Long noncoding RNA LINC01638 contributes to laryngeal squamous cell cancer progression by modulating miR-523-5p/BATF3 axis

Upregulated expression levels of LINC01638 in LSCC tissues

The expression of LINC01638 in LSCC was analyzed by qRT-PCR. Results indicated that LINC01638 level was raised in LSCC tissues compared to normal controls (Figure 1A). Notably, the expression of LINC01638 was upregulated in most of the LSCC tissues compared to their corresponding normal tissues (Figure 1B). Furthermore, LINC01638 expression was positively correlated with clinical stage and lymph node metastasis (Figure 1C, 1D). Then, survival rate was analyzed according to LINC01638 expression in LSCC tissues through the Kaplan-Meier method. Results showed that LINC01638 upregulation indicated a low survival rate (Figure 1E), suggesting that LINC01638 high expression predicted poor outcome.

Figure 1. Upregulated expression levels of LINC01638 in LSCC tissues. (A) LINC01638 expression was upregulated in LSCC tissues compared to adjacent normal tissues. (B) LINC01638 was upregulated in most of the LSCC tissues compared to paired normal tissues. (C) LINC01638 was upregulated in advanced stages of LSCC tissues. (D) LINC01638 level was raised in LSCC tissues with lymph node metastasis. (E) LINC01638 overexpression was associated with a low survival rate. *P<0.05.

LINC01638 knockdown suppressed proliferation, migration and invasion

To analyze the function of LINC01638, two LSCC cell lines (TU177 and AMC-HN-8) were obtained and LINC01638 was knocked down by shRNA (Figure 2A). Using CCK8 assay, we found that LINC01638 knockdown significantly suppressed the proliferation of LSCC cells (Figure 2B). Colony formation assay further demonstrated that the colony number was decreased by LINC01638 silencing (Figure 2C). Additionally, transwell results showed that LINC01638 knockdown markedly reduced the cell migration and invasion (Figure 2D, 2E).

Figure 2. LINC01638 knockdown suppressed proliferation, migration and invasion. (A) LINC01638 shRNA decreased the expression of LINC01638 in TU177 and AMC-HN-8 cells. (B, C) Cell proliferation was evaluated by CCK8 and colony formation assays. (D, E) Transwell migration and invasion assays were performed to test migration and invasion. *P<0.05.

LINC01638 was the sponge for miR-523-5p

To investigate the molecular mechanism of LINC01638, we firstly analyzed its subcellular location by qRT-PCR. Results showed that LINC01638 was located in the cytoplasm (Figure 3A), indicating LINC01638 may be a miRNA sponge. Using bioinformatics analysis (miRDB online tool), five most potential candidates were identified. However, luciferase reporter assay showed that only miR-523-5p mimics inhibited the luciferase activity of LINC01638 (Figure 3B). Thus, we selected miR-523-5p for further validation. Mutant-LINC01638 (LINC01638-MUT) luciferase reporter vector was constructed (Figure 3C). Luciferase reporter assay validated the above conclusion again (Figure 3D). RIP assay further suggested that LINC01638 interacted with miR-523-5p (Figure 3E). Besides, we found that miR-523-5p expression was increased after LINC01638 knockdown (Figure 3F). Afterwards, it was observed that there was a negative correlation between LINC01638 and miR-523-5p expressions in LSCC tissues (Figure 3G).

Figure 3. LINC01638 was the sponge for miR-523-5p. (A) Subcellular location of LINC01638 was analyzed by qRT-PCR. (B) Luciferase reporter assay identified that LINC01638 may be the sponge for miR-523-5p. (C) Predicted binding site and mutated binding site in LINC01638. (D) Luciferase reporter assay was performed. (E) RIP assay was conducted to examine the interaction between LINC01638 and miR-523-5p. (F) Relative expression of miR-523-5p was measured after LINC01638 knockdown. (G) Negative correlation was observed between LINC01638 and miR-523-5p expression in LSCC tissues. *P<0.05.

MiR-523-5p targeted BATF3 directly

Bioinformatics analysis was further conducted to predict the target of miR-523-5p (Figure 4A). BATF3 was identified as the most potential target. Similarly, luciferase reporter vectors were constructed (Figure 4B). Luciferase reporter assay showed that miR-523-5p mimics suppressed the activity of WT-BATF3 reporter (Figure 4C). RIP assay also suggested that miR-523-5p interacted with BATF3 (Figure 4D). Furthermore, qRT-PCR result showed that miR-523-5p mimics suppressed the expression of BATF3 (Figure 4E). To further explore the role of miR-523-5p, CCK8 and transwell assays were performed. Results showed that miR-523-5p mimics significantly repressed the proliferation, migration and invasion of LSCC cells (Figure 4F–4H). Notably, BATF3 overexpression eliminated the effects of miR-523-5p mimics (Figure 4F–4H).

Figure 4. miR-523-5p targeted BATF3 directly. (A, B) Prediction of miR-523-5p targets by bioinformatics analysis. (C) Luciferase reporter assay was performed. (D) RIP assay was conducted to examine the interaction between miR-523-5p and BATF3. (E) miR-523-5p mimics suppressed the expression of BATF3. (F) CCK8 assay was utilized to test proliferation. (G, H) Transwell assay was performed to determine migration and invasion. *P<0.05.

LINC01638 promoted LSCC progression through miR-523-5p/BATF3 pathway

We also noticed that LINC01638 knockdown suppressed BATF3 expression and miR-523-5p inhibitors reversed it (Figure 5A), suggesting that LINC01638 promoted BATF3 expression through inhibiting miR-523-5p.

Figure 5. LINC01638 promoted LSCC progression through miR-523-5p/BATF3 pathway. (A) qRT-PCR analysis of BATF3 expression. (B) CCK8 assay was utilized to test proliferation. (C, D) Transwell assay was performed to determine migration and invasion. (E) Tumor volumes were measured every week. (F) After 5 weeks of injection, tumor weights were determined. *P<0.05.

Interestingly, we also found that overexpression of BATF3 reversed the inhibitory effects of LINC01638 knockdown on proliferation, migration and invasion (Figure 5B–5D). To further demonstrate the function of LINC01638/miR-523-5p/BATF3 axis, animal xenograft assay was performed. Result showed that LINC01638 knockdown suppressed the tumor volumes and weights in vivo (Figure 5E, 5F). Similarly, BATF3 overexpression also reversed the effects of LINC01638 knockdown in vivo (Figure 5E, 5F), indicating that LINC01638 promoted LSCC progression through miR-523-5p/BATF3 pathway.

Patients’ tissues

LSCC tissues and adjacent normal tissues were obtained from Shengjing Hospital of China Medical University. Informed consents were collected from every patient. Clinical data was listed in Table 1. This study was approved by the ethics committee of Shengjing Hospital of China Medical University. Tissues were stored in liquid nitrogen until use.

Cell culture and transfection

LSCC cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured using RPMI-1640 medium (Gibco), supplemented with 10% fetal bovine serum (Gibco). shRNA targeting LINC01638, miR-523-5p mimics, miR-523-5p inhibitors and negative controls were obtained from GenePharma (Shanghai, China). Transfection was performed using Lipofectamine 2000 (Invitrogen/Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s instructions.

qRT-PCR

RNA was extracted using Trizol reagent following the manufacturer’s instructions and transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis kit (Roche, Basel, Switzerland) following the manufacturer’s protocol. Then qPCR was carried out using SYBR-Green Real-Time Master Mix (TaKaRa) on 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative expression was normalized to GAPDH or U6 and calculated based on 2^−ΔΔCt method.

Cell proliferation analysis

For CCK8 assay, cells were seeded into 96-well plates and cultured for indicated time points. Then 10 μL of CCK8 solution (Dojindo Laboratories, Kumamoto, Japan) was added and incubated for 2 h, followed by measurement of absorbance at 450 nm using a Microplate Reader (Bio-Rad, Hercules, CA, USA). For colony formation assay, cells were seeded into 6-well plates and cultured for 14 days. Then colonies were fixed with 70% ethanol and stained with 0.1% crystal violet. Numbers of colonies were finally counted.

Transwell assay

For migration assay, cells were seeded into the non-Matrigel-coated upper chambers (8 μm pore; Corning Costar, Corning, NY, USA) with serum-free medium. The lower chamber was filled with FBS-containing medium. Cells were cultured for 24 h. Then migrated cells in the lower chamber was fixed with 70% ethanol and stained with 0.1% crystal violet. Cell numbers were finally counted using a light microscope. For invasion assay, cells were seeded into Matrigel-coated upper chambers and other steps were the same as migration assay.

RNA immunoprecipitation (RIP) assay

The interactions among LINC01638, miR-523-5p and BATF3 were detected using RIP assay through the EZ-Magna RIP kit (Millipore, Billerica, MA, USA) following the manufacturer’s instructions.

Luciferase reporter assay

The sequences of LINC01638 and BATF3 were inserted into pMIRGLO reporter vector (Promega). For luciferase reporter assay, the luciferase reporter and miR-523-5p mimics were transfected into TU177 cells and cultured for 48 h. Then the relative luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol.

Animal assay

5-week-old female nude (BALB/c-nude) mice from Charles River (Beijing, China) were randomly divided into three groups (5 mice per group). Then TU177 cells (5×10⁶) were subcutaneously injected into the right flanks of the mice. Tumor volumes were measured every week. After 5 weeks, tumor weights were examined. The animal experiment was approved by the ethics committee of Shengjing Hospital of China Medical University.

Statistical analysis

The results were analyzed by GraphPad Prism version 6 and expressed as the means ± standard deviation. The differences between 2 groups were analyzed with the Student’s t-test. Differences between >2 groups were determined by one-way ANOVA followed by Tukey’s post hoc test. Pearson’s correlation analysis was utilized to determine expression correlation in tumor tissues. P<0.05 was considered to indicate a statistically significant difference.