lncRNA ZFPM2-AS1 promotes proliferation via miR-18b-5p/VMA21 axis in lung adenocarcinoma

2.1 Cell lines and cell culture

LUAD cell lines (A549, H1299, H1260, and H520) and human bronchus epithelial cell (16HBE) were all obtained from the American Type Culture Collection (ATCC; Manassas, VA). All cells were cultured in Roswell Park Memorial Institute 1640 medium (KeyGEN; Nanjing, Jiangsu, China), supplemented with 10% fetal bovine serum, 100 mg/mL streptomycin and 100  U/mL penicillin. All cells were cultured in a humid atmosphere containing 5% CO₂ at 37°C.

2.2 Cell transfection

For overexpression, ZFPM2-AS1 and VMA21 were cloned into pcDNA3.1 vectors (Addgene, Cambridge, MA). ShRNAs against ZFPM2-AS1(sh-ZFPM2-AS1#1/2/3) were purchased from Realgene (Nanjing, Jiangsu, China). MiR-18b-5p mimics and NC mimics were obtained from Ambion (GeneCopoeia, Guangzhou, Guangdong, China). All plasmids were transfected into A549 and H1299 cells with Lipofectamine 2000 reagent (Invitrogen, Waltham, MA).

2.3 Quantitative reverse transcription polymerase chain reaction

Total RNA was extracted from cells by TRIzol reagent (Invitrogen). RNAs were reversely transcribed into cDNAs with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA) or TaqMan miRNA Reverse Transcription Kit (Applied Biosystems). The expression levels of ZFPM2-AS1, miR-18b-5p, and VMA21 mRNA were quantified with SYBR Premix Ex Taq (Takara, Dalian, Liaoning, China) on a Roche LightCycler 480 Real-Time PCR System (Applied Biosystems). U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were applied as internal references. The relative expressions were calculated through $$ method. The primer sequences were listed below: ZFPM2-AS1, 5′-TTTCCTACAATGAATCCACCAG-3′ (forward) and 5′-TTTGAGCCACTCTTTGAGG-3′ (reverse); miR-19b-5p, 5′-TGTGCAAATCCATGCAAAACTGA-3′ (forward) and 5′-GTGCAGGGTCCGAGGT-3′ (reverse); VMA21, 5′-GCATCTACACTGAAGACGCTCC-3′ (forward) and 5′-GACCACTGCAACAATAGCAGCG-3′ (reverse); GAPDH, 5′-TATGATGATATCAAGAGGGTAGT-3′ (forward) and 5′-TGTATCCAAACTCATTGTCATAC-3′ (reverse); U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse).

2.4 Western blot analysis

Total protein was extracted with radioimmunoprecipitation assay lysis buffer (Beyotime, China). Extracted proteins were first mixed with loading buffer, separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were then blocked with 5% solution of nonfat milk for 1 hour. Membranes were subsequently incubated with primary antibody against VMA21 (Origene; TA336068) or GAPDH (Origene; TA890003) according to the manufacturer's protocol. Then the membranes were washed three times and incubated with the appropriate amount of secondary antibodies for 1 hour at 37°C.

2.5 Cell counting kit-8 assay

Cells were seeded in 96-well plates at a density of 1 × 10³ per well and cultured for 24, 48, 72 or 96 hours. Cell counting kit-8 (CCK-8) solution was added into 96-well plates after cell growth. Then, the cells were incubated for 4 more hours. After 4 hours, cell supernatant was removed and the optical density value was detected at 450 nm.

2.6 Colony formation assay

After 24 hours of transfection, A549 and H1299 cells were seeded into six-well plates at a density of 1 × 10³ cells per well. The medium was replaced every 72 hours during growth. After 12 days of incubation, cells were fixed with methanol and stained with crystal violet. The colonies were counted when they contained more than 50 cells.

2.7 Luciferase reporter assay

All luciferase reporter vectors (ZFPM2-AS1-WT, ZFPM2-AS1-MUT, VMA21-WT, and VMA21-MUT) were constructed from empty pGL3 vectors (Promega, Madison, WI). A549 and H1299 cells were seeded into 24-well plate and grew until reaching 70% confluence. Cells were then cotransfected with luciferase reporter vectors and NC mimics, miR-18b-5p mimics or miR-18b-5p mimics + pcDNA3.1/ZFPM2-AS1 with Lipofectamine 2000. After 48 hours, luciferase activities were measured with a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.

2.8 RNA immunoprecipitation assay

RNA immunoprecipitation (RIP) assay was carried out with EZ-Magna RIP RNA-binding protein immunoprecipitation kit (Millipore) under the manufacturer's protocol. Then, A549 and H1299 cells were lysed with RNA lysis buffer. Cell lysis solutions were added with magnetic beads coated with anti-Ago2 antibodies (Millipore) or anti-IgG antibodies (Millipore). After 2 hours of incubation at 4°C, co-precipitated RNAs were extracted and examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR).

2.9 RNA pull down

RNA pull-down was carried out to detect the binding capacity between miR-19b-5p and ZFPM2-AS1 or VMA21 in A549 and H1299 cells. ZFPM2-AS1-WT, ZFPM2-AS1-Mut, VMA21-WT, VMA21-Mut, and negative control (NC) were biotinylated and transfected into cells. Cell lysates were then added with streptavidin-coupled magnetic beads for 2 more hours at 37°C. Then, the RNA complex was eluted and miR-18b-5p was quantified through RT-qPCR.

2.10 Statistical analysis

Experiment data were expressed as mean ± SD based on results from three independent experiments. The Student t test and one-way analysis of variance were applied when comparing differences between groups. Survival curves were plotted by the Kaplan-Meier method. Data were considered as statistically significant when P value less than 0.05.

3.1 lncRNA ZFPM2-AS1 promotes proliferation in LUAD

Based on data collected from Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/), we found that the expression of lncRNA ZFPM2-AS1 was positively associated with the survival rate of LUAD patients (Figure 1A). Also, GEPIA showed that ZFPM2-AS1 was highly expressed in LUAD tissues than in normal tissues (Figure 1B). Furthermore, the expression of ZFPM2-AS1 was significantly higher in LUAD cells compared to HBE cells (Figure 1C). For experiment convenience, A549 and H1299 cells lines would be applied in the following research for their relatively high expression of ZFPM2-AS1.

So we performed assays to see if the expression of ZFPM2-AS1 would interfere proliferation of LUAD cells in return. The expression of ZFPM2-AS1 was effectively knocked down in cells transfected with sh-ZFPM2-AS1 (Figure 1D). Sh-ZFPM2-AS1#1 and sh-ZFPM2-AS1#2 would be applied in the following assays due to their relatively high knockdown efficiency. In CCK-8 assay, cells transfected with sh-ZFPM2-AS1 showed a weaker proliferation ability than the negative control group (Figure 1E). Also, cells transfected with sh-ZFPM2-AS1 formed much less colonies than cells transfected with sh-NC (Figure 1F). So based on these assays, lncRNA ZFPM2-AS1 promotes proliferation in LUAD.

3.2 ZFPM2-AS1 binds with and negatively regulates expression of miR-18b-5p

Since the function of ZFPM2-AS1 in LUAD had already been confirmed, we searched for its downstream gene to explore the concrete mechanism. Based on results predicted by starBase v3.0 (http://starbase.sysu.edu.cn/index.php), ZFPM2-AS1 has potential binding sites with 10 miRNAs. Among them, miR-18b-5p has been verified in most types of cancers based on results from Pan-Cancer analysis. So miR-18b-5p was selected for further detection.

We inspected the expression of miR-18b-5p and found its overall expression was lower in LUAD cells (Figure 2A). So we ran a series of test to verify the binding capacity between miR-18b-5p and ZFPM2-AS1. The potential binding site between them is listed in Figure 2B. Luciferase reporter assay showed that luciferase activity of vectors containing wild type ZFPM2-AS1 was lowered by miR-18b-5p mimics, while mutant ZFPM2-AS1 vectors showed no significant response to miR-18b-5p mimics (Figure 2C). Also, the majority of miR-18b-5p was detected from biotinylated wild type ZFPM2-AS1 group instead of biotinylated mutant ZFPM2-AS1 group in RNA pull-down (Figure 2D). Both assays indicated miR-18b-5p and ZFPM2-AS1 could bind at the predicted site. We also detected the influence of ZFPM2-AS1 on miR-18b-5p expression. The overexpression efficiency was verified before further experiments (Figure 2E). Figure 2F shows that overexpression of ZFPM2-AS1 would suppress the expression of miR-18b-5p. So based on these assays, ZFPM2-AS1 binds with and negatively regulates the expression of miR-18b-5p.

3.3 miR-18b-5p binds with and negatively regulates the expression of VMA21

The target gene of miR-18b-5p was searched through PicTar database (https://pictar.mdc-berlin.de). Results showed that 47 mRNAs could bind with miR-18b-5p. Among them, VMA21 had been verified by most experiments at the number of 30. So VMA21 was selected for further detection.

The expression of VMA21 was inspected and we found that its expression in LUAD cells was significantly higher than in HBE cells (Figure 3A). So the binding capacity between VMA21 and miR-18b-5p was verified. The binding site was shown in Figure 3B. In luciferase reporter assay, the luciferase activity of vectors containing wild type VMA21 was suppressed by miR-18b-5p mimics, while mutant VMA21 vectors showed no significant response (Figure 3C). Also, the majority of miR-18b-5p enriched in biotinylated wild type VMA21 group instead of biotinylated mutant VMA21 group (Figure 3D). Furthermore, both mRNA and protein level of VMA21 was decreased by miR-18b-5p mimics (Figure 3E and 3F). So based on these assays, miR-18b-5p binds with and negatively regulates the expression of VMA21.

3.4 ZFPM2-AS1 regulates VMA21 expression by competitively binding with miR-18b-5p

Plenty of research have proved that lncRNAs might competitively bind with miRNAs, thus regulated the target gene expression of miRNAs.11-13 In these circumstances, miRNAs and Ago2 would form RISCs which could bind with both specific lncRNAs and mRNAs. In this way, the expression of mRNAs would be regulated by lncRNAs through miRNAs. Since we have confirmed the binding capacity between miR-18b-5p and ZFPM2-AS1 or VMA21, we would run a series of tests to see if ZFPM2-AS1 regulated VMA21 expression through such ceRNA pattern.

In Figure 4A, RIP assay demonstrated that the expression of miR-18b-5p, ZFPM2-AS1 and VMA21 could all be detected from Ago2 group. The RIP assay indicated that both ZFPM2-AS1 and VMA21 could bind with the RISC formed by miR-18b-5p and Ago2. Furthermore, both the mRNA and protein level of VMA21 could be promoted by overexpression of ZFPM2-AS1 (Figure 4B and 4C). Luciferase reporter assay was also performed to verify such ceRNA axis. In Figure 4D, the luciferase activity of wild type VMA21 was suppressed by overexpression of miR-18b-5p mimics, but was partly rescued by overexpression of ZFPM2-AS1. So based on these assays, ZFPM2-AS1 regulates VMA21 expression by competitively binding with miR-18b-5p.

3.5 ZFPM2-AS1 regulates cell proliferation via miR-18b-5p/VMA21 axis in LUAD

Since the ceRNA axis of ZFPM2-AS1/miR-18b-5p/VMA21 has already been confirmed, we ran a series of rescue assays to see if ZFPM2-AS1 regulated LUAD cell proliferation through this axis. The expression of VMA21 in LUAD cells was promoted for further experiments (Figure 5A). CCK-8 assays revealed that cell proliferation ability was weakened by knockdown of ZFPM2-AS1, but this trend was partly counteracted by overexpression of VMA21 (Figure 5B). Also, in colony formation assay, the number of colonies was decreased in sh-ZFPM2-AS1#1 group while the number in sh-ZFPM2-AS1#1 + pcDNA3.1/VMA21 group was partly restored (Figure 5C). So based on these assays, ZFPM2-AS1 regulates cell proliferation via miR-18b-5p/VMA21 axis in LUAD. The detailed scheme is presented in Figure 6.