TMEM147 Correlates with Immune Infiltration and Serve as a Potential Prognostic Biomarker in Hepatocellular Carcinoma

Background Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and is associated with high mortality. Transmembrane protein 147 (TMEM147) is a seven-transmembrane protein that may mediate immune regulation. However, the relevance of TMEM147 to immune regulation in HCC and the prognosis of HCC patients are unclear. Methods We analyzed TMEM147 expression in HCC by using the Wilcoxon rank-sum test. Real time quantitative PCR (RT-qPCR) and Western blot analysis of tumor tissues and cell lines were used to verify TMEM147 expression in HCC. The influence of TMEM147 on HCC prognosis was assessed using Kaplan–Meier analysis, Cox regression analysis, and a prognostic nomogram. The functions of the TMEM147-related differentially expressed genes (DEGs) were identified by Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and gene set enrichment analysis (GSEA). In addition, we examined the associations between TMEM147 expression and immune infiltration using single-sample gene set enrichment analysis (ssGSEA) and immunofluorescence staining of HCC tissues. Results Our results showed that the expression of TMEM147 was significantly higher in human HCC tissues than in adjacent normal liver tissues, with similar findings in human HCC cell lines. High TMEM147 expression was correlated with T stage, pathological stage, histological grade, race, alpha-fetoprotein level, and vascular invasion in HCC. Moreover, we revealed that high TMEM147 expression was associated with shorter survival times and that TMEM147 could be a risk factor for overall survival, along with T stage, M stage, pathological stage, and tumor status. Mechanistic studies revealed that high TMEM147 expression was linked to the B lymphocyte, antigen response, IL6 signaling pathway, cell cycle, Kirsten rat sarcoma viral oncogene homolog (KRAS) signaling pathway, and myelocytomatosis oncogene (MYC) targets. Correspondingly, TMEM147 expression was positively associated with the infiltration of immune cells, including Th2 cells, follicular helper T cells, macrophages, and NK CD56 bright cells in HCC. Conclusions TMEM147 might be a biomarker for poor prognosis and is related to immune cell infiltration in HCC.


Introduction
Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer death worldwide, with 700,000 related annual deaths in recent years, half of which occurred in China [1,2]. Risk factors for HCC development include viral infections [3], alcohol abuse, and genetic factors [4,5]. With improvements in medical technology, the global morbidity and mortality of HCC have recently decreased [6], but the recurrence rate (>70%) and relapse-related mortality remain high [7]. Additionally, the molecular mechanisms of HCC have not yet been fully elucidated. Thus, there is an urgent need to understand HCC progression, which may lead to discovering a new therapeutic agent to improve patient survival.
As an endoplasmic reticulum (ER) resident protein with seven transmembrane regions, TMEM147 is highly conserved in mammals, but its physiological role is not fully understood. Previous work has shown that TMEM147 is a core component of the nicalin-NOMO protein complex and is essential for embryonic Nodal signaling during embryonic development in vertebrates [9]. Recent research has demonstrated that TMEM147 is a component of a multipass translocon that supports the biogenesis of membrane proteins on the ER-bound ribosomes [10]. Additionally, TMEM147 can mediate immune regulation by binding to galectins of Haemonchus contortus in goats [11]. Downregulation of TMEM147 suppresses cytokineinduced rheumatoid arthritis (RA) by inhibiting the activation of nuclear factor kappa B signaling [12]. Emerging evidence reveals the vital roles of TMEM147 in tumor occurrence and progression. A recent study reported that high expression of TMEM147 may contribute to the development of colon cancer [13]. Similarly, in osteosarcoma, the prognostic marker TMEM147 has been linked to immune cell infiltration and the immunological microenvironment [14]. Another functional illustration revealed that TMEM147 adversely controlled calcium mobilization caused by the cholinergic receptor muscarinic 3 (CHRM3) and interfered with its trafficking to the cell membrane in colon cancer [15]. It's interesting to note that CHRM3 is aberrantly expressed in several malignancies, including HCC, and is regarded to be a prognostic factor for these diseases [16][17][18]. Based on these findings, TMEM147 may play a key role in immune regulation and contribute to tumor progression. However, the functional role of TMEM147 in HCC has not been delineated. Furthermore, whether and how TMEM147 mediates immune regulation in HCC remains unknown.
In the present study, we analyzed RNA sequencing data from The Cancer Genome Atlas (TCGA) database to investigate the expression of TMEM147 and its prognostic value in HCC. Gene Ontology (GO) term enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and gene set enrichment analysis (GSEA) of the HCC dataset from TCGA were applied to further identify the role of TMEM147 in the emergence of HCC. Finally, the associations between TMEM147 expression in HCC and tumor immune cell infiltration were investigated to evaluate the underlying mechanisms of TMEM147. . The primer sequences used in this study for amplifying TMEM147 and β-actin were as follows: TMEM147 (forward, 5′-ACACGCTATGATCTGT ACCACA-3′, reverse, 5′-CAGAGGTGGACGAAGGTCTC-3′) and β-actin (forward, 5′-CATGTACGTT GCTATCCAG GC-3 ′ , reverse, 5 ′ -CTCCTTAATGTCACGCACGAT-3').

Analysis of Differentially Expressed Genes in Patients
with HCC. A total of 374 HCC patients were categorized into the high and low TMEM147 mRNA expression (cutoff value of 50%) groups. Differentially expressed genes (DEGs) between these two groups were identified using the Wilcoxon rank-sum test in the R package "DESeq2" [19]. The threshold criteria of |log2Fold Change| > 1.5 and p < 0:05 were used to identify DEGs. A heatmap was generated to show the top 20 DEGs.
2.5. Functional Enrichment Analyses. The DEGs were subjected to functional enrichment analyses. The GO term enrichment analysis for the biological process (BP), molecular function (MF), and cellular component (CC) categories, as well as KEGG pathway enrichment analysis, was performed by using the R package "cluster Profiler", and the results were visualized with the "ggplot2" package in R [20,21]. 2 Analytical Cellular Pathology 2.6. Gene Set Enrichment Analysis. Novel BPs and pathways differing between the high and low TMEM147 expression groups were identified using GSEA. GSEA was conducted using the R package clusterProfiler (3.14.3) [22]. Each analysis was repeated 5,000 times. In this study, p < 0:05, FDR < 0.25 and absolute normalized enrichment score (|NES|) > 1 were used as the criteria for statistical significance.

Associations between TMEM147 Expression and
Immune Cell Infiltration in HCC. To analyze the associations of TMEM147 expression with immune infiltration, singlesample gene set enrichment analysis (ssGSEA) was performed with the "GSVA" R package [23]. As described previously, we evaluated the relative infiltration levels of 24 immune cell types [24]. The relative enrichment score of each gene expression profile was calculated for each tumor sample. For analysis of associations between immune cell infiltration and TMEM147 expression, the Wilcoxon ranksum test and Spearman correlation analysis were used.

Prognostic Model Construction and Prediction.
HCC patients' clinical outcome data were downloaded from TCGA. Kaplan-Meier analysis was conducted to determine the clinical characteristics associated with overall survival (OS), disease specific survival (DSS), and progression-free interval (PFI). Survival curves were plotted in R (version 3.6.3) using the packages "survmine" and "survival". Furthermore, Cox regression analysis was performed using the packages "survival" and "forestplot" in R [25]. The R package "rms" was used to construct nomograms and generate calibration plots [25]. In addition, receiver operating characteristic (ROC) analysis using the R packages "pROC" and "ggplot" was performed to assess the predictive accuracy of the model. The statistical analyses were performed with the significance level set at 0.05.

TMEM147 Expression across Cancers and in HCC.
We used the Wilcoxon rank-sum test to compare the expression of TMEM147 among multiple cancer types according to TCGA data. TMEM147 expression was significantly higher in 25 types of cancer, including HCC, than in the corresponding normal tissues (Figure 1(a)). As shown in Figure 1(b), TMEM147 exhibited differential mRNA expression in tumor and normal tissues in the HCC dataset of TCGA. In paired samples, the expression of TMEM147 was substantially higher in the HCC samples than in the matched paracancerous samples (Figure 1(c)). We assessed TMEM147 expression in three different GEO datasets (GSE101685, GSE62232, and GSE60502) and validated the high expression level of TMEM147 in HCC (Figures 1(d), 1(e), and 1(f)). The expression of TMEM147 in HCC cell lines (SK-HEP-1, MHCC-97H, SMMC-7721, HepG2, and Hep3B) was significantly higher than in the normal liver epithelial cell line L02 (Figure 1(g)). In addition, we measured the mRNA and protein expression levels of TMEM147 in HCC and paired normal tissues, and the RT-qPCR and Western blot results confirmed the high expression of TMEM147 in HCC (Figures 1(h) and 1(i)).
These results indicated that TMEM147 expression affects HCC initiation and progression.

High TMEM147 Expression Is Correlated with Poor
Prognosis in HCC Patients. We used Kaplan-Meier analysis to establish the relationship between TMEM147 expression and HCC patient prognosis. HCC patients with low TMEM147 expression had higher 10-year OS rates than those with higher TMEM147 expression (HR = 2.08, 95% CI = 1.46-2.97, p < 0:001) (Figure 3   Analytical Cellular Pathology and PFI (HR = 1.53, 95% CI = 1.14-2.05, p = 0:004) (Figure 3(c)) showed that patients in the high TMEM147 expression group had worse outcomes than those in the low TMEM147 expression group. ROC curves for HCC patients were generated to estimate the diagnostic value of TMEM147. Accordingly, the area under the curve (AUC) of TMEM147 was 0.941, indicating strong sensitivity and specificity for HCC diagnosis (Figure 3(d)). Moreover, the prognostic value of TMEM147 for HCC progression was further examined using multivariate Cox regression analysis. As Table 3

Prognostic Model Generation in HCC.
To better predict the prognosis of HCC patients, TMEM147 mRNA expression and other clinicopathological parameters were used to    7 Analytical Cellular Pathology patients. A vertical line was drawn from the total score axis to the outcome axis to determine the 1-, 3-, and 5-year survival rates of patients with HCC. The survival probabilities of HCC patients at 1, 3, and 5 years were approximately 65%, 45%, and 25%, respectively (Figure 3(e)). The nomogram calibration curve for OS demonstrated good consistency in patients with HCC, which indicated that our prediction method was moderately accurate (Figure 3(f)).

Functional Enrichment Analysis of DEGs in HCC.
To explore the mechanisms by which abnormally high TMEM147 expression promotes HCC progression, we compared gene expression in HCC tissues from patients with high and low TMEM147 expression. There were 743 DEGs, among which 610 were downregulated and 133 were upregulated (adjusted p < 0:05, |Log2-fold change| > 1.5) (Figure 4(a)). Heatmaps were generated to show the downregulated and upregulated DEGs in order of prominence (Figure 4(b)). GO term and KEGG pathway enrichment analyses were used to further predict the functions of the TMEM147-related genes in HCC patients (Figure 4(c)). According to GO term enrichment analysis, the TMEM147-related genes were enriched in various BP, cell component (CC), and MF terms, including pattern specification process, regionalization, detoxification of copper ion, presynaptic membrane, presynapse, synaptic membrane, tetrapyrrole binding, steroid hydroxylase activity, and heme binding. Furthermore, KEGG pathway enrichment analysis showed TMEM147-related genes to be enriched in the neuroactive ligand-receptor interaction, mineral absorption, and bile secretion pathways. To identify TMEM147-related signaling pathways in HCC, we performed GSEA of the low and high

Analysis of the Correlations between TMEM147
Expression and Immune Cell Infiltration in HCC. Considering that GSEA showed that there may be an association between TMEM147 and tumor immunity, ssGSEA was used to test whether TMEM147 expression correlates with immune infiltration in HCC (Figures 6(a) and 6(b)).

Discussion
HCC is one of the most common and deadly cancers worldwide [26]. Despite advances in early diagnosis, new targeted drugs, and immune checkpoint inhibitors, HCC patients have a 5-year survival rate of only 18.1%, as most patients have late-stage disease at initial diagnosis [27]. Thus, it is crucial to identify potential prognostic biomarkers of HCC to improve patient outcomes. In our study, TMEM147 mRNA expression was found to be upregulated in HCC tissues. Additionally, HCC patients with high TMEM147 expression had worse prognoses and shorter survival times. Preliminary mechanistic studies revealed that TMEM147 promotes HCC progression in a manner possibly associated with immune infiltration. Our study implies that TMEM147 may be a promising target for HCC treatment.
TMEM147 is a seven-transmembrane protein that significantly impacts the pathology of different diseases and aberrant processes, including tumorigenesis [9,12]. Research has revealed that colon cancer exhibits high mRNA expression of TMEM147, which could thus be involved in the pathogenesis of colon cancer [13]. However, TMEM147 expression and its prognostic value in HCC are unclear. In the present study, through analysis of public TCGA data, we found that TMEM147 was highly expressed in HCC. To verify the above results, we investigated the expression of TMEM147 in HCC samples and HCC cell lines by RT-qPCR and Western blotting. We found that TMEM147 mRNA expression was    Analytical Cellular Pathology significantly increased in HCC tissues and HCC cell lines. This finding indicates that TMEM147 could be considered a diagnostic biomarker for HCC. Furthermore, ROC curve analysis revealed that TMEM147 expression was a reliable diagnostic marker for differentiating HCC tissues from normal liver tissues, with an AUC of 0.941. In addition, higher expression of TMEM147 was significantly correlated with several clinicopathological features, including higher T stage, pathological stage, and histologic grade; higher AFP; White race; and vascular invasion. Based on our findings, we concluded that the expression of TMEM147 could be a novel diagnostic biomarker for HCC.       were independent prognostic factors for OS. In parallel, we constructed a prognostic nomogram based on T and N classification, tumor status, and TMEM147 expression in HCC. According to the calibration plot, the predicted and actual probabilities for 1-, 3-, and 5-year OS agreed well. Thus, the prognostic nomogram based on TMEM147 expression might be clinically valuable for patients with HCC.
Given the close association between TMEM147 expression and HCC progression, we sought to explore the possible functions and mechanisms of TMEM147 in HCC. GSEA indicated that the TMEM147-high phenotype was associated with the MYC targets and KRAS signaling pathway gene sets. Recent studies have revealed that the activation of MYC can contribute to HCC progression by promoting cell proliferation, metastasis, and angiogenesis [28]. There is also evidence that KRAS pathway activation contributes to the initiation and progression of HCC [29]. Therefore, TMEM147 expression might be necessary for  14 Analytical Cellular Pathology HCC tumorigenesis. Interestingly, GSEA also showed that TMEM147 may be involved in signaling pathways that regulate immune and inflammation in HCC, including the B lymphocyte, antigen response, and IL6 signaling pathway gene sets. Our bioinformatics analysis and immunofluorescence staining indicated that high TMEM147 expression was positively associated with infiltration of Th2 cells, TFH cells, NK CD56 bright cells, and macrophages. Th2 cell infiltration has been associated with immunosuppression and poor survival in a number of malignancies [30,31]. In a mouse model of HCC, TFH cell infiltration showed a similar negative correlation to Th2 cell infiltration with regard to survival [32]. Studies on breast, colorectal, and lung cancers have shown that NK CD56 bright cells have very high infiltration levels and promote tumorigenesis [33][34][35]. Macrophages are among the most abundant immune cells infiltrating the tumor microenvironment (TME) in liver cancer and are present at all stages of liver cancer progression [36]. According to earlier research, osteosarcomas with higher TMEM147 expression have less M2 macrophage infiltration and a worse prognosis [14]. In addition to macrophages, neutrophils, DCs, and T cells had significantly different abundances in osteosarcomas with high and low TMEM147 expression, indicating that TMEM147 may mediate the immune microenvironment in osteosarcomas [14]. In fact, the TMEM protein family is heavily involved in the development and immune infiltration of many tumors. For instance, high expression of TMEM204 is linked to high levels of CD8+ and CD4+ T cell, macrophage, neutrophil, and bone marrow dendritic cell infiltration in liver hepatocellular carcinoma (LIHC) [37]. Based on these results, we speculated that TMEM147 may play an essential role in the progression of HCC through a mechanism that regulates immune-related pathways to promote immune infiltration. Although our work indicates the high expression of TMEM147 and its relationship with HCC progression, several key predictions require further functional validation. For instance, our clinical sample size is constrained, and bigger clinical investigations are required to confirm if TMEM147 expression is a prognostic and predictive factor for HCC. In addition, future work is required to design in vivo/in vitro studies to investigate how TMEM147 affects immune infiltration in HCC.

Conclusion
In summary, we found that high TMEM147 expression is closely correlated with poor prognosis in patients with HCC in this study. Overexpression of TMEM147 could play a vital role in the progression of HCC by increasing the infiltration of Th2 cells, TFH cells, NK CD56 bright cells, and macrophages. These findings support TMEM147's role as a prognostic biomarker for HCC via mediation of immune infiltration.

Data Availability
The data underlying this study are freely available from the TCGA dataset (https://portal.gdc.cancer.gov/). The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.