miR-1293 acts as a tumor promotor in lung adenocarcinoma via targeting phosphoglucomutase 5

Analysis of TCGA database and bioinformatics analysis

The expression levels of miR-1293 and phosphoglucomutase (PGM) 5 in LUAD and/or LUSC patients were analyzed in TCGA database using Starbase v3.0 (https://bio.tools/starbase) and/or GEPIA2 (http://gepia2.cancer-pku.cn/) online software. The correlation between miR-1293/PGM5 and patients’ pathologic stage, overall survival, or frequent mutations (eg. KRAS, EGFR, ALK and PIK3CA) as well as the relationship between miR-1293 and PGM5 were assayed in TCGA database via linkedomics (http://www.linkedomics.org/), Starbase v3.0 or GEPIA2 software. The correlations between miR-1293 and these mutations were analyzed by Wilcoxon test. Survival analysis curves were obtained using a Log rank Mantel cox test. RNA expression profiles in GSE118370 were downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), and differentially expressed genes in LUAD tissues were screened by GEO2R with thresholds of |log2 FC (fold-change)| > 2 and P < 0.01. The target genes of miR-1293 were analyzed by TargetScan (version 7.2).

Cell culture

This study used three LUAD cell lines A549, H1299 and H1975, two LUSC cell lines H226 and H520, and one normal lung cell line BEAS-2B. H520 cells were from the American Type Culture Collection (ATCC; Manassas, VA, USA), and other cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). They were all grown in RPMI-1640 medium, containing 10% fetal bovine serum (FBS; Gibco, MD, USA) and 1% penicillin-streptomycin (Gibco) at 37 °C with 5% CO₂.

Plasmid construction and cell transfection

For the PGM5 overexpression study, PGM5 cDNA amplified from BEAS-2B cells was cloned into the pcDNA3.1 vector to generate pcDNA3.1-PGM5 plasmid. All the transfection processes were carried out with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). A total of 2 µg of constructed vector or pcDNA3.1 empty plasmid was transfected into H1299 cells. miR-1293 mimic, miR-1293 antagomir (anti-miR-1293), and their negative controls were synthesized by RiboBio co., LTD (Guangzhou, China). A total of 50 nM of miR-1293 mimic or 100 nM of miR-1293 antagomir was transfected into H1975 and H1299 cells.

Dual-luciferase assay

The 3′-UTR sequences of PGM5 containing miR-1293 binding sites were amplified from BEAS-2B cells and inserted into the pGL3 reporter vector (Promega, Madison, WI, USA). Also, the mutant seed region of PGM5 was cloned into the pGL3 vector. H1975 cells were plated in a 24-well plate and then transfected with miR-1293 mimic or control at a final concentration of 50 nM, and 25 ng of generated PGM5 wild-type (PGM5-WT) or mutant luciferase reporter (PGM5-MUT) using Lipofectamine 3000. A total of 48 h after transfection, cells were harvested to detect luciferase activity using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

Cell proliferation analysis

Cell Counting Kit-8 (CCK-8) was used to examine the proliferation of H1975 and H1299 cells. Cells transfected with indicated vectors were seeded into 96-well plates at a density of 1 × 10³ cells/well. After culturing for 0, 24, 48 and 72 h, respectively, 10 µL of the CCK-8 solution was added to each well. A total of 2 h later, the optical density (OD) value was read by a micro-plate reader (Bio-Tek, Winooski, VT, USA) at 450 nm.

Transwell migration and invasion assay

The migration and invasion of H1975 and H1299 cells were determined by transwell analysis (Yu et al., 2020). For migration assay, the transfected H1975 and H1299 cells were trypsinized and resuspended in RPMI 1640 medium without FBS to adjust the density to 1 × 10⁵ cells/mL, respectively. In the following, 200 μL of the cell suspension without FBS was placed into the upper chamber of transwell (8 μm in pore size, Millipore, Burlington, MA, USA), whereas the lower chamber was filled with 600 μL of RPMI 1640 medium supplemented with 10% FBS. After incubating at 37 °C for 48 h, the upper surface of the membrane was cleaned by cotton swabs, and cells on the lower surface were fixed and stained with 0.1% crystal violet for 30 min. After drying, stained cells were photographed by a light microscope. For invasion analysis, the upper members of transwell inserts were pre-coated with Matrigel, and other operation processes were consistent with the transwell migration assay.

Animal experiments

Twelve 6–8-week-old BALB/c nude mice were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). All mice were raised in specific-pathogen-free conditions under 12/12 cycle of light at room temperature (25–27 °C) and allowed free access to food and water. To investigate the role of miR-1293 in the tumorigenesis of LUAD, H1975 cells (2 × 10⁶) were subcutaneously injected into the right flank of each mouse. One week later, mice were randomly divided into two groups (n = 6 per group): anti-miR-1293 group and anti-NC group. Mice were intratumorally injected with 5 nM miR-1293 antagomir or control, respectively, twice a week for 3 weeks. Tumor volumes were measured every week and calculated with the formula V (mm³) = 1/2 (length × width²). A total of 5 weeks after inoculation, mice were sacrificed by cervical dislocation after inhalational anesthesia with 2% isoflurane, and the xenografted tumors were removed for analysis. A part of tumor tissues was directly stored at −80 °C, which was used to detect the expression of miR-1293 and PGM5. The other part of tumor specimens was fixed in formalin and embedded in paraffin to perform immunohistochemical staining for Ki-67 and MMP9. All procedures involving in animals were reviewed and approved by the Affiliated Cancer Hospital of Nanjing Medical University (No. 2020-005).

Real-time PCR

Total RNA was isolated from cells and tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. cDNA was synthesized by reverse transcription with the reverse transcription kit (Takara, Dalian, China). The relative levels of PGM5 transcripts were detected by Real-time PCR using the SYBR Green mix (Kakara, Dalian, China). The expression of miR-1293 was determined by TaqMan MicroRNA assay kits (Applied Biosystems, Foster City, CA, USA). The relative fold changes of candidate genes were assayed by the 2^−ΔΔCt method, with GAPDH or U6 as an internal control. The primers used were as follows: PGM5, forward, 5′- GATGCTGATGGGGACCGTTA-3′ and reverse, 5′-GCAACGTCCTGAGTCCATCA-3′; GAPDH, forward, 5′-GAAGACGGGCGGAGAGAAAC-3′ and reverse, 5′-CCCAATACGACCAAATCCGTTG-3′; miR-1293, forward, 5′-ACACTCCAGCTGGGTGGGTGGTCTGGAGAT-3′ and reverse, 5′-TGGTGTCGTGGAGTCG-3′; U6, forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′.

Western blot

Cells with different treatments and tumor tissues were lysed with RIPA lysis buffer (Sigma–Aldrich, St. Louis, MO, USA) supplemented with protease inhibitors. Protein concentration was quantified using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Samples were denatured by heating in a water bath at 100 °C for 5 min. Cellular proteins were separated using SDS-PAGE by 10% running gel. After transferring proteins onto the PVDF membrane, the membrane was blocked with 5% defatted milk for 1 h at room temperature, incubated with primary antibodies, including PGM5 (1:500; Novus, St. Louis, MO, USA), MMP2 (1:1,000; Cell Signaling Technology, Danvers, MA, USA) and MMP9 (1:1,000, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, and probed with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000). After that, the blots were detected using enhanced chemiluminescent reagents. The relative levels of proteins were analyzed by densitometric analysis using ImageJ software, with GAPDH as an internal control.

Statistical analysis

All experiments were performed in triplicates and mean ± standard deviation (SD) was calculated. Data were analyzed by the Graphpad Prism 5 software. Student’s t test and one-way analysis of variance (ANOVA) following Bonferroni or Dunnett’s post-test analysis were used to perform all statistical analyses. A P value of <0.05 is considered statistically significant.

miR-1293 expression is elevated in tumor tissues of lung adenocarcinoma patients

miR-1293 expression was assayed in TCGA database using Starbase online software, and the results indicated that compared with normal samples, miR-1293 expression was upregulated in NSCLC samples, including adenocarcinoma (P = 0.0065) and squamous cell carcinoma (P = 2.0 × 10⁻¹²) (Figs. 1A and 1B). Furthermore, miR-1293 expression level was related to the pathologic stage of LUAD patients (Fig. 1C: N = 445; P = 1.916 × 10⁻⁵). Higher miR-1293 expression was correlated with poor overall survival of LUAD patients (Fig. 1D: N = 430; P = 2.621 × 10⁻⁶ or Fig. 1E: N = 503; P = 2.5 × 10⁻⁵). However, we did not find any correlation between miR-1293 expression and the pathologic stage (Fig. 1F: N = 339; P = 0.7166) or overall survival (Fig. 1G: N = 325; P = 0.7199) of LUSC patients. Specially, we further investigated the correlation between miR-1293 expression and the frequent mutations present in LUAD patients and found that miR-1293 expression was positively related to KRAS and PIK3CA mutations, but negatively related to EGFR mutation in LUAD patients, without any correlation with ALK mutation (Figs. 1H–1K). miR-1293 expression was notably elevated in LUAD cell lines, including A549, H1299 and H1975 compared with the normal lung cell line (BEAS-2B). Although miR-1293 expression in LUSC cell line H520 was higher than that in BEAS-2B, we did not find any significant change between H226 and BEAS-2B cells (Fig. 1L). Collectively, these results indicated that miR-1293 was increased in LUAD and elevated miR-1293 might serve as a promising prognostic biomarker for LUAD patients.

miR-1293 promotes the proliferation, migration and invasion of lung adenocarcinoma cells

We further investigated the role of miR-1293 in LUAD cells. The viabilities of H1975 and H1299 cells were significantly promoted after miR-1293 mimic transfection (Figs. 2A and 2B). The numbers of migrated and invasive cells were increased by miR-1293 mimic (Figs. 2C and 2D). Moreover, the mRNA and protein expression levels of MMP2 and MMP9 were elevated by miR-1293 mimic both in H1975 and H1299 cells (Figs. 2E–2G). In contrast, miR-1293 antagomir suppressed the viabilities, decreased cell migration and invasion, and reduced the expression of MMP2 and MMP9 in LUAD cells (Figs. 2A–2G). To exclude the influence of cell proliferation on cell migration and invasion, LUAD cells in the upper chamber were cultured with RPMI 1640 medium without FBS, which were shown in Fig. S1. In general, these data indicated that miR-1293 could facilitate the proliferation, migration and invasion of LUAD cells in vitro.

PGM5 as a target of miR-1293 in lung adenocarcinoma

To predict the target genes of miR-1293 in LUAD, we analyzed three independent datasets: the TCGA data, which revealed genes negatively associated with miR-1293 expression in LUAD; the potential target genes of miR-1293 analyzed by TargetScan; and differentially expressed genes in LUAD tissues from the GSE118370. Overlap of the three datasets was displayed using a Venn diagram. We found that five genes were common to all three datasets (Fig. 3A). Among these genes, PGM5 was selected, because it has been found to be related to tumor progression in some cancers (Jiao et al., 2019; Sun et al., 2019). As shown in Fig. 3B, PGM5 expression showed an inverse correlation with miR-1293 in LUAD patients. We found that PGM5 expression was decreased in tumor tissues of LUAD patients (Fig. 3C). Lower PGM5 expression was related to the poor overall survival of LUAD patients (Fig. 3D). Furthermore, the mRNA and protein expression levels of PGM5 were reduced in LUAD cell lines compared with BEAS-2B cells (Figs. 3E and 3F).

The binding sites between PGM5 mRNA and miR-1293 are shown in Fig. 4A. Subsequently, we constructed wild-type and mutated PGM5 dual-luciferase reporter vectors to validate whether PGM5 was a target of miR-1293 in LUAD. The data demonstrated that miR-1293 dramatically decreased the luciferase activities of PGM5 by directly bounding to its 3′UTR, without any effect on the luciferase activities of PGM5-MUT vector (Fig. 4B). Additionally, we found that miR-1293 significantly decreased the mRNA and protein levels of PGM5 in H1975 and H1299 cells (Figs. 4C–4E). Taken together, these results suggested that PGM5 was a target of miR-1293 in LUAD.

miR-1293 functions via regulating PGM5 expression in lung adenocarcinoma cells

To explore whether miR-1293 inhibited LUAD cells’ proliferation, migration, and invasion via mediating PGM5, we upregulated PGM5 expression in H1299 cells transfected with or without miR-1293 mimic. Western blot assay showed that pcDNA3.1-PGM5 plasmid significantly enhanced PGM5 expression in H1299 cells (Fig. 5A). PGM5 upregulation notably inhibited cell viability and abrogated the promotion of miR-1293 on the viability of H1299 cells (Fig. 5B). Using transwell assays, we found that overexpression of PGM5 decreased the numbers of migratory and invasive cells and reversed the effects of miR-1293 on the migration and invasion of H1299 cells (Figs. 5C and 5D). Also, PGM5 upregulation decreased the expression of MMP2 and MMP9 that was increased by miR-1293 mimic (Figs. 5E and 5F). Therefore, these data further confirmed that miR-1293 functioned via regulating PGM5 expression in LUAD cells.

miR-1293 antagomir suppresses LUAD growth and metastasis via negatively modulating PGM5 level in vivo

The role and mechanism of miR-1293 in LUAD were further evaluated in H1975 xenografted mouse model (Fig. 6A). Administration of anti-miR-1293 significantly reduced miR-1293 expression, accompanied by elevated PGM5 expression in tumor tissues (Figs. 6B and 6C). Tumor volume and Ki-67 expression were reduced by anti-miR-1293 (Figs. 6D–6F). Additionally, MMP9 levels were also decreased in mice treated with anti-miR-1293 (Fig. 6F). Taken together, these results indicated that miR-1293 inhibitor could suppress the growth and metastasis of LUAD via negatively modulating PGM5 level in vivo.