MiR-497-5p down-regulates CDCA4 to restrains lung squamous cell carcinoma progression

Data of mature miRNAs and mRNAs in The Cancer Genome Atlas (TCGA)-LUSC dataset were acquired from TCGA (https://​portal.​gdc.​cancer.​gov/​) on February 12, 2020. After screening, miRNA data (cancer: n = 473; normal: n = 45) and data of mRNA sequences (cancer: n = 497; normal: n = 49) were obtained. Differentially expressed mRNAs (DEmRNAs) were identified by R package “edgeR” (|logFC|> 2, padj < 0.05) [15]. After determining the miRNA, and miRTarBase (http://​mirtarbase.​mbc.​nctu.​edu.​tw/​php/​index.​php), miRDB (http://​mirdb.​org/​), TargetScan (http://​www.​targetscan.​org/​vert_​72/​), starBase (http://​starbase.​sysu.​edu.​cn) /), and mirDIP (http://​ophid.​utoronto.​ca/​mirDIP/​) were used for target gene prediction [16‐20]. The predicted results were intersected with DEmRNAs. The correlation between miRNA and mRNA was calculated. The miRNA-mRNA regulatory pair was determined.

Lung epithelial cells BEAS-2B (ATCC®CRL-9609™), LUSC cells NCI‐H520 (ATCC®HTB-182™), NCI-H1703 (ATCC®CRL-5889™), SK-MES-1 (ATCC®HTB-58™), and EPLC-32M1 (ATCC®CRL-2182™) were obtained from American Type Culture Collection (ATCC). They were grown in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS) (Invitrogen) in a moist incubator.

miR-NC/MiR-497-5p mimic (miR-mimic) (RiboBio; Guangzhou, China) as well as pcDNA3.1-CDCA4/pcDNA3.1(Invitrogen; Carlsbad, CA, USA) were transfected into NCI‐H520 cells using Lipofectamine 3000 (Invitrogen).

Total RNA was extracted using TRIzol kit (Invitrogen). For miR-497-5p detection, complementary DNA (cDNA) was synthesized using TaqMan reverse transcription kit (Applied Biosystems) and amplified using TransScript Green One-Step qRT-PCR SuperMix (Qiagen). For CDCA4 mRNA detection, total RNA was reversely transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Kit used for qPCR Taq was SYBR Premix Ex (Qiagen, Valencia, CA, USA). The relative expression of miR-497-5p and CDCA4 mRNA was analyzed by \(2^{{ - \Delta \Delta {\text{Ct}}}}\) method, and was standardized by U6 or GAPDH. Primers used are shown in Table 1.

In short, transfected NCI‐H520 cells were seeded to 96-well plates (3 × 10³ cells/well), followed by incubation routinely. Next, 20 μL CCK-8 buffer was added to each well at 0/24/48/72/96 h, respectively, followed by incubation for 2 h. Then, optical density (OD) value at 490 nm was measured on a microplate reader.

Tip of a 10 µL sterile pipette was applied to scrape the formed cell monolayer in plates, and phosphate buffer saline (PBS) was used to wash off the separated cells. After 0 and 24 h, wound width was observed and photographed. Wound healing rates of different treatment groups were collected and calculated.

200 µL cell suspension was injected into the upper chamber, which was coated with 1 mg/mL Matrigel gel (356234, BD Corporation, USA). The lower chamber contained 600 µL RPMI-1640 with 10% FBS. After 48 h of incubation at 37 ℃, cells on the lower surface were fixed with 4% formaldehyde for 15 min and stained with 0.5% crystal violet for 30 min. Then, cells passing through the membrane were observed, counted and photographed by a microscope.

Total proteins were isolated using RIPA buffer (Beyotime Institute of Biotechnology, China). After quantified by bicinchoninic acid assay (BCA) (Beyotime Institute of Biotechnology, China), equivalent amount of proteins (30 µg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane. The membrane was then incubated with primary antibodies at 4 ℃ overnight. Primary antibodies included rabbit anti-CDCA4 (Abcam, British) and rabbit anti-GAPDH (Abcam, British). The membrane was then incubated with horseradish peroxidase-conjugated goat anti-rabbit antibodies (Abcam, British). Thereafter, protein signals were detected using the enhanced chemiluminescence western blotting reagent (GE Healthcare Life Sciences, Canada). Finally, Quantity One software (version 4.62; Bio-Rad Laboratories, Inc. USA) was used for gel analysis.

Sequences on wild-type (WT) CDCA4 3’UTR and mutant (MUT) CDCA4 fragments were synthesized by GenePharma (Shanghai, China) and inserted into the pmirGLO luciferase vectors (Promega Corporation) to generate WT-CDCA4 and MUT-CDCA4. NCI‐H520 cells were co-transfected with WT-CDCA4/MUT-CDCA4 (1.6 µg) and miR-mimic/miR-NC (50 nM). Luciferase activity was assessed 48 h after culture using the dual-luciferase reporter assay system (Promega Corporation).

Cells in proper density were seeded to 6-well plates and transfected in standard conditions. Cells were collected and rinsed 3 times with cold PBS, followed by resuspension in 1 × binding buffer. Afterwards, cells were treated with Annexin V-FITC and propidium iodide (PI) for 15 min at room temperature. Finally, cell apoptosis was assessed with flow cytometry (BD Biosciences, San Jose, CA, USA).

Analysis between two groups was Student’s t-test. Analysis among multiple groups were One-way analysis of variance. Student’s t-test was used for post hoc test Pearson correlation coefficient between two genes was calculated. Statistical analysis was conducted on GraphPad Prism 6.0 (La Jolla, CA). Each assay was repeated 3 times. P < 0.05 denoted statistically significant.

Firstly, bioinformatics data suggested remarkably low expression of miR-497-5p in LUSC tissue (Fig. 1A). A study exhibited that overexpressing miR-497-5p restrains progression of non-small cell lung cancer (NSCLC) cells [21]. That is why it was studied here. qRT-PCR result indicated that miR-497-5p in LUSC cell lines was significantly downregulated (Fig. 1B). Nonetheless, this gene was markedly relevant to patient’s survival (Additional file 1: Fig. S1A). Since NCI‐H520 had the lowest level of miR-497-5p, NCI-H520 was selected for later experiments.

NCI‐H520 cell line with overexpressed miR-497-5p was constructed. Based on qRT-PCR, miR-497-5p was markedly increased in the constructed cell line, suggesting it could be used for subsequent experiments (Fig. 2A). Based on CCK8, overexpressing miR-497-5p triggered a conspicuous reduction of the proliferative ability of NCI‐H520 cells compared to that of the negative control (Fig. 2B). Transwell assay and scratch healing method demonstrated that miR-mimic group displayed a conspicuously restrained invasion and migration of cancer cells (Fig. 2C–D). Flow cytometry showed a remarkable elevation of cancer cell apoptosis (Fig. 2E). Taken together, miR-497-5p overexpression repressed cancer-related behaviors.

Downstream targets of miR-497-5p were explored. Firstly, differential analysis was performed for the merged mRNA FPKM and GTEx data, and 2,312 DEmRNAs were obtained (Fig. 3A), among which 2,290 were up-regulated. Then, targets of miR-497-5p were simultaneously predicted using databases, and an intersection of the obtained predicted target genes and the 2,290 up-regulated DEmRNAs was taken to acquire 16 DEmRNAs with binding sites of miR-497-5p (Fig. 3B). CDCA4 possessed the highest Pearson correlation coefficient with the researched miRNA and showed a significantly negative correlation (Fig. 3C). TCGA-LUSC data showed that CDCA4 was highly expressed in LUSC tissue, but survival was not remarkably impacted (Fig. 3D, Additional file 1: Fig. S1B). Moreover, CDCA4 level was not conspicuously different in different T stages and M stages of patients. CDCA4 level of N1 + N2 + N3 patients was higher than N0 patients (p = 0.064; not remarkably different). Stage III and Stage IV patients showed notably higher CDCA4 expression than Stage I and Stage II patients (p = 0.002) (Additional file 1: Fig. S1C). Next, molecular effect of miR-497-5p on CDCA4 was further verified, and their binding sites were shown in Fig. 3E. Subsequently, luciferase method revealed that miR-497-5p upregulation attenuated luciferase intensity of cells with CDCA4-WT instead of CDCA4-MUT, indicating targeting between two genes (Fig. 3F). In addition, levels of CDCA4 protein and mRNA in the miR-497-5p overexpression group were significantly decreased as tested by western blot and qRT-PCR methods (Fig. 3G–H). These data suggested that CDCA4 could serve as a downstream target of miR-497-5p in LUSC.

Cells were transfected to perform rescue assay. The level of CDCA4 was increased notably in miR-NC + oe-CDCA4 group, while it was restored in miR-mimic + oe-CDCA4 (Fig. 4A–B). CCK8 assay results uncovered that the enhanced proliferative ability in LUSC induced by overexpression of CDCA4 could be counteracted by miR-mimic (Fig. 4C). Subsequently, experiments showed that overexpression of CDCA4 noticeably enhanced the invasion and migration in LUSC, while oe-CDCA4 + miR-mimic eliminated these effects (Fig. 4D–E). In addition, flow cytometry results confirmed that CDCA4 significantly inhibited cancer cell apoptosis, while overexpressing miR-497-5p reversed the inhibitory effect (Fig. 4F). In summary, miR-497-5p inhibited LUSC progression by down-regulating CDCA4.