Bioinformatics Analysis and Validation of the Role of Lnc-RAB11B-AS1 in the Development and Prognosis of Hepatocellular Carcinoma

Lnc-RAB11B-AS1 is reported to be dysregulated in several types of cancers and can function as both an oncogene and tumor suppressor gene. To evaluate the potential role of lnc-RAB11B-AS1 in hepatocellular carcinoma (HCC), we investigated and evaluated its expression in HCC based on the data mining of a series of public databases, including TCGA, GEO, ICGC, HPA, DAVID, cBioPortal, GeneMIANA, TIMER, and ENCORI. The data showed downregulation of lnc-RAB11B-AS1 in HCC and was accompanied by the synchronous downregulation of the targeted RAB11B mRNA and its protein. Low expression of lnc-RAB11B-AS1 was associated with shorter overall survival (OS) and disease-free survival (DFS) of HCC patients, PD1/PD-L1 was correlated with low expression of RAB11B. Furthermore, Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed a correlation between immune cell change and non-alcoholic fatty liver disease. The above findings revealed that lnc-RAB11B-AS1 was down-regulated in HCC and closely associated with the clinical stage of the HCC patients, suggesting that lnc-RAB11B-AS1 could be a possible predictor for HCC and a potential new therapeutic target for the treatment of HCC.


Introduction
Hepatocellular carcinoma (HCC) is a common malignant tumor that occurs in the digestive tract with high malignancy, rapid progression, high recurrence rate, and poor prognosis [1]. With approximately 906,000 new cases and 830,000 deaths recorded each year, HCC is the sixth leading cause of cancer incidence worldwide, and the number of associated deaths was ranked third after gastric cancer and esophageal cancer in 2020 [2]. Notably, approximately half of newly diagnosed HCC cases globally occur in China; and as a result, HCC has become the fourth most common malignant tumor and the second most lethal cause of cancer in China, which seriously increases the disease burden [3]. The known risk factors include viral infection, poisonous substances, abnormal metabolism, liver cirrhosis, and smoking [4]. These factors are likely to lead to genetic and epigenetic changes, breaking the internal balance between proto-oncogenes and tumor suppressor genes, and thus resulting in HCC [5]. Research has shown that the sensitivity and specificity of early diagnosis of HCC remain low, and most HCC patients are diagnosed at the middle or even at the late stages. These patients often have severe liver cirrhosis, and it is not suitable for

Data Acquisition and Preprocessing
We searched and downloaded the transcriptomic sequencing data and detailed clinical data of HCC patients from TCGA (https://portal.gdc.cancer.gov/repository, accessed on 3 May 2022) database [18]. A total of 369 HCC cases and 50 normal cases were included in our study after duplicate information was deleted. Among 347 HCC patients with known treatment statuses, 239 patients did not receive treatment (4 of them had another new primary tumor), 18 patients were treated, and the remaining 90 cases were recurrent (13 of them had relapses of other tumors, such as lung and bone cancer). Clinicopathological data included age, sex, clinical stage, histological grade, TNM grade, and survival time. Furthermore, two microarray gene expression datasets, GSE144269 and GSE84402, were obtained from GEO (https://www.ncbi.nlm.nih.gov/geo/, accessed on 5 May 2022) database [19]. It appeared that a great number of lncRNAs might be involved in the occurrence of diseases by affecting the expression and regulation of antisense genes [20,21]. Therefore, the correlation between the expression levels of lnc-RAB11B-AS1 and its protein-coding gene RAB11B was explored in HCC.

Immunohistochemistry (IHC) Assay of RAB11B Protein in HCC Tissues
To verify the results in TCGA and GEO, an IHC assay was performed using HCC tissue microarray purchased from Shanghai Outdo Biotech Company (lot NO. hlivh180su15; Shanghai, China). All experiments followed the manufacturer's instructions. A total of 90 histologically confirmed HCC cases were enrolled in our study, which contain 90 pairs of cancer tissues and adjacent non-cancerous tissues. The protocol was approved by the ethics committee of Shanghai Outdo Biotech Co., Ltd (The approval code of the ethical committee is SHYJS-CP-1901001). Furthermore, these patients underwent surgery from June 2007 to October 2008 and were followed up for 3-5 years with the recurrence time and multiple biochemical indexes recorded, such as TNM stage, pathological grade, and survival time.

Protein-Protein Interactions of RAB11B
GeneMANIA (http://genemania.org/, accessed on 11 June 2022) [22] can be used to search large and publicly available biological datasets for protein-protein, protein-DNA, and genetic interactions of related genes. The default species "Homo sapiens" was selected, and we constructed PPI networks by GeneMANIA online to analyze the interaction between RAB11B and other functional proteins.

Human Protein Atlas (HPA)
HPA (https://www.proteinatlas.org, accessed on 4 August 2022) [23] is a public database containing a large-scale human protein map that not only includes tumor tissues but also covers the protein expression of normal tissues. The new section of this database, "single cell type atlas", includes single-cell RNA sequencing data from 13 human tissues and a total of 192 cell types. It is also available for retrieving genes from different cell types, as well as clustered Uniform Manifold Approximation and Projection plots and expression histograms. On this basis, we explored the expression pattern of RAB11B and other marker genes of cluster cells in different single-cell types. To facilitate the comparison, the total read counts for all genes were calculated and normalized to transcripts per million (nTPM) protein-coding genes for each of the single-cell clusters.

Screening of lnc-RAB11B-AS1 Co-Expressed Genes in HCC and Functional Enrichment Analysis
The main features of the DAVID (https://david.ncifcrf.gov, accessed on 11 June 2022) [24] are functional annotation and information links, which can help investigators identify the pathways that are crucial to biological processes. Using GEPIA 2.0 (http://gepia2.cancer-pku.cn/#index, accessed on 11 June 2022) database [25], we searched for the top 200 genes highly correlated with lnc-RAB11B-AS1. Next, these co-expressed genes were imported to the DAVID for GO and KEGG enrichment analysis.

GSEA Analysis
To analyze the molecular signal pathways potentially regulated by lnc-RAB11B-AS1, GSEA was performed combined with the raw expression profiles from TCGA, as implemented using the "cluserProfiler" R package from the Molecular Signature Database (MSigDB)_v7.0_GMTs and the reference dataset "c5.all.v7.0.entrez.gmt". Based on a 50% cut-off value, the differentially expressed genes (DEGs) between the lnc-RAB11B-AS1 high-and low-expression groups were determined at the transcriptomic level. Enrichment results with normalized p < 0.05 and a false discovery rate of <0.25 were considered statistically significant.

2.
7. An Association between lnc-RAB11B-AS1, RAB11B, and Tumor Immune Cell Infiltration TIMER (https://cistrome.shinyapps.io/timer/, accessed on 18 June 2022) [26] is an integrated database that can be applied to systematically analyze the molecular signatures of tumor-immune interactions, and the levels of six tumor-infiltrating immune subsets can be estimated by the deconvolution method to explore the relationship between the gene expression, mutation, and immune cell infiltrating abundance. Using TIMER, we retrieved a matrix of immune cell infiltration levels in TCGA-LIHC samples, including B cells, CD4 + T cells, CD8 + T cells, neutrophils, macrophages, and dendritic cells. Correlations between the expression of these genes and various tumor-infiltrating immune cells were investigated using the Spearman test, with p < 0.05. Furthermore, xCell integrates the advantages of ssGSEA and can calculate the abundance fraction of 64 immune cell types. We assessed the immune cell infiltration scores for each patient in HCC using normalized gene expression profiles. Immune infiltration scores significantly correlated with gene expression were identified using the "corr.test" function of the R package "psych" (version 2.1.6).

Alternation of lnc-RAB11B-AS1 and RAB11B in HCC
The occurrence and development of malignant tumors are influenced by mutations in specific regulatory genes. The cBioportal database (http://www.cbioportal.org, accessed on 3 August 2022) [27] contains multiple databases with a total of 283 large tumorrelated genomic and proteomic items and provides visualization tools for the analysis of cancer-related genetic data. Following the conditions of data filtering and extraction, we investigated the type and frequency of lnc-RAB11B-AS1 and RAB11B mutations in HCC.

Correlation between Methylation and lnc-RAB11B-AS1 Expression
Aberrant methylation can be a powerful mechanism leading to the loss of gene function; therefore, we analyzed the relationship between lnc-RAB11B-AS1 expression and methylation. We downloaded the methylation data, phenotype information, and clinical data of HCC patients from UCSC Xena (https://xenabrowser.net/datapages/, accessed on 1 August 2022) [28], and 50 HCC patients with "normal-tumor" pairings were selected for further analysis.

Drug Susceptibility Analysis
CellMiner (https://discover.nci.nih.gov/cellminer/CellMiner, accessed on 18 July 2022) [29] database facilitates researchers to integrate and analyze the molecular and pharmacological data from the NCI-60 cancer cell line, which is also used by the National Cancer Institute's Developmental Therapeutics Program to screen more than 100,000 compounds and natural products. We directly selected processed data sets from this database, including RNA-Seq and compound activity (DTP NCI-60). A sensitivity analysis of lnc-RAB11B-AS1 and related drugs was then conducted and we showed scatter plots of the top nine drugs with the most statistical significance.

Prediction of Potential Candidate miRNAs
We used miRWalk (http://mirwalk.umm.uni-heidelberg.de/, accessed on 8 July 2022) [30] and ENCORI (https://starbase.sysu.edu.cn/, accessed on 8 July 2022) [31] database to predict the miRNAs targeted by lnc-RAB11B-AS1 and RAB11B; thus, the same miRNA binding sites of the two genes could be found. To verify the expression pattern and prognosis of candidate miRNAs, the RNA-Seq data of 232 HCC patients and their corresponding clinical information were retrieved from the International Cancer Genome Consortium (ICGC) (https://dcc.icgc.org/, accessed on 10 July 2022) [32], which collected data on 50 different cancer types including abnormal gene expression, somatic mutations, and epigenetic modifications.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism-8.00 and R-4.1.2. To determine the difference in lnc-RAB11B-AS1 expression between HCC and normal groups, we used Wilcoxon rank-sum tests via GraphPad Prism. The relationships between ln-RAB11B-AS1 and clinicopathological features were assessed based on the chi-squared test, which revealed the prognostic significance of lnc-RAB11B-AS1 for HCC. The Kaplan-Meier method was employed for survival analysis using the log-rank test in group comparisons, while the Cox regression model was utilized to investigate the relationship between clinicopathological features and survival. Hazard Ratio (HR) values and 95% CIs were also calculated. All p values were two-tailed, and p < 0.05 indicated statistical significance.

Lnc-RAB11B-AS1 Was Markedly Low Expressed and Positively Correlated with RAB11B in HCC
To explore the expression status of lnc-RAB11B-AS1 and RAB11B in HCC, transcriptome data of the above genes of HCC were retrieved from TCGA. The expression levels of lnc-RAB11B-AS1 and RAB11B were distinctly lower in the single 369 HCC tissues than in the 50 normal tissues (p = 0.032, p = 0.042, respectively, Figure 1A,D). Analysis of GSE144269 and GSE84402 datasets also verified that lnc-RAB11B-AS1 and RAB11B were down-regulated in HCC tissues compared to normal liver tissues (p < 0.001, p = 0.025, respectively, Figure 1B,C,E,F). Furthermore, lnc-RAB11B-AS1 exhibited a positive relationship with RAB11B in TCGA-LIHC (R = 0.54, p < 0.001, Figure 1G). As depicted in Figure 1H,I, similar association patterns between lnc-RAB11B-AS1 and RAB11B were observed in GSE144269 and GSE84402, but only the former showed statistical significance (R = 0.433, p < 0.01; R = 0.521, p = 0.059).

The Effects of Overexpressed lnc-RAB11B-AS1 and RAB11B on Clinicopathological Characteristics
As shown in Table 1, there were more men in the high expression group than women (χ 2 = 9.908, p = 0.002). Highly expressed lnc-RAB11B-AS1 in HCC was associated with a lower level of clinical stage (χ 2 = 4.054, p = 0.044), N stage (χ 2 = 4.605, p = 0.032), and histologic stage (χ 2 = 4.416, p = 0.036). The values of AFP and T-Bil were notably different between lnc-RAB11B-AS1 high and low groups (p = 0.018, p = 0.034, respectively). In addition, we found no significant association between lnc-RAB11B-AS1 expression and age, race, history of hepatocarcinoma risk factors, T stage, M stage, treatment or treatment type, cancer first-degree relative, etc. Therefore, we speculate that high lnc-RAB11B-AS1 expression indicates a better clinical outcome in HCC patients. As for its sense-cognate gene RAB11B shown in Table 2, it was only negatively correlated with the degree of histological grade (χ 2 = 5.920, p = 0.015). In addition, the expression patterns of lnc-RAB11B-AS1 and RAB11B in HCC patients with different clinical stages and histologic grades were visualized through the R package "gg-plot". The histologic grade and clinical stage tended to be higher in patients with a lower expression of the two genes (Figure 2A

The Effects of Overexpressed lnc-RAB11B-AS1 and RAB11B on Clinicopathological Characteristics
As shown in Table 1, there were more men in the high expression group than women (χ 2 = 9.908, p = 0.002). Highly expressed lnc-RAB11B-AS1 in HCC was associated with a lower level of clinical stage (χ 2 = 4.054, p = 0.044), N stage (χ 2 = 4.605, p = 0.032), and histologic stage (χ 2 = 4.416, p = 0.036). The values of AFP and T-Bil were notably different between lnc-RAB11B-AS1 high and low groups (p = 0.018, p = 0.034, respectively). In addition, we found no significant association between lnc-RAB11B-AS1 expression and age, race, history of hepatocarcinoma risk factors, T stage, M stage, treatment or treatment type, cancer first-degree relative, etc. Therefore, we speculate that high lnc-RAB11B-AS1 expression indicates a better clinical outcome in HCC patients. As for its sense-cognate gene RAB11B shown in Table 2, it was only negatively correlated with the degree of histological grade (χ 2 = 5.920, p = 0.015). In addition, the expression patterns of lnc-RAB11B-AS1 and RAB11B in HCC patients with different clinical stages and histologic grades were visualized through the R package "gg-plot". The histologic grade and clinical stage tended to be higher in patients with a lower expression of the two genes (

The Relationship between lnc-RAB11B-AS1 Expression and Prognosis of HCC Patients
The Kaplan-Meier analysis suggested that the upregulated expression of lnc-RAB11B-AS1 was markedly associated with better OS in HCC patients (p = 0.022, Figure 2E). OS was evidently reduced in patients with low RAB11B expression than in patients with high RAB11B expression (p = 0.0028, Figure 2F). As an independent validation dataset, the results of lnc-RAB11B-AS1 and RAB11B in GSE144269 uncovered a similar prognostic trend ( Figure 2G,H). HCC patients with decreased expression of the above two genes experienced a shorter survival time, but no statistical significance was observed (p = 0.12, p = 0.21, respectively) due to the deficiency of samples in the GEO data (n = 68). Furthermore, we evaluated the prognostic significance of RAB11B for HCC using the Kaplan-Meier Plotter database (https://kmplot.com/analysis/, accessed on 25 May 2022) [33]. As we expected, the prognostic data disclosed OS time, disease-specific survival (DSS) time, progression-free survival (PFS) time, and relapse-free survival (RFS) time was longer in HCC patients with a higher RAB11B expression, while those with a low RAB11B expression had a worse survival outcome (All p < 0.01, Figure 2I).

Cox Regression Model Analysis
As detailed in Table 3

External Validation of RAB11B Protein Expression and Clinicopathologic Features of HCC Patients
To verify our results at the protein level, the expression of RAB11B in 90 HCC tissue chips was detected using immunochemistry, and immunostaining was mainly located in the cytoplasm of HCC cells. As was shown in Figure 3A,B, based on the scoring criteria of cytoplasmic staining, the RAB11B expression at the protein level was strikingly decreased in HCC tissues compared with corresponding non-cancerous tissues, which is consistent with the predictive results in public databases. Next, we examined the relationship between RAB11B protein expression and clinicopathological characteristics of the 90 HCC patients (Table 4). We found a negative correlation between the RAB11B expression and the pathology grade, that is to say, HCC patients with a higher expression of RAB11B had a lower pathological grade (χ 2 = 15.691, p < 0.001). In addition, a higher proportion of patients with TB and GGT normal value was observed in the RAB11B high expression group compared to that of the low expression group (χ 2 = 4.107, p = 0.043; χ 2 = 3.903, p = 0.048). In the PD-L1 low expression group, there were more HCC patients with an RAB11B-high expression (χ 2 = 9.357, p = 0.002), and it showed a distinctly negative correlation between them. However, the remaining clinicopathological factors, including sex, age, clinical stage, TNM stage, recurrence, HBsAg, HBcAb, AntiHCV, ALT, AFP, and CTLA4 did not show any statistical significance. We applied a survival analysis to further assess the prognostic significance of RAB11B in HCC, and the results demonstrate that patients with a higher RAB11B expression showed longer OS and DFS, suggesting that RAB11B might also be a protective factor in the development of HCC ( Figure 3C,D).

RAB11B-Associated PPI Network
A total of 20 proteins that have been confirmed or predicted to interact with RAB11B were screened through the GeneMANIA database analysis, including GDI1, TBC1D14, EVI5, TBC1D8B, PCSK9, RAB11FIP1, MYO5B, BICDL1, RAB1B, RAB11A, and so on ( Figure 3E). Different colors of nodes represent different interaction relationships, and physical interactions indicate that the corresponding proteins were connected.

RAB11B Expression in Different Cells of HCC
As was shown in Figure 3F, single-cell data from HPA was visualized using UMAP. Each color represents a single cell cluster identified after clustering analysis, and each scatter represents a cell, which has been divided into a total of 16 kinds of cell groups in the liver. The results revealed that the mRNA level of RAB11B was higher in T-cells and hepatic stellate cells but was lower in kupffer cells and erythroid cells ( Figure 3G). In addition, it disclosed the expression pattern of marker genes in various cell clusters.

Lnc-RAB11B-AS1-Related Genes and Functional Enrichment Analysis
We explored the possible biological function of lnc-RAB11B-AS1 in the pathogenesis and progression of HCC. The lnc-RAB11B-AS1 co-expressed genes were submitted to the DAVID for analysis, and a functional annotation table was obtained. As shown in Figure 4A, the results were visualized using a chordal graph with different colors representing the relationships between related genes and various pathways. In GO Biological Processes, the above genes were mainly involved in mitochondrial ATP synthesis-coupled proton transport, aerobic respiration, mitochondrial electron transport, NADH to ubiquinone, hyaluronan metabolic processes, negative regulation of endopeptidase activity, and longchain fatty acid metabolic processes. In Cellular components, these genes were closely associated with mitochondrial inner membrane, blood microparticles, and extracellular exosomes. Molecular functions were primarily co-regulated with oxidoreductase activity and NADH dehydrogenase (ubiquinone) activity. Additionally, in KEGG analysis, 15 signaling pathways finally showed statistical significance, including in drug metabolismother enzymes, non-alcoholic fatty liver disease (NAFLD), and oxidative phosphorylation ( Figure 4B). The top KEGG enrichment pathway item "NAFLD" is detailed in Figure 4C.

Lnc-RAB11B-AS1-Related Signaling Pathways Obtained by GSEA
HCC patients were divided into two groups according to the expression of lnc-RAB11B-AS1 and approximately 1721 genes were identified after differential analysis using transcriptional profiles retrieved from TCGA, in which 612 DEGs were upregulated and 1109 were downregulated (adjusted p value < 0.05 and |Log2-FC| > 1). The volcano plot depicted in Figure 4D exhibits DEGs, highlighting three up-and downregulated genes with the smallest p-values. Then we performed GSEA between lnc-RAB11B-AS1 high and low groups. The results showed that ATP synthesis-coupled electron transport, axon development, cell adhesion, cell morphogenesis involved in differentiation, and other pathways related to cell-to-cell interactions were significantly enriched ( Figure 4E).

Correlation Analysis between lnc-RAB11B-AS1 and RAB11B Expression, and Tumor-Infiltrating Immune Cells in HCC
Elucidating the connection between gene expression and immune infiltration will provide new horizons for HCC immunotherapy in the future. As shown in Figure 5A Figure 5C. These data further demonstrated that the potential regulatory mechanism of lnc-RAB11B-AS1 and RAB11B in HCC might be brought into effect by influencing immune cell infiltration.
Cells 2022, 11, x FOR PEER REVIEW 5C. These data further demonstrated that the potential regulatory mechanism RAB11B-AS1 and RAB11B in HCC might be brought into effect by influencing i cell infiltration.

Alternation of lnc-RAB11B-AS1 and RAB11B in HCC
A dataset (TCGA, PanCancer Atlas) containing 372 samples with information on genetic alterations was selected for analysis from the cBioPortal database. The results suggested that the incidence of RAB11B mutation was 10% (37/372) while it was only 0.3% in lnc-RAB11B-AS1 ( Figure 5D). The DFS of HCC patients with RAB11B gene alteration tended to be lower than that of patients without gene alteration, but it showed a very slight trend toward significance (p = 0.286; Figure 5E). In addition, in the tumor subgroup analysis, the expression level of the mutation was associated with the histological grade in HCC (p = 0.027; Figure 5F). These findings suggested that the reduction in RAB11B mRNA might link to a poor prognosis for HCC but remains to be verified.

Correlation between mRNA Expression and Methylation of lnc-RAB11B-AS1
We downloaded the methylation signal value matrix of HCC patients from UCSC Xena, and finally obtained the data of 14 methylation sites located in the promoter region of lnc-RAB11B-AS1. Then, six methylation sites with statistical significance were screened through a comparison between 50 pairs of HCC tissues and normal tissues, including cg08667721, cg02020019, cg02868306, cg04663720, cg12028991, and cg04535340. As shown in Figure 5G, the methylation levels of these methylation regions in the tumor group were significantly higher than those in the normal group. In general, the hypermethylation of the promoter region is negatively correlated with gene expression, and the hypermethylation of the lnc-RAB11B-AS1 promoter region may lead to the reduction of transcriptional activity, resulting in its decreased expression in HCC. Moreover, we examined the relationship between lnc-RAB11B-AS1 methylation level and clinical stage in HCC through the "plots" section of the ciBioPortal database ( Figure 5H,I). The methylation level was found to be higher in advanced clinical stages, which was consistent with our previous findings that lnc-RAB11B-AS1 expression was negatively correlated with clinical stage.

Prediction of lnc-RAB11B-AS1 and RAB11B Targeted miRNAs
The Lnclocator database (http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/, accessed on 3 August 2022) [34] was used to predict the subcellular localization of lnc-RAB11B-AS1 and we found it was mainly localized in the cytosol ( Figure 6A). Numerous studies have demonstrated that lncRNAs can affect the post-transcriptional translation process of downstream target genes by interacting with miRNAs. Therefore, we sought to establish a ceRNA network to elucidate the underlying mechanism by which lnc-RAB11B-AS1 regulates RAB11B expression, thus affecting the initiation and development of HCC. Using miRWalk and ENCORI databases, we further performed a prediction of miRNAs targeting lnc-RAB11B-AS1 and RAB11B. The results showed that these two genes had five miRNAs identities in common: hsa-miR-4640-5p, hsa-miR-3918, hsa-miR-30e-5p, hsa-miR-30a-5p, and hsa-miR-4726-5p. Because of the limited information on these miRNAs in TCGA and GEO, we continued to perform data mining in the ICGC database. Hsa-miR-4726-5p expression in HCC tissues was observed to be strikingly higher than that in normal tissues, while the expression status of other miRNAs was the opposite or was not statistically significant ( Figure 6B). The survival curve detailed in Figure 6C revealed that HCC patients with high expression of hsa-miR-4726-5p had a worse prognosis, suggesting that it might be a potential hazard factor for HCC. Moreover, we found a negative relationship between hsa-miR-4726-5p and lnc-RAB11B-AS1 based on the ENCORI database ( Figure 6D). Furthermore, hsa-miR-4726-5p was negatively correlated with RAB11B ( Figure 6E). To summarize, we made an inference that lnc-RAB11B-AS1 functions as an antioncogene that positively regulates its adjacent gene RAB11B by sponging hsa-miR-4726-5p in HCC.

Correlation between lnc-RAB11B-AS1 Expression and Drug Sensitivity
To ensure the reliability of the analysis results, we selected drugs that have been clinically tested and FDA-approved for drug sensitivity analysis. The data revealed high-toweak positive connections between the expression of lnc-RAB11B-AS1 and nelarabine, hydroxyurea, pipobroman, thiotepa, chlorambucil, fluphenazine, triethylenemelamine, and uracil mustard. However, the sensitivity to dasatinib gradually increased when lnc-RAB11B-AS1 expression was elevated ( Figure 6F). Considering these findings, lnc-RAB11B-AS1 might be a novel marker for targeted therapies in HCC.

Correlation between lnc-RAB11B-AS1 Expression and Drug Sensitivity
To ensure the reliability of the analysis results, we selected drugs that have been clinically tested and FDA-approved for drug sensitivity analysis. The data revealed highto-weak positive connections between the expression of lnc-RAB11B-AS1 and nelarabine, hydroxyurea, pipobroman, thiotepa, chlorambucil, fluphenazine, triethylenemelamine, and uracil mustard. However, the sensitivity to dasatinib gradually increased when lnc-RAB11B-AS1 expression was elevated ( Figure 6F). Considering these findings, lnc-RAB11B-AS1 might be a novel marker for targeted therapies in HCC.

Discussion
Thus far, the clinical treatment of HCC has faced a huge bottleneck. Most patients have lost the opportunity for surgical treatment at the time of diagnosis, and chemotherapy remains the main treatment for patients with advanced HCC. With the technological advances in high-throughput sequencing, increasing numbers of functional lncRNAs have been gradually discovered in focused areas [35]. Numerous evidence indicates that lncRNAs are aberrantly expressed in HCC and can be involved in the regulation of initiation, development, and treatment response of HCC. Zhang et al. reported that lnc-SBF2-AS1 was highly expressed in HCC and could promote HCC metastasis by participating in the regulation of the EMY signaling pathway [36], while Yan et al. confirmed that stressresponsive lncRNA ROR knockdown enhanced chemotherapy-induced apoptosis and cytotoxicity [37]. These data all revealed that lncRNAs can not only directly regulate the response of HCC cells to treatment, but also affect the occurrence of HCC by participating in crucial signaling pathways. Lnc-RAB11B-AS1 is a member of the RAS oncogene family and was found to be dysregulated in several tumors, affecting their biological progress and even resistance to therapeutic agents [38]. As an oncogene, Niu and colleagues demonstrated that lnc-RAB11B-AS1 stimulated the development and growth of breast cancer cells in vitro, as well as tumor angiogenesis and distant metastasis of breast cancer, without affecting primary tumor growth in mice [39]. Li and colleagues found that lnc-RAB11B-AS1 is overexpressed in lung cancer tissues and is associated with poor prognosis in lung cancer [13]. Feng et al. detected the expression of lnc-RAB11B-AS1 in 83 gastric cancer tissues and normal tissues using qRT-PCR and found that upregulated lnc-RAB11B-AS1 in gastric cancer tissues was related to higher stage, lymph node metastasis, distant metastasis, and degree of tumor differentiation. Mechanistically, lnc-RAB11B-AS1 may inhibit the proliferation, migration and invasion of gastric carcinoma cells by up-regulating miR-628-3p and increasing the sensitivity to cisplatin [40]. Meanwhile, the carcinostatic action of lnc-RAB11B-AS1 was also investigated in colorectal cancer, endometrial cancer, and osteosarcoma by various experiments [17,41]. The association between lnc-RAB11B-AS1 and HCC progression remains unclear (and relevant studies on it have not yet resolved the matter). More importantly, it has been reported that lnc-RAB11B-AS1 plays a role in osteosarcoma and lung cancer by regulating the expression of its natural antisense transcript RAB11B. Hence, we sought to further explore the regulatory relationship between lnc-RAB11B-AS1 and RAB11B in HCC.
Using TCGA and GEO databases in this study, we found that the expression levels of lnc-RAB11B-AS1 and its sense-cognate gene RAB11B were decreased when compared with paired normal tissues in HCC. Then, an immunohistochemical assay was performed to validate the downregulation of RAB11B protein in HCC tissues. A highly positive relationship was observed between the expression of lnc-RAB11B-AS1 and RAB11B. According to a former study, visualization techniques and descriptive indicators can be used to reflect how survival differs with disease subtype, treatment status, and treatment practice patterns [42]. Through screening the clinicopathological information in the database and referring to relevant literature, we first differentiated HCC patients with different treatment statuses in TCGA, among which 249 were untreated, 18 were treated, 90 were recurrent, and 22 were unreported cases. Subsequently, median survival differences between patients with the above three known statuses were assessed. We found that the overall median survival times of untreated, treated, and recurrent HCC patients were 1.43 (IQR 0.05-2.01) years, 1.48 (IQR 0.10-2.43) years, and 2.43 (IQR 1.02-3.48) years, respectively. While in the ICGC database, we only obtained information on whether these HCC patients were treated and the corresponding treatment patterns (177 were untreated and 63 were treated with chemotherapy, surgery or other therapies). We found that the overall median survival of untreated patients was 2.05 years (IQR 1.39-3.21) and that of treated patients was 2.31 years (IQR 1.56-2.88). Patients diagnosed with recurrent HCC experienced longer survival times than untreated HCC patients, consistent with our hypothesis. Unfortunately, for the GSE144269 and GSE84402 dataset, even after reviewing the original text carefully, we have no information on the treatment status of HCC patients.
A correlation analysis of clinicopathological factors revealed that lnc-RAB11B-AS1 expression was markedly associated with histological grade, clinical stage, AFP, and T-Bil of HCC patients. Our experimental data disclosed a negative correlation between RAB11B expression and pathology grade, T-Bil, GGT, and PD-L1. T-Bil and GGT are both important indicators of liver function in patients and are combined with other objective parameters in clinical diagnosis, such as ALBI score, which is considered a gold standard for the diagnosis of HCC patients [43,44]. Abnormally upregulated T-Bil or GGT is often associated with the occurrence of hepatitis, obstructive jaundice, or cirrhosis. Therefore, lower RAB11B expression in HCC patients may indicate higher values of the above two indicators, which is closely related to the severity of the disease. Additionally, PD-1/PD-L1 is a hot topic in immunotherapy. PD-L1 is the main ligand of PD-1, which is expressed in various cancer cells and exhibits an inhibitory role in the anti-tumor effect of effector T cells, including in gastric, breast, and liver cancer [45]. A recent study reported that the expression of lnc-MIAT was positively correlated with the expression of immune checkpoint molecules in HCC, including PD-1, PD-L1, and CTLA4, influencing the immune microenvironment of HCC [46]. These studies demonstrated the pivotal role of lncRNAs in the tumor microenvironment. In contrast to the functions of the above genes, we found high RAB11B expression may lead to the downregulation of PD-L1, thus inhibiting the progression of HCC. The results of the survival analysis showed that the median survival time of patients with low expression of lnc-RAB11B-AS1 was approximately 45 months, which was less than that identified for patients with high lnc-RAB11B-AS1 expression, at 70 months. Additionally, elevated lnc-RAB11B-AS1 was demonstrated to be a protective factor for HCC in the multivariate Cox regression analysis. The above findings suggest that a low expression of lnc-RAB11B-AS1 indicates an adverse prognosis for HCC and might be a potential molecular marker for prognosis prediction of HCC patients.
Next, we investigated the potential biological function of lnc-RAB11B-AS1 in HCC. It has been shown to exert a carcinostatic action by affecting multiple signaling pathways. GO and KEGG pathway analyses revealed multiple signaling pathways, such as oxidoreductase activity, drug metabolism-other enzymes, NAFLD, and oxidative phosphorylation. NAFLD is one of the most serious risk factors of HCC, while lncRNAs are reported to play a crucial role in NAFLD. Park JG et al. reported that the degree of hepatic steatosis in NAFLD patients was inversely related to the level of lncRNA LeXis in liver tissues [47]. Critical items were identified in GSEA, such as axon development, cell adhesion, and cell morphogenesis. Kong et al. supported the view that the cytoskeletal proteins deregulated by hepatitis B virus X protein (HBx) played a role in cell morphogenesis, adhesion, migration, and proliferation [48]. It is possible that lnc-RAB11B-AS1 downregulation promotes HCC by similar mechanisms.
Immune-infiltrating cells are an important part of the tumor microenvironment. Studies provided evidence that changes in the number of immune infiltrating cells in tumors can positively or negatively affect the prognosis and immunotherapy efficacy of diseases [49]. The change in lnc-RAB11B-AS1 expression was found to be negatively correlated with various immune cells, implying that lnc-RAB11B-AS1 might influence the interaction between immune lymphocytes and malignant tumor cells, and hence regulating the progression of HCC. It appeared that lncRNAs could also participate in the maturation and differentiation of tumor-infiltrating lymphocytes through the regulation of cytokines and participate in the corresponding signaling process of immune regulation, thus affecting the tumor immune function of the organism [50].
DNA sequences can be modified by lncRNAs through changes in chromatin structure, DNA conformation, DNA stability, and gene expression regulation, affecting the occurrence and development of various cancers [51]. Wang et al. also successfully verified that differentially expressed lncRNAs in cancer are modulated by DNA methylation levels using epigenetic data [52]. We further explored the underlying mechanism of lnc-RAB11B-AS1 downregulation in HCC tissues, and the results indicated that DNA hypermethylation in the promoter region of lnc-RAB11B-AS1 leads to its reduced expression in HCC. Furthermore, the methylation level increased with HCC clinical stage and histological grade according to the ciBioPortal database, which was consistent with our previous analysis showing the decreased expression of lnc-RAB11B-AS1 in the advanced stages of HCC.
To our knowledge, lncRNAs regulate signaling pathways and transcription factors through four major modes of action: signal, decoy, guide, and scaffold, thus affecting the biological behavior of cells. The most common mechanism of cytoplasmic lncRNAs is mainly focused on binding miRNAs to act as molecular sponges, interfering with the effects of these miRNAs on target genes. Zhang et al. showed that KCNQ1OT1, the overlapping transcript 1 of lnc-KCNQ1, acted as a competing endogenous RNA for miR-506 and promoted PD-L1 expression in HCC [53]. Wang et al. also confirmed that lncRNA MCM3AP-AS1 promoted the growth of HCC by targeting the miR-194-5p/FOXA1 axis [54]. According to the prediction of public databases, lnc-RAB11B-AS1 is mainly located in the cytosol and exhibits a positive regulatory relationship with RAB11B in HCC, which is in agreement with the results of previous research showing that RAB11B is positively correlated with lnc-RAB11B-AS1 in lung cancer. Therefore, we attempted to establish an lnc-RAB11B-AS1-miRNA-RAB11B axis to test the hypothesis that lnc-RAB11B-AS1 acts as a ceRNA to regulate downstream mRNA expression. Finally, we have clarified the expression of hsa-miR-4726-5p in HCC and its relationships with the two genes and predicted that lnc-RAB11B-AS1 acts as a ceRNA by sponging miR-4726-5p to prevent the progression of HCC via up-regulating RAB11B mRNA. Further experiments are planned for the next step. Moreover, individual characteristics and treatment can be combined to achieve better therapeutic effects. We established a direct link between lnc-RAB11B-AS1 expression and cancer cell sensitivity to chemotherapy, indicating that low lnc-RAB11B-AS1 expression might be sensitive to multiple chemotherapeutic drugs, such as chlorambucil, hydroxyurea, and thiotepa.

Conclusions
In conclusion, this study revealed that low expression of lnc-RAB11B-AS1 was a factor affecting the prognosis of HCC patients. Lnc-RAB11B-AS1 has also shown promise as a potential molecular marker in the prevention and treatment of HCC. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study by the ethics committee of Shanghai Outdo Biotech Co., Ltd for the HCC tissue microarray.

Data Availability Statement:
The data that support the findings of this study are openly available in online databases mentioned in the manuscript, and also available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.