LncRNA ELF3-AS1 is a Prognostic Biomarker and Correlated with Immune Infiltrates in Hepatocellular Carcinoma

Background Expression of long noncoding RNA (lncRNA) ELF3 antisense RNA 1 (ELF3-AS1) is observed in some cancers, while its role in hepatocellular carcinoma (HCC) is unclear. The study aimed to investigate the relationship between ELF3-AS1 and HCC based on database, bioinformatics, and statistical analysis. Methods In this study, Kruskal–Wallis test, Wilcoxon sign-rank test, logistic regression, Kaplan–Meier method, Cox regression analysis, gene set enrichment analysis (GSEA), and immunoinfiltration analysis were used to assess the relationship between ELF3-AS1 expression and clinical characteristics of HCC patients, the relationship between ELF3-AS1 expression and prognosis of HCC patients, and the possible functions of ELF3-AS1 in HCC. Results High expression of ELF3-AS1 in patients with HCC was related to T stage (P < 0.001), gender (P = 0.006), residual tumor (P = 0.008), histologic grade (P < 0.001), adjacent hepatic tissue inflammation (P = 0.011), AFP (P < 0.001), and vascular invasion (P = 0.028). High ELF3-AS1 expression was associated with poor overall survival (OS) (P = 0.001) and DSS (P = 0.047). ELF3-AS1 expression (P = 0.011) was independently correlated with OS in HCC patients. In the high ELF3-AS1 expression group, GPCR-radioligand binding, M phase, Class A/1 (rhodopsin-like receptors), cell cycle checkpoints, translation, mitotic metaphase and anaphase, signaling by robo receptors, keratinization, and rRNA processing were differentially enriched by GESA. ELF3-AS1 expression was associated with immune infiltrating cells. Conclusions ELF3-AS1 expression was associated with poor prognosis in HCC. ELF3-AS1 expression was significantly associated with immune infiltration. ELF3-AS1 is a promising biomarker that can be used for the diagnosis and prognosis of HCC.


Introduction
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths in the world [1]. In the last few years, more than 700,000 people have died each year from HCC, and this number is increasing every year [2]. HCC is a complex neoplastic disease that is highly aggressive, with a pathology that includes changes in tumor cell behavior and the vascular system [3]. Nowadays, surgery is only effective for patients with early HCC [4]. Most patients are diagnosed at an advanced stage, and radiotherapy and chemotherapy are not effective [4]. e high recurrence and metastasis rates of HCC reduce patient survival [5]. Despite some advances in surgical treatment and neoadjuvant therapy, the prognosis of HCC is poor [6]. e lack of useful markers makes it difficult for clinicians to predict the clinical outcome of patients with HCC. erefore, new markers need to be explored to identify HCC patients with poor prognosis. e length of long noncoding RNAs (lncRNAs) is over 200 bp [7]. lncRNAs play a key role in the formation of cancer [8]. Growing evidence for the involvement of aberrant lncRNAs in the development and progression of HCC [4,6,9,10]. erefore, screening for lncRNAs that are clinically relevant to HCC is important for the early diagnosis and effective treatment of HCC. e lncRNA ELF3 antisense RNA 1 (ELF3-AS1) is the antisense transcript for ELF3. ELF3-AS1 was used as an oncogene in bladder cancer (BLCA) [11]. ELF3-AS1 is upregulated in oral squamous cell carcinoma (OSCC) [12]. ELF3-AS1 is upregulated in osteosarcoma (OS) [13]. ELF3-AS1 is upregulated in nonsmall cell lung cancer (NSCLC) [14]. ELF3-AS1 is a prognostic marker and potential therapeutic target for gliomas [15]. However, the correlation between ELF3-AS1 and HCC has not been studied.
In this study, we compared the differences in ELF3-AS1 expression between HCC tumor tissues and normal samples, explored the correlation between ELF3-AS1 expression and clinical characteristics of HCC, and assessed the prognostic value of ELF3-AS1 in HCC. For ELF3-AS1 high and low expression groups, genomic enrichment analysis (GSEA) was performed to explore the possible functions of ELF3-AS1. e possible function of ELF3-AS1 in HCC was explored by immune infiltration analysis. e study may provide new directions for the development of diagnostic and therapeutic strategies for HCC.  [20]. Immunocell algorithm is ssGSEA (algorithm built into the GSVA package). Molecule is ELF3-AS1. Immune cells are 24 immune cells. Data are RNAseq data and clinical data in level 3 HTSeq-FPKM format from the TCGA HCC project. Data filtering is removal of paracancerous tissue. Expression profile data conversion included FPKM format RNAseq data converted to TPM format and log2 transformed for analysis. Other data included markers for 24 immune cells obtained from an article [21]. Table 1 e albumin included 69 (<3.5) (23%) and 231 (≥3.5) (77%). e AFP included 215 (≤400) (76.8%) and 65 (>400) (23.2%). e prothrombin time included 208 (≤4) (70%) and 89 (>4) (30%). e Child-Pugh grade included 219 A (90.9%), 21 B (8.7%), and 1 C (0.4%). e fibrosis Ishak score included 75 0 (34.9%), 31 1/2 (14.4%), 28 3/4 (13%), and 81 5/6 (37.7%). e vascular invasion included 208 no (65.4%) and 110 yes (34.6%). Figure 1(a), ELF3-AS1 was highly expressed in HCC tissues (0.978 ± 0.061 vs. 2.381 ± 0.052, P < 0.001). As shown in Figure 1(b), LF3-AS1 was highly expressed in HCC tissues (P < 0.001), based on 50 HCC tissues and their matched normal liver tissues. As shown in Figure 1(c), the AUC of ELF3-AS1 was 0.904. As shown in Table 2, clinical and gene expression data were collected. As shown in Table 2, the P value of T stage is 0.002, the P value of pathologic stage is 0.003, the P value of gender is 0.008, the P value of race is 0.037, the P value of residual tumor is P � 0.004, the P value of histologic grade is <0.001, the P value of adjacent hepatic tissue inflammation is 0.025, the P value of AFP is <0.001, and the P value of vascular invasion is 0.037. As shown in Figure 2 and Table 3, the P value of T stage is <0.001, the P value of gender is 0.006, the P value of residual tumor is 0.008, the P value of histologic grade is <0.001, the P value of adjacent hepatic tissue inflammation is 0.011, the P value of AFP is <0.001, and the P value of vascular invasion is 0.028. Figure 3, the expression of ELF3-AS1 was positively correlated with poor OS (P � 0.001), diseasespecific survival (DSS) (P � 0.047) of HCC patients. As shown in Table 4, the results of univariate analysis showed that high ELF3-AS1 expression levels (P < 0.001) were associated with T stage (P < 0.001), pathologic stage (P < 0.001), and tumor status (P < 0.001).

Relationship between ELF3-AS1 and Survival of HCC Patients. As shown in
e results of multivariate analysis showed that ELF3-AS1 expression (P � 0.011), pathologic stage (P � 0.013), age (P � 0.013), and tumor status (P � 0.005) were independently correlated with OS in multivariate analysis. e results suggested that increased expression of ELF3-AS1 was associated with poor OS.

ELF3-AS1-Related Pathways.
A dataset of 191 significant differences was enriched in ELF3-AS1 low expression phenotypes. As shown in Table 5 and Figure 4, the top 9 low P-value datasets include GPCR-radioligand binding, M phase, Class A/1 (rhodopsin-like receptors), cell cycle checkpoints, translation, mitotic metaphase and anaphase, signaling by robo receptors, keratinization, and rRNA processing.

Discussion
LncRNAs play a key role in tumorigenesis and progression [22]. ZNF385D-AS2 may be a useful biomarker for prognosis in patients with HCC [9]. High LUCAT1 expression is an independent prognostic factor for HCC [6]. F11-AS1 may serve as a therapeutic target for HCC [4]. GIHCG is a biomarker that can be used to predict the prognosis of patients with HCC [10]. erefore, the study of lncRNAs as new HCC biomarkers and therapeutic targets is very important. ELF3-AS1 is upregulated in OSCC, OS, and NSCLC [12][13][14]. In this study, high ELF3-AS1 expression in HCC was associated with the characteristics including T stage, gender, residual tumor, histologic grade, adjacent hepatic tissue inflammation, AFP, and vascular invasion. High ELF3-AS1 expression was associated with poor OS and DSS.

ELF3-AS1 expression was independently correlated with OS in HCC patients.
ELF3-AS1 mediated BLCA tumorigenesis through enhanced ELF3-AS1/KLF8 signaling [11]. ELF3-AS1 may be regulating GLUT1 to promote the proliferation of OSCC cells [12]. ELF3-AS1 may promote OS cell proliferation by upregulating KLF12 through methylation of the miR-205 gene [13]. ELF3-AS1 promotes cancer cell invasion and migration through downregulation of miR-212 by methylation in NSCLC [14]. ELF3-AS1 is not only an important prognostic marker, but also a potential therapeutic target for glioma [15]. In this study, based on GESA, ELF3-AS1 was related to pathways including GPCR-radioligand binding, M phase, Class A/1 (rhodopsin-like receptors), cell cycle checkpoints, translation, mitotic metaphase and anaphase, signaling by robo receptors, keratinization, and rRNA processing. Immune infiltrating cells in HCC are currently a hot topic, and a major understanding of immune infiltrating cells can support the development of new and emerging immunotherapies. e development and progression of HCC are associated with a unique immune response profile of the liver microenvironment, in which CD4+CD25+Foxp3 regulatory T cells (Tregs) play a key role through their immunosuppressive effects [23]. Immunotherapy enhances the immune response against hepatocellular carcinoma by blocking Treg activity [23]. In this study, we explored the relationship between ELF3-AS1 expression in HCC and various immune infiltrations. ELF3-AS1 expression was associated with infiltration of CD8 T cells, cytotoxic cells, eosinophils, neutrophils, NK cells, Tcm, 17 cells, Treg, Despite some limitations, this is an exploratory study to investigate the relationship between ELF3-AS1 and HCC.

Conclusion
ELF3-AS1 was highly expressed in HCC relative to normal tissue and related to poor OS and DSS. ELF3-AS1 might participate in the development of HCC by pathways including GPCR-radioligand binding, M phase, Class A/1 (rhodopsinlike receptors), cell cycle checkpoints, translation, mitotic metaphase and anaphase, signaling by robo receptors, keratinization, and rRNA processing. ELF3-AS1 expression was associated with immune infiltrating cells. is study investigated the role of ELF3-AS1 in HCC and provided a promising biomarker for the diagnosis and prognosis of HCC.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest relevant to this study.

Authors' Contributions
TC and YP conceived and designed the study. TC, CZ, XW, and YP analyzed the data. TC and YP wrote and revised the paper. All authors read and approved the final manuscript.