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LINC00152 knockdown suppresses tumorigenesis in non-small cell lung cancer via sponging miR-16-5p

Background Non-small cell lung cancer (NSCLC) is one of the most aggressive types of cancer worldwide. It has been reported that long non-coding RNAs (lncRNAs) are involved in the pathogenesis of NSCLC. In addition, LINC00152 is known to be upregulated in NSCLC. However, the mechanism underlying the effect of LINC00152 on NSCLC tumorigenesis remains to be elucidated. Methods In the present study, cell viability, apoptosis and invasion were investigated by CCK-8, flow cytometry and Transwell assays, respectively. Reverse transcription‑quantitative polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels. In addition, the association between LINC00152, microRNA (miR)-16-5p and BCL2-like 2 (BCL2L2) was evaluated using a dual-luciferase assay. Results The results demonstrated that LINC00152-knockdown significantly attenuated the viability of NSCLC cells via promoting cell apoptosis. In addition, the migration and invasion ability of NSCLC cells was also decreased following transfection of cells with LINC00152 siRNA. Furthermore, miR-16-5p inhibitor or BCLCL2-overexpression reversed LINC00152 siRNA-induced NSCLC cell growth inhibition. Conclusions The findings of the present study demonstrated that LINC00152-silencing suppressed NSCLC tumorigenesis via regulating the miR-16-5p/BCL2L2 axis. Therefore, linc00152 has the potential as a molecular marker and may be a potential target for the treatment of non-small cell lung cancer.

however, the underlying mechanism remains unclear and requires further investigation.
MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate mRNA expression (8). It has been reported that miRNAs are involved in tumorigenesis (9). Kim et al. (10) indicated that miR-34a may act as an inhibitor of breast cancer via inhibition of Bcl-2. In addition, high expression of miR-21 was associated with a poor prognosis in patients with breast cancer (11). Therefore, miRNAs may serve as promising biomarkers in human malignancies. Additionally, another study suggested that miR-16-5p may be involved in the development of NSCLC (12), and LINC00152 could sponge miR-16-5p in glioma cells (13). However, the association between LINC00152 and miR-16-5p in NSCLC remains unclear.
The present study aimed to investigate the mechanism by which LINC00152 could regulate the growth of NSCLC cells, with the aim of shedding new light on the treatment of NSCLC. We present the following article in accordance with the MDAR reporting checklist (available at https://jtd. amegroups.com/article/view/10.21037/jtd-22-59/rc).

Cell culture
The NSCLC cell lines (A549, NCI-H838, NCI-H3122 and NCI-H1975), and the human normal lung epithelial BEAS-2B cell line, were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; both Gibco; Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA) and 100 U/mL penicillin at 37 ℃.

CCK-8 assay
The NSCLC cell viability in each group was evaluated using a CCK-8 assay kit (Beyotime Institute of Biotechnology). The optical density values at a wavelength of 450 nm were measured using a microplate reader to determine cell viability.

Cell apoptosis analysis
For the analysis of cell apoptosis, the Annexin V Apoptosis Detection kit (BD Biosciences) was used according to the manufacturer's protocols. The cells were analyzed by flow cytometry (BD Biosciences). The results were analyzed using a flow cytometer (FACS; BD Biosciences) with FlowJo software (v10.6.2; BD Biosciences).

Transwell assay
A 24-well Transwell chamber with an 8-µm pore membrane (Corning Incorporated) was utilized for the Transwell invasion assay. For the invasion assay, the upper chamber was pre-treated with 50 µL Matrigel (12.5 mg/L). NSCLC cells (4×10 4 ) were suspended in 200 µL DMEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA) without FBS. Cell culture medium supplemented with 20% FBS was added into the lower chamber to serve as a chemoattractant. Following incubation for 24 h, the invaded cells were fixed with 4% paraformaldehyde for 10 min at room temperature and stained with 0.25% crystal violet for 5 min at room temperature. Images were captured under a light microscope (magnification, ×200; Olympus Corporation, Tokyo, Japan).

Statistical analysis
All data are expressed as the mean ± standard deviation. Differences between groups were compared using one-way analysis of variance, followed by Tukey's post hoc test, with GraphPad Prism 8 (GraphPad Software Inc., California, USA). P<0.05 was considered to indicate a statistically significant difference.

Results
LINC00152-silencing significantly attenuates the proliferation of NSCLC cells. To investigate the biological effect of LINC00152 on NSCLC, RT-qPCR analysis was performed. As shown in Figure 1A, LINC00152 was notably upregulated in NSCLC cells. Since A549 and NCI-H838 were more sensitive to LINC00152 expression, these two cell lines were selected for further analysis. In addition, NSCLC cell lines were efficiently transfected with si-LINC00152 ( Figure 1B). Subsequently, a CCK-8 assay was performed. The results revealed that LINC00152silencing notably attenuated NSCLC cell proliferation ( Figure 1C,1D). Since the A549 cells were more susceptible to si-LINC00152, A549 cells were selected for subsequent experiments. Additionally, TCGA data analysis revealed that LINC00152 was associated with a poor prognosis in NSCLC ( Figure 1E), and the expression of LINC00152 in LUAD tissues was higher than that in adjacent normal tissues ( Figure 1F). These findings indicated that LINC00152 was upregulated in NSCLC. Knockdown of LINC00152 notably increases apoptosis and inhibits the invasion of NSCLC cells. Subsequently, flow cytometry was performed to measure cell apoptosis. As expected, downregulation of LINC00152 increased the apoptosis of A549 and NCI-H838 cells (Figure 2A,  Figures S1A,S1B). Similarly, NSCLC cell migration and invasion were notably decreased following transfection of A549 cells with si-LINC00152 siRNA ( Figure 2B,2C). Taken together, these findings demonstrated that LINC00152-knockdown could notably increase apoptosis and decrease the migration and invasion ability of NSCLC cells.
LINC00152 inhibits the tumorigenesis of NSCLC via miR-16-5p. To investigate the mechanism underlying   the effect of LINC00152 on NSCLC tumorigenesis, the Starbase database was used. As shown in Figure 3A, miR-16-5p could be the downstream miRNA target of LINC00152. Furthermore, the relative luciferase activity of WT-LINC00152 was significantly decreased in the presence of miR-16-5p mimics ( Figure 3B). In addition, the RT-qPCR results demonstrated that miR-16-5p was markedly downregulated in NSCLC cells ( Figure 3C). Furthermore, the miR-16-5p inhibitor was stably transfected into NSCLC cells ( Figure 3D), and miR-16-5p downregulation partially rescued the antiproliferative effect of si-LINC00152 on A549 cells ( Figure 3E). The aforementioned findings suggested that LINC00152 could attenuate the progression of NSCLC via downregulation of miR-16-5p. LINC00152 indirectly targets BCL2L2. To identify the targets of miR-16-5p, the TargetScan and miRDB databases  were used. The results indicated that miR-16-5p could directly target BCL2L2 ( Figure 4A). In addition, the dualluciferase reporter assay further confirmed these finding ( Figure 4B). Meanwhile, the expression of BCL2L2 in NSCLC cells was significantly increased in the presence of pcDNA3.1-BCL2L2 ( Figure 4C). Furthermore, BCL2L2overexpression significantly reversed the inhibitory effect of si-LINC00152 on NSCLC cell proliferation ( Figure 4D). Consistently, the apoptotic effect of si-LINC00152 on NSCLC cells was markedly inhibited following BCL2L2-   overexpression ( Figure 4E). In summary, LINC00152 could indirectly target BCL2L2.
Overexpression of BCL2L2 significantly attenuates the effect of LINC00152-silencing on the expression of apoptotic proteins in NSCLC cells. Finally, to further investigate the effect of LINC00152 on NSCLC in vitro, Western blot analysis was performed. As expected, the expression levels of BCL2L2 and Bcl-2 in NSCLC cells were significantly decreased by LINC00152-knockdown. This effect was notably reversed by BCL2L2-overexpression ( Figure 5A-5C). By contrast, LINC00152-knockdown notably enhanced the protein expression levels of Bax and caspase-3. However, the effect of si-LINC00152 on the two proteins was markedly suppressed following BCL2L2overexpression ( Figure 5A,5D,5E). The aforementioned results suggested that BCL2L2 overexpression could significantly attenuate the effect of LINC00152-silencing on the expression of apoptotic proteins in NSCLC cells.

Discussion
It has been previously reported that LINC00152 is involved in tumorigenesis (17)(18)(19). For example, a study demonstrated that LINC00152 may serve a key role in the progression of colorectal cancer (20). These findings suggested that LINC00152 could act as an oncogene in several types of cancer. In addition, it has been reported that LINC00152 is upregulated in NSCLC, and that LINC00152 could promote lung adenocarcinoma proliferation via interacting with EZH2 and inhibiting IL24 transcription (7,21). The findings of the present study were consistent with these previous results, further verifying that LINC00152 may serve an important role in the tumorigenesis of NSCLC. miRNAs have multiple biological functions (22). The present study revealed that LINC00152 was associated with miR-16-5p. Several previous studies have reported that miR-16-5p is involved in different types of malignant tumors and that it may act as a tumor suppressor (22)(23)(24). A previous study demonstrated that miR-16-5p could inhibit NSCLC development (12). In addition, Chen et al. (13) reported that LINC00152 could directly bind with miR-16-5p in glioma. The results of the present study were consistent with those of previous studies. However, another study suggested that LINC00152 may regulate the progression of esophageal squamous cell carcinoma via regulating miR-107 (19). This difference may be due to the different tumor type. miRNAs exert their biological functions mainly via regulating the expression of their target mRNAs (9). In the present study, BCL2L2 was revealed to be directly targeted by miR-16-5p. BCL2L2 has been considered to act as an inhibitory factor during the apoptosis of cells, similarly to Bcl-2 (25). Liu et al. (26) confirmed that miR-93 may alleviate the proliferation and induce the apoptosis of trophoblast cells via targeting BCL2L2 in recurrent spontaneous abortion. miR-126-5p has been considered to regulate the tumorigenesis of ovarian cancer via targeting BCL2L2 (27). According to the results of a previous study (13), LINC00152 may promote the tumorigenesis of glioma through sponging miR-16. In addition, BCLCL2 was found to be the direct target of miR-16 in the present study. Therefore, it can be suggested that LINC00152 regulates the expression of BCLCL2 through sponging BCL2L2. However, Yin et al. (28) indicated that PIK3R1 and RAF1 were directly targeted by miR-16-5p. These differences may be attributed to the different types of diseases investigated.
Additionally, the present study demonstrated that LINCC00152-silencing may promote the apoptosis of NSCLC cells. Furthermore, LINC00152-silencing significantly increased the protein expression levels of Bax and caspase-3 and downregulated those of Bcl-2. Bax, caspase-3 and Bcl-2 are considered to be key mediators of cell apoptosis in several diseases (2,29,30). Emerging evidence has supported that Bax and caspase-3 are proapoptotic proteins (31), while Bcl-2 upregulation inhibits cell apoptosis (32). Furthermore, the present study demonstrated that LINC00152-knockdown induced NSCLC cell apoptosis via upregulating Bax and caspase-3, and downregulating Bcl-2.
However, the present study is limited by the fact that the effect of LINC00152 siRNA on NSCLC cell proliferation remains to be further verified. Therefore, further investigations are required in the future.
In conclusion, the results of the present study demonstrated that LINC00152-knockdown inhibited the development of NSCLC via regulating the miR-16-5p/ BCL2L2 axis, thereby providing novel insight into the treatment of NSCLC. Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the noncommercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.