Long noncoding RNA TFAP2A-AS1 exerts promotive effects in non-small cell lung cancer progression via controlling the microRNA-548a-3p/CDK4 axis as a competitive endogenous RNA

In this study, we mainly focus on probing expression profile and detailed functions of long non-coding RNA TFAP2A antisense RNA 1 (TFAP2A-AS1) in non-small cell lung cancer (NSCLC). Moreover, the mechanisms played by TFAP2A-AS1 were unraveled comprehensively. Herein, a notable overexpressed TFAP2A-AS1 in NSCLC was observed by TCGA and our own cohort. An increased TFAP2A-AS1 level displayed a negative correlation with the overall survival of patients with NSCLC. Loss-of-function approaches illustrated that the absence of TFAP2A-AS1 weakened NSCLC cell proliferation, colony formation, migration and invasion in vitro. Also, interference of TFAP2A-AS1 caused in vivo tumor growth suppression. Mechanistically, TFAP2A-AS1 could negative regulate microRNA-584-3p (miR-584-3p) as a competitive endogenous RNA. Furthermore, cyclin-dependent kinase 4 (CDK4), a direct target of miR-584-3p, was positively controlled by TFAP2A-AS1 in a miR-5184-3p-dependent manner. Rescue function experiments corroborated that the anticancer activities of TFAP2A-AS1 deficient on the oncogenicity of NSCLC cells were reversed by downregulating miR-584-3p or overexpressing CDK4. To sum up, TFAP2A-AS1 exhibits cancer-promoting roles in NSCLC through the adjustment of miR-584-3p/CDK4 axis.


Introduction
Lung cancer ranks as the most prevailing human cancer and is the principal reason of tumor-involved deaths around the world [1]. As a predominant type of lung cancer, non-small cell lung cancer (NSCLC) occupies about 80%-85% of all human lung cancer cases, and over 70% of NSCLC cases have developed into middle or advanced stage when symptoms emerge [2]. Since significant progress in terms of basic research, the primary treatment approach, including minimally invasive surgery, radiochemotherapy and targeted therapy, have made commendable advancement in the past decade [3,4]; yet, the clinical efficiency of NSCLC patients remains gloomy [5]. The 5-year survival rate of NSCLC patients at early stage is about 50%, but decreases to a level lower than 5% [6]. Therefore, making great efforts on the mechanisms of NSCLC pathogenesis may help for the identification of promising diagnostic and therapeutic targets.
Long non-coding RNAs (lncRNAs) are described as untranslated transcripts with more than 200 nucleotides [7]. Although they cannot be translated to protein, lncRNAs play a part in the control of genes expression that exert essential roles in mounting physiological and pathological cellular processes [8]. Striking, the expression status of lncRNA presents a notable correlation with a range of human diseases [9]. A substantial number of lncRNAs are deregulated in NSCLC, and contribute to diversified steps during cancer genesis and progression [10][11][12]. The competitive endogenous RNA (ceRNA) theory depicts that lncRNAs have the power to sequester microRNAs (miRNAs), and kept miRNAs away from their target mRNAs, consequently constituting a regulatory network [13]. Thus, lncRNAs and miRNAs have a perfect prospect in the development of novel therapeutic strategies for NSCLC.
Utilizing the cancer genome atlas (TCGA) dataset, we found that TFAP2A antisense RNA 1 (TFAP2A-AS1) was highly expressed in almost all human cancer types, including lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD). Thus, we mainly focus on probing expression profile and detailed functions of TFAP2A-AS1 in NSCLC. Moreover, the molecular events occurred by TFAP2A-AS1 were unraveled comprehensively.

Patients and cell lines
The current research conformed to the Ethical Committee of Jilin Province Cancer Hospital, and was carried out in accordance with the Declaration of Helsinki. After getting the written inform consents, NSCLC tissues were collected from 47 patients with NSCLC in Jilin Province Cancer Hospital. Meanwhile, corresponding adjacent normal tissues were obtained and served as the control.
The inclusion criteria were as follows: (i) patiens who were diagnosed as NSCLC; (ii) was not treated with systemic or local anticancer treatments before the application of surgery; and (iii) agreed to take part in the study. The exclusion criteria were as follows: (i) patients with systemic or local anticancer treatments before surgery; and (ii) did not agree to take part in the study. All samples were stored in liquid nitrogen till needed.
BEAS-2B is a human nontumorigenic bronchial epithelial cell line and cultured in Bronchial epithelial cell growth medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). NSCLC cell lines, A549 and H1299, were grown in RPMI-1640 medium (Gibco). H460 and SK-MES-1, another two NSCLC cell lines, were maintained in F-12K and MEM (Gibco), respectively. Beyond that, the culture conditions of these NSCLC cell lines were the same as those for BEAS-2B. All cell lines used were bought from American Type Culture Collection (Manassas, VA, USA), and grown at 37°C in a humidified incubator containing 5% CO 2 .
qRT-PCR After being extracted utilizing Trizol (Beyotime; Shanghai, China), total RNA was quantified with a NanoDrop 1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). For the determination of TFAP2A-AS1 and CDK4 expression, reverse transcription was operated utilizing the PrimeScript TM RT reagent kit with gDNA Eraser (Takara; Dalian, China) to produce complementary DNA. PCR amplification was then implemented with a TB Green Ò Premix Ex Taq TM II (Takara). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) functioned as the reference control for TFAP2A-AS1 and CDK4. For miR-584-3p quantification, reverse transcription and PCR amplification were undertaken applying miScript Reverse Transcription kit and miScript SYBR Green PCR kit (Qiagen GmbH, Hilden, Germany), respectively. MiR-584-3p was normalized to small nuclear RNA U6. The 2 −ΔΔCq formula was used for genes expression calculation.
Cell counting kit-8 (CCK-8) assay Transfected cells were harvested at 24 h post-transfection, and mixed with complete culture medium to obtain cell suspension. After adjusting the concentration of cell suspension to 2 × 10 4 cells/mL, every well of 96-well plates was covered with 100 µl cell suspension. Prior to being treated with 10 µl CCK-8 solution (Beyotime), cells were cultivated at 37°C with CO 2 for different time periods. Following additional 2 h incubation, the optical density at 450 nm wavelength (OD450) was monitored by means of a microplate reader.

Colony formation assay
The harvested cells were resuspended in complete culture medium, and then inoculated into 6-well plates. Each well was covered with 2 ml cell suspension containing 500 cells. After culturing for 14 days, the culture medium was discarded, and formed colonies were fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet. Finally, the colonies were photographed applying an inverted microscope (Olympus Corp., Tokyo, Japan).

Transwell assay
The Matrigel precoated transwell insets (BD Biosciences) were applied for invasion test. A total of 4 × 10 4 cells resuspended in 200 µl FBS-free medium were plated into the upper compartments. By acting as a chemoattractant, the lower compartments were fulfilled with 600 µl culture medium which was added with 20% FBS. After culturing at 37°C for 24 h, the upper surface of membranes was scrubbed with a cotton swab, whereas the invaded cells were fixed with 4% paraformaldehyde and dyed with 0.1% crystal violet. After snapping with a light microscope (×200 magnification), the amount of invaded cells was counted in five randomly chosen visuals. The uncoated transwell insets were applied in migration test, and followed the same instructions.

Tumor xenograft experiment
The animal procedures conformed to the Animal Care Committee of Jilin Province Cancer Hospital, and were implemented in line with NIH guidelines for the care and use of laboratory animals. The TFAP2A-AS1-targeted short hairpin RNA (shRNA; sh-TFAP2A-AS1) and NC shRNA (sh-NC; GenePharma Co., Ltd., China) were inserted into the lentivirus vectors, after which were transfected into 293T cells (Cell Bank of the Chinese Academy of Sciences; Shanghai, China). 293T cells were cultured in DMEM medium that was added with 10% FBS, 1% Glutamax and 1% Sodium Pyruvate (all from Gibco). The virus supernatant was harvested after 48 h cultivation, and was utilized to infect A549 cells. The stable TFAP2A-AS1ablated cell line was screened out by treating with puromycin.
Male BALB/c nude mice (Shanghai Experimental Animal Center; Shanghai, China), aged 4-6 weeks, were subcutaneously injected utilizing A549 cells overexpressing sh-TFAP2A-AS1 or sh-NC. Tumor volume was recorded weekly by monitoring the width and length of subcutaneous xenografts, and calculated with the formula: volume = 1/2 × length × width 2 . The observation lasted for 4 weeks. As soon as the experiment ends, mice were euthanized, and the tumor xenografts were excised for weighing.

Subcellular fractionation
A Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Belmont, CA) was applied for cell cytoplasm and nucleus separation. qRT-PCR was conducted to determine the TFAP2A-AS1 abundance in cytoplasmic and nuclear RNA.
RNA immunoprecipitation (RIP) RIP assay was executed to delve into the interaction among TFAP2A-AS1, miR-584-3p and CDK4. The whole cell extract was prepared by lysing cells with RIP lysis buffer came from a Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore, Darmstadt, Germany). RIP buffer supplemented with magnetic beads coupled to Argonaut 2 (Ago2) or normal mouse IgG (Millipore) antibodies was adopted to treat the cell lysate at 4°C the whole night. The magnetic beads were collected, probed with Protease K, and were processed for immunoprecipitated RNA extraction. Relative enrichment was examined via qRT-PCR.

Western blotting
After being abstracted utilizing RIPA lysis buffer, the concentration of total protein was detected with a BCA kit (both from Beyotime). Equivalent proteins were divided by 10% SDS-PAGE electrophoresis, and then transferred onto PVDF membranes. Tris Buffered Saline with Tween Ò 20 supplemented with 5% nonfat dried milk was applied for blocking the membranes at room temperature for 2 h. In the next step, the membranes were hatched all night at 4°C with the primary antibodies targeting CDK4 (ab108357; dilution 1:1000) or GAPDH (ab128915; dilution 1:1000; Abcam, Cambridge, MA, USA). Subsequent to three washes utilizing Tris Buffered Saline with Tween Ò 20, the membranes were received cultivation with horseradish peroxidaseconjugated secondary antibody (ab205718; dilution 1:4000; Abcam) and further visualization using Pierce TM ECL Western Blotting Substrate (Thermo Fisher Scientific). Quantity One software (Bio Rad Laboratories, Inc., Hercules, CA, USA) was adopted for analyzing the protein signals.

Statistical analysis
All experiments were repeated for three times. The measurement data are represented as mean ± standard deviation. Survive curves were plotted with the application of Kaplan-Meier method, and compared applying log-rank test. Correlation analysis was conducted utilizing Pearson correlation coefficient. One-way analysis of variance was used in the comparison among multiple groups. Tukey's test was carried for post hoc test. The comparison between two groups was fulfilled via Student's t-test. All statistical analysis was accomplished with the help of SPSS 19.0 (SPSS, Chicago, IL, USA). There was a statistical difference when p < 0.05.
TFAP2A-AS1 deficient disrupts the oncogenicity of NSCLC cells through targeting miR-584-3p/CDK4 axis In this final step, a string of recuse experiments were done to illustrate whether miR-584-3p/CDK4 axis is required for the biological activity of si-TFAP2A-AS1 in NSCLC. Prior to that, the interference efficiency of anti-miR-584-3p in NSCLC cells was verified via qRT-PCR (Fig. 7A). Ablation of TFAP2A-AS1 restricted cell proliferation and colony formation, in which treatment of anti-miR-584-3p counteracted the regulatory effects (Figs. 7B and 7C). In addition, si-TFAP2A-AS1 transfection triggered suppression on NSCLC cell motility was neutralized by inhibiting miR-584-3p (Figs. 7D and 7E). 8A depicted the overexpression of CDK4 by pcDNA3.1-CDK4 in NSCLC cells. Cell proliferation and colony formation was hindered, which was retrieved by pcDNA3.1-CDK4 reintroduction (Figs. 8B and 8C). The ablation of TFAP2A-AS1 led to the diminution of cell migratory and invasive properties, whereas the effects were offset after treatment of pcDNA3.1-CDK4 (Figs. 8D and  8E). In a word, miR-584-3p/CDK4 axis is essential for the actions of TFAP2A-AS1 in NSCLC cells.

Discussion
Ongoing progress of the high throughput gene sequencing technology implies the enrollment of lncRNAs in the realm of cancer genesis and progression [17,18]. LncRNAs have the ability to control the cancer-associated biological activities, thereby having aroused considerable interest over the past years [19]. Regarding tumor therapy, studying the contribution of lncRNAs to cancer pathogenesis is a promising research field, and can aid the identification of attractive therapeutic methods or antitumor drugs [20]. Therefore, we utilized diverse experiments to authenticate whether TFAP2A-AS1 can act as an essential modulator of NSCLC progression. What's more, we explored the interaction among TFAP2A-AS1, miR-548-3p and CDK4.
Considerable evidences suggest that lncRNAs perform core functions in a broad spectrum of biological phenotype in NSCLC. For instance, lncRNAs CCAT1 [21], PART1 [22] and GACAT1 [23] are overexpressed in NSCLC, thus exerting pro-oncogenic actions. Instead, downregulated LINC00476 [24], MEG3 [25] and WT1-AS [26] in NSCLC are certified as anti-carcinogenic lncRNAs. TFAP2A-AS1 is downregulated in breast [27] and gastric [28] cancers, and performs tumor-inhibiting activities in controlling various aggressive phenotypes. But, the detailed roles of TFAP2A-AS1 in NSCLC still keep unclear by the public, so it is extremely urgent and must to make it definite. Herein, a notable overexpressed TFAP2A-AS1 was observed in both NSCLC tissues and cell lines. An increased TFAP2A-AS1 level displayed a negative correlation with the overall survival of patients with NSCLC. Loss-of-function approaches illustrated that the absence of TFAP2A-AS1 weakened NSCLC cell proliferation, colony formation, migration and invasion in vitro. Also, interference of TFAP2A-AS1 caused in vivo tumor growth suppression. Accordingly, these observations offer us with a novel perspective to comprehend NSCLC oncogenesis and progression, which may be helpful for treating this disease.   LncRNAs take part in the transcriptional and posttranscriptional processes and epidemic networks, establishing a complex regulation network [29]. The subcellular distribution of lncRNAs implicates the molecular events mediating their specific roles [30]. Regarding nucleic lncRNAs, they have the function of direct binding with different proteins. Unlike this, lncRNAs located in cell cytoplasm execute their regulatory actions through affecting  the stability presented by mRNAs, which were happened via many methods. Among them, the ceRNA theory becomes the studying hot topic. LncRNAs possess miRNA response elements and can scavenge certain miRNAs from their target mRNAs, thereby generating lncRNA/miRNA/mRNA regulatory pathway.
The molecular events used by TFAP2A-AS1 in NSCLC were revealed in detail too. Initially, subcellular fractionation experiment was conducted, validating that both nucleus and cytoplasm saw TFAP2A-AS1 distribution, with the latter displaying a more part. Thus, we next searched the target of  TFAP2A-AS1. MiR-584-3p shared complementary binding within TFAP2A-AS1. Next, luciferase reporter and RIP assay proved that TFAP2A-AS1 could negative regulated miR-584-3p as a ceRNA. The downstream effector of TFAP2A-AS1/ miR-584-3p axis was also unveiled. Utilizing bioinformatic analysis and experimental confirmation, CDK4 was affirmed as the target of miR-584-3p, which was positively controlled by TFAP2A-AS1in a miR-584-3p-dependent manner. Altogether, we disclosed a novel ceRNA pathway in NSCLC, comprising TFAP2A-AS1, miR-584-3p and CDK4.
MiR-584-3p is lowly expressed in several human cancer types [31][32][33]. Yet, it is uncertain whether miR-584-3p is participated in the pathogenesis of NSCLC. In our current study, we also verified the downregulated miR-584-3p level in NSCLC, and the great contribution of miR-584-3p in the pathogenesis of NSCLC. Previously, miR-584-3p was confirmed to direct target Rho-associated, coiled-coil containing protein kinase 1 [31] in glioma, matrix metallopeptidase 14 [32] in gastric cancer, structure specific recognition protein 1 [33] in colorectal cancer. The target genes of miR-584-3p different in different tumors might be due to the tissue specificity of miRNAs in human cancers, Located on 12q14.1, CDK4 is a silk/threonine protein kinase [34], and met the miR-584-3p target criteria to create its repressing activities in NSCLC. CDK4 performs carcinogenic actions during initation and progression during and is implicated in the regulation of tumor processes [14][15][16]. Herein, rescue function experiments were implemented, which corroborated that the anticancer activities of TFAP2A-AS1 deficient on the oncogenicity of NSCLC cells were reversed by downregulating miR-584-3p or overexpressing CDK4. Therefore, miR-584-3p/CDK4 axis is the downstream effector of TFAP2A-AS1 in NSCLC cells.
Our research had one limitation. The sample size of this study is small. We will collect more samples in our further experiments.
Funding Statement: Our current research was supported by Jilin Province Cancer Hospital.