DDX60 Promotes Migration and Invasion of Head and Neck Squamous Cell Carcinoma Cell through the NF-κ B/IFI27 Signaling Pathway

Background : Despite its significance in multiple cancer types. the function and mechanism of DEXD/H box helicase 60 (DDX60) in head and neck squamous cell carcinoma (HNSCC) remain unreported. Methods : Thirty paired HNSCC tissues and adjoining normal tissues and human normal oral epithelial keratinocytes (HOK) and four HNSCC cells (CAL27, SAS, CAL33, and SCC25) were analyzed for DDX60 expression by Semi-quantitative real-time PCR (SQ RT-PCR) and western blot. To investigate how DDX60 affects HNSCC cell migration and invasion, transwell experiments were performed. The western blot was implemented to understand the interaction among DDX60, Interferon Alpha Inducible Protein 27 (IFI27), and the NF-κ B pathway. Results : Results revealed the upregulation of DDX60 in HNSCC cell lines and tissues. Additionally, patients with upregulated DDX60 expression exhibited a dismal prognosis relative to those with downregulated expression. DDX60 enhanced the migration, invasion, and epithelial to mesenchymal transition (EMT) in HNSCC cells. The results from mechanistic studies revealed that DDX60 could promote the IFI27 expression following the activation of NF-κ B pathway. Conclusion : DDX60 promoted the migratory and invasive capacities of HNSCC cells via the NF-κ B/IFI27 axis.


Introduction
Head and neck cancer (HNC) is among the most prevalent malignant tumors that cause cancer-related deaths, and both its incidence and mortality rates have been rising over the last several years [1,2].Head and neck squamous cell carcinomas (HNSCCs) stem from the squamous epithelium cells of the oral cavity, oropharynx, larynx, and hypopharynx and account for the vast majority of HNC [3].Surgery, radiotherapy, and chemotherapy are the preferred treatment regimens [4].The recurrence and metastasis of HNSCC can affect treatment effects, prognosis, and survival time of patients [5,6].In addition, those diagnosed with HNSCC have a dismal chance of surviving in the long term, although the field of HNSCC treatment and prevention has advanced considerably [7][8][9][10].Hence, conducting in-depth studies is important to explore the mechanisms (from the molecular viewpoint) implicated in the onset and progression of HNSCC.Research on molecular targets should be conducted to achieve targeted HNSCC treatment.
The DEAD-box (DDX) protein family is the largest family of RNA helicases, with 37 members, including DDX5, DDX21, DDX23, and DDX60, all of which have a conserved amino acid sequence ASP-GLUALA-ASP [11].Via their ATP-dependent RNA helicase activity, DDX proteins are capable of interacting with both ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs) and perform important functions in cell life, such as participating in the initiation of translation, the production of mRNA, the splicing and modification of RNA, and the assembly of ribosomes and spliceosomes [12][13][14][15].It is also implicated in the modulation and transcription of genes related to DNA repair, proliferation, cell cycle arrest, apoptosis, and other cell life activities, suggesting that the DDX protein is potentially involved in carcinogenesis [16,17].DEXD/H box helicase 60 (DDX60) is a newly discovered DEAD-box RNA helicase that is upregulated following viral infection and performs an important function in antiviral immunity by promoting the retinoic acid-inducible gene I (RIG-I) receptormediated signaling pathway [18].Recent research has illustrated that DDX60 is highly expressed in oral squamous cell carcinoma (OSCC) and is significantly associated with early recurrence of OSCC, indicating DDX60's potential involvement in tumorigenesis [19].Recently, DDX60 has been implicated in the development of numerous tumors as well as in immunotherapy.However, the function of DDX60 and the mechanism via which it acts in HNSCC remain unreported.Therefore, conducting studies is important to understand the mechanism of HNSCC progression.
In this experiment, we explored the transcription and expression level of DDX60 and its function and mechanism in HNSCC.We also investigated that Interferon Al-pha Inducible Protein 27 (IFI27), which was positively regulated by DDX60, was also regulated by DDX60-mediated activation of NF-κB in HNSCC.Our data indicated that DDX60 shows great promise as a potential HNSCC diagnostic marker or therapeutic target.

Clinical Samples
From January 2015 to December 2022, the samples (30 pairs of HNSCC tissues and matched normal tissues) were obtained from The Third Central Hospital of Tianjin.According to clinical diagnosis, 30 cases of HNSCC were determined, and 30 cases of adjacent normal tissue (more than 2 cm away from cancer tissue) were selected as control group.Inclusion criteria: first-episode patients who had not received other treatment before this surgery; the patient did not receive preoperative radiotherapy, chemotherapy, and intervention, as well as all disease-related management; postoperative pathological diagnosis of HNSCC; patients who have not been diagnosed and treated in other hospitals.Exclusion criteria: previous chemoradiotherapy; patients with multiple primary tumors; liver, kidney, and heart dysfunction combined with other serious diseases; secondary HNSCC.

Semi-Quantitative Real-Time PCR (SQ RT-PCR)
Total RNA was isolated from cancer tissues and cells in each group utilizing TRIzol Reagent (CAS # R1100, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).cDNA was reverse-transcribed with an EasyScript® One-Step gDNA Removal and cDNA Synthesis Super Mix (CAS # AW311-02, TransGen Biotech, Beijing, China).An SYBR Green qPCR Mix Kit (CAS # BL697A, Biosharp, Beijing, China) was employed to perform SQ RT-PCR.The 2 −∆∆Ct method was applied to calculate the relative expression.β-actin was used as the internal reference for DDX60, IFI27, Vimentin, E-cadherin and N-cadherin.Supplementary Table 1 summarizes the primers used for SQ RT-PCR.

Transwell Experiment
The Transwell experiment was conducted following a previously reported method [20].In brief, for Transwell migration experiments, we resuspended the transfected cells (2 × 10 4 ) in serum-free DMEM (200 µL) and inoculated them in each chamber of the upper chamber (CAS # 3470, Corning Incorporated, Corning, NY, USA).For the Transwell invasion experiment, Matrigel (40 µL, 1 mg/mL, CAS # 356234, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was added in the upper chamber, and then DMEM without serum (200 µL) was used to resuspend the transfected cells (5 × 10 4 ), followed by inoculating them in each chamber of the upper chamber.In the bottom chamber, 24-well plates were filled with DMEM complete medium (600 µL) supplemented with 20% FBS.Then, 4% paraformaldehyde (PFA) was used to fix cells in the bottom chamber for 20 min after 48 h of incubation.Cell staining was performed with 0.1% crystal violet for 5 min.Transwell cells were randomly assigned to three areas for shooting and counting analyses.

Statistical Analysis
The experiments were repeated three times.The format of mean ± standard error of the mean (SEM) was used to present the collected data.The data were statistically analyzed, and GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA) was applied for plotting.Student's t-tests were used to understand the differences between the two subgroups.Multiple subgroups were compared via a one-way analysis of variance and Tukey post-hoc test.Based on the distribution patterns of indicators, Pearson or Spearman correlation tests were used to estimate the association between variables.Using the survival package, univariate and multivariate Cox regression models were used to assess the survival significance of variables (p < 0.05; statistically significant).

The Expression Level of DDX60 was Upregulated in HCC and was Correlated with a Dismal Prognosis
We explored the expression level of DDX60 and verified its regulatory mechanism in HNSCC to investigate the role of DDX60.First, the Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn/)database was searched to predict the DDX60 mRNA level in pan-cancers.Through analysis, DDX60 was upregulated in several cancers as well as HNSCC (Fig. 1A).In addition, the Encyclopedia of RNA Interactomes (ENCORI; https://starbase.sysu.edu.cn/)database was searched to further verify the mRNA level of DDX60 in HNSCC (Fig. 1B).The survival curve was analyzed via GEPIA and ENCORI, and patients who were suffering from HNSCC and were characterized by high mRNA levels of DDX60 exhibited a lower 10-year overall survival rate than patients with HN-SCC who were characterized by a lower expression level of DDX60 (Fig. 1C,D).Thirty pairs of tissues (HNSCC tumor and adjoining normal tissues) were used to study the DDX60 mRNA levels.The results revealed the importance of upregulated DDX60 in HNSCC tissues.The DDX60 mRNA level was increased in HNSCC tissues as opposed to the adjoining non-tumor tissues (Fig. 1E).Moreover, HNSCC tissues also exhibited DDX60 upregulation as op- posed to normal tissues (Fig. 1F).To investigate the role of DDX60 in HNSCC, four cell lines originated from tongue were selected: CAL27, SAS, CAL33, and SCC25.We also studied the mRNA and protein levels of DDX60 in human normal oral epithelial keratinocytes (HOK) and these four HNSCC cells.Results showed that both DDX60 mRNA and protein levels were found to be elevated in the four HN-SCC cell lines relative to the normal cell line (Fig. 1G,H).

DDX60 Enhanced the Migratory and Invasive Capacities of HNSCC Cells
Tumor initiation, invasion, and metastasis all require the involvement of epithelial-mesenchymal transition (EMT), one of the hallmarks of malignant tumors [21][22][23].Therefore, we initially investigated whether DDX60 affected EMT in HNSCC cells.Transwell migration and invasion assay results revealed that DDX60 overexpression could enhance cell migration and invasion abilities of HN-SCC cells, whereas DDX60 expression was silenced, and these processes were inhibited (Fig. 2A-D).Thus, we hypothesize that DDX60 promoted EMT in HNSCC cells.

DDX60 Regulated the EMT Markers in HNSCC Cells
EMTs are characterized by the absence of epithelial cell markers (e.g., E-cadherin) and upregulation of mesenchymal cell markers (like vimentin and N-cadherin) [24].We investigated how DDX60 affected EMT markers, such as Vimentin, E-cadherin, and N-cadherin.SQ RT-PCR results showed that forced overexpression of DDX60 remarkably elevated the mRNA levels of N-cadherin and Vimentin while reducing those of E-cadherin (Fig. 3A-D).Conversely, DDX60 knockdown could reduce the mRNA levels of N-cadherin and Vimentin while increasing those of E-cadherin (Fig. 3A-D).In addition, increased protein levels of N-cadherin and Vimentin but reduced those of E-cadherin were observed after DDX60 overexpression, whereas the opposite results occurred by downregulation of DDX60 in HNSCC cells (Fig. 3E-I).

DDX60 Positively Regulated the IFI27 Expression in HNSCC Cells
To further explore the potential mechanism of DDX60 in HNSCC, we performed the correlation analysis using GEPIA.According to the findings, IFI27 (interferon alpha inducible protein 27) mRNA level was considerably elevated in HNSCC tissues in comparison to the normal tissues, and IFI27 was positively corrected with DDX60 (Fig. 4A,B).Patients with HNSCC who had high levels of IFI27 mRNA had a lower 10-year overall survival rate in comparison to those with lower levels (Fig. 4C).Furthermore, the above prediction results were verified by SQ RT-PCR (Fig. 4D,E).Moreover, overexpression or knockdown of DDX60 promoted or inhibited IFI27 at the protein and mRNA levels (Fig. 4F-I).

DDX60 Regulated the EMT via Upregulation of IFI27 in HNSCC Cells
Based on the above results, we ask whether DDX60 regulated the EMT through the IFI27-mediated mechanism.A rescue experiment was implemented to validate the hypothesis, and the results illustrated that silencing IFI27 in HNSCC cells attenuated the DDX60-induced upregulation of N-cadherin and Vimentin mRNA levels and the Ecadherin inhibition (Fig. 5A-E).In addition, a similar phenomenon has been observed at the protein level (Fig. 5F-K).Then, further research was conducted to determine whether DDX60 influenced the migratory and invasive ca-pacities of HNSCC cells via modulation of IFI27.The analysis showed that HNSCC cells' migratory and invasive capacities were remarkably reduced upon DDX60 knockdown, whereas co-transfection with IFI27 overexpressing plasmid removed this inhibition (Fig. 5L-N).Thus, we hypothesize that these processes can be promoted by DDX60 via the upregulation of the IFI27 in HNSCC cells.

DDX60 Upregulated IFI27 via NF-κB-Mediated Mechanism in HNSCC Cells
We delved further into the molecular mechanism via which DDX60 upregulates IFI27 in HNSCC cells.When comparing patients with high and low DDX60 expression, GSEA revealed that the NF-κB pathway was significantly enriched in the former group (Fig. 6A), indicating that the NF-κB pathway might be activated by DDX60.
To further our understanding of how DDX60 works in HNSCC cells, we examined how changed DDX60 expression affected the protein levels of p65 and p-protein levels, two key molecular components in the NF-κB pathway.We discovered that the protein level of p65 did not change significantly regardless of DDX60 levels (Fig. 6B-D).However, while p-p65 protein levels rose remarkably during DDX60 overexpression, they dropped remarkably after DDX60 knockdown (Fig. 6B-D).The direct transcription of IFI27 is induced by the NF-κB pathway activation, and previous research has shown that the promoter region of IFI27 contains NF-κB-p65 binding sites [25].Therefore, we investigated whether DDX60 could upregulate IFI27 via activating the NF-κB pathway.We discovered that DDX60 overexpression could significantly increase the p-p65 protein level.A significant reduction in the p65 and p-p65 protein levels was also observed, while p65 expression levels were also inhibited in DDX60 overexpressed cells (Fig. 6E-H).In addition, reducing p65 protein levels could attenuate the promoting effect of DDX60 overexpression on IFI27 expression (Fig. 6E-I).The results indicated that IFI27 expression levels in HNSCC cells may be upregulated by DDX60 via the NF-κB pathway (Fig. 6J).

Discussion
DDX60, an important regulatory molecule, was initially proven to be involved in RNA-mediated processes via hydrolysis [26].It is also associated with antiviral activities, radiosensitivity of cancer cells, and immunotherapies [26][27][28].However, the role of DDX60 in cancers such as HNSCC, is still much unknown.Herein, we studied how DDX60 affected HNSCC progression.The results revealed that the upregulation of DDX60 levels in primary tissues and cell lines was associated with HNSCC.The findings matched those from a search of online databases.We showed that HNSCC cells' ability to migrate and invade was enhanced by DDX60, an oncogene.
The associated mechanism was studied, and the results revealed that DDX60 could upregulate IFI27 in HNSCC cells.IFI27, which is stably induced by interferon, is a vital member of the FAM14 family and is implicated in the biological processes of numerous malignancies [28][29][30].Prognosis in pancreatic cancer patients is strongly correlated with IFI27 expression level, and its upregulation is linked to a dismal prognosis [31].Existing literature has shown that EMT may occur in ovarian cancer cells when IFI27 is upregulated [32].Downregulation of IFI27 by activating transcription factor 3 (ATF3) could inhibit the growth and migration of tongue squamous cell carcinoma cells [33].Li et al. [25] demonstrated that a disintegrin-like metalloproteinase with thrombospondin motifs-16 (ADAMTS16) could upregulate IFI27 expression by activating the NF-κB pathway in gastric carcinogenesis.We showed that DDX60 was another regulator of IFI27 in HNSCC cells.
The NF-κB pathway is a classic signaling pathway.Notably, the onset and advancement of HNSCC are affected when the NF-κB pathway is abnormally activated.This pathway can be activated by several protein molecules, such as DNA-damage-inducible transcript 4 (DDIT4), mucosaassociated lymphoid tissue lymphoma translocation protein 1 (MALT1), and ADAMTS16 [25,34,35].Activation of NF-κB pathway could trigger NF-κB-dependent gene expression and play an essential role in cancers [36].Herein, we observed that the upregulation of DDX60 can significantly increase the p-p65 protein level in HNSCC cells.However, the level of p-p65 reduced significantly following the knockdown of DDX60.Furthermore, we discovered that DDX60 regulated the IFI27 expression by activating the NF-κB pathway in HNSCC cells.These results indicated that DDX60 was another activator of the NF-κB pathway, which could lead to IFI27 expression in HNSCC cells.
Our study found that DDX60 has better sensitivity in HNSCC.It is concluded that DDX60 has good diagnostic efficacy in patients with HNSCC, and the results suggest that DDX60 protein can be used as a potential biomarker and therapeutic target for HNSCC.However, due to research limitations, such as sample size or potential biases, our research still has some shortcomings, and we hope that subsequent studies can further determine the above results by expanding the sample size and classification research.However, our study provides a basis for further research on the function and mechanism of DDX60 in HNSCC.

Conclusion
We found that DDX60, as an oncogene in HNSCC cells, can activate the NF-κB pathway, thereby increasing the expression of IFI27.Our results provided an experimental basis for understanding the tumorigenic mechanism of DDX60 associated with HNSCC.The results can potentially help identify new HNSCC biomarkers and targets for targeted therapy.work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity.

Fig. 4 .
Fig. 4. DDX60 was positively corrected with interferon alpha inducible protein 27 (IFI27) in HNSCC cells.(A) The relationship between DDX60 and the mRNA level of IFI27 was established by the GEPIA database.(B) IFI27 mRNA levels in HNSCC tissues and control tissues were predicted utilizing data from the GEPIA database.(C) A correlation between IFI27 mRNA levels and HNSCC patient survival was predicted using the GEPIA database.(D) IFI27 mRNA level was measured by SQ RT-PCR in 30 matched pairs of HNSCC and paraneoplastic tissues.(E) Analysis of the correlation between DDX60 and IFI27 according to their mRNA levels in 30 pairs of HNSCC and its paraneoplastic tissues detected by SQ RT-PCR.(F) The mRNA levels of DDX60 and IFI27 in CAL27 and SAS of HNSCC cells were detected by SQ RT-PCR.(G) The expression levels of DDX60 and IFI27 in CAL27 and SAS of HNSCC cells were detected by western blot.(H,I) Quantification of DDX60 (H) and IFI27 (I) protein levels in (G).*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.TPM, transcript per million.