Long non-coding RNA GATA6-AS1 upregulates GATA6 to regulate the biological behaviors of lung adenocarcinoma cells.

BACKGROUND: Lung adenocarcinoma (LUAD) is known to be one of the leading causes of cancer-related deaths globally. In recent decades, long non-coding RNAs (lncRNAs) have been indicated to exert pivotal regulating functions in multiple biological behaviors in the initiation and development of LUAD. However, the functional mechanism of lncRNA GATA binding protein 6 antisense RNA 1 (GATA6-AS1) in LUAD has not been explored.
METHODS: In the current study, GATA6-AS1 expression in LUAD tissues was revealed. Meanwhile, GATA6-AS1 expression in LUAD cells was investigated via RT-qPCR analysis. After A549 and H1975 cells were transfected with GATA6-AS1 overexpression plasmids, EdU and colony formation assays, TUNEL assays and flow cytometry analyses, as well as wound healing and Transwell assays were conducted to detect cell proliferation, apoptosis, migration and invasion. Afterwards, bioinformatic tools, western blot analyses, dual-luciferase reporter assays, and RNA immunoprecipitation (RIP) assays were performed to investigate the correlation of microRNA-4530 (miR-4530), GATA6-AS1 and GATA6.
RESULTS: We found that GATA6-AS1 expression was low-expressed in LUAD tissues and cells. Furthermore, the upregulation of GATA6-AS1 suppressed the proliferative, migration and invasion abilities, as well as promoted apoptotic rate of A549 and H1975 cells. Moreover, the mechanistic investigations revealed that GATA6-AS1 upregulated the expression of its cognate sense gene GATA6 by binding with miR-4530, thereby modulating the malignant progression of LUAD cells.
CONCLUSIONS: GATA6-AS1 repressed LUAD cell proliferation, migration and invasion, and promoted cell apoptosis via regulation of the miR-4530/GATA6 axis, indicating GATA6-AS1 as a new prognostic biomarker for LUAD.

Introduction

Lung cancer is the most common cause of tumor-related deaths with a 5-year survival rate of 5%, and it is estimated that there are 228,820 new cases and 135,720 deaths in 2020 in the United States [1]. From the perspective of histology, small-cell lung cancer and non-small cell lung cancer are two main types of lung cancer [2]. NSCLC accounts for approximately 85% of all lung cancer cases, while lung adenocarcinoma (LUAD) is the main subtype of non-small cell lung cancer [3, 4]. Although the fact that great improvements have been achieved for the diagnosis and treatment of LUAD, the mortality rates of patients diagnosed at advanced stage remain relatively high [5]. Additionally, as the common therapeutic method for LUAD clinically, chemotherapy can only prolong the survival time of LUAD patients without permanent cure [6]. Therefore, it is of the essence to comprehend the molecular mechanism underlying LUAD for the purpose of identifying novel effective diagnostic biomarkers.

Long non-coding RNAs (lncRNAs) are longer than 200 nucleotides with limited or without protein-coding capacity [7]. Increasing studies suggested that lncRNAs serve as vital regulators in a series of cellular behaviors implicated in the tumorigenesis and development of malignancies [8]. For example, MAGI2-AS3 suppresses breast cancer by silencing DNA methylation of MAGI2 [9]. LncRNA-SOX2OT facilitates LUAD cell invasion and migration via miR-122-5p-mediated activation of PKM2 [10]. LINC00858 downregulation represses cell growth and triggers cell apoptosis of gastric cancer by reducing WNK2 promoter methylation [11]. Recently, the novel lncRNA GATA binding protein 6 antisense RNA 1 (GATA6-AS1) was reported to exert tumor suppressive function in gastric cancer [12]. The expression of GATA6-AS1 was downregulated in 483 LUAD tissues compared to 387 normal tissues based on GEPIA database. Nevertheless, the functions and mechanism of GATA6-AS1 in the occurrence and development of LUAD are little known.

MicroRNAs (miRNAs) are evolutionarily conserved and endogenous RNAs with approximately 22 nucleotides in length, and have no protein-coding potential [13]. Increasing evidence has indicated that miRNAs play critical regulatory roles in many biological processes of cancers, such as cell proliferation, apoptosis and migration [14]. MicroRNA-4530 (miR-4530) was previously indicated to promote malignant development in breast carcinoma [15]. In addition, it has been revealed that lncRNAs can function as competing endogenous RNAs (ceRNAs) for miRNAs to modulate the expression of downstream mRNAs [16, 17]. In addition, ectopic expressions of lncRNAs lead to the abnormality of ceRNA regulatory network, in which lncRNAs competitively interact with miRNAs to regulate expression patterns of target genes, thus inducing carcinogenesis and cancer growth [18]. For example, lncRNA ACTA2-AS1 promotes cervical cancer development through serving as a ceRNA for miR-143-3p to upregulate SMAD3 expression [19]. Hence, the interaction between GATA6-AS1 and miR-4530 was to be explored in our study.

The aim of the present research is to elucidate the expression levels and biological mechanism of GATA6-AS1 in LUAD cells. Furthermore, the regulatory function of the GATA6-AS1/miR-4530/GATA6 network on the malignant behaviors of LUAD was demonstrated. These findings were able to provide new insights for the pathogenesis of LUAD.

Materials and methods

Bioinformatics analysis

LncLocator (http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/) [20] was used for determination of the subcellular location of GATA6-AS1. miRDB database [21] was used to reveal the miRNAs (181 miRNAs, data not shown) that potentially bind with GATA6 and the binding site of GATA6 and miR-4530. Regrna2 database [22] was used to reveal the miRNAs (57 miRNAs, data not shown) that potentially bind with GATA6-AS1 and the binding site of GATA6-AS1 and miR-4530.

Clinical LUAD tissue collection

Thirty-five clinical LUAD tumor tissues and paired adjacent non-tumor tissues were collected from Liaocheng People’s Hospital. All the tissues were collected during surgical procedures and stored in liquid nitrogen or at − 80 °C for future use. Written informed consents were signed by all the patients and the study was approved by the Ethics Committee of Liaocheng People’s Hospital. All methods were carried out in accordance with relevant guidelines and regulations. Clinical characteristics of LUAD patients were provided in Table 1.

Table 1

Clinical characteristics of LUAD patients

Variables	Cases (n)
Age (years)
≤ 60	26
> 60	9
Sex
Female	21
Male	14
Differentiation
High	11
Medium or low	24
T Staging
T1 + T2	20
T3 + T4	15
Cervical lymph node metastasis
N0	27
N +	8
Distant metastasis
M0	34
M1	1
Smoking
Yes	21
No	14

LUAD: lung adenocarcinoma

Cell culture

The four LUAD cell lines (A427, A549, H1975, HCC827) and human lung cell line (BEAS-2B) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). All the cells were maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA), added with 10% heat-inactivated fetal bovine serum (Gibco) in a moist air atmosphere with 5% CO2 at 37 °C.

Cell transfection

For the purpose of overexpressing GATA6-AS1 or GATA6, the pcDNA3.1-GATA6-AS1 or pcDNA3.1-GATA6 vectors were purchased from Invitrogen (Carlsbad, CA, USA), while the empty pcDNA3.1 vector served as a negative control (NC). The specific short-hairpin RNAs (shRNAs) targeting GATA6 (sh-GATA6#1: 5’-CCGGCACCACAACTACCACCTTATGCTCGAGCATAAGGTGGTAGTTGTGGTGTTTTTTG-3′ and sh-GATA6#2: 5′-CCGGATTCCCATGACTCCAACTTCCCTCGAGGGAAGTTGGAGTCATGGGAATTTTTTTG-3′) and negative control (sh-NC) were synthesized by GenePharma. (Shanghai, China) for GATA6 knockdown. MiR-4530 mimics and corresponding NC mimics were also purchased from GenePharma. A549 and H1975 cells went through transfection for approximately 48 h using Lipofectamine 2000 (Invitrogen), followed by being collected and utilized for following experiments.

RNA extraction and reverse transcription quantitative PCR (RT-qPCR)

Total RNA was isolated and extracted from LUAD cells by TRIzol reagent (Invitrogen) according to the recommendations of manufacturer. Next, RNA concentration was detected and then reverse transcribed to single-stranded complementary DNA with a Reverse Transcription System Kit (Takara, Dalian, China). Subsequently, RT-qPCR reactions were carried out using Universal SYBR Green Master (Roche, Basel, Switzerland). The quantifications of GATA6-AS1, GATA6, and miR-4530 were measured by the 2−ΔΔCt method [23]. GAPDH and U6 acted as the internal references for normalization, respectively. Primer sequences were listed in Table 2.

Table 2

Relative primer sequences for PCR

Gene	Sequences
GATA6-AS1	Forward: 5′-CCTGGAGAGTTTCAGAAAGGA-3′
	Reverse: 5′-ACGCCTCTTGTCCTAAAGTC-3′
miR-4530	Forward: 5′-CCCAGCAGGACGGGAG-3′
	Reverse: 5′-CTCTACAGCTATATTGCCAGCCAC-3′
U6	Forward: 5′-CTCGCTTCGGCAGCACA-3′
	Reverse: 5′-AACGCTTCACGAATTTGCGT-3′
GATA6	Forward: 5′-AGACTTGCTCTGGTAATAGCA-3′
	Reverse: 5′-CTGTAGGTTGTGTTGTGGG-3′
GAPDH	Forward: 5ʹ-GCATCCTGGGCTACACTG-3ʹ
	Reverse: 5ʹ-TGGTCGTTGAGGGCAAT-3ʹ

EdU assay

For conducting the assay, the Cell-Light EdU DNA Cell Proliferation Kit (RiboBio, Guangzhou, China) was adopted based on the specification of manufacturer. Transfected cells were cultured with EdU for 2 h, fixed with 4% paraformaldehyde, stained with Apollo Dye Solution and mounted with DAPI (Sigma-Aldrich, St. Louis, Missouri, USA). Finally, samples were imaged under a fluorescence microscope (Leica, Mannheim, Germany) and were manually counted. EdU positive cells were calculated as the number of EdU (green) positively stained cells/the number of DAPI (blue) positively stained cells in several randomly selected fields.

Colony formation assay

The treated cells (with the density of 1 × 103 cells/well) were plated into 6-well plates for 2 weeks of incubation, and the medium was replaced every 3 days. Thereafter, the cells were immobilized by paraformaldehyde for 30 min and stained with crystal violet (Aladdin, China) for 20 min at room temperature. After purification by phosphate buffered saline (PBS) twice, the viable cells were visualized and counted from 5 randomly identified fields under a microscope (Nikon, Tokyo, Japan).

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay

The cell apoptosis was investigated using the TUNEL apoptosis assay kit (Beyotime, Shanghai, China) as instructed by the manufacturer. In short, the LUAD cells were cleared with PBS for three times and mounted with 4% paraformaldehyde for 30 min. Next, the abovesaid cells were treated with the PBS possessing 0.3% Triton X-100 at room temperature for 5 min. Subsequently, the TUNEL detection solution was added to the cells. Apoptotic cells were manually counted in 5 randomly selected fields under a light microscope (Olympus Corporation). TUNEL positive cells were calculated as the number of TUNEL (green) positively stained cells/the number of DAPI (blue) positively stained cells.

Flow cytometry analysis

Flow cytometry was conducted to examine cell apoptotic ability utilizing an Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (BioLegend, San Diego, CA, USA). After 48 h of transfection, cells were extracted by trypsin without EDTA, and washed thrice with phosphate-buffered saline. After centrifugation and resuspension in 100 μL of flow cytometry binding buffer, the cells were treated with 5 μL of Annexin-V-FITC and 5 μL of propidium iodide solution in the dark for 15 min. The apoptotic ratio was measured with a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Wound healing assay

The transfected cells were embedded into a 6-well plate with 1 × 103 cells per well and cultivated overnight. Thereafter, a scratch was created with 10 μL pipette tip as the cells were grown to approximately 90% confluence. After being purified thrice with PBS, the cells were imaged from 5 fields in each group via a light microscope (Olympus, Tokyo, Japan). Twenty-four hours later, images were captured again at the same fields.

Transwell invasion assay

The cell invasion assay was performed with a 24-well Transwell chamber (Corning, NY, USA). Transfected A549 and H1975 cells (4 × 104 cells) were plated into the Matrigel-precoated upper chamber. The lower chamber was filled with 600 µL RPMI-1640 containing 10% FBS. After incubation for 24 h, cells on the upper side of the membrane were removed using clean swabs, and cells on the underside were captured under a Leica DM IL LED inverted microscope. The number of invaded cells was counted in 5 randomly selected fields.

Western blot analysis

Total protein was separated and collected from the transfected cells with a lysis buffer (Thermo Scientific, Massachusetts, USA). The protein concentration was detected by the BCA (bicinchoninic acid) Protein Assay kit (Thermo Scientific). The proteins were isolated with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by transferring onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Afterwards, the membrane was sealed with 5% non-fat milk for 10 min and incubated for 12 h at 4 °C with primary antibodies of anti-GAPDH (1:2500, ab9485, Abcam) and anti-GATA6 (1:1000, ab175927, Abcam). Subsequently, the PVDF membrane was washed with TBST buffer for three times and probed with HRP-conjugated secondary antibody for 1 h at room temperature. At last, the immunoreactive bands were observed via chemiluminescence (Millipore) and were quantified using ImageJ 1.52 software. GAPDH was used for normalization.

Subcellular fractionation assay

The nuclear and cytoplasmic parts of transfected cells were divided and rinsed according to the protocol of the Cytoplasmic & Nuclear RNA Purification Kit (Norgen). The expression levels of GAPDH, U6 and GATA6-AS1 in nuclear and cytoplasm fractions of cells was analyzed via RT‐qPCR.

RNA immunoprecipitation (RIP) assay

An RIP assay was conducted with the Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore). Moreover, cell lysates were collected in RNA immunoprecipitation assay (RIPA) lysis buffer (Millipore) with magnetic beads. Next, anti-Ago2 (ab186733, Abcam) or anti-IgG (ab205718, Abcam) was coincubated for 12 h with magnetic beads at 4 °C for the purpose of acquiring immunoprecipitation complex. Later, the immunoprecipitated RNA was gathered and purified using TRIzol reagent (Takara, Dalian, China). Finally, the results were detected by RT-qPCR.

Dual‑luciferase reporter assay

Partial sequences of GATA6-AS1 and GATA6 3′ untranslated region (3′UTR) including wide type or mutant type miR-4530 binding sites were inserted into dual-luciferase reporter vector (pmirGLO; Promega, Madison, WI, USA) to produce GATA6-AS1-WT, GATA6-AS1-MUT and GATA6-WT, GATA6-MUT. Subsequently, the constructed reporter plasmids were respectively transfected into treated cells with miR-4530 mimics. Afterwards, a dual-luciferase reporter assay system (Promega) was adopted to determine the luciferase activity of cells.

Statistical analysis

The statistical analysis was performed utilizing Graphpad Prism 5.02 software (La Jolla, CA, USA). All data were obtained from at least three independent experiments and showed as the means ± standard deviation. Student’s t-test was utilized to measure differences statistically between two groups, while statistical differences among multiple groups were compared by one-way analysis of variance followed by Tukey's post hoc test. The p < 0.05 was indicative of significant difference.

Results

The GATA6-AS1 was low-expressed in lung adenocarcinoma and GATA6-AS1 upregulation inhibited cell growth, migration and invasion

To investigate the functions of GATA6-AS1 in LUAD, the expression pattern of GATA6-AS1 in LUAD tissues was discovered from the database of GEPIA (http://gepia.cancer-pku.cn/index.html). The finding indicated that GATA6-AS1 expression was significantly decreased in LUAD tissues (n = 483) compared with adjacent noncancerous tissues (n = 347) (Fig. 1a). Thereafter, RT-qPCR revealed that GATA6-AS1 expression was downregulated in 35 LUAD tissues compared to that in 35 non-tumor tissues (Fig. 1b). GATA6-AS1 expression was lower in LUAD cells (A427, A549, H1975 and HCC827) than normal human lung cells (BEAS-2B) (Fig. 1c). Since GATA6-AS1 expression was decreased in A549 and H1975 cells, the two cell lines were selected for the subsequent assays. Later, the expression of GATA6-AS1 was significantly elevated in A549 and H1975 cells with transfection of pcDNA3.1-GATA6-AS1, as suggested by RT-qPCR (Fig. 1d). Afterwards, an EdU assay was conducted to analyze the suppressive effect of overexpressed GATA6-AS1 on the proliferative ability of A549 and H1975 cells (Fig. 1e). Moreover, a colony formation assay was carried out, showing that cell proliferation was inhibited after increasing GATA6-AS1 expression (Fig. 1f–g). Next, we carried out the TUNEL assay to detect the influence of upregulated GATA6-AS1 on cell apoptosis. The findings showed that the upregulation of GATA6-AS1 led to a significant increase of the percentage of apoptotic cells (Fig. 1h). Furthermore, the results of flow cytometric analysis suggested that GATA6-AS1 overexpression increaseed the apoptotic rate of A549 and H1975 cells (Fig. 1i). Later, wound healing assay and Transwell invasion assay were conducted to examine whether overexpressed GATA6-AS1 affects migration and invasion capacities of A549 and H1975 cells. The findings demonstrated that GATA6-AS1 overexpression repressed cell migration and invasion compared with negative control group (Fig. 1j–l).

Fig. 1

The downregulated GATA6-AS1 suppressed the proliferation and migration, as well as induced the apoptosis of LUAD. a GATA6-AS1 expression in 483 LUAD tissues and 437 adjacent normal tissues was indicated from GEPIA database. b Expression of GATA6-AS1 in 35 LUAD tissues and non-tumor tissues was detected by RT-qPCR. c RT-qPCR revealed the expression of GATA6-AS1 in LUAD cells and human lung cells. d The overexpression efficiency of GATA6-AS1 was depicted through RT-qPCR. e The EdU assay was performed to evaluate effects of upregulated GATA6-AS1 on proliferative ability of A549 and H1975 cells. f, g The colony formation assay demonstrated cell proliferation after transfection with overexpressed GATA6-AS1. h The influence of GATA6-AS1 overexpression on the apoptosis of LUAD cells was examined by the TUNEL assay. i Flow cytometric analysis depicted the apoptotic rate of A549 and H1975 cells upon the upregulation of GATA6-AS1. j–k The wound healing assay was conducted to determine cell migration affected by overexpressed GATA6-AS1. l Transwell assay was performed to reveal invasion of LUAD cells transfected with pcDNA3.1 or pcDNA3.1-GATA6-AS1. *p < 0.05, **p < 0.01

GATA6 expression was downregulated and positively regulated by GATA6-AS1 in lung adenocarcinoma cells

As the above-mentioned experiments revealed that GATA6-AS1 served as a tumor suppressor in LUAD, GATA6, the cognate sense transcript of GATA6-AS1, drew our attention. We firstly searched the database of GEPIA, finding that GATA6 was low-expressed in LUAD tissues (n = 483) compared to adjacent noncancerous tissue samples (n = 347) (Fig. 2a). GATA6 was downregulated in 35 clinical LUAD tissues compared to that in 35 non-tumor tissues (Fig. 2b). In addition, the results of RT-qPCR and western blot revealed the downregulation of GATA6 expression in LUAD cells compared with normal human lung cells at both mRNA and protein levels (Fig. 2c). Afterwards, we wondered if GATA6-AS1 exerts regulatory effects on GATA6. RT-qPCR analysis revealed that the expression of GATA6 was promoted in A549 and H1975 cells upon the transfection of pcDNA3.1-GATA6-AS1 (Fig. 2d). Meanwhile, the increased protein level of GATA6 in A549 and H1975 cells after overexpressing GATA6-AS1 was depicted by western blot (Fig. 2e). Thereafter, the putative subcellular location was revealed from LncLocator database, suggesting that GATA6-AS1 was mainly located at cytoplasm of LUAD cells (Fig. 2f), indicating the posttranscriptional regulation of GATA6-AS1 on GATA6. This result was further supported by a subcellular fractionation assay, which showed the most percentage of GATA6-AS1 in the cytoplasm of A549 and H1975 cells (Fig. 2g).

Fig. 2

As the cognate sense transcript of GATA6-AS1, GATA6 was low-expressed in LUAD cells. a GEPIA database showed the expression of GATA6 in 483 LUAD tissue samples and 347 corresponding normal tissue samples. b Expression of GATA6 in 35 LUAD tissues and non-tumor tissues was detected by RT-qPCR. c The mRNA expression and protein expression of GATA6 in LUAD cells and normal lung cells were detected by RT-qPCR and western blot. d RT-qPCR analysis was performed to reveal the overexpression efficiency of GATA6 in A549 and H1975 cells. e Western blot analysis showed the expression of GATA6 at the protein level in A549 and H1975 cells after upregulation of GATA6. f LncLocator database predicted the subcellular location of GATA6-AS1. g The main distribution of GATA6-AS1 in A549 and H1975 cells was determined utilizing the subcellular fractionation assay. *p < 0.05, **p < 0.01

GATA6-AS1 interacted with miR-4530 to upregulate GATA6 expression

The ceRNA mechanism is a typical posttranscriptional mechanism, and lncRNAs were widely reported to regulate their cognate sense transcripts via the ceRNA mechanism [24-27]. We hypothesized that GATA6-AS1 regulates GATA6 via the ceRNA mechanism. Subsequently, an RIP assay was conducted to investigate whether GATA6-AS1 exists in RNA-induced silencing complexes (RISCs). As illustrated by Fig. 3a, the abundant enrichment of GATA6-AS1 suggested that GATA6-AS1 might act as the molecular sponge of specific miRNA in the ceRNA regulatory network. Next, two miRNAs (miR-4530 and miR-1520) sharing potential binding sites with GATA6-AS1 were screened out with the screening condition of miRDB [21] and RegRNA2 [22] databases (Fig. 3b). Afterwards, the expression levels of miR-4530 and miR-1200 were examined using RT-qPCR, suggesting that miR-4530 was overexpressed in LUAD cells (Fig. 3c, d). Moreover, RT-qPCR analysis revealed that the expression of miR-4530 was elevated in A549 and H1975 cells with transfection of miR-4530-mimics (Fig. 3e). In addition, the expression level of GATA6 was suppressed by miR-4530 overexpression and then reversed by upregulated GATA6-AS1 in A549 and H1975 cells, as indicated by RT-qPCR (Fig. 3f). GATA6-AS1 can suppress the expression of miR-4530, as revealed in Fig. 3g. Overexpression of miR-4530 rescued the stimulating effects of GATA6-AS1 on GATA6 expression (Fig. 3h). Thereafter, the binding sequences between GATA6-AS1 and miR-4530, or between GATA6 and miR-4530 were depicted via prediction from miRDB and RegRNA2 (Fig. 3i). Subsequently, a dual‑luciferase reporter assay revealed that miR-4530 upregulation inhibited the luciferase activities of GATA6-AS1-WT and GATA6-WT, while the luciferase activities of GATA6-AS1-MUT and GATA6-MUT were unchanged following the same transfection in A549 and H1975 cells (Fig. 3j, k). Later, as shown in the RIP assay, the enrichment of GATA6-AS1, miR-4530 and GATA6 was increased in anti-Ago2 precipitated products in A549 and H1975 cells, indicating that GATA6-AS1, miR-4530 and GATA6 co-existed in RISC (Fig. 3l). In conclusion, the GATA6-AS1 served as a ceRNA against miR-4530 to upregulate GATA6.

Fig. 3

MiR-4530 was the downstream molecule of GATA6-AS1. a The RIP assay verified that GATA6-AS1 existed in A549 and H1975 cells. b Suitable miRNAs were selected with the help of bioinformatic tools. c, d The expressions of miR-4530 and miR-1200 in LUAD cells and normal lung cells were detected by RT-qPCR. e RT-qPCR analysis measured the overexpression efficiency of miR-4530 in A549 and H1975 cells. f GATA6 expression in A549 and H1975 cells after the transfection of miR-4530-mimics and miR-4530-mimics + pcDNA3.1/GATA6-AS1 was investigated using RT-qPCR. g MiR-4530 expression in A549 and H1975 cells transfected with pcDNA3.1/GATA6-AS1 was detected by RT-qPCR. h GATA6 expression in A549 and H1975 cells transfected with pcDNA3.1, pcDNA3.1/GATA6-AS1, or cotransfected with pcDNA3.1/GATA6-AS1 + miR-4530 mimics was detected by RT-qPCR. i The possible binding sites of miR-4530 and GATA6-AS1 or GATA6 were predicted using bioinformatic tools. j, k The dual‑luciferase reporter assay was conducted to detect the interaction of miR-4530 and GATA6-AS1 or GATA6 in A549 and H1975 cells. l The co-existence of GATA6-AS1, miR-4530 and GATA6 in RISC was validated by the RIP assay. *p < 0.05, **p < 0.01

GATA6-AS1 modulated the biological processes of lung adenocarcinoma cells via GATA6

For exploring the underlying functional mechanism of GATA6-AS1 in LUAD, a series of rescue assays were conducted. First, the expression of GATA6 was repressed significantly in A549 and H1975 cells after the knockdown of GATA6 (Fig. 4a). Subsequently, an EdU assay revealed that the GATA6-AS1 upregulation-reduced apoptotic ability of A549 and H1975 cells was restored by silencing GATA6 (Fig. 4b, c). Additionally, the inhibited cell proliferation by GATA6-AS1 overexpression was countervailed after depleting GATA6, as suggested by a colony formation assay (Fig. 4d). Later, the TUNEL assay was performed to illustrate the inhibitory effect of GATA6 knockdown on cell apoptosis previously promoted by upregulated GATA6-AS1 (Fig. 4e). Furthermore, the apoptotic rate of A549 and H1975 cells increased by overexpressed GATA6-AS1 and then suppressed by GATA6 depletion, as elucidated by flow cytometry analysis (Fig. 4f). Thereafter, wound healing and Transwell assay revealed that the GATA6-AS1 overexpression-inhibited migration and invasion capacities of A549 and H1975 cells were counteracted after downregulation of GATA6 (Fig. 4g–h). Therefore, GATA6-AS1 inhibited malignant progression of LUAD cells via upregulating GATA6.

Fig. 4

GATA6-AS1 mediated the proliferation, migration, invasion, apoptosis of LUAD cells by GATA6. a The RT-qPCR analysis measured the knockdown efficiency of GATA6 in A549 and H1975 cells. b, c The EdU assay analyzed the influence of GATA6 silence on proliferative rate of A549 and H1975 cells reduced by overexpressed GATA6-AS1. d The proliferative cells decreased by GATA6-AS1 upregulation was affected by depleting GATA6, as suggested by the colony formation assay. e The TUNEL assay was conducted to examine how GATA6-AS1 overexpression-suppressed cell apoptosis was neutralized after downregulated GATA6. f Flow cytometry analysis investigated the change of apoptotic rate in A549 and H1975 cells after transfected with upregulated GATA6-AS1 and then upregulated GATA6-AS1 plus silencing GATA6. g–h The effects of GATA6 knockdown on GATA6-AS1 upregulation-repressed cell migration and invasion were measured utilizing the wound healing and transwell assays. **p < 0.01

Discussion

As a main histological class of non-small-cell lung cancer, LUAD accounts for a large proportion of cancer-related deaths globally [1]. The molecular studies on LUAD are complex [28]. Smoking is recognized as the major pathogenic factor of LUAD [29]. Although great progresses have been made about therapeutic methods for patients with LUAD [30], the prognosis of this disease remains unsatisfactory due to tumor metastasis and late diagnosis. Therefore, it is urgently required to figure out novel and effective biomarkers for the diagnosis and treatment of LUAD, improving the clinical outcomes.

Emerging evidence has elucidated that a great number of lncRNAs are regulatory factors in the initiation and development of LUAD [31]. For example, lncRNA HOTAIRM1 suppresses LUAD cell proliferation and invasion via the miR-498/WWOX axis [32]. LncRNA PIK3CD-AS2 facilitates malignant progression of LUAD by YBX1-mediated inhibition of p53 pathway [33]. LncRNA FBXL19-AS1 promotes tumor growth and migration via sponging miR-203a-3p in LUAD [34]. It was reported that GATA6-AS1 served as one of the top 10 lncRNAs representing some of the highest clinical diagnostic values for lung squamous cell carcinoma [35]. In the current study, we explored the regulating function of GATA6-AS1 in LUAD. The expression level of GATA6-AS1 was validated to be downregulated in LUAD tissues and cells. Furthermore, loss-of-function assays illustrated that GATA6-AS1 overexpression inhibited cell proliferation, migration, invasion, and induced cell apoptosis in LUAD. Overall, these findings exhibited that GATA6-AS1 exerted tumor suppressive function in the cellular processes of LUAD. Previously, GATA6-AS1 was indicated to suppress proliferation and migration of LUAD cells by binding with miR-543 to upregulate RKIP [36] and by sponging miR-324-5p to increase the expression of FBXO11 and SP1 [37]. GATA6-AS1 is associated with the favorable prognosis of lung squamous cell carcinoma [38].

Even though numerous lncRNAs function in trans via RNA-RNA or RNA–protein interaction, more and more studies have demonstrated that some lncRNAs loci act in cis to modulate expression levels of nearby genes [39]. Previous research has revealed that lncRNAs with the feature of high syntenic conservation across species are related to neighboring transcription factors across the genome, such as PTV1 and MYC, GATA6-AS1 and GATA6, LINC00261 and FOXA2, PITRM1-AS1 and KLF6 [40]. Moreover, GATA6 has been identified as an antioncogene in lung cancer [41-43]. GATA6 exerts suppressive effect in lung cancer by inducing cell senescence [44]. GATA6 transcriptionally inactivates Shh to inhibit lung squamous cell carcinoma cell proliferation and migration [45]. We speculated that GATA6 might exert important functions in LUAD together with GATA6‑AS1 and demonstrated that GATA6 showed low expression in LUAD cells. Moreover, rescue experiments indicated that GATA6 knockdown neutralized the biological behaviors of LUAD cells caused by overexpressed GATA6-AS1. Contradictorily, Yan Xu et al. revealed that silencing of GATA6 exerts anti-oncogenic effects in LUAD [46]. Inhibition of GATA6 reduces the proliferation of Kras mutant LUAD tumors in mouse models [47]. GATA6 activates PCAT1 to maintain stemness of non-small cell lung cancer cells [48].

MiR-4530, as the downstream molecule of GATA6-AS1, was high-expressed in LUAD cells. In the ceRNA mechanism, GATA6-AS1 bound with miR-4530 to upregulate the expression of GATA6, thereby modulating the malignant phenotypes of LUAD cells.

Conclusions

In conclusion, our findings elucidated that GATA6-AS1 suppresses proliferative, migration, invasion abilities and motivated apoptotic capacity of LUAD cells. We innovatively revealed that GATA6-AS1 bound with miR-4530 to inhibit the degradation of GATA6 caused by miR-4530. GATA6-AS1 exerted its suppressive effects on LUAD cells by the miR-4530/GATA6 signaling pathway, suggesting GATA6-AS1 as a potential molecular marker for LUAD (Additional file 1).

Additional file 1. Original western blotting protein bands.