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

Non-small cell lung cancer (NSCLC) is the most deadly cancer worldwide. LncRNA KCNQ1OT1 has been reported to be involved in the progression of various tumors, including NSCLC. However, the precise mechanism of KCNQ1OT1 in NSCLC requires further investigation. The expression levels of KCNQ1OT1, miR-129-5p and JAG1 were detected by qRT-PCR or western blot. Kaplan–Meier survival analysis was used to assess the correlation between KCNQ1OT1 expression and the overall survival of NSCLC patients. CCK-8 assay was used to measure cell viability. Cell migration and invasion were detected by transwell assay. The targets of KCNQ1OT1 and miR-129-5p were predicted by bioinformatics, which was confirmed by dual-luciferase reporter assay or pull-down assay. KCNQ1OT1 expression was significantly enhanced, while miR-129-5p expression was dramatically reduced in NSCLC tissues and cells. Higher KCNQ1OT1 shortened overall survival and was positively associated with tumor stage and lymph node metastasis. KCNQ1OT1 knockdown inhibited proliferation, migration and invasion of NSCLC cells. Inhibition of miR-129-5p attenuated the inhibition of NSCLC cell viability, migration and invasion induced by KCNQ1OT1 knockdown. In addition, JAG1 was confirmed as a target of miR-129-5p. Knockdown of JAG1 reversed the effects of miR-129-5p knockdown on NSCLC progression. KCNQ1OT1 regulated JAG1 expression by sponging miR-129-5p in NSCLC cells. KCNQ1OT1 induced proliferation, migration and invasion of NSCLC cells by sponging miR-129-5p and regulating JAG1 expression, indicating that KCNQ1OT1 was a therapeutic target for NSCLC.

Long noncoding RNAs (lncRNAs) are non-protein coding transcripts longer than 200 nucleotides in length [4]. Increasing evidence has shown that lncRNAs exerted significant regulatory effects on various biological processes and gene expression through multiple mechanisms [5]. KCNQ1 overlapping transcript 1 (KCNQ1OT1) was involved in the regulation of numerous genes within the kcnq1 domain [6]. LncRNA KCNQ1OT1 has been reported to play roles in the pathogenesis of various cancer, such as colorectal cancer [7], breast cancer [8] and tongue cancer [9]. In previous studies, KCNQ1OT1 facilitated progression of cholangiocarcinoma (CCA) by sponging miR-140-5p and regulating SOX4 expression [10]. However, the mechanism of KCNQ1OT1 in NSCLC still needs further research.
MicroRNAs (miRNAs) are non-protein-encoded short noncoding RNAs composed of 18-25 nucleotides. The roles of miRNAs in epigenetic regulation had progressed, in which miRNAs regulated the protein levels of target mRNAs [11]. A previous study revealed that miR-129-5p was down-regulated, and inhibited cell proliferation and EMT by negatively regulating HMGB1 in gastric cancer [12]. Moreover, the role of miR-129-5p in NSCLC have also been studied. LncRNA NNT-AS1 was a carcinogen in non-small cell lung cancer (NSCLC), and acted as a competing endogenous RNA (ceRNA) by targeting miR-129-5p in lung cancer [13]. However, the relationship between KCNQ1OT1 and miR-129-5p in the progression of NSCLC has not been elucidated. Jagged1 (JAG1) has been reported to play a role in multiple types of cancer [14]. Previous studies have shown that JAG1 regulated tumor progression, including NSCLC. Tang et al. found that miR-377-3p suppressed cell proliferation and invasion via targeting JAG1 in ovarian cancer [15]. Wang et al. indicated that miR-26b was a tumor inhibitor through binding to JAG1 in cervical cancer [16].
In this study, we measured the expression levels of KCNQ1OT1, miR-129-5p and JAG1 in NSCLC tissues and cells. In addition, we explored the potential interactions between KCNQ1OT1 and miR-129-5p or miR-129-5p and JAG1. In conclusion, this study may provide novel therapeutic targets for NSCLC treatment.

Tissue samples
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.

Quantitative real-time PCR
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:

CCK-8 assay
The viability of cells was detected with Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). First, 100 µl of cell suspension (1.0 × 10 5 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).

Transwell assay
Cell migration and invasion ability were detected using transwell assay. For cell migration, the transfected cells were plated (1.0 × 10 5 cells per well) in the upper chamber of a 24-well transwell containing 8 μm polycarbonate membrane (Millipore, Billerica, MA, USA). The serumfree 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.

Dual-luciferase reporter assay
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
RNA pull-down assay was performed as previously described [17]. Biotin-labeled wild-type KCNQ1OT1 (Bio-KCNQ1OT1), mutant KCNQ1OT1 (Bio-KCN-Q1OT1-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.

Western blot assay
After transfection, A549 and H460 cells were harvested and lysed in RIPA lysis buffer (Thermo Fisher Scientific

Statistical analysis
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 (Graph-Pad, San Diego, CA, USA). At P-value < 0.05, the difference was considered to be statistically significant.

KCNQ1OT1 was upregulated in NSCLC tissues and cells and correlated with poor prognosis
In order to verify the differential expression of KCN-Q1OT1 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.

Inhibition of miR-129-5p reversed the effects of KCNQ1OT1 knockdown on proliferation, migration, invasion of NSCLC cells
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.

MiR-129-5p targeted JAG1 in NSCLC cells
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.

KCNQ1OT1 regulated JAG1 by sponging miR-129-5p in NSCLC cells
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

Discussion
With the great achievements in diagnosis and treatment, the survival time of NSCLC has increased, but the prognosis of NSCLC is still poor [18]. In recent years, the studies suggested that lncRNAs dysregulation was associated with the progression of cancers, including NSCLC [19]. To elucidate the molecular mechanism of lncRNAs in the prognosis of NSCLC is meaningful.
Recent studies exhibited that lncRNA KCNQ1OT1 was upregulated in various cancers. A previous study suggested that KCNQ1OT1 facilitated tumor growth by competitively sponging miR-504 and up-regulating cyclin-dependent kinase 16 (CDK16) in hepatocellular carcinoma [20]. A previous research revealed that KCNQ1OT1 was upregulated in early stage lung cancer and was associated with prognosis in LC patients by suppressing cell proliferation [21]. Furthermore, Dong et al. demonstrated that KCNQ1OT1 was markedly upregulated in NSCLC tissues and cells, and promoted NSCLC cells proliferation, migration, and invasion by MiR-129-5p targeted JAG1 in NSCLC cells. a The putative binding sites of miR-129-5p and JAG1. b, c Luciferase activity was examined in A549 and H460 cells co-transfected with wt-JAG1 or mut-JAG1 and miR-129-5p mimic or NC mimic. d The protein level of JAG1 in A549 and H460 cells transfected with NC inhibitor or miR-129-5p inhibitor. e The protein level of JAG1 in A549 and H460 cells transfected with NC mimic or miR-129-5p mimic. *P < 0.05 regulating the KCNQ1OT1/miR-27b-3p/HSP90AA1 axis [22]. Consistent with previous study, the level of KCNQ1OT1 in NSCLC tissues and cell lines was dramatically higher than that in non-tumor tissues and cells. These results revealed that KCNQ1OT1 might exhibit vital roles in NSCLC development and progression.
It has been reported that lncRNAs could be used as competitive endogenous RNAs (ceRNAs) [23]. This study showed that KCNQ1OT1 could competitively bind to miR-129-5p. MiR-129-5p has been reported as an anti-tumor role in many cancers. In ovarian cancer, miR-129-5p was significantly downregulated in OC tissues and cells, and miR-129-5p acting as tumor suppressor inhibited cell proliferation and promoted apoptosis of OC cell by attenuating the effects of PCAT-1 [24]. In osteosarcomas, MALAT1 increased stem cell-like properties by regulating the expression of RET via sponging miR-129-5p [25]. Moreover, the effects of miR-129-5p in NSCLC have also been reported. MiR-129-5p suppressed NSCLC stemness and chemoresistance by targeting DLK1 [26]. In our study, we demonstrated that miR-129-5p was dramatically down-regulated in NSCLC tissues and cells. Meanwhile, miR-129-5p had binding sites with KCNQ1OT1. Inhibition of miR-129-5p could abolish the effects of KCNQ1OT1 knockdown on the progression of NSCLC.
JAG1 is a Notch ligand that plays a vital role in a variety of signaling pathways [27]. Recent evidence suggests that JAG1 was an oncogene in NSCLC by inducing cell metastasis [28]. In this study, we found that JAG1 was inhibited by miR-129-5p in NSCLC, and JAG1 knockdown reversed the effects of miR-129-5p inhibition. In short, KCNQ1OT1 regulated JAG1 expression by sponging miR-129-5p in NSCLC cells.

Conclusion
In conclusion, this study suggested that KCNQ1OT1 and JAG1 were upregulated, while miR-129-5p was down-regulated in NSCLC tissues and cells. Knockdown of KCNQ1OT1 significantly inhibited proliferation, migration and invasion of NSCLC cells. Meanwhile, miR-129-5p inhibition reversed the effects of KCNQ1OT1 knockdown on the progression of NSCLC. In a word, we found that KCNQ1OT1 promoted the NSCLC progression by regulating the KCNQ1OT1/miR-129-5p/JAG1 axis, which provides therapeutic targets for NSCLC.

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