ZCCHC24 is a Prognostic-related Biomarker and Correlated with Immune Inltrates in Gastric Cancer and Lung Squamous Cell Carcinoma

Background: ZCCHC24 is one of alternative splicing factors which affect immune cell inltration, but the mechanisms whereby it drives immune inltration in cancer remain uncertain. Methods: ZCCHC24 expression was analyzed via the Oncomine database and Tumor Immune Estimation Resource (TIMER) site. We evaluated the inuence of ZCCHC24 on clinical prognosis using Kaplan-Meier plotter, the PrognoScan database and Gene Expression Proling Interactive Analysis (GEPIA). The correlations between ZCCHC24 and cancer immune inltrates was investigated via TIMER. In addition, correlations between ZCCHC24 expression and gene marker sets of immune inltrates were analyzed by TIMER and GEPIA. Results: ZCCHC24 signicantly impacts the prognosis of diverse cancers via The Cancer Genome Atlas (TCGA). elevated ZCCHC24 expression was signicantly correlated with poor OS and progression-free survival (PFS) in gastric cancers (OS HR = 2.11, P = 6.3e-12; PFS HR = 2.03, P = 3.1e-11). Moreover, ZCCHC24 signicantly impacts the prognosis of diverse cancers via The Cancer Genome Atlas (TCGA). Specically, high ZCCHC24 expression was correlated with worse OS and PFS in Stage, T stage, N stage, M stage, Lauren classication and differentiation of gastric cancer patients. ZCCHC24 expression was positively correlated with inltrating levels of CD4+ T(cid:0)CD8+ T cells, Macrophages, Neutrophils and dendritic cells (DCs) in stomach adenocarcinoma (STAD) and lung squamous cell carcinoma (LUSC). ZCCHC24 expression showed strong correlations with diverse immune marker sets in STAD and LUSC. Conclusions: These ndings suggest that ZCCHC24 is a key factor which governs immune cell recruitment to gastric cancer and lung squamous cell carcinoma, potentially playing a vital role in governing immune cell inltration and thus representing a valuable prognostic biomarker in gastric cancer and lung squamous cell carcinoma patients. and the other to the zf-3CxxC superfamily(19). ZCCHC superfamily proteins are involved in biological functions, such as cell growth, differentiation, DNA recognition, RNA packaging, transcriptional activation, regulation of protein and and lipid roles and tumorigenesis LOC1499503 a in identied fusion partner of congenital glioblastoma multiforme(23), to as a novel oncogene of papillary thyroid carcinoma(24, 25). ZCCHC13, involved in on Additionally, cancer-specic AS changes have been recognised as important signatures to predict the treatment ecacy in recent years(32). Research showed that changes in the expression of AS factor ZCCHC24 contribute to phase-specic AS during reprogramming(33). These ndings suggest that ZCCHC24 has multifaceted functional roles in immune cell inltration. and markers of monocytes, TAMs, and M1 and M2 macrophages to those in TIMER (Table 3). These ndings suggest that ZCCHC24 may regulate Macrophage polarisation in STAD and LUSC. High ZCCHC24 expression relates to the high inltration level of DCs in STAD and LUSC. DC markers also showed signicant correlations with the ZCCHC24 expression. These results further reveal the strong relationship between ZCCHC24 and DC inltration. DCs can increase the levels of tumour metastasis via enhancing regulatory T cell (Treg) responses and suppressing CD8+ T cell cytotoxicity.(55) Further work will be necessary to establish whether ZCCHC24 plays a key role in regulating DC inltration and tumour metastasis. A signicant correlation exists between ZCCHC24 and markers of Neutrophil. Neutrophils can also promote tumour progression, metastasis and angiogenesis directly (56). Further work will be necessary to establish whether ZCCHC24 plays a key role in regulating Neutrophil inltration. We further observed a signicant correlation between ZCCHC24 and markers of Tregs and exhausted T cells, indicating that ZCCHC24 may play a role in immune escape in gastric cancer and lung squamous cell carcinoma. However, further work will be needed to conrm the mechanisms underlying such escape. We also observed a signicant correlation between ZCCHC24 and markers of multiple T cell markers (Th1, Th2, Tfh and Th17), including STAD and LUSC. These ndings can be indicative of a potential mechanism in which ZCCHC24 regulates T cell functions in STAD and LUSC. Altogether, these results highlight the capability of ZCCHC24 to potentially regulate immune cell recruitment and activation in STAD and LUSC. Therefore, the ndings of this study further conrm that ZCCHC24 is specically correlated with immune inltrating cells in STAD and LUSC and thus plays a vital role in immune escape in gastric cancer and lung squamous cell carcinoma microenvironment. ZCCHC24 expression was also a reliable predictor of the presence of lymph node metastasis in GC patients, indicating that ZCCHC24 may be a valuable prognostic indicator of metastatic progression in GC tumour types. We further found that the degree of ZCCHC24 expression correlated with the expression of several different markers of immune cell subsets within tumours, thus highlighting a possible role for ZCCHC24 in the immunological interactions in GC and LUSC, making it a valuable biomarker worthy of further research in this type of cancer. ZCCHC2 expression in GC correlated with a higher T stage HR in OS. These results together thus suggest that ZCCHC24 may have value as a GC prognostic biomarker. In this study,The expression of ZCCHC24 correlated with the degree of immune inltration in multiple cancer types, and particularly in GC and LUSC .We found that ZCCHC24 expression was moderately positively correlated with the degree of Macrophages, CD4+ T cells and DCs inltration, and weakly positively correlated with the degree of CD8+ T cells, Neutrophil inltration in STAD and LUSC. Moreover, the correlation between ZCCHC24 expression and the marker genes of immune cells implicate that ZCCHC24 can control immune cell inltration and interactions within the tumour microenvironment in STAD and LUSC. First, gene markers of M1 macrophages showed weak and gene markers of M2 macrophages showed positive correlations with ZCCHC24 expression. These results reveal the potential regulating role of ZCCHC24 play a role in regulating TAM polarization. In addition, our results indicated that ZCCHC24 levels in STAD and LUSC to correlate with markers of Treg cells and T cell exhaustion, This suggests that ZCCHC24 has the potential to activate Tregs. Morever, ZCCH24 expression also show signicant correlations with DC markers and Neutrophil markers. These results reveal the potential regulating role of ZCCHC24 in tumour metastasis. Furthermore, we found that expression of ZCCHC24 correlated with that of multiple T cell markers (Th1, Th2, Tfh and Th17) in STAD and LUSC. These correlations could be indicative of a potential mechanism where ZCCHC24 regulates T cell functions in STAD and LUSC. Together, these results highlight the ability of ZCCHC24 to potentially regulate immune cell recruitment and activation in STAD and LUSC.


Introduction
Gastric cancer (GC) is the most common malignancy in worldwide, and it is particularly prevalent in East Asia (1). Despite advances in diagnostic imaging, surgical techniques and systemic therapy, GC remains to be the third leading cause of cancer-related deaths worldwide, and its prognosis is still poor (2). The interactions between the external environment and a series of factors concerning genetics and epigenetics cause the onset of GC and lead to the deregulation of the signalling pathways that may induce the onset of cancer (3,4). Lung squamous cell carcinoma (LUSC) is one of the most common histological subtypes of non-small-cell lung cancer (NSCLC), it remain leading cause of cancer related death worldwide for a long period (5). The development and progression of GC and LUSC are regulated by immunological mechanism (5,6) .The immune in ltration of tumours is correlated with poor prognosis, which involves macrophages, neutrophils and regulatory T cells (7)(8)(9).The cytotoxic T lymphocytes, CD8 + and DCs of tumour in ltration are generally associated with favourable Gastric and Lung cancer outcomes (5,(12)(13). Although inverse prognosis prediction values are available for several subsets of these immune cells, high ratios of Foxp3+/CD4 + and Foxp3+/CD8 + provide a poor prognosis in tumours (14,15), whereas high Th1/Th2 ratios in tumours are associated with good prognosis(16). Many different immunotherapies have been proposed to treat this cancer type. Inhibitors of Cytotoxic programmed death ligand-1(PD-L1), programmed death-1(PD-1) and T lymphocyteassociated antigen 4 (CTLA4) show promising antitumor effects in malignant melanoma and NSCLC,However, anti-CTLA4, anti-PD-1 and anti-PD-L1 did not show great e cacy in GC (17,18).
Zinc nger CCHC-type 24 (ZCCHC24) is a member of the ZCCHC superfamily of proteins, which includes two zinc nger domains: one associated with the CCHC-type superfamily and the other to the zf-3CxxC superfamily (19). ZCCHC superfamily proteins are involved in biological functions, such as cell growth, differentiation, DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly and lipid binding (20). Each member plays diverse roles in cellular physiological function and disease aetiology. 2. ZCCHC2 regulates retinoblastoma tumorigenesis by inhibiting the activity of c-My (21); ZCCHC7 is disrupted by the 3 sequences of FSHD region gene 1 family, member B, whereas LOC1499503 is a potential genetic target in acute lymphoblastic leukaemia (22); ZCCHC8 is identi ed as a fusion partner of ROS1 in congenital glioblastoma multiforme (23), ZCCHC12 is referred to as a novel oncogene of papillary thyroid carcinoma(24, 25). ZCCHC1, ZCCHC5, ZCCHC9, ZCCHC13, ZCCHC20 and ZCCHC21 are related to various solid tumours, such as breast, lung, liver, colorectal and renal cancers (21). ZCCHC24 is also involved in the regulation of apoptosis. However, the research on ZCCHC24 in cancer is limited.
Most ZCCHC superfamily proteins participate in RNA metabolism, including transcriptional elongation, polyadenylation, translation, pre-messenger RNA splicing, RNA export, RNA degradation, microRNA and ribosomal RNA biogenesis and post-transcriptional gene silencing (27). Alternative splicing (AS) is one of the most important mechanisms of post-transcriptional regulation. This mechanism is a regulated process by which RNA precursors are effectively spliced and joined and can generate great biodiversity (28). Studies demonstrated the physiological contribution of AS to tissue-identity acquisition, organ development and tissue physiology; meanwhile, studies of AS demonstrated its involvement in multiple pathologies, including cancer(28). Growing evidence demonstrates that AS plays an important role in immune microenvironment formation (30,31). AS alterations may not only affect immune cell in ltration but also regulate tumor-associated immune cytolytic activity. Additionally, cancer-speci c AS changes have been recognised as important signatures to predict the treatment e cacy in recent years (32). Research showed that changes in the expression of AS factor ZCCHC24 contribute to phase-speci c AS during reprogramming (33). These ndings suggest that ZCCHC24 has multifaceted functional roles in immune cell in ltration.

Oncomine Database Analysis
The expression level of the ZCCHC24 gene and prognostic outcome in various types of cancers was identi ed in the Oncomine database (https://www.oncomine.org/resource/login.html) (34). The threshold was determined according to the following values: P-value of 0.001, fold change of 1.5, and gene ranking of all.

PrognoScan Database Analysis
The PrognoScan database is designed to facilitate meta-analyses of gene prognostic value by comparing the relationship between gene expression and relevant outcome including overall survival (OS) in a wide range of published cancer microarray data sets (35). We therefore used this database to assess the relationship between ZCCHC24 expression and patient outcome(http://www.abren.net/Progn.oScan/).

Kaplan-Meier Plotter Database Analysis
The Kaplan-Meier plotter offers a means of readily exploring the impact of a wide array of genes on patient survival in 21 different types of cancer, with large sample sizes for the breast (n = 6,234), ovarian (n = 2,190), lung (n = 3,452) and gastric (n = 1,440) cancer cohorts. The correlation between ZCCHC24 expression and survival in blander, ovarian, lung and gastric cancers was analyzed by Kaplan-Meier plotter (http://kmplot. com/analysis/) (36). The hazard ratio (HR) with 95% con dence intervals and log-rank P-value were also computed.

TIMER Database Analysis
TIMER is a comprehensive resource for systematic analysis of immune in ltrates across diverse cancer types (https://cistrome.shinyapps.io/timer/). These gene markers are referenced in prior studies (40)(41)(42). The correlation module generated the expression scatter plots between a pair of user de ned genes in a given cancer type, together with the Spearman's correlation and the estimated statistical signi cance. ZCCHC24 was used for the xaxis with gene symbols, and related marker genes are represented on the y-axis as gene symbols. The gene expression level was displayed with log2 RSEM.

Gene Correlation Analysis in GEPIA
GEPIA is an online database which facilitates the standardized analysis of RNA-seq data from 9,736 tumour samples and 8,587 normal control samples in the TCGA and GTEx data sets (http://gepia.cancer-pku.cn/index.html) (43) .We therefore employed this database to assess the link between ZCCHC24 expression and patient prognosis in multiple tumour types, and we further assessed the link between ZCCHC24 expression and the expression of particular markers associated with immune cell in ltration of tumours.

Statistical Analysis
The PrognoScan, Kaplan-Meier plotter, TIMER and GEPIA databases were used for generating Survival curves. The results generated in Oncomine are displayed with P-values, fold changes, and ranks. The results of Kaplan-Meier plots, PrognoScan, and GEPIA are displayed with HR and P or Cox Pvalues from a log-rank test. Spearman's correlation analyses were used to gauge the degree of correlation between particular variables, with the following values being used to judge the strength of correlation: 0.00-0.19 "very weak," 0.20-0.39 "weak," 0.40-0.59 "moderate," 0.60-0.79 "strong," 0.80-1.0 "very strong." P-values <0.05 were considered statistically signi cant.

Results
The mRNA Expression Levels of ZCCHC24 in Different Types of Human Cancers We rst used Oncomine database to assess the expression of ZCCHC24 in multiple tumour and normal tissue types. This analysis revealed that the ZCCHC24 expression was higher in Colorectal Cancer, Esophageal Cancer, Gastric cancer, lymphoma tumors, Pancreatic Cancer and Sacorma compared to the normal tissues. In addition, we also found that relative to normal tissue controls, ZCCHC24 expression was lower in Bladder, Breast, Cervical, Colorectal, Head and neck, Kidney, Leukemia, Liver, Lung, Ovarian and Prostate cancers ( Figure 1A). The detailed results of ZCCHC24 expression in different cancer types are summarized in Supplementary Table 1. We examined ZCCHC24 expression using the RNA-seq data of multiple malignancies in TCGA to further evaluate ZCCHC24 expression in human cancers. We found that the expression of ZCCHC24 was signi cantly elevated relative to normal controls in HNSC (cholangiocarcinoma) and SKCM metastasis (Skin Cutaneous Melanoma). In constrast, ZCCHC24 expression was signi cantly lower in BLCA (bladder urothelial carcinoma), BRCA (breast invasive carcinoma), CHOL(cholangiocarcinoma),COAD (colon adenocarcinoma), KICH (kidney chromophobe), LICH Liver hepatocellular carcinoma ,LUAD (lung adenocarcinoma), LUSC(lung squamous cell carcinoma ), PRAD(prostatic cancer),READ (rectum adenocarcinoma), STAD (stomach adenocarcinoma), THCA (thyroid carcinoma),and UCEC (uterine corpus endometrial carcinoma) compared with adjacent normal tissues( Figure 1B).

Prognostic Potential of ZCCHC24 in Cancers
We investigated whether ZCCHC24 expression was correlated with prognosis in cancer patients. The relationships between the expression of ZCCHC24 and cancer patient outcome using the PrognoScan database (Supplementary Table 2).Notably, We found that multiple cancer types exhibited a signi cant association between patient prognosis and ZCCHC24 expression including Blood,Breast, Head and neck, Ovarian, Brain and Colon cancer ( Elevated ZCCHC24 expression is linked to prognosis in gastric cancer patients exhibiting lymphatic metastasis As we found ZCCHC24 expression to be linked with poor gastric cancer patient prognosis, we next investigated the relationship between the ZCCHC24 expression and clinical characteristics of gastric cancer patients via Kaplan-Meier plotter databases. Overexpression of ZCCHC24 was associated with worse OS and PFS in male and female patients as well as two types of Lauren classi cation and differentiation (P < 0.05). Speci cally, high ZCCHC24 mRNA expression was correlated with worse OS and PFS in stage 1 to 4,each N and M0-1 stage of gastric cancer patients (Table 1). In addition, high ZCCHC24 expression has the highest HR values of N1 of OS and PFS in the four N categories. The most common type of metastasis is lymph node metastasis and is directly linked with patients' prognosis in gastric cancer (44). These results suggest that ZCCHC24 expression level can impact the prognosis in gastric cancer patients with lymph node metastasis.

Correlation Analysis Between ZCCHC24 Expression and Immune Marker Sets
We next further explored the link between ZCCHC24 expression and levels of immune cell in ltration based on sets of immunological markers in STAD and LUSC using the TIMER and GEPIA databases. We analyzed the correlations between ZCCHC24 expression and immune marker genes of different  Table 2 and Figures 4). In contrast, ZCCHC24 expression correlated with just 17 of these markers in LIHC (Table 2).We therefore further assessed the relationship between ZCCHC24 expression and these markers in STAD and LUSC using the GEPIA database revealing similar correlations between ZCCHC24 and markers of monocytes, TAMs, and M1 and M2 macrophages to those in TIMER (  (56). Further work will be necessary to establish whether ZCCHC24 plays a key role in regulating Neutrophil in ltration. We further observed a signi cant correlation between ZCCHC24 and markers of Tregs and exhausted T cells, indicating that ZCCHC24 may play a role in immune escape in gastric cancer and lung squamous cell carcinoma. However, further work will be needed to con rm the mechanisms underlying such escape. We also observed a signi cant correlation between ZCCHC24 and markers of multiple T cell markers (Th1, Th2, Tfh and Th17), including STAD and LUSC. These ndings can be indicative of a potential mechanism in which ZCCHC24 regulates T cell functions in STAD and LUSC. Altogether, these results highlight the capability of ZCCHC24 to potentially regulate immune cell recruitment and activation in STAD and LUSC. Therefore, the ndings of this study further con rm that ZCCHC24 is speci cally correlated with immune in ltrating cells in STAD and LUSC and thus plays a vital role in immune escape in gastric cancer and lung squamous cell carcinoma microenvironment.  Discussion ZCCHC24, which is characterised with the consensus sequence CX2-C-X4-H-X4-C, has a high-a nity binding to single-stranded nucleic acids, especially single-stranded RNAs(18). increasing literatures have implicated the ZCCHC superfamily in the regulation of RNA metabolism. AS is a ubiquitous process by which a single pre-mRNA can generate diverse mature mRNAs and expand the protein diversity, providing the potential for functional and regulatory complexity in cells (57).The importance of AS on tumour immunity increasingly gains support, and the increased analysis of AS events has demonstrated the independent oncogenic effects that may be relevant to the suppressive immune microenvironment in cancers (58). The current study reports that ZCCHC24 contributes to the phase-speci c patterns of AS during reprogramming (59).
In this study, we found that ZCCHC24 expression correlated with patient prognosis in several types of cancer and a particularly strong correlation between high ZCCHC24 expression and a poor STAD and LUSC prognosis. This elevated ZCCHC24 expression was also a reliable predictor of the presence of lymph node metastasis in GC patients, indicating that ZCCHC24 may be a valuable prognostic indicator of metastatic progression in GC tumour types. We further found that the degree of ZCCHC24 expression correlated with the expression of several different markers of immune cell subsets within tumours, thus highlighting a possible role for ZCCHC24 in the immunological interactions in GC and LUSC, making it a valuable biomarker worthy of further research in this type of cancer.
In this report, we examined the expression levels of ZCCHC24 and systematic prognostic landscape in different types of cancers using independent datasets in Oncomine and 33 types of TCGA data in GEPIA. The differential expression of ZCCHC24 between cancer and normal tissues was observed in many types of cancers. Based on the Oncomine database, we found that ZCCHC24 was highly expressed in Colorectal Cancer, Esophageal Cancer, shown to be correlated with poor prognosis of gastric cancer in stage, T stage, N stage, M stage, Lauren classi cation and differentiation. Elevated ZCCHC2 expression in GC correlated with a higher T stage HR in OS. These results together thus suggest that ZCCHC24 may have value as a GC prognostic biomarker.
In this study,The expression of ZCCHC24 correlated with the degree of immune in ltration in multiple cancer types, and particularly in GC and LUSC .We found that ZCCHC24 expression was moderately positively correlated with the degree of Macrophages, CD4+ T cells and DCs in ltration, and weakly positively correlated with the degree of CD8+ T cells, Neutrophil in ltration in STAD and LUSC. Moreover, the correlation between ZCCHC24 expression and the marker genes of immune cells implicate that ZCCHC24 can control immune cell in ltration and interactions within the tumour microenvironment in STAD and LUSC. First, gene markers of M1 macrophages showed weak and gene markers of M2 macrophages showed positive correlations with ZCCHC24 expression. These results reveal the potential regulating role of ZCCHC24 play a role in regulating TAM polarization. In addition, our results indicated that ZCCHC24 levels in STAD and LUSC to correlate with markers of Treg cells and T cell exhaustion, This suggests that ZCCHC24 has the potential to activate Tregs. Morever, ZCCH24 expression also show signi cant correlations with DC markers and Neutrophil markers.
These results reveal the potential regulating role of ZCCHC24 in tumour metastasis. Furthermore, we found that expression of ZCCHC24 correlated with that of multiple T cell markers (Th1, Th2, Tfh and Th17) in STAD and LUSC. These correlations could be indicative of a potential mechanism where ZCCHC24 regulates T cell functions in STAD and LUSC. Together, these results highlight the ability of ZCCHC24 to potentially regulate immune cell recruitment and activation in STAD and LUSC.
In summary,ZCCHC24 may be an important regulator of immune cell in ltration and a valuable prognostic biomarker in gastric cancer and lung adenocarcinoma patients.