ELOVL6 promotes the progression of head and neck squamous cell carcinoma via activating WNT/β‐catenin pathway

This study was to explore the role of ELOVL6 in the development of head and neck squamous cell carcinoma (HNSCC). Considering its previously identified oncogenic role in hepatocellular carcinoma. ELOVL6 gene expression, clinicopathological analysis, enrichment analysis, and immune infiltration analysis were based on the data from Gene Expression Omnibus and The Cancer Genome Atlas, with additional bioinformatics analyses performed. Human HNSCC tissue microarray and cell lines were used. The expression of ELOVL6 in HNSCC was detected by quantitative polymerase chain reaction, immunohistochemistry assay, and western blot analysis. The proliferation ability of HNSCC cells, invasion, and apoptosis were evaluated using cell counting kit‐8 method, Transwell assay, and flow cytometry, respectively. Based on the data derived from the cancer databases and our HNSCC cell and tissue studies, we found that ELOVL6 was overexpressed in HNSCC. Moreover, ELOVL6 expression level had a positive correlation with clinicopathology of HNSCC. Gene set enrichment analysis showed that ELOVL6 affected the occurrence of HNSCC through WNT signaling pathway. Functional experiments demonstrated that ELOVL6 knockdown inhibited the proliferation and invasion of HNSCC cells while promoting apoptosis. Additionally, compound 3f, an agonist of WNT/β‐catenin signaling pathway, enhances the effect of ELOVL6 on the progression of HNSCC cells. ELOVL6 is upregulated in HNSCC and promotes the development of HNSCC cells by inducing WNT/β‐catenin signaling pathway. ELOVL6 stands a potential target for the treatment of HNSCC and a prognosis indicator of human HNSCC.

Despite its known associations, the role of ELOVL6 in cancer progression, particularly in HNSCC, remains largely unexplored.In this study, we utilized microarray data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases examine the expression of ELOVL6 in HNSCC samples.Our analysis revealed high expression of ELOVL6 in HNSCC, with patients exhibiting elevated ELOVL6 expression experiencing poorer survival outcomes.KEGG analysis highlighted a close association between ELOVL6 and the WNT signal pathway.Given the pivotal role of WNT signaling in maintaining cancer stem cell (CSC)   properties in a variety of cancers, [15][16][17] including HNSCC, 18 we hypothesized ELOVL6 contributes to cancer progression through its regulatory influence on the WNT/β-catenin signaling pathway.
Our study represents the first comprehensive analysis of the correlation between ELOVL6 and HNSCC, shedding light on the molecular mechanism underlying the occurrence and development of this malignancy and enhancing our understanding of HNSCC pathogenesis.

| Data source and preprocessing
Data sets were acquired by establishing the screening criteria for the species type as "Homo sapiens," from GEO database of National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/geo/).Keywords such as "Head and neck squamous cell carcinoma" (HNSCC) were employed in the search.Then select the study was narrowed down to "Expression profiling by array" to obtain the series matrix files.Additionally, mRNA expression profiles and corresponding clinical information of 546 HNSCC patients were downloaded from the TCGA database (https://portal.gdc.cancer.gov/projects/TCGA-HNSC).Following data normalization, the data sets were merged.Subsequently, incomplete clinical data and samples with a 0-day follow-up duration were excluded, resulting in 499 HNSCC samples that constituted the primary cohort.The clinical factors considered included gender, stage, age, grade, T-phase, M-phase, N-phase, survival status, and the number of days of survival.RNA-Seq and clinical data were retained for subsequent analysis.R (3.6.3 version) and R Bioconductor software package were implanted for data analysis.

| Gene set enrichment analysis (GSEA)
GSEA was performed on using normalized RNA-Seq data obtained from TCGA. 19A number of 1000 permutations were employed.The analysis focused on exploring possible biological functions of ELOVL6.The GSEA created a list of all gene permutations associated with ELOVL6 expression.Then the samples were categorized into high ELOVL6 groups and low ELOVL6 groups.The aim was to discern potential functions and survival differences using GSEA.Multiple genome substitutions were performed for each test.The degree of ELOVL6 expression served a phenotypic marker.Pathway enrichment in each phenotype was classified based on normalized enrichment scores (NES) and nominal p values.

| Immune infiltrates analysis
The potential relationship between ELOVL6 expression and tumor-infiltrating immune cells (TIICs) was evaluated using TIMER-related modules.TIMER is a comprehensive resource for immune infiltration analysis in various cancer types (https:// cistrome.shinyapps.io/timer/). 20Time utilized deconvolution a recently published statistical method called deconvolution to infer TIIC prevalence from gene expression profiles. 21To approximate TIIC abundance, TIMER database used TCGA data from 10,897 samples of 32 cancers.The investigation focused on the ELOVL6 expression in HNSCC, exploring its relevance to the abundance of TIICs (including B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells) through gene modules.TIMER generated a graph illustrating gene expression levels against tumor purity.supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotics (penicillin-streptomycin). Cells were cultured in a 5% carbon dioxide incubator at 37°C.
To knock down ELOVL6, Oligobio (Beijing) synthesized three small interfering RNAs (RNAi) (Table 1) and one negative control (shCtrl) targeting reverse splicing junction site of ELOVL6.RNAi or shCtrl at a concentration of 50 ng was introduced into BR-V121 and FD-LSC-1 cells with a liposomal RNAiMAX transfection agent (Invitrogen) following the manufacturer's instructions.Virus infection was confirmed by assessing 80% cell infection, and testing was performed 48 h after infection.

| RNA extraction and quantitative polymerase chain reaction (qRT-PCR)
Total RNA was extracted using Trizol (Sigma) according to the manufacturer's protocol, and its quality was examined using a Nanodrop (Thermo).cDNA was obtained by reverse transcription using Hiscript QRT supermix for qPCR (Vazyme).Following cDNA synthesis, qRT-PCR was performed with SYBR Green Mastermixs (Vazyme).GAPDH served as the reference gene.Expression levels were quantified by the 2 − ΔΔCt method.

| Western blot analysis
The centrifugal machine was used to cool the temperature to 4°C.
The six-well plate was removed, and the cells underwent two washes with phosphate-buffered saline (PBS).RIPA lysis buffer containing protease inhibitor cocktail (Abcam) was added at the rate of 150 μL per well.Total protein was extracted and the supernatant was collected after centrifugation at 12000 rpm at 4°C for 20 min.The protein concentration was determined by Beyotime (China).Then 20 μg of protein was mixed with 5×SDS loading buffer in each lane, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to PVDF membrane (Millipore).At room temperature, the membranes were enclosed in PBS with 3% BSA buffer for 1 h and incubated overnight in a primary antibody shaker diluted with 3% BSA buffer at 4°C.On the following day, the film was washed with TBST and incubated with specific secondary antibodies in a room-temperature shaker for 2 h.The film was washed with TBST.Protein expression was detected by an ECL chemiluminescence kit (Beyotime).

| Cell counting kit-8 (CCK-8) assay
Cell proliferation rate was determined by CCK-8 colorimetric method.2000 cells were seeded into Wells in 96-well plates (Corning) and cultured in a cell incubator.At specific time points, CCK-8 solution (10 μL; Sigma) was added and incubated with the cultured cells in a hatchery for 2 h, followed by absorbance (450 nm) analysis using an enzyme-linked immunodetector (Tecan infinite).

| Transwell assay
Transwell chamber (Corning) was used to detect the invasion ability of the cells.After digestion of logarithmic growth cells by trypsin, cell suspensions were prepared using serum-free medium.The cells were counted by blood cell counting method, and 50,000 cells in each group were inoculated into a transwell chamber for invasion test.The lower chamber received 600 μL of 30% FBS culture medium, and the setup was incubated at 37°C for 48 h.Subsequently, the small chamber culture medium was discarded.The chamber was fixed with 4% paraformaldehyde (Sigma) for 10 min, and 0.1% crystal violet for another 10 min.Finally, the invading cells were photographed using T A B L E 1 The target sequences of ELOVL6-interfered shRNA.

Target number
Target sequence | 1081 an inverted microscope.Cell counts were performed using the Image J software.Two 100x visual fields and three 200x visual fields were selected for each experiment, and entire process was repeated in three biological experiments.

| Immunohistochemistry (IHC) assay
An immunohistochemical study of ELOVL6 was performed on the HNSCC paraffin-embedded tissue microarray (HN049La01), which was purchased from Zhongke Guanghua (Xi'an) and contained 43 cases of laryngeal cancer and six cases of adjacent laryngeal cancer.
The tissue chips were dewaxed with xylene, followed by sequential washes, and rehydrated with 100%, 75%, and 50% ethanol and PBS (Servicebio).For antigen extraction, the slides were placed in 50 mm Tris-HCl buffer at pH 9.0, heated in a decock pressure cooker for 20 min, then remained in the buffer for 15 min, and inhibited the endogenous peroxidase activity with 3% catalase (Absin) in PBS.
Nonspecific binding was blocked with 3% normal goat serum (Absin) for 30 min.The tissue microarray was then incubated with anti-ELOVL6 antibody (1:150, Rabbit, cat.no.ab69857, Abcam) to phosphorylate AKT (cell signaling) for 1 h at room temperature.The immunoassay was performed using DAB staining system according to the manufacturer's instructions (Bioaitech

| Overexpression of ELOVL6 in HNSCC tissues
In this study, we obtained four HNSCC-related gene expression profiling data sets directly from GEO website: GSE30784, GSE23036, GSE33205, and GSE59102.GSE30784 consists of 44 normal tissues, 167 cancer tissue samples, and 17 dysplasia tissue samples.Among these, 17 abnormal tissue samples were excluded.GSE23036 comprises five normal tissues and 63 cancer tissue samples.
GSE33205 consists of 25 normal tissue samples and 44 cancer tissue samples.GSE59102 consists of 13 adjacent tissues and 29 cancer tissue samples.We divided the four data sets into two groups: the first group includes GSE30784, while the second group encompasses GSE23036, GSE33205, and GSE59102.The data from the second group underwent integration and batch correction.
Finally, the ELOVL6 differences between the two groups of data are analyzed.The results from both groups are consistent, revealing a significant disparity in ELOVL6 expression (p < 0.01) between normal and tumor tissues.ELOVL6 is highly expressed in tumor tissue (Figure 1A).
Furthermore, immunohistochemical analysis of HNSCC tissue chips (Figure 1B) demonstrated high ELOVL6 expression in tumor tissues, a finding further validated by statistical analysis (p < 0.01, Figure 1C).

| Relationship between ELOVL6 expression and clinicopathology in HNSCC
We selected 502 cases of HNSCC from the TCGA database.The association between the expression levels of ELOVL6 and the clinical-pathologic characteristics of HNSCC patients was assessed.
The expression of ELOVL6 was strongly correlated with tumor tissue grade (p < 0.01, Figure 2A).Survival analysis showed that HNSCC patients with high-expression ELOVL6 had a worse prognosis than those with low-expression ELOVL6 (p < 0.01, Figure 2B).
Univariate Logistic regression analysis, treating ELOVL6 expression as a well-defined covariate, indicated the association with suggest that patients with ELOVL6 high-expression HNSCC are more likely to develop tumors with higher grades than those with low expression (Table 2).
The relationship between expression of ELOVL6 and clinicopathological characteristics in HNSCC microarray was examined by Mann-Whitney U test, and it was found that there was a significant difference between ELOVL6 expression and tumor T-phase (p < 0.05).Spearman correlation analysis revealed that the expression of ELOVL6 gene was positively correlated with the T-phase (p < 0.05), indicating the expression of ELOVL6 gene increased with the deepening of tumor malignancy.

| Analysis of survival outcomes and variables
We used Cox analysis to explore the relationship between ELOVL6 expression and OS along with other variable characteristics in patients with HNSCC.Single-factor correlation analysis highlighted that stage, T-phase, M-phase, N-phase, and ELOVL6 mRNA expression significantly correlated with OS.The subsequent multivariate analysis, presented as a forest plot in Figure 2C, identified ELOVL6 expression, T-phase, M-phase, and N-phase as independent prognostic factors in patients with HNSCC (Table 3).

| GSEA investigation of ELOVL6
To explore the potential biological functions of ELOVL6, KEGG pathway analysis was conducted.GSEA showed significant differences in the enrichment of high levels of ELOVL6 in KEGG pathways (p < 0.050).According to the standardized enrichment score (NES), we identified highly enriched signaling pathways.KEGG pathway enrichment analysis revealed nine categories positively associated with elevated ELOVL6 levels, as detailed in Table 4.These categories include WNT signaling pathway, fatty acid metabolism, biosynthesis of unsaturated fatty acids, certain cancers, RNA degradation, cell cycle, insulin signaling pathway, and tight junction (Figure 3A).

| Relationship between ELOVL6 expression and tumor-infiltrating immune cells
It was established in the previous study that independent tumorinfiltrating lymphocytes play a key role in predicting OS and sentinel lymph node status. 24We used TIMER to analyze the possible correlation between ELOVL6 expression and immune infiltration level in HNSCC.
The expression of ELOVL6 is positively correlated with CD4 + T cells and negatively correlated with B cells, CD8 + T cells, macrophages, neutrophils, and dendritic cells, as shown in Figure 3B.All these results suggest that ELOVL6 plays a key role in the immune invasion of HNSCC.

| Upregulation of ELOVL6 expression in HNSCC cell lines
We analyzed the expression of ELOVL6 in human HNSCC cell lines (FD-LSC-1, Hep-2, HN4) and normal oral epithelial cells (HOK) using Western blot and qRT-PCR analyses.The results demonstrated that the expression of ELOVL6 was significantly upregulated in FD-LSC-1, Hep-2, and HN4 cells, especially in FD-LSC-1 cells (Figure 1D,E).
FD-LSC-1 cell line was selected for further experiments.To establish stable ELOVL6 knockdown FD-LSC-1 cell lines, we transfected them with an ELOVL6 knockdown viral vector.

| ELOVL6 gene knockdown inhibited the proliferation and invasion of HNSCC cells and promoted apoptosis
ELOVL6 gene knockdown inhibits the proliferation and invasion of HNSCC cells.As expected, ELOVL6 levels were significantly reduced in RNAi transfected cells as measured by qRT-PCR and western blot assay (Figure 4A,B).Through CCK-8 assay, we found that ELOVL6 knockdown significantly inhibited the proliferation of FD-LSC-1 cells (Figure 4C).In addition, Transwell assay data showed that ELOVL6

| DISCUSSION
ELOVL6 is a microsomal enzyme that plays a crucial role in the elongation of saturated and monounsaturated fatty acids. 11Fatty acid metabolism (FAM), serve as a cancer marker, plays an important role in tumor initiation and carcinogenesis.The expression of ELOVL6 is notably inhibited by polyunsaturated fatty acids in the diet. 12The expression of ELOVL6 is prominently observed in various cancer types, including nonalcoholic steatohepatitis-related hepatocellular carcinoma, 25 lung squamous cell carcinoma 26 lung adenocarcinoma, 27 breast cancer, 28 and bladder cancer. 29Cancer tissues exhibit a high abundance of phospholipids containing elongation acyl chains, with ELOVL6 being the principal enzyme responsible for fatty acid elongation in cancer. 26This elongation has also been detected in nonalcoholic steatohepatitis (NASH) associated with hepatocellular carcinoma. 25,30I G U R E 5 The expression of ELOVL6 in tumors was significantly increased, as revealed in a comprehensive analysis of GEO database in various tumors, particularly in HNSCC.Furthermore, cell experiment studies demonstrated that the overexpression of ELOVL6 in HNSCC cells was significantly higher compared to that in HOK cells.Immunohistochemical analysis of HNSCC tissue chips also demonstrated that the expression of ELOVL6 protein was significantly upregulated in head and neck squamous cell carcinoma.These findings suggested that ELOVL6 may play an important role in regulating tumor progression.
In addition, ELOVL6 overexpression has been found to be associated with axillary lymph node metastasis and reduced diseasefree survival in breast cancer, suggesting an association between ELOVL6 overexpression and poor prognosis. 28We utilized TCGA Previous studies have implicated that ELOVL6 is involved in fatty acid metabolic pathways, and increased fat generation is an early and common event in the development of cancer. 31Lipogenesis, particularly certain enzymes associated with it, has been recognized as a potential target for cancer therapy. 32WNT signaling is an important regulator of lipogenesis or insulin secretion in the melaninproducing pathway. 33Junjvliekea 34 found that ELOVL6 regulates the WNT signaling pathway to regulate lipid metabolism in bovine adipocytes.Our KEGG pathway analysis revealed that the The model of the mechanism of ELOVL6 regulating WNT/β-catenin in head and neck squamous cell carcinoma (HNSCC).
upregulation of ELOVL6 primarily correlates with the WNT signaling pathway, thereby exerting control over the initiation and progression of cancer cells.This finding underscores the pivotal role of the WNT signaling pathway as a downstream target influenced by ELOVL6 in HNSCC.To further support this finding, we demonstrated that ELOVL6 promotes HNSCC cells growth through the WNT/β-catenin pathway using CCK8 assay, Western blot, and Transwell assay (Figure 6).The upregulation of ELOVL6 expression affects the WNT/ β-catenin signaling pathway, which is critical for tissue development and homeostasis, by regulating its endogenous stem cells.The WNT signaling pathway is known to regulate CSC generation in HNSCC. 35The abnormal activation of WNT/β-catenin signaling plays a crucial regulatory role in the pathogenesis of HNSCC, 36 promoting neoplastic transformation in head and neck tissues. 37Furthermore, it modulates the process of epithelial-mesenchymal transition in laryngeal squamous cell carcinoma, contributing to tumor development.
While no previous studies have identified the role of ELOVL6 in tumor immune response, our CIBERSORT analysis showed that the expression level of ELOVL6 was positively correlated with the infiltration level of immune cells, especially CD4 + T cells.In antitumor immune response, CD4 + T cells are very important. 38HNSCC is characteristic of highly immune-infiltrating, especially in oropharyngeal tumors. 39However only 15% of patients with metastatic HNSCC respond to anti-PD-1 therapy. 40Studies have found that PD-1 + and ICOS + TILs are enriched in tumor-reactive CD4 + Th cells in HNSCC, with CD4 + and CD8 + T cells playing complementary roles in antitumor responses. 41In HNSCC, tumor-infiltrating activated CD4 + T cells have been associated with a favorable prognosis. 42The high presence of CD4 + T cells is an important indicator of positive prognosis in patients with non-small cell lung cancer (NSCLC). 43ese findings collectively suggest that ELOVL6 may exert a pivotal role in modulating the tumor immune response, thereby rendering it a promising target for immunotherapeutic interventions.writing-review and editing.Liang Gong: Project administration.

2. 4 |
Cell culture and transfection HNSCC cell lines FD-LSC-1, Hep-2, HN4, and normal oral epithelial cells HOK were all purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai).HOK cells were cultured in HOK cell culture medium (Sciencell), and FD-LSC-1, Hep-2, and HN4 were cultured and maintained in Dulbecco's modified Eagle's medium (BasalMedia) Apoptosis detection was carried out by Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (eBioscien).Apoptosis analysis was performed for one ELOVL6 RNA-interference group, one control group, and one MOCK group.Ten thousand cells were collected in each group.The three groups of cells were simultaneously planted into 6 cm cell culture dishes and were harvested once they reached 80% confluence.Cold PBS (Servicebio) was used to wash cells and added 1x binding buffer to form a single-cell suspension.The cells were double stained with fluorescein Annexin V-FITC and propidium iodide (PI).After staining, these tubes of cell suspension were analyzed with flow cytometry (BD) within 1 h.Forward scatter (FSC) and side scatter (SSC) were set as scattering gating to distinguish cells of different sizes and shapes.Circle the most concentrated cell groups and eliminate cell fragments.Application of fluorescence labeling: fluorescent dyes Annexin V and PI were used to label apoptotic cells and distinguish the stages of apoptosis.Cells labeled as Annexin V positive, PI negative, or weak were considered to be apoptotic.Annexin V and PI positive quality control cell was performed to determine the location of the cross-quadrant before the experiment.Flow cytometry and FLOWJO software were used to analyze the data to determine the percentage of early and apoptotic cells in each group.

F
I G U R E 2 ELOVL6 expression and the association among clinicopathologic factors.(A) Grade; (B) survival analysis; (C) multivariate Cox analysis of ELOVL6 expression and other clinicopathological variables.*p < 0.05, ***p < 0.001.T A B L E 2 ELOVL6 expression associated with clinical pathological characteristics (logistic regression).
knockdown significantly reduced the number of invasive cells (Figure 4D,E).We used flow cytometry and PI/AV co-staining to evaluate the effect of ELOVL6 knockdown on apoptosis in FD-LSC-1 cells.ELOVL6 knockdown significantly increased the apoptosis rate of FD-LSC-1 cells compared with the shCtrl and MOCK groups (Figure 4F,G).ELOVL6 knockdown promoted apoptosis of FD-LSC-1 cells through WNT/β-catenin signaling pathway.To further investigate the mechanism of ELOVL6 inhibiting apoptosis, we performed Western blot analysis on genes related to WNT/β-catenin signaling pathway.It was found that ELOVL6 knockdown resulted in downregulation of wnt5a, c-myc, β-catenin, and COX2 protein expressions (Figure 4H,J).

F
I G U R E 3 (A) Enrichment plot from gene set enrichment analysis (GSEA).(B) Correlations between ELOVL6 expression and immune infiltration levels.F I G U R E 4 Knockdown of ELOVL6 prevented cell proliferation and induced cell apoptosis in head and neck squamous cell carcinoma (HNSCC).(A) The protein expression of ELOVL6 was significantly declined in FD-LSC-1 cells transfected with ELOVL6 knockdown by quantitative polymerase chain reaction (qRT-PCR) assay.(B) The protein expression of ELOVL6 was significantly declined in FD-LSC-1 cells transfected with ELOVL6 knockdown by western blot assay.(C) ELOVL6 knockdown obviously inhibits cell proliferation in FD-LSC-1 cells.(D, E) ELOVL6 knockdown obviously inhibits cell invasion ability (magnification, 100x).(F, G) ELOVL6 knockdown significantly promoted cell apoptosis in FD-LSC-1 cells.(H, I) ELOVL6 knockdown inhibits the protein expression level of wnt5a, c-myc, β-catenin, and COX2 compared with in control group.The original pictures of western blot analysis of (B) and (H) are in the supplementary file "original data."*p < 0.05, ***p < 0.001,****p < 0.0001.

3. 8 |
Figure 5A).It is proved that ELOVL6 promoted cell proliferation by regulating WNT/β-catenin pathway.At the same time, Transwell assay showed that ELOVL6 knockdown significantly reduced the cell metastasis rate, while compound 3f promoted the cell metastasis rate (p < 0.001, Figure5B,C).The regulatory role of ELOVL6 in the WNT/ β-catenin pathway facilitates the invasive potential of HNSCC cells, as evidenced by experimental validation.Further western blot analysis of genes related to WNT/β-catenin signaling pathway showed that ELOVL6 knockdown decreased the expression of wnt5a, c-myc, β-catenin, COX2 proteins, while compound 3f increased these proteins (Figure5D,E).It was demonstrated that ELOVL6 promotes protein expression by regulating WNT/β-catenin ELOVL6 affected cell progression by regulating WNT/β-catenin pathway.(A) ELOVL6 knockdown significantly inhibits cell proliferation, while compound 3f enhanced the effect of ELOVL6 on cell proliferation in FD-LSC-1 cells.(B, C) ELOVL6 knockdown obviously inhibits cell invasion ability, while compound 3f enhances the effect of ELOVL6 on cell invasion ability in FD-LSC-1 cells (magnification, 100x).(D, E) ELOVL6 overexpression obviously inhibits WNT/β-catenin pathway, while compound 3f enhanced the effect of ELOVL6 on epithelialmesenchymal transition (EMT) progression in FD-LSC-1 cells, The original pictures of Western blot of analysis (D) are in the supplementary file "original data."**p < 0.01, ***p < 0.001, ****p < 0.0001.
data to analyze the association between ELOVL6 and clinicopathological features, survival time, function, immune invasion, and expression of HNSCC.Understanding whether the hightened expression of biomarkers in tumors is directly related to HNSCC will contribute to unraveling the mechanism underlying observed clinical survival patterns.In our study, the expression of ELOVL6 in HNSCC was related to clinicopathological factors (Grade), survival time, and poor prognosis.Univariate analysis found that ELOVL6 expression, as a clear-cut variable, was associated with clinicopathological factors with poor prognosis, with tumor-Stage, T-phase, N-phase, and M-phase potentially playing an indispensable role in the further progression of tumors.Both univariate and multivariate analysis also showed that ELOVL6 was closely related to OS with significantly decreased survival rates observed in cases of high expression of ELOVL6.This was consistent with the findings of Martin A.26 IHC analysis of ELOVL6 further highlighted that the expression of ELOVL6 increased significantly at high T stage.This suggests that elevated expression of ELOVL6 is associated with poor prognosis.To the best of our knowledge, this study represents the first report of an association between ELOVL6 expression and the OS of HNSCC patients.Our in vitro experiments indicate that ELOVL6 plays a crucial role in the pathogenesis and progression of head and neck squamous cell carcinoma (HNSCC).We generated stable ELOVL6 knockdown cell lines (shELOVL6-1, shELOVL6-2, shELOVL6-3) and shCtrl cell lines to investigate the function of ELOVL6 in head and neck squamous cell cancer cells.In this study, CCK8 experiment showed that ELOVL6 could promote the proliferation of FD-LSC-1 cells in vitro.Additionally, Transwell assay and Flow cytometry experiment demonstrated that ELOVL6 gene promoted HNSCC invasion while inhibiting apoptosis.These findings underscore the importance of considering ELOVL6 as a potential biomarker or indicator, and therapeutic targets that determine prognosis.
Elevated expression of ELOVL6 is associated with advanced grade, stage, and tumor status compared ELOVL6 low expression in patients with HNSCC.ELOVL6 appears to impact tumorigenesis mechanisms and tumor immunological progression in HNSCC.Understanding the molecular mechanism by which ELOVL6 activates WNT/β-catenin signaling pathway in HNSCC will increase our knowledge of the biological foundations of HNSCC.However, future experiments and clinical trials are necessary to validate these findings.With a deeper comprehension of its functional scope, ELOVL6 holds the potential to emerge as an efficacious tool for the diagnosis and treatment of HNSCC, facilitating the integration of ELOVL6 in the prognosis evaluations for HNSCC.Consequently, this may pave the way for biomarker therapy to be considered a promising avenue for future treatment of HNSCC.AUTHOR CONTRIBUTIONS Ruoya Wang: Data curation; formal analysis; investigation; methodology; writing-original draft; writing-review and editing.Xianzhi Liu: Data curation; investigation; methodology; writing-original draft; writing-review and editing.Xiyao Li: Data curation; investigation; methodology; writing-original draft; writing-review and editing.Ming Qian: Formal analysis; methodology; writing-review and editing.Xi Yang: Formal analysis; methodology; writing-review and editing.Qichuan Jiang: Formal analysis; methodology; writingreview and editing.Yijie Wang: Formal analysis; methodology; writing-review and editing.Hao Liu: Data curation; investigation; writing-review and editing.Jianguo Chen: Formal analysis; investigation; methodology; writing-original draft; writing-review and editing.Xuefeng Wang: Formal analysis; writing-original draft; Correlation between overall survival and multivariable characteristics in The Cancer Genome Atlas (TCGA) patients via Cox regression and multivariate survival model.Gene sets enriched in phenotype high.
T A B L E 3 T A B L E 4Note: Gene sets with NOM p < 0.05 is considered as significant.Abbreviations: NES, normalized enrichment score; NOM, nominal.