ZFC3H1 provides a reference to evaluate the prognosis and treat prostate adenocarcinoma: A study based on TCGA data

Prostate adenocarcinoma (PRAD) is a common tumor with a high mortality rate, and advanced PRAD remains incurable. Exploring biomarkers related to PRAD prognosis could provide valuable information for clinical treatment. Zinc nger C3H1 domain-containing protein (ZFC3H1) is a large protein (1989 amino acids) that is expressed in many human tumors, playing an important role in RNA turnover and translation. In the present study, we rst evaluated the expression prole of ZFC3H1 using bioinformatic analysis of public datasets from The Cancer Genome Atlas database. We then predicted the target genes of ZFC3H1, performed gene ontology enrichment analysis, and constructed a protein–protein interaction network. We also assessed the expression proles of these genes and evaluated their prognostic signicance. We also transfected a ZFC3H1 small interfering RNA (siRNA) into PRAD cells and investigated their migration and invasive capabilities through wound healing and transwell invasion assays. found many including were notably lower than in corresponding and in the high group showed poor We hypothesized that the low have be inhibitory effect on The results also that the levels (encoding M-phase phosphoprotein 6) and MRPS31 (encoding mitochondrial ribosomal protein lower high MPHOSPH6 and Knockdown of ZFC3H1


Results
We found that the expression levels of ZFC3H1 in many cancers, including PRAD, were notably lower than in corresponding non-cancerous tissues, and patients in the high ZFC3H1-expression group showed poor survival. We hypothesized that the low expression of ZFC3H1 in tumor tissue might have be an inhibitory effect on the autoimmune system. The results also showed that the expression levels of MPHOSPH6 (encoding M-phase phosphoprotein 6) and MRPS31 (encoding mitochondrial ribosomal protein S31) were lower in PRAD tissues than in non-cancerous tissues, and the survival time of patients with high MPHOSPH6 and MRPS31 expression was poor. To further demonstrate the role of ZC3H1 in PRAD, we did RNA transfection. Knockdown of ZFC3H1 signi cantly inhibited PRAD cell migration and invasion.
Conclusion ZFC3H1 is closely related to PRAD's migration and invasion ability, and could represent a new marker for PRAD prognosis and provide a reference for the development of new therapies to treat PRAD.

Background
Cancer is the second most common cause of death (8.97 million deaths) after ischemic heart disease [1].
Prostate adenocarcinoma (PRAD) is the second most common cancer causing cancer mortality in men [2]. The 5-year survival rate of PRAD is about 70-100%; however, for patients with distant metastatic cancer, the 5-year survival rate is only 30% [3]. Among the most prevalent solid tumors with advanced disease, PRAD has the fewest therapeutic options, and advanced PRAD remains incurable [4,5].
Therefore, accurately evaluating the prognosis of PRAD is vital.
Zinc nger C3H1 domain-containing protein (ZFC3H1), also known as CCDC131 (coiled-coil domain containing protein), or PSRC2 (proline/serine-rich coiled-coil protein), is a large protein (1989 amino acids), is a large proteins with a C3H1-type zinc nger as the central, including ve reversed transcribed peptide(TRP) repeats and six half-reversed transcribed peptide (HAT) repeats in the region of C-terminal. It is a central factor in the retention and degradation of polyadenylated RNA, and is involved in the processing of a wide range of RNAs, playing a crucial role in the degradation of nuclear RNAs [6][7][8][9].
Garland et al. reported a disruptive relationship between excess RNA and polycomb repressive complex 2 (PRC2) upon depletion of ZFC3H1 in mouse ESCs [10]. In addition, there are also reports that the translation was signi cantly inhibited by depletion of ZFC3H1 [11]. The reason may be that ZFC3H1 plays an important role in promoting turnover of unstable nuclear RNAs and preventing their cytoplasmic transport and global translational repression. When its activity decreases, normally unstable RNAs accumulate and are transported to the cytoplasm, thus inhibiting translation globally [12]. ZFC3H1 is also reported to play an important role in regulating cytokine production and RNA decay [13][14][15]. Considering that advanced PRAD depends on protein synthesis to maintain its survival and accelerate metabolism to promote growth [16], we hypothesized that ZFC3H1 would correlate with the prognosis of PRAD and play an active role in the progression of PRAD.
In the present study, based on The Cancer Genome Atlas (TCGA) database, we aimed to analyze the expression levels of ZFC3H1 and its target genes in PRAD, explore the role of ZFC3H1 in PRAD, and nally determine whether ZFC3H1 could provide a reference to evaluate PRAD prognosis and treatment by detecting its in uence on the migration and invasion of PRAD cells.

ZFC3H1 expression in PRAD samples from the TCGA database
The TCGA is a central repository of multidimensional experimental cancer data, comprising data pertaining to more than 30 types of human tumors. We obtained the expression pro le of ZFC3H1 in different human cancers and corresponding non-cancerous tissues, including PRAD, as well as its correlation with the prognosis of patients with PRAD, via the Gene Expression Pro ling Interactive Analysis (GEPIA) tool (http://gepia.cancer-pku.cn/), which is based on the TCGA database.

Prediction and screening of target genes
The target genes of ZFC3H1 were predicted using three databases: STRING (https://string-db.org/), BioGRID (https://thebiogrid.org/), and IntAct (https://www.ebi.ac.uk/intact/). To improve the accuracy of the prediction results and to construct Venn diagrams, we chose target genes that overlapped between at least two of the three databases. Enrichment analysis of overlapping target genes in the ZFC3H1 signaling pathway Enrichment analysis of overlapping target genes in the ZFC3H1 signaling pathway was performed using Metascape (http://metascape.org/gp/index.html). We analyzed the function of these genes in tumor biological processes via gene ontology (GO) enrichment analysis.
Identifying node degree genes via protein-protein interaction (PPI) network analyses of overlapping target genes PPI analysis was performed using overlapping target genes of ZFC3H1 through Metascape, which identi ed 26 node degree genes.
Prognostic signi cance of the chosen node degree genes To prevent any errors resulting from the use of a single database, the 26 node degree genes were analyzed using the starbase (http://starbase.sysu.edu.cn/index.php) tool to observe whether their expression levels varied between PRAD and corresponding non-cancerous tissues, and whether these genes had any in uence on the prognosis of PRAD.
Cells transfected for 48h were collected, and the cell concentration was adjusted to 10 5 cells/ml. 100ul cell suspension was inoculated in transwell chamber. After incubation for 24 h, the cells were xed with methanol for 10 min and stained with 0.1% crystal violet for 10 min. The number of migrated cells was counted and photographed under an inverted phase contrast microscope (Olympus; ×40 magnification).

ZFC3H1 expression in PRAD samples from the TCGA database
As shown in Fig. 1A and Fig. 1B, the expression of ZFC3H1 was low in many human cancers, such as PRAD, adrenocortical carcinoma, colon adenocarcinoma, lung adenocarcinoma, etc, but showed high expression only in thymoma. However, unexpectedly, as shown in Fig. 1C, the survival time of patients with PRAD and high expression of ZFC3H1 was poor. These results indicated that ZFC3H1 might enhance the progression of PRAD.

Prediction and screening of target genes
Analysis using the STRING, BioGRID, and IntAct databases led to the identi cation of 71, 103, and 98 genes, respectively. We selected genes that were overlapping between at least two of the three databases as target genes, which produced a shortlist of 81 potential target genes of ZFC3H1. A Venn diagram was then constructed for these target genes (Fig. 2). Enrichment analysis of overlapping target genes in the ZFC3H1 signaling pathway GO enrichment analyses was performed to determine the functions of the 81 target genes in tumor biological processes (Fig. 3). The putative target genes of ZFC3H1 were found to be enriched in certain cell processes, such as 28S ribosomal subunit, mitochondrial, regulation of mRNA metabolic process, mRNA processing, chromosome separation, mRNA surveillance pathway, RNA 3'-end processing, cell cycle phase transition, nucleic acid transport, nucleotide-excision repair, etc.
Identifying node degree genes via PPI network analyses of overlapping target genes To explore the interaction between the 81 overlapping target genes, a PPI network was constructed using Metascape (Fig. 4). From the PPI network, 26 node degree genes were identi ed: APP , C1D, CDC5L,  CSNK2A1, CUL7, EXOSC1, EXOSC5, EXOSC7, HNRNPK, MOV10, MPHOSPH6, MRPS11, MRPS14, MRPS23,  MRPS24, MRPS25, MRPS31, MRPS35, MRPS5, MRPS7, MTREX, PTCD3, RALY, SRRT, UPF1, and ZC3H3. Prognostic signi cance evaluation of the twenty-six node degree genes The twenty-six node degree genes were analyzed using the starbase tool to determine whether there were differences in their expression levels between PRAD and corresponding non-cancerous tissues. The expression levels of APP, CDC5L, MPHOSPH6, MRPS31, and MTREX in PRAD tissues were observed to be lower than those in corresponding non-cancerous tissues (Fig. 5A). In addition, we found that the overall survival time of patients in the MPHOSPH6 and MRPS31 high expression groups was lower than that of patients in the respective low expression groups (Fig. 5B).

ZFC3H1 siRNA reduced PRAD cell migration and invasive capability
There has been much evidence that ZFC3H1 is associated with cell mRNA translation. To verify whether silencing ZFC3H1 could reduce the capability of migration and invasion in PRAD cells, western blotting determined the interference e ciency of ZFC3H1 siRNA ( Figure 6C). The experimental results of wound healing showed that, compared with that of the negative control siRNA, ZFC3H1 knockdown reduced PRAD cell migration and motility markedly ( Figure 6A). The transwell assay also showed the number of invaded was signi cant decrease following transfected with ZFC3H1 siRNA ( Figure 6B). These results indicated that inhibiting ZFC3H1 suppressed PRAD cell migration and invasion signi cantly.

Discussion
Given the high mortality and intractability of advanced PRAD, it is necessary to develop tools or markers to evaluate the prognosis of PRAD. The majority of PRAD initially depends on the androgen receptor (AR) pathway; therefore, the targeted inhibition of androgen biosynthesis therapy has been widely used. However, due to the frequent occurrence of drug resistance and progression of PRAD, new treatments need to be developed [16]. ZFC3H1, as a central factor in the retention and degradation of polyadenylated RNA, is involved in the processing of a wide range of RNAs, and plays a crucial role in the degradation of nuclear RNAs [6][7][8][9]. In addition, the depletion of ZFC3H1 resulted in a signi cant inhibition of translation [11]. Thus, the above studies show that ZFC3H1 has marked research value. To further understand the correlation between ZFC3H1 and the prognosis of PRAD, in the present study, we carried out bioinformatic analyses based on data from the TCGA database, and validated the results using human PRAD cells.
First, we found that ZFC3H1 expression levels in many cancers, including PRAD, were signi cantly lower compared with those in corresponding non-cancerous tissues. In addition, the survival time of patients with PRAD with high ZFC3H1 expression groups was shorter than that in patients with low ZFC3H1 expression.
Second, we predicted 81 target genes of ZFC3H1 and overlapping target genes were examined via GO enrichment analyses to provide clues to functional roles of these genes in the biological processes of PRAD. A PPI network was constructed, and the most signi cant node degree genes (n = 26) were selected. We found that the expression levels of APP, CDC5L, MPHOSPH6, MRPS31, and MTREX in PRAD tissues were notably lower than those in corresponding non-cancerous tissues, and the patients in the MPHOSPH6 and MRPS31 high-expression groups showed much shorter overall survival than patients in the respective low-expression groups. These ndings supported the hypothesis that ZFC3H1 plays an active role in PRAD development.
To further demonstrate the effect of ZFC3H1 on the prognosis of PRAD, PRAD cells were transfected with an siRNA targeting ZFC3H1. Western blotting showed that the interference effect is remarkable. Subsequently, wound healing and transwell invasion assays showed that ZFC3H1 knockdown signi cantly inhibited PRAD cell migration and invasion abilities.

Conclusions
In summary, we reported that ZFC3H1 is closely related to the migration and invasion ability of PRAD. ZFC3H1 or its regulated genes might provide new biomarkers for PRAD prognosis and provide a reference for the development of new therapies to treat PRAD. However, the low expression of ZFC3H1 in PRAD has not yet been explained and further studies are warranted.

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