LncRNA KCNQ1OT1 promotes cell proliferation, migration and invasion via regulating miR-129-5p/JAG1 axis in non-small cell lung cancer

All NSCLC tissues and the corresponding adjacent normal tissues were obtained from patients who underwent surgical resection at the Guangdong Second Provincial General Hospital. All patients did not undergo treatments prior to surgery. All patients were categorized according to the Eighth Edition of the American Joint Committee on Cancer TNM Staging System for lung cancer. This research was approved by the Ethics Committee of the Guangdong Second Provincial General Hospital, and written informed consent was collected from all participants. All tissue samples were immediately frozen in liquid nitrogen and then stored at − 80 °C until RNA extraction. The clinical characteristics of patients are summarized in Table 1.

Human lung epithelial cell line (BEAS-2B) and the NSCLC cell lines (A549, H1299, H460, H446 and H1975) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA) and were grown in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C containing 5% CO₂.

Small interfering RNA (siRNA) against KCNQ1OT1 (si-KCNQ1OT1, 5′-GGUAGAAUAGUUCUGUCUU-3′; si-KCNQ1OT1#2, 5′-GCCAAUAGCAACUGACUAA-3′; si-KCNQ1OT1#3, 5′-GCCACAUCUAACACCUAUA-3′) and the negative control siRNA (si-NC), KCNQ1OT1 overexpression plasmid (pcDNA-KCNQ1OT1) and the control (pcDNA-NC) were purchased from RiboBio (Guangzhou, China). The miR-129-5p mimic and the mimic negative control (NC mimic), miR-129-5p inhibitor and the negative control (NC inhibitor), siRNA against JAG1 (si-JAG1, 5′-GGCCAAGCCUUGUGUAAAU-3′) and the corresponding negative control (si-NC) were synthesized by Genelily BioTech (Shanghai, China). Cells were transfected by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s requirements.

Total RNA was isolated from tissues and cells using TRIzol reagent (Invitrogen) by the protocols of the manufacturer. The first strand cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kits (Thermo Fisher Scientific). The expression levels were detected using SYBR Green Mixture (Takara, Dalian, China). GAPDH or U6 was used as the endogenous control. Primers as follows: KCNQ1OT1 (forward, 5′-AGGGTGACAGTGTTTCATAGGCT-3′; reverse, 5′-GAGGCACATTCATTCGTTGGT-3′), miR-129-5p (forward, 5′-ACCCAGTGCGATTTGTCA-3′; reverse, 5′-ACTGTACTGGAAGATGGACC-3′), JAG1 (forward, 5′-GTCCATGCAGAACGTGAACG-3′; reverse, 5′-GCGGGACTGATACTCCTTGA-3′), GAPDH (forward, 5′-TCGCCAGCCGAGCCACATC-3′; reverse, 5′-CGTTCTCAGCCTTGACGGTGC-3′), U6 (forward, 5′-CGATACAGAGAAGATTAGCATGGC-3′; reverse, 5′-AACGCTTCACGAATTTGCGT-3′).

The viability of cells was detected with Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). First, 100 µl of cell suspension (1.0 × 10⁵ cells per well) were seeded into 96-well plates and cultured at 37 °C. Then, 10 µl CCK-8 solution was added to each well after incubation for 48 h. After 4 h, the absorbance was read at 450 nm using Microplate Reader (Bio-Rad, Hercules, CA, USA).

Cell migration and invasion ability were detected using transwell assay. For cell migration, the transfected cells were plated (1.0 × 10⁵ cells per well) in the upper chamber of a 24-well transwell containing 8 μm polycarbonate membrane (Millipore, Billerica, MA, USA). The serum-free DMEM was added into the upper chamber, and DMEM containing 10% FBS as a chemoattractant was added to the lower chamber. Then, cells were cultured for 48 h at 37 °C, and the cells migrated to the lower surface were fixed with methanol and stained with 0.1% crystal violet. At last, the cells were counted with microscopy. For cell invasion, transwell chambers were coated with Matrigel (Millipore), and other experimental procedures were performed as previously described.

The putative binding sites of KCNQ1OT1 and miR-129-5p or miR-129-5p and JAG1 were predicted by LncBase Predicted v.2 or StarBase v2.0. The fragments of KCNQ1OT1 containing wild-type (wt) or mutant (mut) binding sites of miR-129-5p were inserted into pGL3 luciferase reporter plasmids (Promega, Madison, WI, USA), and were co-transfected with miR-129-5p mimic or NC mimic into A549 and H460 cells using Lipofectamine 2000 (Invitrogen). In addition, the sequences of JAG1 3′UTR containing wild-type (wt) or mutant (mut) binding sites of miR-129-5p was cloned into pGL3 vectors (Promega). A549 and H460 cells were co-transfected with miR-129-5p mimic or NC mimic and corresponding luciferase reporter vector using Lipofectamine 2000 (Invitrogen). After the transfection for 48 h, Dual-Luciferase Reporter Assay System (Promega) was used for luciferase activity analysis according to the manufacturer’s instructions.

RNA pull-down assay was performed as previously described [17]. Biotin-labeled wild-type KCNQ1OT1 (Bio-KCNQ1OT1), mutant KCNQ1OT1 (Bio-KCNQ1OT1-MUT) and the control (Bio-NC) were purchased from RiboBio. Briefly, the biotinylated RNA was incubated with total RNA extracted from A549 and H460 cell lysates overnight at 4 °C. Then, the biotin-coupled RNA complexes were pulled down using M-280 Streptavidin Dynabeads (Invitrogen) at room temperature for 2 h. After RNA isolation, the abundance of miR-129-5p was detected by qRT-PCR.

After transfection, A549 and H460 cells were harvested and lysed in RIPA lysis buffer (Thermo Fisher Scientific). Total protein was quantified using the BCA Protein Assay Kit (Pierce, Appleton, WI, USA) with the protocols of the manufacturer. Then, the proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Furthermore, the membranes were blocked by 5% non-fat milk (Nestlé, Shuangcheng, China) for 2 h at 37 °C, and then incubated with primary antibodies against JAG1 (1:2000; Abcam, Cambridge, UK) or GAPDH (1:2000; Abcam) overnight at 4 °C. Subsequently, the membranes were washed three times with TBST and interacted with horseradish peroxidase (HRP)-conjugated secondary antibody (1:4000; Abcam) for 2 h at room temperature. Finally, the protein bands were visualized via enhanced chemiluminescence system (Thermo Fisher Scientific) and quantitated by ImageJ software (National Institutes of Health, Bethesda, MD, USA). GAPDH was regarded as an endogenous reference.

All data were displayed as the mean ± standard deviation (SD) from three independent experiments. Differences between the two groups were analyzed by Student’s t test, and multiple groups were analyzed by one-way analysis of variance (one-way ANOVA). Statistical analysis was performed using Graphpad Prism 7.0 software (GraphPad, San Diego, CA, USA). At P-value < 0.05, the difference was considered to be statistically significant.

In order to verify the differential expression of KCNQ1OT1 in NSCLC tissues and cells, qRT-PCR was performed to detect expression levels. The results revealed that KCNQ1OT1 expression was significantly upregulated in NSCLC tissues compared to adjacent non-tumor tissues (Fig. 1a). Similarly, the expression of KCNQ1OT1 was dramatically higher in NSCLC cells (A549, H1299, H460, H446 and H1975) than that in BEAS-2B cells (Fig. 1b). Furthermore, Kaplan–Meier survival analysis showed that higher KCNQ1OT1 expression resulted in poor overall survival compared with lower KCNQ1OT1 expression (Fig. 1c). Finally, we also investigated the correlation between KCNQ1OT1 expression levels and clinical pathological features. The data indicated that KCNQ1OT1 expression was not associated with patient age, gender, smoking and histology, but was correlated with tumor stage and lymph node metastasis (Table 1). All these data suggested that KCNQ1OT1 expression was related to NSCLC prognosis and might play crucial roles in NSCLC development and progression.

To investigate the effects of KCNQ1OT1 on NSCLC progression, A549 and H460 cells were transfected with si-KCNQ1OT1, si-KCNQ1OT1#2, si-KCNQ1OT1#3 or si-NC. First, qRT-PCR results showed that the si-KCNQ1OT1 group had the most significant down-regulation after transfection with si-KCNQ1OT1, si-KCNQ1OT1#2 or si-KCNQ1OT1#3, so si-KCNQ1OT1 was selected for subsequent research (Fig. 2a and Additional file 1: Figure S1). CCK-8 assay and transwell assay exhibited that KCNQ1OT1 knockdown dramatically suppressed cell viability (Fig. 2b), migration (Fig. 2c) and invasion (Fig. 2d) in A549 and H460 cells. These data demonstrated that KCNQ1OT1 knockdown blocked cell proliferation, migration and invasion of NSCLC cells.

To verify whether KCNQ1OT1 could act as a ceRNA by competitively binding miRNAs in NSCLC, we predicted that KCNQ1OT1 had putative binding sites with miR-129-5p by LncBase Predicted v.2 (Fig. 3a). For further validation, dual-luciferase reporter assay was performed. The results showed that cells co-transfected with wt-KCNQ1OT1 and miR-129-5p mimic had strikingly lower luciferase activity than other co-transfected complexes (Fig. 3b, c). Moreover, RNA pull-down assay further confirmed that KCNQ1OT1 bound to miR-129-5p (Fig. 3d). Besides, the overexpression efficiency of KCNQ1OT1 was determined by qRT-RCR (Fig. 3e and Additional file 2: Figure S2). Furthermore, KCNQ1OT1 overexpression significantly reduced miR-129-5p expression, and KCNQ1OT1 knockdown strikingly increased miR-129-5p expression in A549 and H460 cells (Fig. 3f, g). In addition, miR-129-5p expression was remarkably down-regulated in NSCLC tissues and cells (Fig. 3h, j), and was negatively correlated with KCNQ1OT1 expression in NSCLC tissues (Fig. 3i). Also, the overexpression efficiency and suppression efficiency of miR-129-5p were determined by qRT-PCR (Fig. 3k). These results revealed that KCNQ1OT1 directly bound to miR-129-5p in NSCLC.

To further investigate the effects of miR-129-5p on NSCLC development, A549 and H460 cells were transfected with si-NC + NC inhibitor, si-KCNQ1OT1 + NC inhibitor or si-KCNQ1OT1 + miR-129-5p inhibitor. The results showed that transfection with miR-129-5p inhibitor attenuated the increase in miR-129-5p expression caused by KCNQ1OT1 silencing (Fig. 4a). Moreover, simultaneous knockdown of KCNQ1OT1 and miR-129-5p reversed the inhibitory effects of KCNQ1OT1 depletion on cell proliferation (Fig. 4b), migration (Fig. 4c) and invasion (Fig. 4d) of A549 and H460 cells. These results indicated that inhibition of miR-129-5p could abrogate the inhibitory effects of KCNQ1OT1 knockdown on NSCLC progression.

StarBase v2.0 predicted that miR-129-5p might bind to JAG1 (Fig. 5a). Then, dual-luciferase reporter assay was performed to verify whether JAG1 was a target for miR-129-5p. The results suggested that miR-129-5p mimic significantly decreased the luciferase activity in A549 and H460 cells transfected with wt-JAG1, whereas luciferase activity could not be regulated when the binding sites were mutated (Fig. 5b, c). To further explore whether JAG1 was regulated by miR-129-5p, western blot results revealed that miR-129-5p inhibition obviously increased the protein level of JAG1, while miR-129-5p mimic markedly reduced the protein level of JAG1 in A549 and H460 cells compared to the negative control (Fig. 5d, e). These data suggested that JAG1 was a target gene of miR-129-5p.

To further determine the effects of miR-129-5p on NSCLC progression, A549 and H460 cells were transfected with NC inhibitor, miR-129-5p inhibitor, miR-129-5p inhibitor + si-NC or miR-129-5p inhibitor + si-JAG1. Firstly, western blot analysis revealed that transfection with si-JAG1 alleviated the elevation in JAG1 protein expression caused by miR-129-5p knockdown (Fig. 6a). Moreover, CCK-8 assay showed that down-regulation of miR-129-5p obviously increased cell viability, while the effect was eliminated by silencing of JAG1 (Fig. 6b). Transwell assay revealed that knockdown of miR-129-5p remarkably promoted cell migration and invasion, whereas the impact was reversed after transfection with si-JAG1 (Fig. 6c, d). These results indicated that miR-129-5p might suppress cell proliferation, migration and invasion in NSCLC by modulating JAG1.

The western blot results revealed that KCNQ1OT1 knockdown significantly reduced the protein levels of JAG1, whereas the miR-129-5p inhibitor restored the JAG1 expression in A549 and H460 cells (Fig. 7a, b). Moreover, JAG1 expression was overtly increased in NSCLC tissues and was positively correlated with KCNQ1OT1 expression (Additional file 3: Figure S3A and AB). These results suggested that KCNQ1OT1 modulated JAG1 expression by regulating miR-129-5p in NSCLC cells.