LncRNA PRRT3-AS1 exerts oncogenic effects on nonsmall cell lung cancer by targeting microRNA-507/homeobox B5 axis

Long noncoding RNAs (lncRNAs) act as key regulators controlling complex cellular behaviors in nonsmall cell lung cancer (NSCLC). We investigated the expression of lncRNA PRRT3 antisense RNA 1 (PRRT3-AS1) in paired samples of NSCLC and adjacent normal tissues from a patient cohort in our hospital using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and found that it was significantly higher in NSCLC tissue than in normal tissue, consistent with The Cancer Genome Atlas database. Furthermore, functional investigation revealed that lncRNA PRRT3-AS1 depletion inhibited NSCLC-cell proliferation, colony formation, invasion, and migration, whereas its overexpression exerted the opposite effects. Moreover, PRRT3-AS1 knockdown suppressed in vivo NSCLC growth. Investigation of downstream mechanisms using RNA immunoprecipitation and luciferase reporter assay revealed that lncRNA PRRT3-AS1 acted as a competing endogenous RNA by adsorbing microRNA-507 (miR-507) and enhanced the expression of its target gene, homeobox B5 (HOXB5), in NSCLC. Furthermore, miR-507 downregulation or HOXB5 upregulation eliminated the cancer-inhibiting effects of lncRNA PRRT3-AS1 depletion in NSCLC cells. To conclude, the lncRNA PRRT3-AS1/miR-507/HOXB5 pathway acts as a promoter of malignant characteristics in NSCLC, and this newly identified competing endogenous RNA pathway may be an effective diagnostic, prognostic, and therapeutic target in NSCLC.


Introduction
Among all cancers in humans, lung cancer has the highest incidence and mortality rate globally [1], with approximately 2.1 million lung cancer cases and over 1.8 million mortalities annually. Nonsmall cell lung cancer (NSCLC) is the predominant type of lung cancer, accounting for approximately 85% of all lung cancer cases [2]. Although advances in therapeutics have significantly improved the prognosis of NSCLC patients in recent decades, the therapeutic efficacy remains unsatisfactory, and many NSCLC patients still die [3]. The NSCLC pathogenesis and development is a complicated multistep process involving many risk factors and genes, and the underlying molecular mechanisms are poorly understood [4]. Therefore, determining the mechanisms underlying NSCLC pathogenesis is vital to identify effective targets for NSCLC diagnosis, prevention, and management.
Several lncRNAs are known to be key regulators of NSCLC malignancy. We investigated the expression and detailed functions of PRRT3 antisense RNA 1 (PRRT3-AS1) in NSCLC, in addition to the downstream mechanisms.

Materials and Methods
Clinical tissue samples We obtained NSCLC tissues from 53 patients in our hospital who had not undergone chemotherapy or radiotherapy before surgery. Immediately after tissue excision, all tissue samples were immersed and stored in liquid nitrogen until further analysis. This research was approved by the ethics committee of the Affiliated Zhongshan Hospital of Dalian University. In addition, all participating patients provided written informed consent.

Cell cultures
We purchased human NSCLC cell lines A549, H460, SK-MES-1, and H1299 from the American Type Culture Collection (ATCC; Manassas, VA, USA). Bronchial epithelial cell growth medium (Lonza; Walkersville, MD, USA) was adopted for culturing a human nontumorigenic bronchial epithelial cell line BEAS-2B (ATCC). Cell lines A549 and H1299 were grown in RPMI-1640 medium (Gibco). SK-MES-1 and H460 cell lines were grown in F-12K and MEM (Gibco), respectively. Each of the above culture media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco). All cells were grown in a humidified atmosphere of 5% CO 2 at 37°C.

Quantitative reverse transcription polymerase chain reaction
We used the TRIzol Ò reagent (Invitrogen) to extract total RNA. Total RNA was reverse-transcribed by using the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa; Dalian, China). With the obtained cDNA as a template, we performed quantitative polymerase chain reaction (PCR; qPCR) to determine miR-507 expression by using the Mir-X miRNA quantitative reverse transcription PCR (qRT-PCR) TB Green Ò Kit (TaKaRa). U6 served as the endogenous control for miR-507. To measure PRRT3-AS1 and HOXB5 expression, we used the PrimeScript Ò RT Reagent Kit for cDNA synthesis and SYBR Ò Premix Ex Taq TM II (TaKaRa) for qPCR. PRRT3-AS1 and HOXB5 levels were normalized to GAPDH the internal control, and reported as log-fold changes using the 2 −ΔΔCq formula.
Cell Counting Kit-8 and colony formation assays We inoculated transfected NSCLC cells into 96-well plates with a density of 2000 cells per well and incubated them at 37°C in a 5% CO 2 atmosphere. After incubating with 10 µL of the cell counting kit-8 (CCK-8) solution (Beyotime), we estimated the proliferation rate by the measurement of optical density at 450 nm. We performed the assay every day until day 4 and plotted the results as a growth curve.
For colonies, a cell suspension was prepared, and 2 ml of the cell suspension containing 600 cells was added into each well of 6-well plates. After 14 days of cultivation, the newly formed colonies were fixed with 100% methanol and stained with 0.1% crystal violet. The cells in the colonies were manually counted using an inverted microscope (x200 magnification).

Transwell cell migration and invasion assays
We explored the invasive ability of NSCLC cells using Transwell inserts (BD Biosciences). We added 100 µL of Matrigel (BD Biosciences) into the inner sides of the Transwell inserts and performed polymerization by incubating them at 37°C for 2 h. Next, we loaded the upper chambers with 100 μL of FBS-free culture medium containing 4 × 10 4 of transfected cells, and plated 600 μL of culture medium supplemented with 10% FBS into the lower chambers. We allowed the NSCLC cells to penetrate the pores in the submembrane surface for 24 h, then fixed them with 100% methanol, and stained them with 0.1% crystal violet. The cells that invaded the lower chambers were counted in five randomly chosen regions (in each well) under an inverted microscope. For the migration assay, we did not apply Matrigel, but the remaining steps were the same as those in the Matrigel invasion assay.

Xenograft tumor assay
The short hairpin RNA (shRNA) targeting PRRT3-AS1 (sh-PRRT3-AS1), and NC shRNA (sh-NC) were designed and chemically generated by GenePharma. We inserted the shRNAs into a lentiviral vector to transfect the shRNAs into H460 cells. The PRRT3-AS1-knockdown H460 cells were selected by cultivating them with puromycin. Next, a total of 8 BALB/c nude mice (age, 4-6 weeks) were obtained from HFK Bioscience (Beijing, China) and randomly divided into sh-PRRT3-AS1 and sh-NC groups. A total of 2 × 10 6 PRRT3-AS1-knockdown H460 cells were subcutaneously inoculated into each mouse in the sh-PRRT3-AS1 group. We monitored the width and length of tumor xenografts from 1 week after cell inoculation and then every 5 days. Thirty-two days later, all mice were anesthetized by means of cervical dislocation, and the tumor xenografts were completely detached and weighed.
All animal experiments were approved by the Animal Care and Use Committee of Affiliated Zhongshan Hospital of Dalian University. The humane endpoints were tumor diameter >1.5 cm, tumor ulceration, abnormal feeding, weight loss, ascites and cachexia. Meanwhile, no anesthesia was applied in the animal experiments.

Fluorescence in situ hybridization
The subcellular location of PRRT3-AS1 in NSCLC cells was examined using the Fluorescent In Situ Hybridization (FISH) Kit (RiboBio Co., Ltd.,). In detail, after washing with phosphate-buffered saline, H460 and A549 cells were fixed with 4% paraformaldehyde, followed by the hybridization treatment specifically targeting PRRT3-AS1 (RiboBio Co., Ltd.) at 37°C without light. On the next day, DNA staining was performed using Hoechst solution. The cells were, imaged under a confocal laser scanning microscope (Leica; Solms, Germany).

RNA immunoprecipitation assay
We performed a RNA immunoprecipitation (RIP) assay using the Magna RIP RNA-Binding Protein Immunoprecipitation kit (Merck-Millipore; Bedford, MA, USA). Briefly, we harvested 80% confluent NSCLC cells and lysed them using the RIP lysis buffer. Next, we performed Argonaute 2 (Ago2) immunoprecipitation by incubating the cell lysate with magnetic beads conjugated with anti-Ago2 antibody or normal immunoglobulin G (IgG) (control; Merck Millipore). Ago2 is an important element of RNA-induced silencing complex, and it can promote the degradation of target mRNAs via its catalytic activity in gene silencing processes induced by miRNAs. After overnight incubation at 4°C, we extracted the immunoprecipitated RNA and evaluated the abundance of lncRNA PRRT3-AS1, miR-507, and HOXB5 mRNA using qRT-PCR.

Western blotting
We performed total protein extraction and quantification using the Pierce RIPA lysis buffer and the Pierce TM bicinchoninic acid (BCA) Kit (both from Thermo Fisher Scientific; MA, USA), respectively. Protein samples were separated by 10% SDS-PAGE and blotted onto PVDF membranes. After blocking them with 5% defatted milk powder, the membranes were incubated at 4°C overnight with the primary antibodies against Hoxb5 (ab109375) or Gapdh (ab128915), followed by incubation with the HRP-conjugated anti-rabbit secondary antibody (ab6721; all from Abcam, USA). Finally, the bands of target proteins were developed by treatment with an enhanced chemiluminescence reagent (Beyotime).

Statistical analysis
All experiments were independently repeated three times. Results are reported as the mean ± 1 SD. The betweengroup differences were compared using Student's t-test or ANOVA. p < 0.05 was considered statistically significant.

Results
PRRT3-AS1 promotes the aggressive behaviors of NSCLC cells PRRT3-AS1 expression in human cancers was first investigated by analyzing The Cancer Genome Atlas dataset. We found that this gene was upregulated in nearly all human cancer types (Fig. 1A). Additionally, PRRT3-AS1 was the 47 th overexpressed lncRNA in lung adenocarcinoma (LUAD; Fig. 1B). Also, relative to control samples, PRRT3-AS1 was obviously overexpressed in lung squamous cell carcinoma and LUAD samples in the TCGA database (Fig. 1C). Next, we collected paired samples of NSCLC and adjacent normal tissues from 53 patients and determined the lncRNA PRRT3-AS1 levels in both sets. The qRT-PCR analysis revealed that PRRT3-AS1 was strongly expressed in NSCLC tissues (Fig. 1D). In addition, all four NSCLC cell lines manifested relatively higher lncRNA PRRT3-AS1 levels than the BEAS-2B (non-tumor control) cells (Fig. 1E).
HOXB5 is directly targeted by miR-507 in NSCLC cells and controlled by the lncRNA PRRT3-AS1/miR-507 axis Next, we determined the roles of miR-507 overexpression (Fig. 4A) in NSCLC cells. We found that miR-507 enhanced tumor-suppressing activities in NSCLC cells, affecting multiple aggressive behaviors (Figs. 4B-4E). Since HOXB5 (Fig. 5A) was predicted as a potential target of miR-507, we next analyzed whether miR-507 directly targeted HOXB5 in NSCLC cells. We found that miR-507 overexpression decreased the luciferase activity of the WT-HOXB5 vector in NSCLC cells, whereas no change was observed for the MUT-HOXB5 luciferase vector (Fig. 5B). In addition, our data showed a significant decrease in HOXB5 levels (Figs. 5C and 5D) in miR-507-overexpressed-NSCLC cells. All these results together demonstrate that HOXB5 is a downstream target of miR-507 in NSCLC cells.
In addition, the introduction of the miR-507 inhibitor or pc-HOXB5 offset the impaired H460 cell migration and invasion (Fig. 7C) induced by PRRT3-AS1 downregulation. Moreover, the cell migration and invasion abilities of cells transfected with pc-PRRT3-AS1 significantly increased, while those of cells transfected with miR-507 mimic or si-HOXB5 decreased this effect (Fig. 7D). To conclude, lncRNA PRRT3-AS1 worsened the oncogenicity of NSCLC, at least, in part, by targeting the miR-507/HOXB5 regulatory axis.

PRRT3-AS1 depletion impairs growth of NSCLC cells in vivo
Xenograft tumor assay was performed to illustrate whether lncRNA PRRT3-AS1 affected NSCLC tumor growth in vivo. In contrast to tumors in the sh-NC group, the sh-PRRT3-AS1-transfected subcutaneous tumors grew significantly slower (Figs. 8A and 8B). Also, tumor weight was much lower in the sh-PRRT3-AS1 group in comparison with that in the sh-NC group (Fig. 8C). Furthermore, lncRNA PRRT3-AS1 (Fig. 8D) and HOXB5 (Fig. 8E) levels were reduced in tumor xenografts obtained from the sh-PRRT3-AS1 group, and lncRNA PRRT3-AS1-depleted tumor xenografts manifested clearly higher miR-507 levels (Fig. 8F). Taken together, these results indicated that downregulation of PRRT3-AS1 hampered NSCLC tumor growth in vivo.

Discussion
Many lncRNAs have been considered as crucial promoters or inhibitors of NSCLC progression [25], but more recently, several studies have highlighted other important roles played by lncRNAs in regulating complex cellular behaviors in NSCLC [35][36][37]. Therefore, finding novel cancerassociated lncRNAs and exploring their regulatory actions in NSCLC is necessary to identify promising targets for NSCLC treatment. To date, 33,829 lncRNAs have been verified in the human genome according to the ENCODE database [38]; however, for most of these, the biological role and working mechanisms are still unknown. Our aim in this study was to investigate the specific functions of the lncRNA PRRT3-AS1 in NSCLC and the underlying downstream mechanisms.
PRRT3-AS1 is overexpressed in prostate cancer, and the knockdown of PRRT3-AS1 suppresses cell viability, migration, and invasion, while promoting cell apoptosis [39]. However, the expression pattern, clinical implications, and functions of PRRT3-AS1 in NSCLC are still unknown. Our RNA expression analysis results revealed that PRRT3-AS1 was strongly expressed in NSCLC tissues, consistent with the information about PRRT3-AS1 in the TCGA database. Loss-and gain-of-function assays to determine comprehensively the biological functions of lncRNA PRRT3-AS1 in NSCLC progression showed that it played a pro-oncogenic role in NSCLC; PRRT3-AS1 knockdown evidently inhibited NSCLC cell growth and motility in vitro, whereas PRRT3-AS1 overexpression exerted the opposite effects. These results suggest that PRRT3-AS1 could be considered as a possible target for NSCLC diagnosis, prognosis, and management.
Next, we determined the mechanisms underlying the effect of lncRNA PRRT3-AS1 on malignant properties. The extensive research on the potential mechanisms of lncRNA function has mostly been dependent on the cellular localization of lncRNAs. Nuclear lncRNAs directly bind to proteins and regulate gene expression at the transcriptional level [40]. In contrast, one of the critical roles of lncRNAs distributed in the cell cytoplasm is to partake, as ceRNAs, in the post-transcriptional regulation of gene expression [41]. According to the ceRNA theory, lncRNAs adsorb specific miRNAs, disabling the interaction of miRNAs with their target mRNAs [41]. Using two different methods, lncLocator prediction and fluorescence in situ hybridization, we determined that the subcellular localization of lncRNA PRRT3-AS1 was primarily in the cytoplasm of NSCLC cells, offering a theoretical basis for lncRNA PRRT3-AS1 functions as a ceRNA.
Our study had two limitations. Firstly, we did not explore the effect of PRRT3-AS1 on metastasis in vivo. Secondly, other ways may be involved in the mechanisms underlying the oncogenic roles of PRRT3-AS1 in NSCLC. We will resolve the two limitations in the near future.
To conclude, PRRT3-AS1, which is overexpressed in NSCLC, upregulates HOXB5 expression by sequestering miR-507 and aggravates the oncogenicity of NSCLC. Our findings advance the understanding of NSCLC pathogenesis and strongly suggest the PRRT3-AS1/miR-507/HOXB5 pathway as a promising novel therapeutic target. Funding Statement: The authors received no specific funding for this study.

Conflicts of Interest:
The authors declare that they have no conflicts of interest to report regarding the present study.