LncRNA ASAP1-IT1 enhances cancer cell stemness via regulating miR-509-3p/YAP1 axis in NSCLC

Non-small cell lung cancer (NSCLC) is a major cause of cancer-related death worldwide, and cancer stem cell is responsible for the poor clinical outcome of NSCLC. Previous reports indicated that long noncoding RNAs (lncRNAs) play important roles in maintaining cancer stemness, however, the underlying mechanisms remain unclear. This study investigates the role of ASAP1 Intronic Transcript 1 (ASAP1-IT1) in cancer cell stemness of NSCLC. The expression of ASAP1-IT1, microRNA-509-3p (miR-509-3p) and apoptosis-/stemness-related genes was analyzed by qRT-PCR in NSCLC tissues, cancer cells and spheres of cancer stem cells. Knockdown of ASAP1-IT1 or overexpression of miR-509-3p in NSCLC cells by infection or transfection of respective plasmids. Sphere formation and colony formation were used to detect NSCLC stem cell-like properties and tumor growth in vitro. Luciferase reporter assays, RNA immunoprecitation (RIP) and qRT-PCR assays were used to analyze the interaction between lncRNA and miRNA. The expression of expression of regulated genes of ASAP1-IT1/miR-509-3p axis was evaluated by qRT-PCR and Western blot. The NSCLC xenograft mouse model was used to validate the role of ASAP1-IT1 in NSCLC stemness and tumor growth in vivo. ASAP1-IT1 was up-regulated in NSCLC tissues, cancer cells, and in spheres of A549-derived cancer stem cells. Downregulation of ASAP1-IT1 or overexpression of miR-509-3p significantly decreased cell colony formation and stem cell-like properties of A549-dereived stem cells with decreased expression of stem cell biomarkers SOX2, CD34, and CD133, and suppressing the expression of cell growth-related genes, Cyclin A1, Cyclin B1, and PCNA. Furthermore, knockdown of ASAP1-IT1 or overexpression of miR-509-3p repressed tumor growth in nude mice via reducing expression of tumorigenic genes. ASAP1-IT1 was found to interact with miR-509-3p. Moreover, overexpression of ASAP1-IT1 blocked the inhibition by miR-509-3p on stem cell-like properties and cell growth of A549-dereived stem cells both in vitro and in vivo. Finally, the level of YAP1 was regulated by ASAP1-IT1 and miR-509-3p. YAP1-involved ASAP1-IT1/miR-509-3p axis promoted NSCLC progression by regulating cancer cell stemness, and targeting this signaling pathway could be is a promising therapeutic strategy to overcome NSCLC stemness.


Background
Non-small cell lung cancer (NSCLC) is the major type of lung cancer which is a leading course of cancer-related death worldwide [1]. Although the treatment of NSCLC has been improved, the 5-years survival rate remains low owing to cancer relapse after surgery or radiotherapy [2,3]. Therefore, it is critical to better understand the molecular mechanisms of NSCLC progression and find out biomarkers to identify indolent and aggressive tumors, to provide novel therapeutic strategies.
Increasing evidence have demonstrated that the recurrence of NSCLC is mostly induced by cancer stem cells in tumors [4][5][6]. Cancer stem cells are involved in cancer initiation, proliferation, invasion, and differentiation, resulting in occurrence of aggressive and metastatic cancers [7]. Simultaneously, cancer stem cells can bring about multi-drug resistance in NSCLC [8]. Thus, it is interesting and meaningful to investigate the association of cancer stem cell and NSCLC progression, that would create therapeutic strategies.
In this study, we investigate the role of ASAP1-IT1, and the association of ASAP1-IT1, miR-509-3p and YAP1 in NSCLC progression and cancer cell stemness. each participant signed an informed consent form. All the patients were given no treatment before surgery. The collected specimens were stored in a liquid nitrogen tank.

Cell maintenance and transfection
The A549 and A549/R (cisplatin resistant) cells were obtained from China Infrastructure of Cell Line Resource (Beijing, China), and cultured in DMEM (Dulbecco's Modified Eagle Medium) media (WISENT, Nanjing, Jiangsu, China) in a CO 2 incubator at 37 °C. The DMEM media was supplemented with 10% FBS (fetal bovine serum), 100 mg/mL streptomycin and 100 U/mL penicillin (WISENT). The FBS was bought from Thermo Fisher Scientific (Waltham, Massachusetts, USA). All the other cell culture materials were bought from WISENT.

Hematoxylin-eosin (H&E) staining
The NSCLC specimens were fixed in 4% paraformaldehyde, embedded in paraffin and subjected to sectioning. Then, H&E staining was performed as described previously [10]. All the chemicals and reagents were bought from Servicebio (Wuhan, Hubei, China).

RNA extraction and qRT-PCR (quantitative real time polymerase chain reaction)
The total RNA was extracted using TRIzol reagent (Life Technologies, Rockville, MD, USA). The reverse transcription kit (Thermo Fisher Scientific) was applied to synthesize cDNA (complementary DNA) from the total RNA. Quantitative RT-PCRs were used to evaluate the levels of miRNAs, lncRNAs, or mRNAs using the SYBR Premix Ex Taq II (TaKaRa, Dalian, China). The qRT-PCR was carried out under the thermal cycling conditions: 95 °C × 5 min, and 40 cycles of 95 °C × 30 s, 60 °C × 30 s, and 72 °C × 1 min. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the internal control for RNA and lncRNA, and U6 was used as the internal control of miRNAs. The relative RNA levels were calculated following the 2 −ΔΔCT method. The PCR primers were list in Table 1. All experiments were performed in quadruplicate, and each assay was repeated independently for 3 times.

Western blot
Total protein samples were prepared from the tissues or cells using RIPA (radio immunoprecipitation assay) buffer (Beyotime) for 30 min. The lysates were centrifuged at 12,000×g for 15 min to obtain the supernatants, and they were de-natured at 98 °C for 20 min. 50 μg proteins were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). All the prepared gels were transferred to the 0.22 μm PVDF (polyvinylidene difluoride) membranes (Thermo Fisher Scientific) for 2 h, and the proteins-carried PVDF

Table1
The used PCR primers in this study

Sphere formation and colony formation of cancer stem cells
Cancer stem cells were sorted from A549 cells or transfected-A549 cells by sphero-cyst medium [10], and the sorted cancer stem cells from A549 were named as A549derived stem cells. The 3 × 10 4 cells were seeded in 6-well ultra-low cluster plates (Thermo Fisher Scientific). They were maintained in DMEM/F12 serum free medium (Thermo Fisher Scientific) containing epidermal growth factor (20 ng/mL), beta-fibroblast growth factor (20 ng/ mL), insulin (4 μg/mL), and B27 (2%). All reagents were from Sigma. Finally, the number of spheres was counted under an inverted microscope (Leica, Oskar-Barnack-Straße, Germay) post 10 days incubation. The biomarkers of cancer stem cells SOX2, CD34, and CD133 were used to identify the cancer stem cells using qRT-PCR and Western blot. The A549 cancer stem cells were isolated using magnetic bead kit, and 3 × 10 4 stem cells were seeded in sixwell plates with ultra-low adhesion. They were cultured in sphere formation media for a week, and post another 20 days, the number of spheres was calculated under a microscope.

Cell apoptosis and cell cycle analysis
To determine cell apoptosis of A549-dereived stem cells and other cells, cells were collected at 80 g × 4 °C × 5 min for annexin-V/FITC (fluoresceine isothiocyanate)/PI (propidium iodide) staining after 48 h infection with the indicated plasmids or miRNAs. Briefly, cells were incubated with annexin-V and PI for 10 min and 5 min, respectively, in a dark room using the annexin V-FITC/ PI staining kit (Beyotime, Beijing, China). Finally, the cell cycle was evaluated using the Beckman Coulter Navios EX flow cytometry (Beckman, Shanghai, China).
For analyzing cell cycle, the cells were cultured in 12-well plates (Thermo Fisher Scientific). After transfection with the indicated miRNAs or plasmids for 48 h, they were subjected to suspension and fixation in 75% ethanol at 4 °C for 12 h. Then, they were washed with PBS (phosphate-buffered saline) for 3 times. Afterwards, they were resuspended in 500 μL staining buffer containing propidium iodide (5 mg/mL)/RNase (10 mg/mL) at 37 °C for 30 min in a dark room. Finally, cell apoptosis rate was evaluated using the flow cytometry. Each treatment group consisted four wells, and each assay was repeated for 3 times independently.

Cell counting kit-8 (CCK-8) assay
The A549-dereived stem cells or A549 cells were plated into a 96-well plate (Thermo Fisher Scientific) at the density of 4 × 10 3 cells per well for determination of cell viability. CCK-8 reagent (Sangon, Shanghai, China) were added into medium after the cells were cultured 36 h. After incubation for 1 h at 37 °C, the formation of watersoluble formazan was examined in a microplate reader (Bio-Rad) with the light length of 450 nm. All experiments were performed in quadruplicate, and each assay was repeated independently for 3 times.

RNA immunoprecipitation (RIP) assay
RIP assay was conducted to examine the interaction between ASAP1-IT1 and miR-509-3p using the RIP RNA-binding protein immunoprecipitation kit Magna (Millipore, Massachusetts, USA). Briefly, the cell lysate was incubated with anti-Ago2 (argonaute-2), and anti-IgG was used as a negative control. The antibodies were purchased from Bioworld Technology (Nanjing, China). At last, the collected immunoprecipitated RNA samples were used to determine ASAP1-IT1 and miR-1301-3p content by qRT-PCR analysis. All experiments were performed in quadruplicate, and each assay was repeated independently for 3 times.

Mouse tumorigenesis assay
The A549-dereived stem cells were infected with the mentioned shRNAs or plasmids as indicated. Post 48 h infection, the A549-dereived stem cells were collected at 80 g × 4 °C × 5 min. Afterwards, 4 × 10 6 cells were inoculated into 5-week-old nude mice. The BALB/c nude mice were bought from the Model Animal Research Center of Nanjing University (Nanjing, China), and divided randomly with 3 mice in each group. The animal experiments were approved by the research ethics committee of Chengdu Medical College (approved animal protocol No. CMC20LM22). The tumor volume was calculated every 3 days using the formula, tumor volume = (length × width 2 )/2. All the tumor-carried nude mice were sacrificed on the 24th day post inoculation.

Statistical analysis
Statistical analysis was conducted using SPSS software package (version 20.0, SPSS Inc., NY, USA) and GraphPad Prism 6 (GraphPad Software, CA, USA). A p value < 0.05 was considered statistically significant. The data were obtained from three independent experiments, and all data were expressed as mean ± standard deviation (S.D.). The statistical significance was examined using Two-tailed Student's t-test for two-group comparisons and one-way analysis of variance (ANOVA) test with post-hoc analysis for multi-group comparisons.

Knockdown of ASAP1-IT1 suppressed cell growth and reduced cisplatin resistance in cancer stem cells, and promoted apoptosis
Cancer stem cells (CSCs) are closely associated with chemo-resistance, which often reduces therapeutic effects of anti-cancer drugs, such as cisplatin. To examine whether ASAP1-IT1 serves as a dominant factor of cisplatin resistance in NSCLC cells, A549 cells with ASAP1-IT1-knockdown were incubated with 4 μM cisplatin. The qRT-PCR data showed that ASAP1-IT1 was increased after incubation with cisplatin in a timedependent manner ( Fig. 2A). Also, ASAP1-IT1 was upregulated in A549/R cells compared with that in A549 cells (Fig. 2B). ASAP1-IT1-knockdown A549 cells were then incubated with cisplatin at different concentration, and the CCK-8 assays demonstrated that increasing concentration of cisplatin significantly decreased cell viability of A549 cells with ASAP1-IT1-knockdown (Fig. 2C). Moreover, annexin V-FITC/PI assays and qRT-PCR analysis confirmed that ASAP1-IT1 knockdown obviously increased apoptotic cells of A549 by elevating expression of Bax and caspase-3, and inhibiting Bcl-2 expression (Fig. 2D-F). Knockdown of ASAP1-IT1 also repressed colony formation of A549-dereived stem cells (Fig. 2G,  H), and silencing ASAP1-IT1 expression significantly decreased expression of cell growth-associated genes including Cyclin A1, Cyclin B1, and PCNA (Fig. 2I).

Overexpression of ASAP1-IT1 blocked the effect of miR-509-3p on cancer stem cells both in vitro and in vivo
Based on the above results, overexpression of miR-509-3p reduced the stemness, cisplatin resistance and growth, and promoted apoptosis of A549-dereived stem cells both in vitro and in vivo. Analysis of sphere formation indicated that overexpression of ASAP1-IT1 significantly overturned the effect of miR-509-3p in sphere formation and restored expression of stemness-associated genes including SOX2, CD44, and CD133 (Fig. 5A, B). ASAP1-IT1 overexpression reversed miR-509-3pinduced apoptosis by restoring miR-509-3p-controlled expression of apoptotic genes (Fig. 5C, D). Similarly, ASAP1-IT1 overexpression abolished the suppressive effect of miR-509-3p on cell growth by recovering miR-509-3p-mediated expression of cell growth-related genes including Cyclin A1, Cyclin B1, and PCNA (Fig. 5E, F).

Discussion
Increasing evidence have demonstrated that cancer stem cells participate in the aggressive and destructive behaviors of lung cancers including NSCLC, a main cause of cancer-induced death worldwide [2,3,35,36]. The cancer stem cells significantly increase the recurrence of NSCLC after surgery or radiotherapy [4][5][6]. The cancer stem cells possess the properties of self-renewal, cancer initiation, and cancer progression [7]. Cancer stem cells also aggravate multi-drug resistance in lung cancer [8]. Therefore, exploring cancer stem cell-related potential molecular mechanism in NSCLC progression is critical, as it would contribute to the treatment of patients with NSCLC. It has been reported that a variety of lncRNAs are involved in the progression of NSCLC [14,15,37,38]. As reported in previous studies, ASAP1-IT1 is upregulated in NSCLC and promotes cancer proliferation, invasion and migration [22]. Consistent with this report, our preliminary sequencing data shows that ASAP1-IT1 was one of the top 20 up-regulated lncRNA in NSCLC with poor clinical outcome (data not shown). Moreover, ASAP1-IT1 was validated to be upregulated in NSCLC tumors, cancer cells, and A549 cell spheres. Cancer stem cells  Down-regulation of ASAP1-IT1 could be a good strategy to fight against NSCLC. LncRNAs exert their activity in cancers often by functioning as sponges of miRNAs [15,43]. In this study, ASAP1-IT1 was firstly predicted to interact with miR-509-3p. The interaction between ASAP1-IT1 and miR-509-3p was proved by luciferase RIP assays. Previous studies have revealed that miR-509-3p inhibits tumor growth in multiple types of cancers. For example, miR-509-3p improves sensitivity of cancer cells to platinum in ovarian cancer [23,24,44], and it depresses cell proliferation and invasion via down-regulating X-linked inhibitor of apoptosis in glioma [28]. MiR-509 promotes hepatoma progression by activating NF-κB (nuclear factor kappa B) signaling pathway. However, the role of miR-509-3p in the stemness of NSCLC has not been investigated. In consistent with previous studies, miR-509-3p was significantly down-regulated in NSCLC tissues, NSCLC cells, A549 spheres, and in A549/R cells. Overexpression of miR-509-3p suppressed sphere formation and cell growth, and increased cell apoptosis of A549-dereived stem Fig. 6 YAP1 is regulated by miR-509-3p and ASAP1-IT1. A The potential target of miR-509-3p is predicted by TargetScan. B qRT-PCR analysis of YAP1 expression in NSCLC tissues and adjacent tissues, *p < 0.01 compared with adjacent tissues. C qRT-PCR analysis of YAP1 in A549 spheres, *p < 0.01 compared with parental group. D Luciferase activity of pmirGLO-YAP1-3′UTR-WT (wild type) or pmirGLO-YAP1-3′UTR-Mut (mutant) was modulated by miR-509-3p and ASAP1-IT1 in A549 cells, *p < 0.01 compared with Mock + pcDNA, φp < 0.01 compared with miR-509-3p + pcDNA, Фp < 0.01 compared with miR-509-3p + ASAP1-IT1. E YAP1 mRNA levels in A549 cells analyzed byqRT-PCR, *p < 0.01 compared with Mock + pcDNA, φp < 0.01 compared with miR-509-3p + pcDNA, Фp < 0.01 compared with miR-509-3p + ASAP1-IT1. F Western blot analysis on YAP1 protein in A549 cells cells. Also, overexpression of miR-509-3p decreased expression of SOX2, CD44, and CD133, and enhanced expression of Bax and caspase-3 while repressed Bcl-2 expression in cancer cells. Furthermore, overexpression of miR-509-3p suppressed tumor growth in nude mice. These results implied that miR-509-3p was a tumor suppressor, and overexpression of miR-509-3p could offer a novel approach to prevent NSCLC progression. Furthermore, overexpression of ASAP1-IT1 significantly blocked overexpression of miR-224-3p-mediated inhibition of cancer stem cell-like properties and cell growth of A549-dereived stem cells both in vitro and in vivo. Additionally, overexpression of ASAP1-IT1 abolished miR-509-3p-induced cancer cell apoptosis of A549-dereived stem cells both in vitro and in vivo, indicating that the interaction between ASAP1-IT1 and miR-509-3pis involved in regulation of cancer cell stemness and NSCLC progression.