IncRNA AC004943.2 regulates miR‐135a‐5p and PTK2/P13K axis to promote laryngeal squamous cell carcinoma progression

Abstract Long noncoding RNAs (lncRNAs) are involved in regulatory processes in laryngeal squamous cell carcinoma (LSCC) at posttranscriptional epigenetic modification level. Yet, the function and underlying mechanism behind lncRNA AC004943.2 in LSCC is still obscure. Therefore, the potential role of AC004943.2 in LSCC progression was investigated. The expression of gene or protein was tested by real‐time quantitative polymerase chain reaction and western blot. MTT, colony formation, wound healing, and transwell experiments were applied to detect LSCC cell viability, proliferation, migration and invasion, respectively. The interaction among AC004943.2, miR‐135a‐5p, and protein tyrosine kinase 2 (PTK2) were analyzed by bioinformatics prediction and luciferase assay. AC004943.2 was highly expressed in LSCC cells compared with normal human bronchial epithelial cells, while miR‐135a‐5p was lowly expressed. AC004943.2 knockdown or miR‐135a‐5p overexpression inhibited LSCC cell viability, proliferation, migration and invasion. Mechanistically, AC004943.2 increased PTK2 expression in LSCC cells by sponging miR‐135a‐5p. Furthermore, miR‐135a‐5p knockdown inverted the inhibitory effect of AC004943.2 silencing on LSCC cell malignant behaviors. MiR‐135a‐5p upregulation attenuated the PTK2/PI3K pathway to inhibit progression of LSCC. AC004943.2 facilitated the cancerous phenotypes of LSCC cells by activating the PTK2/PI3K pathway through targeting miR‐135a‐5p, which furnished a therapeutic candidate for LSCC treatment.

LncRNAs can play a crucial role in the majority of key cellular processes involved in the maintenance of cellular homeostasis by regulating various molecular mechanisms. 56][7] Kong et al. pointed out that lncRNA AC016773.1 and C00299 participated in LSCC progression. 8Cao et al. identified that lncRNA IGKJ2-MALLP2 inhibited LSCC progression by sponging miR-1911-3p. 6terestingly, it has been reported that lncRNA AC004943.2was upregulated in LSCC. 9Nevertheless, the exact role and the mechanism of lncRNA AC004943.2 in LSCC is still unknown.
MicroRNAs (miRNAs) were a type of endogenous noncoding small RNAs with around 22 nucleotides in length, which are ubiquitously expressed and participate in modulating gene expression.
Recent studies showed that miRNAs dysregulation play critical roles in carcinogenesis.As widely described, miRNAs are involved in LSCC progression. 10For instance, miR-29a upregulation inhibited LSCC progression by targeting STAT3. 11In addition, miR-125b could suppress LSCC cell growth and migration by targeting STAT3. 12Of note, it has been widely described that miR-135a-5p can function as an orchestrator of various cellular processes, including the differentiation, regeneration, and tumorigenesis. 13,14MiR-135a-5p downregulation was observed in several cancers, and its low expression was linked to poor overall prognosis in gastric cancer.6][17] Herein, by using the bioinformatic prediction, we noticed that AC004943.2had potential binding sites to miR-135a-5p, and miR-135a-5p had potential binding sites to protein tyrosine kinase 2 (PTK2).The function and the underlying mechanism of miR-135a have not been explored yet in LSCC.
In this study, we performed a detailed investigation of AC004943.2 in regulating the miR-135a-5p/PTK2 axis to promote LSCC progression.Our results extend the understanding of AC004943.2 in the progression of LSCC, which provides effective targets for LSCC diagnoses and therapy.

| Real-time quantitative polymerase chain reaction
The total RNA was extracted using TRIzol reagent (Invitrogen).For mRNA, cDNA was generated by the High-Capacity RNA-to-cDNA Kit (Applied Biosystems, USA).The SYBR qPCR master mix (TOYOBO, Japan) was used to measure the fluorescence intensity of PCR reactions.To detect the expression of miRNA, quantitative PCR were measured by TaqMan Universal Master Mix II (Applied Biosystems).

| Wound healing assay
Cells were inoculated and grown to reach 90% confluence, and then scratched in the single layer with the tip of pipette.Cells were subsequently put in an incubator for additional 4 h, and the images were captured.Digital photographs were obtained from a Carl Zeiss light microscope (Axio Observer A1, Germany), and the scratch area was measured by Image-J software.

| Transwell assay
Cells were transfected with indicated plasmids and planted in the upper compartment containing Matrigel-coated membrane (Cat.No.

| Colony formation assay
Cells were inoculated for colony forming assay.After 14 days of cell growth, the cell clones were formed and examined.The clones were then immobilized with menthol and dyed with 0.1% crystal violet at ambient temperature.Then, all colonies were preserved with ethanol, and they were dyed with crystal violet for 2 h.Finally, they were captured under a Carl Zeiss light microscope (Axio Observer A1).

| Western blot
Protein samples were extracted by using ice-cold lysis buffer (Beyotime, China) containing protease inhibitors (Cell Signaling Technology, Boston, USA).The protein concentrations were determined using a BCA assay.Protein samples (20 μg) were fractionated on 10% SDS-PAGE and wet-transferred onto PVDF membranes.The membranes were incubated with specific antibodies against anti-PTK2 (ab271836), PI3K (ab302959), p-PI3K (ab278545), and GAPDH (ab9485) overnight.All these antibodies were purchased from Abcam (Cambridge, UK).The membranes were then incubated with HRP-labeled corresponding secondary antibodies at room temperature for 1 h.Finally, protein expression was performed using a chemiluminescent instrument (Bio-Rad, USA).

| Statistical analysis
GraphPad Prism 7 (GraphPad, USA) was used to examine the statistical data.Data were presented as mean � standard deviation (SD) from at least 3 independent experiments.Student's t-test was used to examine the differences between the two groups, while one-way ANOVA followed by Tukey's post hoc test was applied to analyze the differences across multiple groups.The p values less than 0.05 were regarded as significant.

| AC004943.2 promoted LSCC progression
To examine the potential function of AC004943.

| MiR-135a-5p targeted PTK2
To explore the mechanism of miR-135a-5p in LSCC, we used the Starbase database to search for miR-135a-5p potential targets.As revealed in Figure 5A, miR-135a-5p had potential binding sites to PTK2.Dual luciferase reporter assay results indicated that miR-135a-5p mimics/inhibitor transfection remarkedly inhibited/promoted the luciferase activity of the PTK2 WT group, whereas neither miR-135a-5p mimics and not its inhibitor had any effect on the luciferase activity of the PTK2 MUT group (Figure 5B), suggesting that PTK2 directly bounded with miR-135a-5p.Furthermore, we detected PTK2 in LSCC cells.As depicted in Figure 5C,D, PTK2 was highly expressed in TU686 and AMC-HN-8 cells compared to 16HBE cells.It was also turned out that miR-135a-5p overexpression significantly reduced PTK2 mRNA and protein levels in LSCC cells (Figure 5E,F).To sum up, these data implied that miR-135a-5p downregulated PTK2 expression in LSCC cells.

| DISCUSSION
LSCC is the most prevalent head and neck squamous cell carcinomas (HNSCC). 18Despite significant advances in LSCC therapy across the world, the 5-year prognosis for LSCC patients remains unsatisfactory. 19Hence, it is critical to understand carcinogenesis mechanisms in order to create novel targeted therapeutics for LSCC.In this study, we found that AC004943.2upregulation promoted LSCC progression.Mechanistically, AC004943.2facilitated LSCC development by activating the PTK2/PI3K pathway through sponging miR-135a-5p.
Numerous studies have reported that lncRNAs play essential roles in LSCC progression. 20,21It has been widely documented that lncRNAs acted as miRNAs sponges and competed with mRNAs, 22 and the lncRNA-miRNA-mRNA network was essential for LSCC progression.For instance, the lncRNA FGD5-AS1/miR-497-5p axis modulated SEPT2 to promote LSCC progression and enhanced cisplatin resistance. 20Li et al. reported that lncRNA SNHG20 accelerated the progression of LSCC by sponging miR-140. 23A previous study demonstrated that lncRNA AC004943.2was overexpressed in LSCC. 9Nonetheless, the exact function and the mechanism of lncRNA AC004943.2 in LSCC remain unclear.Our study revealed that AC004943.2was highly expressed in LSCC cells, which was consistent with previous research. 9In addition, AC004943.2silencing markedly suppressed LSCC cell viability, proliferation, and migration and invasion.Our results confirmed that AC004943.2upregulation promoted the progression of LSCC.
5][26] Erkul et al. found that miR-21 acted as a crucial factor in diagnosis and serve as a possible biomarker in LSCC. 27The function of miR-135a-5p in cancers has been widely studied. 28,29However, further research is needed to determine the exact function and the mechanism of miR-135a in LSCC.Our findings discovered that miR-135a-5p was lowly expressed in LSCC, and its overexpression could suppress LSCC cell malignant behaviors.microenvironments. 30PTK2 is highly expressed in a variety of cancer types. 30Notably, previous investigations have demonstrated that PTK2 was involved in the progression of LSCC.For instance, AURKA revived dormant LSCC via activating the PTK2/PI3K/Akt pathway. 31CK1 facilitated LSCC tumorigenesis and progression by mediating the PTK2 signaling pathway. 32We also found that PTK2 was overexpressed in LSCC cells.MiRNAs are negative posttranscriptional regulators of target gene expression. 33We confirmed that PTK2 was the target gene of miR-135a-5p.Furthermore, we found that miR-135a-5p overexpression markedly reduced PTK2 expression in LSCC cells.Taken together, we firstly discovered that miR-135a-5p suppressed LSCC progression via attenuating the PTK2/PI3K pathway.
In summary, our findings demonstrated that AC004943.2increased PTK2 via sponging miR-135a-5p and thus activated the PTK2/PI3K pathway to promote the tumorigenesis of LSCC.Our research is expected to provide valuable insights into elucidating the molecular mechanism of AC004943.2 in LSCC.

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JOURNAL OF CELL COMMUNICATION AND SIGNALING#354230, Thermo Fisher Scientific) and incubated in DMEM without serum.The lower compartment was incorporated with FBScontaining medium.Invading cells in the down chamber were fixed with methanol after cultivation.The invaded cells were dyed with crystal violet and captured using a Carl Zeiss light microscope (Axio Observer A1).