Long noncoding RNA POU3F3 enhances cancer cell proliferation, migration and invasion in non-small cell lung cancer (adenocarcinoma) by downregulating microRNA-30d-5p

A total of 80 patients with NSCLC (adenocarcinoma) were enrolled in Guangdong Provincial Hospital of Integrated Traditional Chinese and Western Medicine between May 2016 and May 2018. Inclusion criteria: 1) patients who received pathological examinations and malignant tumors were confirmed by 3 experienced pathologists; 2) patients with complete medical record; 3) patients were willing to participate. Exclusions criteria: 1) patients with benigned lung tumors; 2) patients with other severe diseases. All patients signed written informed consent. This study was approved by the Ethics Committee of Guangdong Provincial Hospital of Integrated Traditional Chinese and Western Medicine.

Biopsy was performed on all patients to collect tumor and adjacent healthy tissue specimens. The specimens were confirmed by 3 experienced pathologists. All in vitro experiments in this study were performed using NCI-H23 and NCI-H522 cell lines, which were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). ATCC-formulated RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) was used to cultivate both cell lines in an incubator at 37 °C with 5% CO₂.

Total RNAs were extracted using Trizol reagent (Invitrogen, USA). Reverse transcription was performed using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) and PCR reactions were prepared using the Applied Biosystems™ Power SYBR™ Green PCR Master Mix. To detect miR-30d-5p precursor, miRNAs were extracted using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific). MystiCq® microRNA cDNA Synthesis Mix (Sigma-Aldrich, St. Louis, MO, USA) was used to synthesize cDNA, and miScript SYBR Green PCR Kit (QIAGEN) was used to prepare PCR reaction systems. Primers of lncRNA POU3F3, miR-30d-5p, as well as GAPDH and U6 endogenous controls were provided by Sangon (Shanghai, China). PCR reactions were performed on ABI PRISM 7500 qRT-PCR machine (Applied Biosystems, Rockford, IL, USA). Data normalizations were performed based on 2^-ΔΔCT method. Primer sequences were: 5′-AATCACTGCAATTGAAGGAAAAA-3′ (forward) and 5′-CCTTGTTTTCCAACCCTTAGACT-3′ (reverse) for POU3F3; 5′-GTCTCCTCTGACTTCAACAGCG-3′ (forward) and 5′-ACCACCCTGTTGCTGTAGCCAA-3′ (reverse) for GAPDH; 5′-GTTGTTTGTAAACAUCCCC-3′ (forward) and 5′-GTAGCAGCAAACATCTGACTG-3′ (reverse) for precursor miR-30d-5p. Forward primer of miR-30d-5p was 5′-TGTAAACATCCCCGACTGG-3′. Universal mature miRNA reverse primers and U6 primers were from the kit. The expression of pri-miR-30d-5p was determined by TaqMan™ Pri-miRNA Assay (Assay ID Hs03302839_pri; ThermoFisher).

POU3F3 gene (NCBI Accession: NR_037883.1) was inserted into pcDNA3.1 vector to establish POU3F3 expression vector. Vector construction service was provided by Sangon. Scrambled miRNA negative control 2 and hsa-miR-30d-5p miRNA Mimic were purchased from Sigma-Aldrich. Cell transfections were performed using Invitrogen™ Lipofectamine™ 2000. Transfection Reagent with 10 nM vectors and 40 nM siRNAs. Cells treated with Lipofectamine 2000 reagent only and cells transfected with empty vector were used as control and negative control cells, respectively. Cells were harvested at 24 h after transfection and overexpression of POU3F3 and miR-30d-5p were confirmed by qRT-PCR before subsequent experiments.

Cell proliferation assay was performed at 24 h after transfection using the Cell Counting Kit 8 (ab228554, Abcam). ATCC-formulated RPMI-1640 medium containing 10% FBS was used to prepare single cell suspensions with cell concentration at 3 × 10⁴ cells/ml. Each well of a 96-well plate was filled with 0.1 ml cell suspension and cells were cultivated in an incubator at 37 °C with 5% CO₂, followed by addition of 10 μl CCK-8 solution every 24 h until 96 h. Cells were then cultivated for an additional of 4 h, and optical density was measured at 450 nm.

In vitro, Transwell cell migration and invasion assays were performed to test cell migration and invasion abilities at 24 h after transfections. Cell suspensions (3 × 10⁴ cells/ml) were prepared using serum-free ATCC-formulated RPMI-1640 medium. Cell suspensions were transferred into upper Transwell chamber (0.1 ml/well), and the lower chamber was filled with ATCC-formulated RPMI-1640 medium containing 20% FBS. Cells were cultivated for 2 h, followed by staining of upper chamber membranes with 0.5% crystal violet (Sigma-Aldrich, USA) at room temperature for 20 min. An optical microscope was used to observe the stained cells. Matrigel (10%, 356,234, Millipore, USA) was used to coat upper chamber membrane for 6 h at 37 °C before invasion assay.

The results of qRT-PCR showed that, compared to adjacent healthy tissues, POU3F3 was significantly upregulated (Fig. 1a), while miR-30d-5p was significantly downregulated (Fig. 1b) in tumor tissues (p < 0.05). It is worth noting that expression levels of POU3F3 and miR-30a-5p were not significantly changed with the increasing of clinical stages (data not shown). Chi-squared test showed that expression levels of POU3F3 in NSCLC tissues were not significantly associated with patients’ gender, age, AJCC stages and smoking or drinking habits (Table 1, all p > 0.05).

Correlations between the expression levels of POU3F3 and miR-30d-5p were analyzed by Pearson’s correlation coefficient. POU3F3 and miR-30d-5p were significantly and inversely correlated across tumor tissues (Fig. 2a, r = − 0.87, p < 0.0001), but not in adjacent healthy tissues (Fig. 2b, r = − 0.15, p = 0.18).

To further investigate interactions between POU3F3 and miR-30d-5p, POU3F3 and miR-30d-5p were overexpressed in both NCI-H23 and NCI-H522 cell lines, followed by detecting the expression levels of POU3F3 and miR-30d-5p by RT-qPCR. As shown in Fig. 3a, overexpression of POU3F3 resulted in the downregulation of miR-30d-5p in both cell lines (Fig. 3a, p < 0.05), while overexpression of miR-30d-5p did not affect the expression of POU3F3 (Fig. 3b). It was worth noting that we also analyzed the expression of miR-30d-5p in cells transfected with POU3F3 overexpression, which also showed downregulated miR-30d-5p precursor and pri-miR-30d-5p (data not shown), indicating that POU3F3 downregulated miR-30d-5p at transcription level. In addition, RNA-RNA interaction predicted by IntaRNA 2.0 (http://​rna.​informatik.​uni-freiburg.​de/​IntaRNA/​Input.​jsp) revealed no strong binding site of miR-30d-5p on POU3F3 (data not shown). Moreover, preliminary dual-luciferase activity assay (co-transfection of miR-30d-5p + POU3F3 or NC miRNA + POU3F3) showed that miR-30d-5p and POU3F3 may not directly interact with each other (data not shown).

In vitro cell proliferation assay results showed that, in comparison to un-transfected cells (control, C) and cells transfected with empty vectors (negative control, NC), cells with the overexpression of POU3F3 showed enhanced proliferation ability. In contrast, cells with the overexpression of miR-30d-5p showed inhibited proliferation ability (Fig. 4, p < 0.05). In addition, in comparison to cells with the overexpression of POU3F3 only, cells with the overexpression of both POU3F3 and miR-30d-5p showed significantly reduced cell proliferation ability (p < 0.05).

In vitro cell migration and invasion assay results showed that, compared with C and NC groups, cells with the overexpression of POU3F3 showed increased migration (Fig. 5a) and invasion (Fig. 5b) rates of both NCI-H23 and NCI-H522 cell lines (p < 0.05). In contrast, cells with the overexpression of miR-30d-5p showed decreased migration (Fig. 5a) and invasion (Fig. 5b) of both NCI-H23 and NCI-H522 cell lines (p < 0.05). In addition, compared with cells with the overexpression of POU3F3 only, cells with the overexpression of both POU3F3 and miR-30d-5p showed significantly inhibited cell migration and invasion ability (p < 0.05).