Long Noncoding RNA LINC01554 Inhibits the Progression of NSCLC Progression by Functioning as a ceRNA for miR-1267 and Regulating ING3/Akt/mTOR Pathway

Objectives This study focused on the biological functions and mechanisms of action of LINC01554 in nonsmall cell lung cancer (NSCLC). Methods The expression and prognostic values of LINC01554 in NSCLC were evaluated using The Cancer Genome Atlas datasets. MTT, colony formation, wound healing, transwell, and in vivo assays were performed to investigate the role of LINC01554 in NSCLC. The related protein expression levels were measured via western blotting. Bioinformatic analysis was conducted to predict targeted genes. The relationship between LINC01554, microRNA- (miR-) 1267, miR-1267, and inhibitor of growth family member 3 (ING3) was analysed via a dual-luciferase reporter assay. Results LINC01554 expression was downregulated in NSCLC and associated with NSCLC prognosis. LINC01554 overexpression suppressed NSCLC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Bioinformatic and dual-luciferase reporter assays demonstrated that LINC01554 expression directly targeted miR-1267 expression, which in turn directly acted on ING3. An miR-1267 mimic significantly reduced ING3 expression, whereas an miR-1267 inhibitor observably elevated its expression. LINC01554 overexpression increased ING3 expression, whereas this effect was counteracted by the miR-1267 mimic. LINC01554 overexpression also significantly suppressed the expression of phosphorylated protein kinase B (Akt) and phosphorylated mammalian target of rapamycin (mTOR) expression; this effect was abrogated by the miR-1267 mimic. Mechanistically, LINC01554 overexpression repressed the growth, migration, invasion, and epithelial-mesenchymal transition (EMT) of NSCLC cells through the regulation of the miR-1267/ING3 axis via regulation of the Akt/mTOR signalling pathway. Conclusions We provide the first evidence of the involvement of the LINC01554/miR-1267 axis in NSCLC proliferation and metastasis through the ING3Akt/mTOR pathway. Thus, LINC01554 may serve as a novel therapeutic target for NSCLC.


Introduction
Lung cancer is the most common malignancy, accounting for approximately 11.6% of all malignant tumours worldwide [1,2]. Nonsmall cell lung cancer (NSCLC) accounts for approximately 80-85% of all lung cancers. The main subtypes of NSCLC are lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) [3]. Despite considerable advances in diagnostic techniques and treatment strategies, the prognosis of NSCLC patients remains poor, with the 5-year overall survival rate being as low as 15% [4,5]. Thus, there is an urgent need to understand the detailed molecular mechanism associated with NSCLC proliferation and metastasis and explore novel therapeutic targets for metastatic NSCLC treatment.
Recent evidence has highlighted the key role of ncRNAs (noncoding RNA) in tumorigenesis [6]. Two typical ncRNA subtypes are miRNAs (microRNAs) and long chain noncoding RNAs (lncRNAs) [7,8]. It is believed that the interaction between lncRNAs and miRNAs may play an important role in tumorigenesis. Based on lncRNA-miRNA-mRNA network, important regulatory pathways and therapeutic targets can be revealed. For instance, the lncRNA JPX is associated with poor prognosis and progression of NSCLC through direct interaction with microRNA-(miRNA, miR-) 145-5p and CCND2 [9]. The lncRNA MIR17HG inhibits proliferation, migration, and invasion of NSCLC cells by adsorbing miR-142-3p via sponging, thereby upregulating its expression and simultaneously downregulating Bach-1 expression [10]. In addition, the expression of the lncRNA SBF2-AS1 is markedly elevated in NSCLC tissues, and it regulates NSCLC cell migration and invasion by modulating the miR-362-3p/growth factor receptor-bound protein 2 axis [11]. The expression of long intergenic nonprotein coding RNA 1554 (LINC01554), located on chromosome 5q15, was found to be downregulated in hepatocellular carcinoma (HCC) where it was involved in cancer progression [2,12,13]. However, the expression and biological functions of LINC01554 in NSCLC remain unclear.
The bioinformatics analysis shows that predicted that miR-1267 and inhibitor of growth family member 3 (ING3) were the downstream target of LINC01554. Further gene set enrichment analysis (GSEA) analysis found that LINC01554 inhibited the Akt/mTOR pathway based on the TCGA databases. Thus, in the current study, we explored whether LINC01554 could regulate NSCLC cell proliferation, migration, invasion, and EMT via functioning as a (competitive endogenous RNA) ceRNA for miR-1267 and by regulating the ING3/Akt/mTOR pathway.
Vectors such as pcDNA3.1-LINC01554, the empty pcDNA3.1 vector, si-negative control (NC), si-LINC01554-1, and si-LINC01554-2 were synthesised by GenePharma (Shanghai, China) along with the miR-1267 mimic/inhibitor and NC mimic/inhibitor. Transfections were conducted using Lipofectamine 3000 (Invitrogen, CA, USA) following standard protocols. The culture medium was replaced with complete RPMI-1640 medium 6 h after transfection. Cells were collected after 48 h of transfection, and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was performed to detect LINC01554 and miR-1267 expression for the analysis of transfection efficiency.
2.5. Colony Formation Assay. As previously described, cells were seeded into six-well plates (1 × 10 2 cells/well) and cultured [17]. Colonies with more than 50 cells were fixed using 4% formaldehyde for 15 min at 37°C. The colonies were subsequently stained using crystal violet (Beyotime, Shanghai, China). After 15 min, colonies were counted and photographed under a microscope (Bethesda, MD, USA).
2.6. Wound Healing Assay. Cells were plated into six-well plates containing serum-free medium until they reached at least 80% confluency. The cell layer was then scratched using a sterile 200 μL pipette tip. Cells were washed with phosphate-buffered saline to remove any debris, and the cell culture medium was replaced. After 48 h of culture, representative images were obtained using an inverted light microscope (Olympus Corporation) [18].

Transwell Invasion Assay.
Transwell chambers (Millipore, Billerica, MA, USA) containing Matrigel were used to determine the invasive ability of cells [16]. A total of 1 × 10 5 cells in RPMI-1640 serum-free growth medium (Gibco, USA) were seeded in the upper wells, whereas the lower wells were filled with the same medium containing 10% serum. After 24 h incubation at 37°C, the cells invading the lower side of the chamber were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 10 min. Images were captured with a microscope (Olympus Corporation).

LINC01554 Overexpression Suppressed NSCLC Cell
Proliferation In Vitro. We tested LINC01554 expression level in normal 16HBE cells and NSCLC cells and found it to be significantly downregulated in H23 and H1299 cells as compared with that in 16HBE cells (Figure 2(a), P < 0:05). As shown in Figure 2(b), LINC01554 expression was notably elevated and suppressed following transfection with LINC01554-overexpressing vectors and small-interfering RNAs (siRNAs; si-LINC01554-1 and si-LINC01554-2), respectively (P < 0:05). The results of MTT and colony formation assays revealed that cell viability and proliferation were suppressed in LINC01554 overexpression group but elevated in the si-LINC01554 transfection group (Figures 2(c) and 2(d), P < 0:05).

LINC01554 Overexpression Suppressed NSCLC Cell
Migration, Invasion, and EMT In Vitro. As shown in Figures 3(a) and 3(b), the cell invasion capacity was significantly inhibited by LINC01554 overexpression (P < 0:05). Western blot analysis results of EMT-related protein expression in H23 and H1299 cells showed that the expression of E-cadherin evidently increased but that of snail and vimentin was markedly downregulated upon LINC01554 overexpression (Figure 3(c), P < 0:05). Nevertheless, LINC01554 knockdown exerted the opposite effect on NCI-H520 cells.

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BioMed Research International we initially predicted the miRNA targets of LINC01554 using the StarBase tool. In addition, we found the expressions of miRNAs that were significantly upregulated from TCGA-LUSC and TCGA-LUAD databases. The significantly upregulated miRNAs overlapped with the predicted miRNA targets of LINC01554, and one potential target miRNA (miR-1267; Figure 4(a)) was identified. Moreover, the dual-luciferase reporter gene assay results showed that the luciferase activity of the wild-type pGL3-promoter LINC01554 3-UTR was significantly decreased in the miR-1267 mimic group, whereas that of the mutant-type pGL3-promoter LINC01554 3-UTR remained unchanged (Figure 4(b), P < 0:05). We used TargetScan and StarBase tools to identify the potential downstream target genes of miR-1267. We also collected the genes whose expressions were significantly downregulated from TCGA-LUSC and TCGA-LUAD databases. The significantly downregulated genes overlapped with the predicted downstream target genes of miR-1267, and one potential downstream target gene (ING3; Figure 4(c)) was identified. The luciferase activity markedly decreased when the wild-type pGL3promoter ING3 3-UTR and miR-1267 mimic were cotransfected into cells, as compared with the luciferase activity in negative control cells (Figure 4(d), P < 0:05). We overexpressed and inhibited the expression of miR-1267 using the miR-1267 mimic and inhibitor (Figure 4(e)), respectively, and detected the expression of ING3 via western blot analysis. As shown in Figure 4(f), the miR-1267 mimic significantly reduced ING3 expression, and the miR-1267 inhibitor observably elevated ING3 expression (P < 0:05).
3.6. miR-1267 Overexpression Reversed the LINC01554 Upregulation-Induced Tumour-Suppressive Effects on NSCLC Cells. To determine whether LINC01554 contributes to NSCLC progression by targeting miR-1267, we further investigated the function of miR-1267 in NSCLC cells. The MTT assay result indicated that LINC01554 overexpression led to a considerable decrease in the proliferation ability of H23 and H1299 cells, and this effect was abrogated following the cotransfection with pcDNA3.1 LINC01554 and miR-1267 mimic ( Figure 5(a), P < 0:05). Wound healing and transwell invasion assays revealed that the migration and invasion of NSCLC cells were significantly reduced by LINC01554 overexpression. However, the migration and invasion capabilities of cells were dramatically enhanced following cotransfection with pcDNA3.1 LINC01554 and miR-1267 mimic (Figures 5(b) and 5(c), P < 0:05). Likewise, as presented in Figure 5  To understand the underlying mechanism of LINC01554 in NSCLC progression, gene set enrichment analysis (GSEA) was performed on TCGA-LUSD datasets. The results showed that LINC01554 expression was associated with PI3K-Akt-mTOR signalling (Figures 6(a) and 6(b)), thereby indicating that the PI3K-Akt-mTOR pathway might be involved in the growth-inhibiting effect of LINC01554. The overexpression of LINC01554 significantly elevated ING3 expression but suppressed p-Akt and p-mTOR expressions. However, the total level of Akt and mTOR protein remained unchanged ( Figure 6(c), P < 0:05). Moreover, H23 and H1299 cells were cotransfected with LINC01554 and miR-1267 mimic; as expected, the expression of ING3, p-Akt, and p-mTOR was recovered. The miR-1267 mimic blocked the effect of the upregulated LINC01554 (Figure 6(d), P < 0:05).

Discussion
NSCLC has seriously endangered human health, but due to the complexity of the pathogenesis of NSCLC, there is no effective treatment in clinic [12]. Previous studies have confirmed that dysregulation in lncRNA expression plays a key role in many biological processes in different cancers, including NSCLC. In our study, results showed that NSCLC cell proliferation could be inhibited by overexpressed LINC01554 both in vivo and in vitro. The above results were in accordance with the previous study [2,12,19,20], which indicated that LINC01554 played a tumour suppressive role in NSCLC development.
Recently, the research on lncRNA-miRNA-mRNA ceRNA network provides a basis for a better understanding of the role of lncRNA-miRNA interaction in mRNA regulation and NSCLC development [21,22]. miRNAs can interact with target mRNA to degrade mRNA or inhibit mRNA translation [7]. Compared to miRNAs, lncRNAs are longer ncRNAs that play a role through more diverse mechanisms [23]. In addition to directly targeting mRNAs, it has also been proved to have the function of ceRNAs, which interact with miRNAs and indirectly regulate mRNAs [24]. Based on bioinformatic analysis and dual-luciferase assay results, we confirmed that LINC01554 directly targets miR-1267 and that miR-1267 in turn regulates ING3 expression. miR-1267 was reported to be upregulated in breast cancer, and its expression was significantly related to cancer grade and stage [25]. A recent study stated that the lncRNA SNHG11 is involved in the progression of gastric cancer through regulation of the expression of catenin-β1 and autophagyrelated 12 via miR-1276 [26]. However, the biological function of miR-1267 in NSCLC progression has not been clarified. Notably, in this study, we found that miR-1267 overexpression reversed the LINC01554 upregulationinduced tumour-suppressive effects on NSCLC cells and in line with the previous study.
As a crucial inhibitor of the growth gene family, ING3 is usually abnormally expressed in both the cytoplasm and nucleus of tumour cells and has been shown to be involved in the progression of many tumours. Studies have shown that ING3 regulates cell proliferation, apoptosis, and cell cycle arrest in gastric cancer by inactivating the PI3K/Akt pathway and promoting cell death [27]. Downregulation of ING3 expression and its transfer to the cytoplasm can regulate cell cycle arrest, senescence, and apoptosis in head and neck squamous cell carcinoma [28]. ING3 level is markedly decreased in breast cancer where its expression is closely correlated with cancer prognosis [29]. These results are consistent with our results, which indicate that LINC01554 might directly regulate ING3 through miR-1267 and modulate the proliferation, migration, invasion, and EMT of NSCLC cells.
Abnormal expression of lncRNAs has been also reported to be involved in the progression of NSCLC through inactivation or activation of the PI3K/Akt signalling pathway. For instance, the lncRNA HULC facilitates NSCLC cell growth and suppresses cell apoptosis by stimulating the PI3K/Akt signalling pathway [30]. HOXB-AS3 stimulates NSCLC cell proliferation, migration, and invasion by dysregulating the PI3K/Akt signalling pathway [31]. The lncRNA TBX5-AS1 modulates tumour progression in NSCLC by suppressing the PI3K/Akt signalling pathway [32]. To identify the downstream signal pathway of LINC01554 in anti-NSCLC, we performed a GSEA analysis. The results showed that LINC01554 could inhibit Akt/mTOR pathway. Collectively, overregulation of LINC01554 inhibits cell viability, migration, and proliferation by inhibiting the Akt/mTOR pathway.
In summary, we report for the first time that LINC01554 directly regulates ING3 expression through miR-1267 and thus affects the proliferation, migration, invasion, and EMT of NSCLC cells by regulating the Akt/mTOR pathway. LINC01554 may function as a novel prospective therapeutic target for the treatment of NSCLC.

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
The animal experiments in this study were approved by the Animal Care and Use Committee of Chinese PLA General Hospital (S2021-001-12).