DDX1 is a prognostic biomarker and correlates with immune infiltrations in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is known to be the fifth most common cancer and is the third cause of cancer-related mortalities worldwide. Thus, it is considered a huge threat to people’s health [1]. Surgical resection and liver transplantation are the most suitable methods for treatment of early-stage HCC. However, for non-resectable patients, radiofrequency ablation (RFA) and stereotactic body radiation therapy (SBRT) are deemed as first-line local treatment options [2, 3]. Nonetheless, many patients with HCC treated with resection or local ablation show early relapse after treatment [4]. In the past decade, immunotherapy has become popular for successfully treating various cancers [5‐8]. Interestingly, cumulative evidence revealed that immune-mediated mechanisms are deeply involved in the progression of HCC [9]. This suggests the great potential of immune-based therapies for treating patients with HCC. However, few studies report only satisfactory immunotherapy efficacy for HCC. The failure of immunotherapy could be attributed to the complex composition of the tumor microenvironment (TME). Thus, it is important to understand the tumor immune microenvironment (TIME) for identifying pivotal therapeutic targets and prognostic biomarkers for HCC.

DDX1 is a member of the DEAD-box RNA helicase family and participates in various biological processes, such as mRNA translation, tRNA splicing, rRNA processing, microRNA maturation, and repair of DNA double-strand breaks (DSBs) [10‐14]. In addition to its role in transcriptional regulation, a recent study reported that DDX1 interacts with eIF3a and eIF4b to decrease insulin translation [15]. Furthermore, some studies found that DDX1 can inhibit viral replication [16, 17]. Moreover, researchers have found that DDX1 promotes tumorigenesis in various carcinomas, such as retinoblastoma, neuroblastoma, testicular carcinoma, colorectal cancer, and breast cancer [18‐21]. Nevertheless, the underlying mechanism of DDX1 in the progression of HCC remains obscure. In this research, we tried to revealed the potential role of DDX1 in the TIME of HCC.

The DEAD-box RNA helicase family is known to play critical roles in various RNA metabolic processes. Recent studies indicate that DDX members are dysregulated in multiple cancers and function as key players in tumor progression [30]. Hu et al. identified that DDX51 regulates cellular proliferation in esophageal squamous cell carcinoma through the PI3K/AKT/mTOR pathway [31]. Jiang et al. proved that knock-down of DDX46 caused a significant reduction in cell invasion and migration in osteosarcoma [32]. DDX1 activates the transcription of 12p stem cell genes in testicular tumorigenesis [19]. Tanaka et al. proved the role of DDX1 in promoting colorectal tumorigenesis in vitro and in vivo [20]. Researchers have proved that high DDX1 expression is associated with improvement in local control, distant metastatic-free survival, and OS when compared with low DDX1 expression in node-negative and early-stage patients with breast cancer [33]. However, the role of DDX1 in HCC is not yet determined. Therefore, in this study, we focused on determining the potential prognostic value of DDX1 in HCC.

We acquired HCC samples from the TCGA database to explore the role of DDX1 in HCC progression. The results reveal that high DDX1 expression is associated with a poor prognosis of HCC. On analyzing the relationship between DDX1 and clinicopathologic features we found that DDX1 may serve as an adverse prognostic factor in HCC. Patients with high DDX1 expression may present an advanced T stage, histologic grade, and pathological stage.

According to molecular profiles and clinical outcomes, HCC has a highly heterogeneous nature, which present a formidable challenge to an accurate diagnosis and treatment [34]. Luckily, previous studies have proved that molecular subtype stratification could overcome the hurdles caused by tumor heterogeneity [35, 36]. In this research, 240 HCC samples downloaded from ICGC dataset were divided into high and low DDX1-expression subgroups, the KM survival analysis revealed that patients with high levels of DDX1 are at higher risk of suffering a poor prognosis. This results were consistent with the findings from TCGA dataset.

As liver carcinoma is typically associated with high malignancy, it is usually diagnosed at a late stage, posing a challenge for radical surgery. In order to improve the outcome of HCC, early diagnosis and treatment are crucial. As we known, tumorigenesis has been demonstrated to result from multiple gene mutations, patients with gene mutations are known to have a poor prognosis, In this research,we accessed the alteration percentage of DDX1 in HCC. After analyzing three independent datasets, we found the percentage of DDX1 alteration to be 1.9%. Furthermore, no significant differences was observed in OS between the mutation and unmutation DDX1 groups. In addition to gene mutations, numerous epigenetic changes, such as DNA methylation and histone modifications, contribute to tumor development [37‐39]. Nowadays, since DNA methylation is tissue-specific and stable, detecting abnormal DNA methylation in liquid biopsies has been shown to be a promising biomarker for cancer diagnosis [40, 41]. A previous study indicated that HCC is caused by DNA methylation [42]. For example, Kuo et al. found that patients with higher IRAK3 methylation had worse prognosis [43]. However, no study has examined the relationship between DDX1 methylation and the oncologic outcome of HCC. Hence, in this study, we carried out methylation analysis and evaluated the correlation of methylation level with prognosis. We identified six CpG sites and two of them with hypermethylation were associated with poor prognosis. Besides, a previous study demonstrated that the hypomethylation status of oncogenes could also promote tumor development [44]. In this study, using UALCAN we found that HCC tissues had lower levels of DDX1 promoter methylation than normal tissues (P < 0.05). Further analysis indicated that the high tumor grade was linked to low promoter methylation levels. Thus, DDX1 methylation examination has the potential to be developed as a screening tool for predicting tumor status and progression; however, further in vivo and in vitro experiments are needed.

TME is a complicated assembly of the tumor, immune, stromal, and extracellular components [45]. Previous studies revealed that TME facilitates the progression of HCC, thus indicating that it could be exploited as a potential therapeutic target [46]. The importance of immune cell infiltration in the TME has been recognized for various solid tumors [47, 48]. In this study, we determined the association between DDX1 expression and immune cell infiltration in HCC through the TIMER database. The results revealed that DDX1 is positively associated with dendritic cells, B cells, macrophages, and T cells. In addition, we verified the positive correlation between DDX1 expression and immune cell infiltration using the QUANTISEQ algorithm. M2 macrophages and Tregs were higher in the high DDX1 expression group compared with the low DDX1 expression group. Several studies indicate that macrophages are essential components of the TME and play key roles in tumor progression [49]. Liu et al. [50] found that M2 macrophages target miR-149-5p/MMP9 signaling pathway thereby facilitating HCC progression. Yeung et al. demonstrated that M2 macrophages are related to the adverse prognosis of liver cancer and promote HCC invasion by promoting Epithelial mesenchymal transformation(EMT) [51]. Another research revealed that M2-polarized macrophages promote EMT of HCC cells and accelerate tumor progression through the TLR4/STAT3 signaling pathway [52]. Similarly, Tregs are also critical components of TME. Jiang et al. proved that Tregs are closely associated with the prognosis of HCC. They secrete TGF-β1 which triggers EMT, thereby enhancing tumor invasiveness [53]. Shen et al. [54] put forth that TGF-β could drive Treg cell polarization to promote the progression of HCC. These results suggest that DDX1 plays a vital role in the TIME of HCC. However, additional experimental and theoretical studies are needed to validate the relationship between DDX1 and tumor infiltration.

To explore the potential biological functions of DDX1, we identified the DEGs in HCC samples and conducted GO and KEGG enrichment analyses. The results showed that DDX1 was mainly related to cell cycle and RNA editing. In addition, since DEAD box proteins function by interacting with other proteins, they are susceptible to being regulated by their partners and their microenvironment [55]. Hence, using STRING and GEPIA database, we extracted 50 DDX1-binding proteins and the top 100 DDX1-related genes. Intersection analysis of the two groups identified HNRNPU, TARDBP, and HNRNPK as the common hits. TARDBP is an RNA-binding protein involved in the cell cycle of HCC tumor cells, and its expression level is related to an advanced stage and high grade of HCC [56]. The CDK2 protein is known as an essential role in cell cycle regulation [57]. Liang et al. have found HNRNPU enhances CDK2 transcription, thereby promoting HCC development [58]. GSEA analysis also identified significant KEGG pathways associated with the cell cycle.

In summary, this study is the first to identify DDX1 as a diagnostic and prognostic biomarker of HCC. The results indicate that DDX1 has an important role in TIME and is involved in the regulation of the cell cycle of HCC. Therefore, targeted DDX1 therapy is a potential treatment strategy for patients with HCC.