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Abstract

BACKGROUND:

It has been discovered that lncRNA ARAP1-AS1 is upregulated and operates as a tumor promoter in many cancers. However, its pattern of expression and potential mechanism in lung adenocarcinoma (LUAD) is still unknown.

METHODS:

The levels of lncRNA ARAP1-AS1, miR-8068, and CEACAM5 expressions in LUAD cell lines and tissues were assessed by conducting western blot and RT-qPCR analyses. MiR-8068’s potential targeting relationships with lncRNA ARAP1-AS1 and CEACAM5 were ascertained by performing bioinformatics analysis. The interaction of lncRNA ARAP1-AS1 with miR-8068 was validated by means of by RIP and luciferase reporter experiments. CCK-8, cell adhesion, and Transwell migration experiments were conducted to study how lncRNA ARAP1-AS1 affects LUAD cell migration, adhesion, and proliferation. To confirm the function of lncRNA ARAP1-AS1 in vivo, a tumor formation experiment was executed.

RESULTS:

An elevated expression of lncRNA ARAP1-AS1 was observed among the LUAD cells and tissues. The overexpression of lncRNA ARAP1-AS boosted cell proliferation, adhesion, and migration in LUAD and also favored in vivo tumor growth. MiR-8068 was found to be lncRNA ARAP1-AS1’s target gene. MiR-8068 overexpression partially antagonized lncRNA ARAP1-AS1’s promotive effect on proliferation, viability, and adhesion. Meanwhile CEACAM5 could alleviate the miR-8068-induced inhibition of tumor growth. The negative correlation of miR-8068 with lncRNA ARAP1-AS1 or CEACAM5 was also revealed.

CONCLUSION:

To upregulate CEACAM5 expression lncRNA ARAP1-AS1 targeted miR-8068, thus promoting the progression of LUAD. This indicates that the lncRNA ARAP1-AS1/miR-8068/CEACAM5 axis has potential as a therapeutic target in LUAD treatment.

Keywords

ARAP1-AS1 miR-8068 CEACAM5 lung adenocarcinoma

1. Introduction

Lung cancer is the second most commonly diagnosed cancer and leading cause of cancer death worldwide [1]. Lung adenocarcinoma (LUAD) represents the primary histological type of lung cancer with unsatisfactory prognosis [2, 3]. In spite of the developments in novel cancer therapies, LUAD still has a 5-year survival rate that is below 20% [4]. Previous research have revealed numerous long non-coding RNAs (lncRNAs) participating in LUAD progression [5, 6, 7]. However, their types and specific mechanisms have yet to be explored completely. Hence, searching for robust biomarkers may help tailor targeted therapeutic treatments for patients with LUAD.

LncRNAs endogenous RNAs that do not code for proteins and contain over 200 nucleotides are involved in numerous malignant cancers. They perform a pivotal role in various cellular functions by regulating gene expression, translation, and chromatin organization [8]. In the recent years, several lncRNAs have been found to be dysregulated in LUAD cells, wherein they served as tumor promoters or suppressors. For example, lncRNA ZFPM2-AS1 induces LUAD by negatively regulating miR-18b-5p and upregulating VMA21 expressions [9]. The pyroptosis-related lncRNA FAM83A-AS1 exerts a carcinogenic effect on LUAD through the miR-150-5p/MMP14 axis [10]. Meanwhile lncRNA LINC00968 suppresses cell invasion, migration, and proliferation in LUAD by targeting the miR-22-5p/CDC14A axis [7]. It has been documented that the lncRNA ARAP1 antisense RNA 1 (ARAP1-AS1) promotes tumorigenesis and metastasis in many cancer [11, 12, 13]. One study documented a high expression of ARAP1-AS1 in lung cancer [14]. Nonetheless, the detailed mechanism of lncRNA ARAP1-AS1 remains unknown.

LncRNAs that influence cancer occurrence and development via gene regulation have complex mechanisms [15]. This includes binding to targeting miRNAs that promote or suppress tumorigenesis. For instance, studies have shown a variety of miRNA-regulated pathways that participate in cancer progression. The highly expressed miR-3529/miR-150/miR-31/miR-30d were found to inhibit the malignancy of LUAD [10, 16, 17, 18]. As for miR-8068, its upregulation is detectable in autoimmune hepatitis, a liver inflammation status related to liver cancer. MiR-8068 is also associated with the pathogenicity of SARS-CoV-2 and host immune responses. However, its role in lung cancer has yet to be reported.

CEACAM5 is located at chromosome 19q13.2 and consists of 10 exons. CEACAM5 is responsible for encoding a cell surface glycoprotein, which represents the founding member of the carcinoembryonic antigen (CEA) protein family. The encoded protein has been utilized as a clinical biomarker for colorectal, gastrointestinal, and lung cancers, as it may promote tumor development via its involvement in cell adhesion [19, 20, 21]. In addition, the encoded protein may modulate cell polarity, differentiation, and apoptosis [22]. Nevertheless, how CEACAM5 regulates LUAD remains obscure.

In this research, we intended to study the effects of lncRNA ARAP1-AS1 on LUAD. Further, bioinformatics analyses clarified the interaction of miR-8068 with CEACAM5 and lncRNA ARAP1-AS1. This novel interaction axis was validated in vitro. Our findings may contribute in uncovering the molecular mechanisms involved in LUAD and help identify novel biomarkers for its targeted therapies.

2. Materials and methods

2.1 Collection of tissue specimens

Thirty-eight sets of lung cancer and normal adjacent tissues were gathered from patients suffering from LUAD. The Ethics Committee of The First Affiliated Hospital of Chengdu Medical College granted approval to the protocols used in this study. All participants who agreed to the usage of their specimens in the study provided a written informed consent. The execution of this study complied with the World Medical Association’s Declaration of Helsinki.

2.2 Cell culture

The human normal lung cell BEAS-2B and four human LUAD cell lines (A549, PC9, Calu-3, DV-90) were bought from the Chinese Academy of Sciences, Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). DMEM (Invitrogen, USA) with an added fetal bovine serum (FBS; 10%; Hyclone, USA) was used in culturing all cells. The cultures were incubated in a 37 ${}^{\circ}$ C humidified environment containing 5% CO ${}_{2}$ (Forma Scientific, UK).

2.3 RNA extraction and RT-qPCR

The cells and tissues were treated with the TRIZOL reagent (ThermoFisher, USA) to extract their total RNAs. Afterward, cDNA were synthesized by subjecting the total RNAs to reverse transcription using an RT primer and AMV reverse transcriptase (Takara, Japan). MiRNA, lncRNA, and mRNA were quantified with Taqman probes (ThermoFisher, USA). RT-qPCR was accomplished in an ABI 7300 Sequence Detection Systems (Applied Biosystems) with a Taqman PCR Kit (ThermoFisher, USA). The endogenous controls adopted for the expressions of miR-8068, CEACAM5, and lncRNA ARAP1-AS1 were U6 and GAPDH. Table 1 lists the primer sequences.

Table 1
Real-time PCR primer sequences

Gene name	Sequence
ARAP1-AS1	Forward 5’-AGCCACATAAATTCAGCAG-3’
	Reverse 5’-CGATGTAGTAGGATTCCTTT-3’
miR-8068	Forward 5’-TGTTTGTTGTAAGGATCGT-3’
	Reverse 5’-CAGTGCGTGTCGTGGAGT-3’
CD24	Forward 5’-ACAGCCAGTCTCTTCGTGGT-3’
	Reverse 5’-CCTGTTTTTCCTTGCCACAT-3’
GAPDH	Forward 5’-GACTCATGACCACAGTCCATG-3’
	Reverse 5’-AGAGGCAGGGATGATGTTCT-3’
U6	Forward 5’-GCTTCGGCAGCACATATACTAAAAT-3’
	Reverse 5’-CGCTTCACGAATTTGCGTGTCAT-3’
EPCAM	Forward 5’-CTCTTTAAGGCCAAGCAGTG-3’
	Reverse 5’-TCGCTCAGAGCAGGTTATTT-3’
CEACAM5	Forward 5’-AGGCCAATAACTCAGCCAGT-3’
	Reverse 5’-GGGTTTGGAGTTGTTGCTGG-3’

2.4 Cell transfection

Synthgene, China synthesized the miR-8068 mimic (miR-8068), mimic NC (miR-NC), pcDNA-CEACAM5 overexpression vector (OE-CEACAM5) its empty vector (OE-NC), siRNA targeting ARAP1-AS1(si-ARAP1-AS1), and its negative control (si-NC). First, 1 $\times$ 10 ${}^{6}$ PC9 and A549 cells were inoculated on each well of 96-well plates. Subsequently, 180 $\mu$ L DMEM (Life Technologies, Invitrogen) was added into each well, followed by a 24-hour incubation at 37 ${}^{\circ}$ C in an environment containing 5% CO ${}_{2}$ . For cell transfection, Lipo2000 (Invitrogen, China) was used for the transfection of miR-8068 mimic/mimic NC OE-CEACAM5/OE-NC, and si-ARAP1-AS1/si-NC into the PC9 and A549 cells. All samples were collected after the 48-hour transfection. Lastly, the transfection efficiency was ascertained by means of qRT-PCR.

For the ectopic expression of lncRNA ARAP1-AS1, lentiviral particles containing pLenti-CMV-lncRNA ARAP1-AS1 (OE-lnc) and empty pLenti-CMV (OE-NC) were produced by OBio, Shanghai, China. The lentiviral expression vectors were transfected into 293T cells, followed by a 7-week puromycin maintenance. The lentiviruses produced were then utilized to infect A549 and PC9 cells. Forty-eight hours later, RT-qPCR was performed to analyze the cells.

2.5 Localization within the cytoplasm and nucleus

Following the manufacturer’s instructions, NE- PERTM Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Scientific) was employed to locate lncRNA ARAP1-AS1 within the A549 and PC9 cell lines. The cytoplasmic and nuclear components of the cells were separated and collected. RT-qPCR was conducted to estimate lncRNA ARAP1-AS1 expressions within the cell cytoplasm and nucleus. GAPDH was adopted as the cytoplasmic positioning control while U6 was utilized as the nuclear positioning control.

2.6 Cell counting Kit-8 (CCK-8) assay

The proliferation and viability of the cells were evaluated by means of the CCK-8 experiment. On 96-well plates, a 100 $\mu$ L cell suspension containing 2 $\times$ 10 ${}^{3}$ cells was added into each well. The cells were then kept at 37 ${}^{\circ}$ C in a humidified incubator with 5% CO ${}_{2}$ for specified durations. The proliferation of cells was examined at four time points after inoculation: 0 ${}^{\text{th}}$ , 24 ${}^{\text{th}}$ , 48 ${}^{\text{th}}$ , and 72 ${}^{\text{nd}}$ hours. At each time point, 10 $\mu$ L CCK-8 solution (Syngene, Nanjing, China) was added into each well, and the mixtures were incubated for 2 more hours at 37 ${}^{\circ}$ C. A microplate reader (Thermofisher, USA) was utilized in measuring the absorbance in each well at 450 nm.

2.7 Cell adhesion assay

A 96-well culture plate was pre-coated with 30 $\mu$ g/well collagen I (Sigma-Aldrich), maintained at 4 ${}^{\circ}$ C for a 12-hour coating and then air-dried at room temperature. Serum-free DMEM (Life Technologies, Invitroge) with 0.1% BSA was used in resuspending the transfected cells Next, every well of the pre-coated plate was loaded with 100 $\mu$ l cell suspension and then the cells were maintained at 37 ${}^{\circ}$ C for 20 min. Afterward, the cells that failed to adhere were rinsed off with 100 $\mu$ l DMEM while the adherent cells were incubated for 4 hours at 37 ${}^{\circ}$ C in DMEM containing 10% FBS. After incubation, each well containing a mixture was supplied with 10 $\mu$ l of MTT substrate (ATCC) and then subjected to an additional 2-h incubation at 30 ${}^{\circ}$ C. Thereafter, a 100 $\mu$ l lysis buffer was added to treat the fixed cells. Lastly, to assess the adhesive capability of the cells, a microplate reader (Thermofisher, USA) was utilized in taking the readings at a 570 nm wavelength.

2.8 Transwell migration assay

Transwell inserts (Corning, Inc. USA) and 24-well plates were used for this assay. A549 and PC9 cells ( $\sim$ 100 $\mu$ l; 1 $\times$ 106 cells/ml) in 200 $\mu$ l medium were added onto the inserts while the wells were filled with 300 $\mu$ l medium containing 30% FBS. After 24 h passed, the cells that failed to migrate were cleared away. Meanwhile, the rest of the cells were subjected to fixation and then staining for 10 min with 75% methanol and 0.1% crystal violet, respectively. The cells were photographed and counted under a light inverted microscope (Olympus Corporation, USA).

2.9 Dual-luciferase reporter assay

The relationship of miR-8068 with the target genes was tested by means of the luciferase reporter experiment. MiR-8068’s wild-type (WT) and mutated (MUT) binding domains in each target gene were amplified and subsequently inserted into psiCHECK-2 vectors (Synthgene Biotech, Nanjing, China). Four luciferase reporter plasmids were then constructed: ARAP1-AS1-WT ARAP1-AS1-Mut, CEACAM5-3’-UTR-Mut and CEACAM5-3’-UTR-WT The A549 and PC9 cell lines were inoculated on 24-well culture plates and then allowed to reach 60% confluence. A Lipofectamine 2000 (Thermofisher, USA) was used in transfecting the A549 and PC9 cell lines with a combination of a luciferase reporter plasmid (0.5 $\mu$ g) and 100 nM of either a miR-8068 mimics or NC mimics. After 48 hours of transfection, a Dual-Luciferase Reporter System (Promega, Shanghai, China) was utilized for the detection of luciferase activities, which were then normalized to Renilla signals.

2.10 RNA-binding protein immunoprecipitation (RIP) assay

The RIP assay was carried out with an EZ-Magna RIP Kit (EMD Millipore) to identify RNA-protein interactions. In brief, PC9 and A549 cells were lysed in a RIP lysis buffer supplemented with cocktail (Roche Diagnostics). The cell supernatants were then extracted and then maintained at 4 ${}^{\circ}$ C overnight with primary antibodies against IgG and Ago2 (EMD Millipore) and protein A/G magnetic beads. Thereafter, all the RNAs were purified with RNase-free DNase I (Promega) and Proteinase K, and the expressions of miR-8068 and ARAP1-AS1 were determined via RT-qPCR.

2.11 Tumor formation experiment in nude mice

BALB/c nude mice with thymus-deficiency ( $n=$ 10; 18–20 g and 4–6 weeks old) were bought from Shanghai Shrek Experimental Animal Center (Shanghai, China). The mice were divided into groups (OE-NC and OE-lnc) of 5. The groups were kept in separate cages within the animal room, wherein the temperature and humidity were kept constant. The mice were also subjected to an alternating 12-hour light and 12-hour dark cycle The mice were subcutaneously inoculated with 1 $\times$ 10 ${}^{7}$ A549 cells/ml that were stably transfected with either OE-lnc or OE-NC Afterward, the mice were continuously raised under normal conditions for 6 weeks to form the tumor. At the end of the experiment, the mice were euthanized by means of cervical dislocation after CO ${}_{2}$ asphyxiation. The transplanted tumor was excised and fixed for observation and weighing. The Ethics Committee of The First Affiliated Hospital of Chengdu Medical College granted approval to this animal experiment. This animal study observed the institutional and national guidelines for animal care and use.

2.12 Western blot

A RIPA lysis buffer supplemented with PMSF was utilized to lyse the cells at 4 ${}^{\circ}$ C and extract their total proteins. The extracted proteins were tested for protein concentration with a BCA assay kit (Santa Cruz, California, USA) and then electrophoresed through a 10% SDS-PAGE gel. The separated proteins were moved to PVDF membranes that were subsequently sealed by adding skimmed milk and incubating at room temperature for an hour. Thereafter, these membranes were maintained overnight at 4 ${}^{\circ}$ C with the prepared primary antibodies against CEACAM5 (cat#HPA019758, 1:10000, Millipore sigma) and GAPDH (cat#ab9485; 1:1000; Abcam). Next, the anti-goat HRP secondary antibody (31402, 1:500) and anti-mouse HRP secondary antibody (31430, 1:500), both from Invitrogen, were added and incubated at 37 ${}^{\circ}$ C for 1 h for the recognition of the primary antibodies. Lastly, an enhanced chemiluminescence substrate (ECL; PierceTM ECL Plus, ThermoFisher, MA, USA) was applied for the visualization of the blots.

2.13 Statistical analysis

The statistical analyses of the data were performed in SPSS 18.0 (SPSS, Inc.). Each experiment was conducted thrice, and the collected data were indicated in the following format: mean $\pm$ standard deviation (SD). Statistical differences between the experimental and control groups were tested by applying the Student $t$ -test. On the other hand, one-way ANOVA and Tukey’s post-hoc test were applied for the analysis of statistical differences among the three or more groups. A $P<$ 0.05 indicated statistical significance.

3. Results

3.1 ARAP1-AS1 overexpression boosts the growth of LUAD cells in vivo and in vitro

We first assessed lncRNA ARAP1-AS1 expression among the LUAD cell lines and in the 38 sets of normal and LUAD tissues. Relative to the normal lung cell BEAS-2B, all the tumor cell lines (A549, PC9, DV-90, and Calu-3) exhibited an upregulation of ARAP1-AS1 (Fig. 1A). The highest ARAP1-AS1 levels were observed among the A549 and PC9 cells, hence they were selected for the succeeding experiments. The LUAD and adjacent normal tissues were evaluated for ARAP1-AS1 expression via RT-qPCR. As shown in Fig. 1B, ARAP1-AS1 had a significantly higher expression in LUAD tissues than in normal ones. The proportions of ARAP1-AS1 expression within the cytoplasm and nuclei of PC9 and A549 cell lines were detected. The results showed that ARAP1-AS expression was mainly enriched in cytoplasmic part of A549 and PC9 (Fig. 1C). To investigate ARAP1-AS1’s oncogenic ability and role in LUAD progression, its overexpression vector, knockdown vector, and negative control were transfected into the PC9 and A549 cell lines. The transfection efficiency is validated in Fig. 1D, showing that ARAP1-AS1 overexpression vector led to the increase of ARAP1-AS1 expression, whereas ARAP1-AS1 knockdown vector led to the decrease of ARAP1-AS1 expression in PC9 and A549 cells The CCK-8 assay revealed that ARAP1-AS1 overexpression significantly promoted the viability of the PC9 and A549 cell lines, but ARAP1-AS1 knockdown inhibited the viability (Fig. 1E). Moreover, the outcomes of the Transwell migration experiment suggested that the overexpression of ARAP1-AS1 stimulated cell migration more than the NC group, whereas ARAP1-AS1 impaired cell migration (Fig. 1F). It was demonstrated in the cell adhesion assay that the overexpression of ARAP1-AS1 in the PC9 and A549 cell lines strengthened tumor cell adhesion, but ARAP1-AS1 knockdown showed the opposite effect (Fig. 1G). Collectively, lncRNA ARAP1-AS1 exerted oncogenic effects on LUAD growth in vitro. We also conducted an in vivo experiment in mice to examine how ARAP1-AS1 affected LUAD tumor growth. The outcomes revealed that mice with ARAP1-AS1 overexpression had LUAD tumors that are larger in appearance and measurements than those from the NC mice (Fig. 2A and B). Moreover, the tumors from the OE-lnc group were heavier than the tumors from the OE-NC group (Fig. 2C). Our findings evidence that ARAP1-AS1 operates as a tumor oncogene during the progression of LUAD.

Figure 1.

LncRNA ARAP1-AS1 overexpression promotes cell viability, proliferation, and adhesion. (A) LncRNA ARAP1-AS1 expression was quantified in normal lung cells (BEAS-2B) and all LUAD cell lines (A549, PC9, Calu-3, and DV-90) via RT-qPCR. ${}^{**}P<$ 0.001 vs. BEAS-2B. (B) LncRNA ARAP1-AS1 expression level was analyzed in LUAD tumors and normal tissues by conducting RT-qPCR. (C) The localization of lncRNA ARAP1-AS1 within the nuclei and cytoplasm of the PC9 and A549 cells was ascertained via RT-qPCR. (D) PC9 and A549 cell lines were transfected with the lncRNA ARAP1-AS1 overexpression vector (OE-lnc), its negative control (OE-NC), si-ARAP1-AS1 (si-lnc) or si-NC. The transfection efficiency was assessed by means of RT-qPCR. ${}^{**}P<$ 0.001 vs. OE-NC. ${}^{{\#}{\#}}P<$ 0.001 vs. si-NC. (E) The proliferation of the PC9 and A549 transfected with OE-lnc, OE-NC, si-ARAP1-AS1 (si-lnc) or si-NC was evaluated with the aid of CCK-8. ${}^{**}P<$ 0.001 vs. OE-NC. ${}^{{\#}{\#}}P<$ 0.001 vs. si-NC. (F) PC9 and A549 cells transfected with OE-lnc, OE-NC, si-ARAP1-AS1 (si-lnc) or si-NC were assessed for their migrative abilities via the Transwell migration assay. ${}^{**}P<$ 0.001 vs. OE-NC. ${}^{{\#}{\#}}P<$ 0.001 vs. si-NC. (G) Cell adhesion experiment was conducted to gauge the adhesion of PC9 and A549 cells transfected with OE-lnc, OE-NC, si-ARAP1-AS1 (si-lnc) or si-NC. ${}^{**}P<$ 0.001 vs. OE-NC. ${}^{{\#}{\#}}P<$ 0.001 vs. si-NC.

Figure 2.

LncRNA ARAP1-AS1 boosts tumor growth in vivo. Nude mice were subcutaneously injected with infected A549 cells. (A) Photograph of the xenograft tumors, 6-weeks after the subcutaneous implantation of A549 cells. (B) Growth curves of xenograft tumors. (C) Weight of xenograft tumors on the 6 ${}^{\text{th}}$ week after the implantation. ${}^{**}P<$ 0.001 vs. OE-NC.

Figure 3.

LncRNA ARAP1-AS1 directly interacts with miR-8068. (A) The promising ARAP1-AS1 binding site in the region of miR-8068 was identified by starBase. (B) The relative luciferase signals in PC9 and A549 cells that had a combined transfection of ARAP1-AS1-WT/MUT luciferase reporter vectors and a miR-8068 mimic or mimic NC. ${}^{**}P<$ 0.001 vs. miR-NC. (C) The coprecipitation of miR-8068 and ARAP1-AS1 in PC9 and A549 cells was analyzed via the RIP assay. ${}^{**}P<$ 0.001, vs. Anti-IgG. (D) MiR-8068 levels in LUAD tumors and normal lung tissues were analyzed by conducting RT-qPCR. (E) MiR-8068 expression levels in BEAS-2B cells and LUAD cells (A549 and PC9), as quantified by RT-qPCR. ${}^{**}P<$ 0.001 vs. BEAS-2B. (F) The association of ARAP1-AS1 with miR-8068, as analyzed by Pearson correlation coefficient.

Figure 4.

The suppression of cell viability, migration, and adhesion by miR-8068 upregulation is counteracted by ARAP1-AS1 overexpression. PC9 and A549 cells were transfected with ARAP1-AS1 overexpressing vectors (OE-lnc), OE-NC, miR-8068 mimic (mimic), mimic-NC, and OE-lnc $+$ mimic. (A) MiR-8068 expression was determined in transfected cells by carrying out RT-qPCR. (B) The proliferation of the transfected cells was assessed by means of the CCK-8 experiment. (C) The migration of the transfected cells was evaluated via Transwell migration experiments. (D) Cell adhesion assay was carried out to assess the ability of the transfected cells to adhere. ${}^{*}P<$ 0.05, ${}^{**}P<$ 0.001 vs. OE-NC. ${}^{\#}P<$ 0.05, ${}^{{\#}{\#}}P<$ 0.001 vs. mimic-NC, ${}^{\&}P<$ 0.05, ${}^{{\&}{\&}}P<$ 0.001 vs. OE $+$ mimic.

Figure 5.

CEACAM5 is a miR-8068 target gene. (A) Three genes (CD24, EPCAM, CEACAM5) were related to cell proliferation and cell adhesion based on STRING analysis. (B) RT-qPCR analysis of the expression levels of CD24, EPCAM, and CEACAM5 in tumoral and normal tissues. (C) TargetScan predicted the promising miR-8068 binding sites in CEACAM5 3’UTR. (D) Relative values of the luciferase signals were assessed in PC9 and A549 cells that had a combined transfection of a CEACAM5 3’UTR-WT/MUT luciferase reporter vector and a miR-8068 mimic or mimic NC. ${}^{**}P<$ 0.001 vs. miR-NC. (E) The expression levels of CEACAM5 in BEAS-2B cells and LUAD cells (A549 and PC9) as quantified by RT-qPCR. ${}^{**}P<$ 0.001 vs. BEAS-2B. (F) Pearson correlation coefficient revealed that CEACAM5 was inversely correlated with miR-8068. (G) Pearson correlation coefficient revealed that CEACAM5 was postively correlated with ARAP1-AS1. (H) The expression of CEACAM5 in the LUAD cells transfected with ARAP1-AS1 overexpressing vectors (OE-lnc), OE-NC, miR-8068 mimic (mimic), mimic-NC, and OE-lnc $+$ mimic was quantified by means of western blot analysis. ${}^{**}P<$ 0.001 vs. OE-NC. ${}^{{\#}{\#}}P<$ 0.001 vs. mimic-NC, ${}^{{\&}{\&}}P<$ 0.001 vs. OE $+$ mimic.

Figure 6.

MiR-8068 tones down CEACAM5 expression and exerts a suppressive effect in LUAD progression. CEACAM5 overexpression vectors (OE-CEACAM5), OE-NC, miR-8068 mimic (mimic), mimic-NC, and OE-CEACAM5 $+$ mimic were introduced into the PC9 and A549 cells. (A) The expression of CEACAM5 in the abovementioned transfected cells was quantified by means of western blot analysis. (B) The proliferation of the transfected cells was analyzed by means of the CCK-8 assay. (C) Transwell migration assay was conducted to assess the migration of the transfected cells. (D) Cell adhesion assay was used to assess the adhesion of the transfected cells. ${}^{*}P<$ 0.05, ${}^{**}P<$ 0.001 vs. OE-NC. ${}^{\#}P<$ 0.05, ${}^{{\#}{\#}}P<$ 0.001 vs. mimic-NC. ${}^{\&}P<$ 0.05, ${}^{{\&}{\&}}P<$ 0.001 vs. OE $+$ mimic.

Figure 7.

The mechanism of ARAP1-AS1/miR-8068/CEACAM5 axis in LUAD.

3.2 ARAP1-AS1 directly interacts with miR-8068

We conducted bioinformatics analyses in starBase (http://starbase.sysu.edu.cn) to search for the potential target miRNA of ARAP1-AS1. As shown in Fig. 3A, we found a promising binding domain shared by ARAP1-AS1 and miR-8068. In view of this, we conducted luciferase reporter and RIP assays to investigate the relationship of lncRNA ARAP1-AS1 with miR-8068. Our findings showed that miR-8068 overexpression diminished the luciferase activity in cells containing the wild-type binding site of ARAP1-AS1. Meanwhile, no significant changes in luciferase activity were observed among the PC9 and A549 cells containing a mutated ARAP1-AS1 binding site (Fig. 3B). RIP assay further proved that ARAP1-AS1 could bind to miR-8068 by the AGO2 protein (Fig. 3C). In addition, the results of the RT-qPCR analysis indicated a significant miR-8068 downregulation in LUAD tissues when compared to that in normal lung tissues (Fig. 3D). Such downregulation was also observed in both the PC9 and A549 cell lines when compared to the miR-8068 expression in BEAS-2B cells (Fig. 3E). MiR-8068’s association with ARAP1-AS1 was analyzed, revealing that ARAP1-AS1 was capable of negatively regulating miR-8068 expression in LUAD cells (Fig. 3F). To sum, ARAP1-AS1 can target miR-8068 in LUAD cells.

3.3 MiR-8068 is involved inARAP1-AS1-mediated oncogenicity of LUAD in vitro

To investigate miR-8068’s relevance in the ARAP1-AS1-mediated LUAD progression, RT-qPCR was carried out to quantify miR-8068 expression in the PC9 and A549 cell lines transfected with OE-lnc and miR-8068 mimic. Overexpressing ARAP1-AS1 hampered the miR-8068-mimic-induced stimulation of miR-8068 expression in LUAD cells Fig. 4A. In the functional analyses, CCK-8 results revealed that exogenous miR-8068 significantly decreased the cell viability (Fig. 4B). However, this decrease in viability was rescued by the simultaneous transfection of miR-8068 mimic and OE-lnc. Furthermore, a similar trend was observed when the combined transfection of the two restored to normal levels the suppressed LUAD cell migration effected by the miR-8068 mimic (Fig. 4C). The cell adhesion analysis demonstrated that the miR-8068 mimic also lessened the cell adhesion, and yet this inhibitory effect was offset by ARAP1-AS1 overexpression (Fig. 4D). To conclude, the miR-8068 mimic inhibits the LUAD progression induced by the overexpression of ARAP1-AS1.

3.4 MiR-8068 targets CEACAM5

We investigated the molecular mechanism underlying the ARAP1-AS1-driven LUAD progression, by utilizing the GEPIA website (http://gepia.cancer-pku.cn/) in screening for target genes related to LUAD. Based on the data from GEPIA, there were 247 upregulated genes in LUAD, screened with the following parameters: logFC $>$ 2 and adj.P $<$ 0.01. The 247 upregulated genes were uploaded to STRING and, it was found that three genes (CD24, EPCAM, CEACAM5) were related to cell proliferation and cell adhesion (Fig. 5A). Among these genes, we chose CEACAM5 as a potential miR-806 target as it exhibited the most pronounced difference in relative levels when the LUAD tumoral and normal tissues are compared (Fig. 5B). The putative binding site of miR-8068 and CEACAM5 3’UTR are shown in Fig. 5C. The luciferase reporter experiment demonstrated that the overexpression of miR-8068 weakened the luciferase activity in cells carrying WT-CEACAM5 binding sites while no considerable changes in luciferase activity were observed among those with the mutant (Fig. 5D). This indicates the importance of this binding site in miR-8068’s function. The PC9 and A549 cell lines manifested significantly higher CEACAM5 expressions than the BEAS-2B cells (Fig. 5E). In addition, it was revealed that miR-8068 expression was inversely correlated with that of CEACAM5 (Fig. 5F), and ARAP1-AS1 expression was positive correlated with CEACAM5 expression in LUAD tissues (Fig. 5G). Western blot analysis confirmed that ARAP1-AS1 overexpression enhanced the expression of CEACAM5 protein, and miR-8068 mimic reduced the expression of CEACAM5 protein (Fig. 5H). Hence, CEACAM5 might have an involvement in the miR-8068induced tumor suppression.

3.5 MiR-8068 tones down CEACAM5 expression and exerts its suppressive effect on LUAD progression

In view of the abovementioned findings, we further verified the mechanism of the miR-8068/CEACAM5 axis in LUAD progression by transfecting CEACAM5/miR-8068 NC mimics, CEACAM5 overexpression vector, miR-8068 mimic, and a combination of CEACAM5 and miR-8068 mimic into A549 and PC9 cells. Western blot analysis uncovered that miR-8068 overexpression remarkably suppressed the expression levels of CEACAM5 protein (Fig. 6A). This outcome, however, was reversed by overexpressing CEACAM5. The results of the CCK-8 and Transwell migration experiments indicate that a high CEACAM5 expression could promote cell viability and migration (Fig. 6B and C). Nonetheless, these promotive effects may be counteracted by the introduction of exogenous miR-8068. Moreover, the adhesion analysis revealed trends that are similar with that of the proliferation assessment (Fig. 6D). Altogether, these findings evidence the crucial role of lncRNA ARAP1-AS1 in LUAD tumorigenesis, executed via the regulation of the miR-8068/CEACAM5 axis.

4. Discussion

LUAD is the most prevalent lung cancer and is linked to high morbidity and poor prognosis worldwide [3]. Clinically, the targeted treatment based on molecular alteration, which drives cancer development and maintenance, has made progress in LUAD [23]. Studies have reported multiple lncRNAs involved in the progression and prognosis of LUAD [16, 24]. Hence, dissecting the roles of these lncRNAs in LUAD is important to the development of personalized therapies. Our present study demonstrated that lncRNA ARAP1-AS1 acted as an oncogenic driver of LUAD by partly regulating the miR-8068/CEACAM5 axis (Fig. 7). Our findings might provide a novel target for LUAD intervention.

Evidence have shown the strong association of altered LncRNAs with LUAD. For example, LncRNA LINC01287 stimulates the proliferation and inhibits the apoptosis of LUAD cells by upregulating RNASEH2A after competitively binding with miR-3529-5p [16]. The novel lncRNA, ARAP1-AS1, was previously found be to a tumor promoter in many cancers. For example, ARAP1-AS1 activates the Wnt/ $\beta$ -catenin signaling pathway’s oncogenic action, which led to colorectal cancer progression [13]. Furthermore, ARAP1-AS1 epigenetically modulates the expression of dual-specificity phosphatase 5 (DUSP5), a well-known anti-oncogenic protein, thus resulting in the promotion of cervical cancer [25]. Here, we observed an elevated lncRNA ARAP1-AS1 expression in LUAD cells and tissues. Moreover, the overexpression of the lncRNA stimulated the adhesion, migration, and proliferation of LUAD cells as well as in vivo tumorigenesis. Our findings were consistent with a previous study in lung cancer [14], wherein it was revealed that knocking down ARAP1-AS1 inhibited cell proliferation by inducing G0/G1 cell cycle organization in vitro and in vivo. Overall, these suggest that ARAP1-AS1 serves as tumor promoter in LUAD.

As lncRNAs harbor miRNA response elements (MREs), lncRNAs may serve as ceRNAs to inhibit miRNA expression [26]. Our findings suggest that miR-8068 was ARAP1-AS1’s target miRNA. We identified and validated the negative regulatory association between miR-8068 and ARAP1-AS1 in LUAD cells. The re-upregulation of miR-8068, accomplished by transfecting miR-8068 mimics into the A549 and PC9 cell lines, considerably repressed the viability, adhesion, and migration of cells. The oncogenic effect of ARAP1-AS1 in LUAD progression antagonized the suppressive effect of miR-8068. This indicates that ARAP1-AS1 may serve as a progress promoter in LUAD by interacting miR-8068. MiR-8068’s differential expression has been detected in serval neurodegenerative disorders as well as in osteoarthritis. In cancers, bioinformatics analyses indicate that miR-8068 might be involved in the kidneys’ clear cell renal carcinoma. Nevertheless, no study has reported its role in LUAD malignancy. To our knowledge, this study has been the first to reveal miR-8068’s inhibitory role in cancer progression, shedding new light on the specific molecular regulatory mechanism of cancer.

Through bioinformatics analyses, we screened out CEACAM5 as a potential miR-8068 target gene. The CEACAM gene family mutations have been reported to contribute in numerous cancers, such as breast, colon, gastric, and lung cancers [27, 28, 29]. Among them, CEACAM5 is a commonly recognized biomarker of cancer occurrence and recurrence. A recent study found that CEACAM5 upregulation could stimulate the migration and proliferation of tumor cells to induce non-small-cell lung cancer [29]. Comparable results were presented in our research. We studied the expression levels of CEACAM5 mRNA in LUAD tissues and further demonstrated that its overexpression augmented the oncogenic potential of LUAD cells. However, the overexpression of miR-8068 mimics neutralized the effect of CEACAM5 on LUAD cell malignancy. Our findings evidence that miR-8068 executes a vital role in LUAD progression through CEACAM5 regulation.

More detailed mechanisms and signal pathways of lncRNA ARAP1-AS1 in lung cancer requires further studies. Also, the functions of the miR-8068/CEACAM5 axis needs further verification through clinical investigations.

5. Conclusion

We presented the elevated expression of lncRNA ARAP1-AS1 in LUAD cells and tissues, which was also accompanied by an upregulated CEACAM5 and downregulated miR-8068 expressions. The overexpression of miR-8068 could partly antagonize the stimulating influence of ARAP1-AS1 on the adhesion, migration, and viability of LUAD cells. Furthermore, CEACAM5 overexpression can inhibit the miR-8068-induced suppression of proliferation among the LUAD cell lines. Our findings evidence that the lncRNA ARAP1-AS1/miR-8068/CEACAM5 axis is a promising therapeutic target in LUAD treatment.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethics approval

The Ethics Committee of The First Affiliated Hospital of Chengdu Medical College (Chengdu, China) approved this research. This study was executed in accordance with the World Medical Association’s Declaration of Helsinki. All patients submitted signed informed consents.

The Ethics Committee of The First Affiliated Hospital of Chengdu Medical College granted approval to this study. The execution of the study observed the national and institutional guidelines for animal care and use. All animal experiments had been conducted in strict adherence to the ARRIVE guidelines.

Consent for publication

Consent for publication was obtained from all participants.

Competing interests

The authors declare that they have no conflicts of interests.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by The First Affiliated Hospital of Chengdu Medical College High-level Talents Research Start-up Fund Project (CYFY-GQ26).

Authors’ contributions

Conception: XFZ.

Interpretation or analysis of data: ZQW and XFZ.

Preparation of the manuscript: HW.

Revision for important intellectual content: XBW and ZQW.

Supervision: all authors.

All authors have read and approved this manuscript.

Footnotes

Acknowledgments

None.

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