at a 1:5 ratio. The resultant solution was used to coat 24-well plates (0.2 ml/well) and incubated at 37°C for 5 h. HDLECs were cultured in DMEM in wells at a density of ~5×104 cells/well. When experiment was finished (6 h after incubation), an inverted microscope (magnification, ×400) was used to observe and capture images. Mean tube lengths were quantified (normalized to the control) using the Image-Pro® Plus software (Media Cybernetics, Inc.).

Western blotting

Total protein was extracted from A549 cells using 1× RIPA buffer (Beyotime Institute of Biotechnology). The protein concentration of supernatant was determined using the bicinchoninic acid method. Approximately 50 µg protein/lane were separated by 10% SDS-PAGE. The separated proteins were then transferred to a nitrocellulose membrane (EMD Millipore). Blocking was performed using 5% non-fat milk for 1 h at room temperature. Subsequently, the immunoblotted membrane was incubated with the following primary antibodies: Anti-HDAC7 (cat. no. ab50212, 1:1,000, Abcam), anti-VEGFA (cat. no. ab51745, 1:1,000, Abcam) and anti-GAPDH (cat. no. GTX124502, 1:1,000, GeneTex Inc.) overnight at 4°C. The following day, the membrane was washed with PBS-Tween-20 (0.05%) (PBS-T) and incubated with secondary antibodies for 1 h at room temperature. anti-mouse IgG-HRP (cat. no. SE131, 1:2,000, Beijing Solarbio Science & Technology Co., Ltd.), anti-rabbit IgG-HRP (cat. no. SE134, 1:2,000. Beijing Solarbio Science & Technology Co., Ltd.). After washing with PBS-T, protein signal intensity was measured using an ECL exposure system.

The Cancer Genome Atlas (TCGA) analysis

The gene expression datasets of lung tumors were downloaded from the Genomic Data Commons (GDC) lung adenocarcinoma TCGA datasets (160 cancer and 369 normal tissues). The datasets used were originally from api.gdc.cancer.gov/data/. For stage plot analysis, we grouped the patients according to stage I, II, III and IV lung cancer. Then, we input genes (OIP5-AS1, miR-140-5p, HDAC7, VEGFA) expression data into GraphPad Prism (GraphPad Software) to draw the stage plot. For Kaplan-Meier plot analysis, the clinical information of survival was used to draw survival curve using GraphPad Prism software.

Statistical analysis

GraphPad Prism 8.0 (GraphPad Software, Inc.) was used to perform statistical analysis. The data of three replicates were calculated as the mean ± SD. Comparisons between two or more experimental groups were analyzed using unpaired Student's t-test and one-way ANOVA with Tukey's post hoc test. Lung tumor tissues and paired adjacent normal tissues of TCGA were evaluated using paired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

OIP5-AS1 promotes NSCLC metastasis

To determine the role of OIP5-AS1 in NSCLC metastasis, TCGA datasets of NSCLC were analyzed. OIP5-AS1 was significantly upregulated in lung tumor tissues compared with normal tissues (Fig. 1A). Moreover, higher levels of OIP5-AS1 were observed in advanced stages of NSCLC tumors (stage III and IV) compared with lower stages of tumors (stage I and II) (Fig. 1B). Low levels of OIP5-AS1 were associated with higher overall survival rate of patients with NSCLC (Fig. 1C).

Figure 1. OIP5-AS1 promotes NSCLC metastasis. (A) Bioinformatic analysis of The Cancer Genome Atlas datasets showed OIP5-AS1 levels in human normal lung tissues (n=369) or lung adenocarcinoma tumors (n=160). (B) Stage plot showing that OIP5-AS1 levels were associated with advanced stages of patients with NSCLC. F=3.62, P=0.0135. (C) Kaplan-Meier plot showed the overall survival rate of patients with lung adenocarcinoma. P=0.014, n (high) =80, n (low) =80. (D) Reverse transcription-quantitative PCR showing OIP5-AS1 levels in H1299 or A549 cells transfected with si-NC or si-OIP5-AS. (E) Representative microscopy images (400× magnification) and (F) quantification (normalized to the control) of lymphatic vessel formation in human dermal lymphatic endothelial cells cultured with conditioned medium of H1299 or A549 cells transfected with si-NC or si-OIP5-AS1. (G) Transwell assay showing invasion of H1299 or A549 cells transfected with si-NC or si-OIP5-AS1 (400× magnification). Data are presented as the mean ± standard deviation. *P<0.05; ***P<0.001. OIP5-AS1, Opa-interacting protein 5 antisense RNA 1; NSCLC, non-small cell lung cancer; si, small interfering; NC, negative control; TPM, Transcripts Per Million.

Next, OIP5-AS1 knockdown in H1299 and A549 cells was performed, and RT-qPCR analysis showed that OIP5-AS1 levels significantly decreased in si-OIP-AS1 groups compared with si-NC groups (Fig. 1D). Compared with si-NC groups, OIP5-AS1 knockdown led to a significant reduction in lymphatic vessel formation ability in HDLECs incubated with H1299 or A549 CM (Fig. 1E and F). Likewise, OIP5-AS1 knockdown attenuated the invasion of H1299 or A549 cells compared with the si-NC group (Fig. 1G). These data showed that OIP5-AS1 plays a role in NSCLC progression.

miR-140-5p rescues OIP5-AS1-induced NSCLC metastasis

miRNAs were reported to function in several cancer types by being sponged by lncRNAs (24–26). Based on these findings, the present study aimed to identify potential miRNAs responsible for OIP5-AS1-enhanced NSCLC progression. miR-140-5p was identified as the candidate gene using bioinformatics analysis (Fig. 2A). To validate the findings, a dual-luciferase reporter assay was performed using A549 cells, as this is the most used cell line in previous stduies investigating lung cancer (27,28). The results demonstrated that the miR-140-5p mimic significantly reduced the luciferase activity of A549 cells compared with NC mimic, whereas miR-140-5p inhibitor significantly increased luciferase activity compared with the NC inhibitor group. There is no significant alteration in mutant OIP5-AS1-expressing A549 cells (Fig. 2B). Furthermore, miR-140-5p mimic significantly reduced OIP5-AS1 expression levels compared with the NC mimic group, while miR-140-5p inhibitor significantly increased OIP5-AS1 expression levels compared with the NC inhibitor group. The miR-140-5p mimic greatly increased miR-140-5p levels and the miR-140-5p inhibitor downregulated miR-140-5p expression (Fig. 2C). Clinically, it was found that miR-140-5p levels were significantly lower in NSCLC tumors compared with normal tissues (Fig. 2D).

Figure 2. miR-140-5p rescues OIP5-AS1-regulated NSCLC metastasis. (A) Bioinformatics analysis showing the binding site of OIP5-AS1 by miR-140-5p. Complementary sequences are shown in bold. (B) Luciferase activity in A549 cells transfected with NC mimic, miR-140-5p mimic, NC inhibitor and miR-140-5p inhibitor. (C) RT-qPCR showing miR-140-5p and OIP5-AS1 expression levels in A549 cells transfected with NC mimic, miR-140-5p mimic, NC inhibitor and miR-140-5p inhibitor. (D) Bioinformatic analysis of The Cancer Genome Atlas datasets showing miR-140-5p levels in human normal lung tissues (n=369) or lung adenocarcinoma tumors (n=160). (E) RT-qPCR showing miR-140-5p levels in A549 cells transfected with si-NC, si-OIP5-AS1 or si-OIP5-AS1 + miR-140-5p inhibitor. (F) Representative microscopy images (400× magnification) and (G) quantification (normalized to the control) of lymphatic vessel formation in human dermal lymphatic endothelial cells cultured with conditioned medium of A549 cells transfected with si-NC, si-OIP5-AS1 or si-OIP5-AS1 + miR-140-5p inhibitor. (H) Transwell assay showing invasion of A549 cells transfected with si-NC, si-OIP5-AS1 or si-OIP5-AS1 + miR-140-5p inhibitor (400× magnification). Data are presented as the mean ± standard deviation. *P<0.05; **P<0.01 and ***P<0.001. miR, microRNA; OIP5-AS1, Opa-interacting protein 5 antisense RNA 1; NSCLC, non-small cell lung cancer; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; TPM, Transcripts Per Million.

To investigate the role of miR-140-5p in OIP5-AS1-regulated metastasis, miR-140-5p levels were examined in A549 cells treated with si-OIP5-AS1, si-OIP5-AS1 plus miR-140-5p inhibitor. si-OIP5-AS1 increased miR-140-5p levels by 4-fold and miR-140-5p inhibitor restored miR-140-5p levels compared with the si-NC (Fig. 2E). Transfection with si-OIP5-AS1 attenuated the lymphatic vessel formation ability in HDLECs cultured with A549 medium, which was partly rescued by miR-140-5p inhibitor (Fig. 2F and G). OIP5-AS1 knockdown reduced cell invasion compared with the si-NC group. Cell invasion was rescued by miR-140-5p inhibitor (Fig. 2H). Taken together, these data demonstrated that miR-140-5p is a mediator of OIP5-AS1-regulated NSCLC metastasis.

HDAC7 is involved in miR-140-5p-modulated metastatic phenotypes of NSCLC

The aforementioned findings led to the investigation of the downstream targets of miR-140-5p in NSCLC. TargetScan was used and predicted the epigenetic regulator HDAC7 as a potential candidate (Fig. 3A). The dual-luciferase reporter assay results showed that compared with their respective NC groups, miR-140-5p mimic significantly reduced luciferase activity of A549 cells containing the wild-type 3′-UTR of HDAC7, whereas miR-140-5p inhibitor significantly increased luciferase activity. There is no significant alteration in mutant 3′-UTR of HDAC7-expressing A549 cells (Fig. 3B). Compared with their respective NC groups, western blotting results demonstrated miR-140-5p mimic attenuated HDAC7 protein levels, while miR-140-5p inhibitor increased HDAC7 protein levels (Fig. 3C). In addition, HDAC7 levels were significantly higher in NSCLC tumors compared with normal adjacent tissues (Fig. 3D).

Figure 3. HDAC7 is involved in miR-140-5p-modulated metastatic phenotypes of non-small cell lung cancer. (A) Bioinformatics of the putative binding site at the 3′-UTR of HDAC7 by miR-140-5p. Complementary sequences are shown in bold. (B) Luciferase activity in A549 cells transfected with NC mimic, miR-140-5p mimic, NC inhibitor and miR-140-5p inhibitor. (C) Western blotting showing HDAC7 expression levels in A549 cells transfected with NC mimic, miR-140-5p mimic, NC inhibitor and miR-140-5p inhibitor. GAPDH was the internal control. (D) Bioinformatic analysis of The Cancer Genome Atlas datasets showing HDAC7 levels in human normal lung tissues (n=369) or lung adenocarcinoma tumors (n=160). Reverse transcription-quantitative PCR showing HDAC7 levels in A549 cells transfected with (E) pcDNA6 and pcDNA6-HDAC7 and (F) NC mimic, miR-140-5p mimic and miR-140-5p mimic + HDAC7. (G) Representative microscopy images (400× magnification) and (H) quantification (normalized to the control) of lymphatic vessel formation in human dermal lymphatic endothelial cells cultured with conditioned medium of A549 cells transfected with NC mimic, miR-140-5p mimic and miR-140-5p mimic + HDAC7. (I) Transwell invasion assay showing invasion of A549 cells transfected with NC mimic, miR-140-5p mimic and miR-140-5p mimic + HDAC7 (400× magnification). Data are presented as the mean ± standard deviation. *P<0.05; **P<0.01 and ***P<0.001. HDAC7, human dermal lymphatic endothelial cells; miR, microRNA; NC, negative control; 3′UTR, 3′-untranslated region; TPM, Transcripts Per Million.

To evaluate whether HDAC7 plays a role in miR-140-5p-regulated metastasis, HDAC7 was elevated by ~24-fold in pcDNA6-HDAC7-transfected A549 cells compared with pcDNA6-transfected A549 cells. Meanwhile, miR-140-5p mimic downregulated HDAC7 expression and HDAC7 overexpression in miR-140-5p mimic-transfected A549 cells resulted in 4–5-fold upregulation of HDAC7 compared with the NC mimic (Fig. 3E and F). Compared with the NC mimic group, miR-140-5p mimic significantly decreased the lymphatic vessel formation ability of HDLECs incubated with A549 medium, which was rescued by HDAC7 overexpression (Fig. 3G and H). miR-140-5p mimic reduced the number of invaded cells, and this trend was enhanced by HDAC7 (Fig. 3I). The results suggested that HDAC7 plays a role miR-140-5p-inhibited NSCLC metastasis.

VEGFA acts as an effector for HDAC7-regulated NSCLC metastasis

It was recently reported that HDAC7 contributed to cancer progression via regulating VEGFA expression levels (21). VEGFA has also been shown to be implicated in cancer metastasis (29,30). Thus, it was hypothesized that VEGFA acted as a downstream effector for the OIP5-AS1/miR-140-5p/HDAC7 axis in NSCLC cells. RT-qPCR and western blotting were performed to analyze VEGFA expression levels in HDAC7-knockdown A549 cells (Fig. 4A and B). HDAC7 expression was markedly reduced in A549 cells transfected with si-HDAC7 compared with si-NC. Consistent with previous results, HDAC7 knockdown resulted in downregulation of VEGFA levels compared with the si-NC group. VEGFA expression levels were significantly upregulated in NSCLC tumors compared with normal adjacent tissues (Fig. 4C).

Figure 4. VEGFA functions as an effector for HDAC7-regulated non-small cell lung cancer metastasis. (A) RT-qPCR showing HDAC7 and VEGFA levels in A549 cells transfected with si-NC and si-HDAC7. (B) Western blotting of HDAC7 and VEGFA levels in A549 cells transfected with si-NC and si-HDAC7. GAPDH was the internal control. (C) Bioinformatic analysis of The Cancer Genome Atlas datasets showing VEGFA levels in human normal lung tissues (n=369) or lung adenocarcinoma tumors (n=160). RT-qPCR showing VEGFA levels in A549 cells transfected with (D) pcDNA6 and pcDNA-VEGFA and (E) si-NC, si-HDAC7 and si-HDAC7 + VEGFA. (F) Representative microscopy images (400× magnification) and (G) quantification (normalized to the control) of lymphatic vessel formation in human dermal lymphatic endothelial cells cultured with conditioned medium of A549 cells transfected with si-NC, si-HDAC7 and si-HDAC7 + VEGFA. (H) Transwell invasion assay showing invasion of A549 cells transfected with si-NC, si-HDAC7 and si-HDAC7 + VEGFA (400× magnification). Data are presented as the mean ± standard deviation. *P<0.05; **P<0.01 and ***P<0.001. HDAC7, human dermal lymphatic endothelial cells; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; NC, negative control; HDAC7, human dermal lymphatic endothelial cells; VEGFA, vascular endothelial growth factor A; TPM, Transcripts Per Million.

To assess the function of VEGFA in NSCLC, VEGFA was overexpressed in HDAC7-depleted A549 cells (Fig. 4D and E). HDAC7 knockdown impaired lymphatic vessel formation of HDLEC cells incubated with A549 medium; however, VEGFA overexpression significantly restored this phenotype (Fig. 4F and G). In addition, si-HDAC7 cells reduced invasion ability compared with the si-NC group, which was reverted by VEGFA overexpression (Fig. 4H). The results suggested that VEGFA plays a role in OIP5-AS1/miR-140-5p/HDAC7 axis-induced tumor metastasis of NSCLC.

Discussion

The present study demonstrated that OIP5-AS1 negatively interacted with miR-140-5p, thereby regulating HDAC7 and VEGFA expression levels (Fig. 5). miR-140-5p functioned as an effector for the biological role of OIP5-AS1 in lung cancer cell metastasis. HDAC7 and VEGFA rescued OIP5-AS1/miR-140-5p-related tumor phenotypes, and it was observed that the OIP5-AS1/miR-140-5p/HDAC7 axis was dysregulated in TCGA lung tumors.

Figure 5. Model of OIP5-AS1-modulated NSCLC metastasis. OIP5-AS1 promoted HDAC7 and VEGFA expression via binding miR-140-5p, subsequently contributing to NSCLC metastasis. OIP5-AS1, Opa-interacting protein 5 antisense RNA 1; NSCLC, non-small cell lung cancer; HDAC7, human dermal lymphatic endothelial cells; VEGFA, vascular endothelial growth factor A; miR-140-5p, microRNA-140-5p.

Generally, patients with NSCLC are treated with chemotherapy agents, such as cisplatin, for most cases (31). Although the overall survival rate of patients with NSCLC has improved recently, the therapeutic effect of chemotherapy reaches a plateau with a 20% response rate and median survival of 8–10 months (32). More notably, molecular targeted therapy is one of the most novel strategies for NSCLC treatment, such as epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement (33). Gefitinib and crizotinib are the two targeted drugs for EGFR and ALK tyrosine kinase inhibitors, respectively (33). In the present study, it was confirmed that OIP5-AS1 exerted a role in NSCLC progression, suggesting that OIP5-AS1 may have value as a diagnostic biomarker and therapeutic target for NSCLC treatment.

LncRNAs function as endogenous miRNA decoys in cancer to modulate the expression of downstream target genes (34). It was shown that OIP5-AS1 acts as a competitive endogenous RNA for sponging miRNAs, such as miR-338-3p (6), miR-448 (9) and miR-378a-3p (10). In the present study, we demonstrated that OIP5-AS1 negatively regulated miR-140-5p by complementary binding.

A study indicated that miR-140-5p suppressed the migration, proliferation and invasion of NSCLC cells (14). The lncRNA HANR negatively regulated miR-140-5p to aggravate the proliferation, migration and invasion of NSCLC (35). Consistently, the present study found that miR-140-5p inhibited the invasion and lymphatic vessel formation ability of A549 cells. In addition, miR-140-5p levels were relatively lower in human clinical lung tumors tissues compared with normal tissues. Additionally, miR-140-5p was shown to partly restore OIP5-AS1-induced phenotypes.

HDAC7 is a member of the HDAC family. Commonly, HDAC7 upregulates the acetylation of histones (especially H3) in an enzymatic activity-dependent manner, thereby activating the expression of target genes (36). However, HDAC7 also exerts non-enzymatic dependent activity, instead functioning via interaction with other transcriptional factors (18,37). The present data showed that HDAC7 positively regulated VEGFA, which is in agreement with previously published study (21). Caslini et al (21), indicated that HDAC7 reduced H3K27ac, a super enhancer-related gene, levels in human breast cancer cells. Caslini et al reported that HDAC7-knockdown influenced chromatin configuration and blocked the expression of oncogenes. Moreover, the present results confirmed the regulatory relationship between HDAC7 and VEGFA using RT-qPCR and western blot analysis.

VEGF was shown to induce angiogenesis in a VEGFR2-dependent manner, which is important for tumor metastasis (38). Recently, homeoprotein Six1, a member of Six family of homeodomain transcription factors, enhanced colorectal cancer metastasis and angiogenesis via upregulating VEGF (39). miR-195 inhibited VEGF to regulate squamous cell lung cancer metastasis and angiogenesis (40). Sinha et al (41) suggested that VEGF was involved in breast cancer metastasis and angiogenesis via the focal adhesion kinase/matrix metalloproteinase-9 signaling pathway. In agreement with the present data, it was also observed that VEGFA promoted lung cancer invasion and lymphatic vessel formation.

In summary, the present study suggested the oncogenic role of lncRNA OIP5-AS1 in NSCLC metastasis via miR-140-5p at the cellular and clinical levels. HDAC7 and VEGFA are two downstream effectors for mediating OIP5-AS1/miR-140-5p-regulated NSCLC metastasis. The present findings may provide an improved understanding of NSCLC progression and accelerate the development of diagnostic and therapeutic strategies for the treatment of NSCLC.

Acknowledgements

Not applicable.

Funding

The study was supported by Project jointly built by HENAN Medical Science and Technology Public Relations Plan (grant no. 2018020837).

Availability of data and materials

The datasets analyzed in this study are available in The Cancer Genome Atlas, (https://cancergenome.nih.gov/).

Authors' contributions

JM conceived and designed the experiments, and wrote the manuscript. GL, WW, PX and DY performed the experiments. HB and RL analyzed the data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References