Prognostic significance of lncRNA AP004608.1 in prostate cancer

This study aimed to screen and determine the value of AP004608.1 expression as a biomarker for Prostate cancer (PCa) survival. We investigated the expression and prognosis of AP004608.1 through bioinformatics analysis. Low AP004608.1 expression predicted favorable Overall survival (OS) and Progression-free survival (PFS) in PCa patients, according to the Cancer Genome Atlas (TCGA) database. Cox regression demonstrated that low AP004608.1 expression were in-dependent biomarkers for OS. Moreover, Gene Expression Omnibus (GEO) database was utilized to verify the prognostic role of AP004608.1 in PCa, and the similar results were reached. A meta-analysis revealed that low AP004608.1 expression was closely relevant to better OS. AP004608.1 could constitute a promising prognostic biomarker, and probably plays an important role in PCa.


Introduction
PCa is the second most common solid tumor in men and the fifth cause of cancer mortality (1,2). The incidence and mortality rates of shows an extreme geographical variation, and increases progressively with the age of the worldwide population (2)(3)(4)(5)(6). In addition to the above two unmodifiable factors, the dysregulation of hormonal pathways due to several modifiable environmental factors, leads to an increased high-grade risk (7)(8)(9)(10).
Although the death rate from has decreased as diagnostic and therapeutic procedures have improved, the early diagnosis and prognosis of individual patients varied substantially due to the tumor's heterogeneity (11)(12)(13)(14). Hence, finding a novel biomarker with high accuracy is critical for achieving individualized PCa diagnosis and prognosis assessment.
The current clinical approaches in diagnosis include digital rectal examination (DRE), prostate-specific antigen (PSA) measurement and prostate biopsies (15). However, the effectiveness of DRE can only reach 5% -30%, and is contingent on the experience and skill of the examiner (16). PSA is an organ but not a cancer-specific marker, its sensitivity ranges between 67.5% and 80%, therefore, about 20-30% of could not be diagnosed (3). Liquid biopsy based on circulating tumor cells (CTCs), extracellular vesicles (EVs), circulating tumor DNA (ctDNA) and RNA (ctRNA) has emerged as an attractive and promising strategy complementary to invasive tissue biopsy to guide diagnosis and treatment (3,(17)(18)(19). What's more, liquid biopsy showed a significant potential to modify PCa management since ability to represent comprehensive information and follow-up the progression of PCa.
Long non-coding RNA (lncRNA), a class of biologically functional non-coding RNAs longer than 200 bases, has become a hotspot in the molecular biology in PCa. The abnormal expression of lncRNA is closely related to the progression, metastasis and prognosis of PCa (20-23). AP004608.1 is a newly discovered lncRNA, and is abnormally expressed in PCa, pancreatic and lung adenocarcinoma (24)(25)(26)(27). However, the potentially target mRNAs, the clinical and prognostic significance of AP004608.1 in PCa is still unknown, and its functional role in PCa has never been documented.
In this study, we first screened and determined AP004608.1 expression as a biomarker for PCa survival. We used bioinformatics to analyze RNA sequencing (RNA-Seq) data from tissue gene expression profiles in the TCGA database, mined new genes closely related to prognosis, analyzed the relation of low AP004608.1 expression and OS and PFS in PCa patients, and assessed the prognostic significance of AP004608.1 expression as an in-dependent biomarker for OS (28)(29)(30)(31). Then, we validated the prognostic role of AP004608.1 using GEO database. In addition, we performed a meta-analysis and revealed that low AP004608.1 expression was closely relevant to better OS.

Materials and methods
Differentially expressed genes mining from public databases 489 PCa tissue samples and 51 normal tissue samples mRNA data were downloaded from the TCGA database (https://portal. gdc.cancer.gov/) and PCa from the GEO database (https://www. ncbi.nlm.nih.gov/geo/) Expression profile microarray data (GSE6956) was downloaded for a total of 89 cases. The Wilcox Test method was used to screen for differential genes in tumor and normal tissues, with the screening condition: |log FC>2| (log FC=log2 mean tumor sample expression -log2 mean normal sample , log FC>0 indicates up-regulated genes, log FC<0 indicates downregulated genes), significance of corrected gene expression differences P<0.05. Using R scripts, the common differentially expressed genes were screened.

Independent prognostic genes screening
Differential genes were filtered using the KM method (discrete algorithm) and the cox (continuous algorithms) in turn. The survival genes were the differential genes that had both derived P < 0.05 and a standard deviation < 0.1. Then, P values were determined by comparing survival genes to other clinical indicators (age, T-stage, M-stage, and N-stage) and survival genes were considered independent prognostic genes if P<0.05. Moreover, Roc Filter was used to assess the accuracy of genes as prognostic genes. 0.7≤AUC<1 was used as the standard in this study.
The expression analysis and clinical relevance of AP004608.1 TCGA data, including gene expression data (HTSeq-FPKMA) for 551 cases, methylation data (IIIumina Human Methylation 450) for 553 cases, survival data, clinical indicators (age, T-stage, M staging, N staging), and tumor progression-free survival (PFS) data, were downloaded using the UCSC-XENA tool (https://xena.ucsc.edu/). The P of single gene difference between normal and tumor samples was calculated (P<2.22×10 -16 ) and plotted in box plots; OS plots of overall survival curves for AP004608.1 high and low expression groups were plotted; subject work characteristic (ROC) curves were plotted to interpret AP004608.1 prognostic factors accuracy of patient survival over large spans (3, 5, 10 years).

Meta-analysis
On the relationship between the AP004608.1 and PCa, data from the GEO database and the TCGA database were used. The overall prognostic significance of the AP004608.1 in PCa was assessed using meta-analysis. To investigate the link between the expression of the AP004608.1 and the prognosis of PCa patients, combined hazard ratio (HR) and 95% confidence intervals (CIs) were determined using R-language scripts. The Q (I2) test was used to analyze heterogeneity between the two datasets, and in this study, I2 = 0%, P>0.05 heterogeneity was low, so a fixedeffect model was chosen for combination and forest map.

Statistical analysis
R was used to conduct all of the analyses (v.4.1.0). The charts were analyzed using "GraphPad" program. For the study of statistical paired data, the Wilcoxon signed-rank test and the ttest were utilized. A statistically significant difference was defined as P<0.05.

Results
PCa patients' prognosis-related differential gene screening The RNA-Seq database of 489 columns of PCa tissue samples and 51 columns of normal prostate tissue samples was downloaded from the TCGA database and screened for differential genes using R, yielding 3664 genes that were significantly differentially expressed between PCa tissue and normal prostate tissue, including 2220 genes with upregulated expression and 1444 genes with downregulated expression. Kaplan-Meier and Cox survival analysis revealed that 41 genes were significantly differentially expressed between PCa tissue and normal prostate tissue (P<0.05), as shown in Table 1. Five independent prognostic genes, IGHV7-81, AP004608.1, AP000844.2, SNORD6 and LRRC31, were further screened by R script based on the survival gene screening. Meanwhile, the AUC in ROC curves were used to verify the accuracy of their prognostic genes. The AUC for AP004608.1, which reached 0.8525, was the highest of these five prognostic genes, indicating that this gene was also more accurate as a prognostic gene (Table 2). Finally, we found that the difference between AP004508.1 gene and two variables of T-stage and Nstage were more significant, LRRC31 only differed significantly from T-stage, and the remaining three genes were not significantly different from the clinical correlation variables, so we chose AP004608.1 as the -related differential gene for further study.
The clinical and prognostic value of AP004608.1 expression according to TCGA database The RNA-Seq of the AP004608.1 from the TCGA database (51 cases in the normal prostate group and 489 cases in PCa tissues) revealed that the mRNA expression of the AP004608.1 was low in normal prostate tissues and highly expressed in PCa tissues, with a statistically significant difference (P<2.22×10 -16 ) as shown in Figure 1A. Figure 1B showed the OS curves for the high and low expression groups of the AP004608.1, with statistically significant variations in OS between the two groups (P<0.05). The ROC curves showed that using the AP004608.1, the accuracy of predicting patient survival for large spans (3, 5 and 10 years) was 0.779, 0.795, and 0.568, respectively ( Figure 1C), while the accuracy of predicting patient survival for small spans (1, 2 and 3 years) was 0.982, 0.722, and 0.779, respectively ( Figure 1C). Univariate screening of relevant prognostic genes based on P<0.05 and HR >1 was performed for age, T-stage, M-stage, N-stage and AP004608.1expression, and then multifactorial analysis was performed for age, T staging, N staging, and AP004608.1 expression, and it was discovered that P<0.05 for AP004608.1, indicating that AP004608.1 could be used as a key prognostic gene in PCa independently of other clinical features ( Figures 1D, E). As illustrated in Figure 1F, PFS curves for the high and low expression groups of the AP004608.1 were plotted, and there was no significant difference in PFS between the two groups (P=0.916). The GEO database's AP004608.1 RNA-Seq (PCa expression profile microarray data of 89 cases) revealed that AP004608.1 mRNA was lowly expressed in normal prostate tissues and highly expressed in PCa tissues, with a statistically significant difference (P<0.05) between the two groups, as shown in Figure 1G. The AP004608.1 was also found to be a critical prognostic gene for prostate cancer treatment.

The correlation analysis of AP004608.1 expression and clinical indicators
Age, M stage, T stage, and N stage were used to group four factors, and the association between each component and AP004608.1 expression was calculated. If P<0.05, the factor was connected with AP004608.1 expression, and if P>0.05, it was not correlated with AP004608.1 expression. Age, M-stage, and T-stage III/IV were not connected with AP004608.1 expression (P>0.05), as shown in Table 3 and Figure 2. T-stage II/III, II/IV, and N-stage were correlated with AP004608.1 expression (P<0.05).  Meta-analysis and predictive performance of AP004608.1 expression Meta-analysis was used to analyze the importance of the AP004608.1 in the overall prognosis of PCa patients using data from the TCGA and GEO datasets. To analyze the connection between AP004608.1 expression and the prognosis of PCa patients, the combined HR and 95% C were determined. This gene has a combined HR of 1.22 (>1), indicating that it is a highrisk gene for PCa development and has a good link with prostate cancer prognosis (Figure 3).

Discussion
In our study, we analyzed the clinical and prognostic role of AP004608.1 expression in PCa according to TCGA database. For the first time, we discovered the strongly negative association between AP004608.1 expression and OS of PCa. We found that AP004608.1 expression was closely associated with a series of significant features, including histological type and molecular type (24,27,32). Cox regression models established the critical role of low AP004608.1 expression in the favorable prognosis of patients with PCa. In addition, this study also verified the AP004608.1 expression in the GEO database and confirmed the important role of AP004608.1 expression in the prognosis of PCa patients. Meta-analysis of 551 prostate cancer patients from the TCGA database and 89 prostate cancer patients from the GEO database revealed that AP004608.1 expression is an independent predictive factor in PCa patients' OS. Our analyses confirmed the close correlation AP004608.1 expression and clinical indicators (Age, T-stage, M-stage, N-stage) of PCa, which is to say, low AP004608.1 expression was closely relevant to better OS. Collectively, our analyses emphasized that AP004608.1 is a promising biomarker for predicting prognosis of patients with PCa.
The identification of several novel biomarkers in tumor tissues, serum, and even urine was facilitated by the advances in chip technology and next-generation high-throughput sequencing (NGS). However, only a few biomarkers have been approved for use by the US Food and Drug Administration (FDA) (PSA in 1994, PHI in 2012, and PCA3 in 2012). due to its limited tumor specificity, more effective diagnostic/prognostic indicators for PCa are still lacking in clinical practice (22,23). Thus, for the clinical diagnosis and treatment of prostate cancer, it is critical to discover the markers that are associated with PCa diagnosis and prognosis (24). Among these novel biomarkers, lncRNA has received more and more attention and has become the hotspot of PCa research. For example, the abnormal expression of lncRNA T1 and OIP5-AS1 is related to PCa progression through AKT/NF-kB signaling and ferroptosis resistance respectively (33,34). lncRNA NEAT1 and T6 were reported to promotes bone metastasis in PCa (35,36). However, there is currently insufficient evidence that which lncRNA can function as a biomarker of PCa diagnosis and prognosis. AP004608.1 is a newly discovered lncRNA, and is abnormally expressed in PCa, pancreatic and lung adenocarcinoma (13)(14)(15)(16). We based the AP004608.1 expression in PCa tissues and normal prostate tissues on the clinical diagnosis and prognosis of PCa on the basis of RNA-Seq differential gene screening. According to a series of bioinformatics investigations, Cox regression modeling and Meta-analysis of 640 PCa patients from the TCGA and GEO databases, the results herald that AP004608.1 is expected to be a new target and prognostic factor for PCa treatment. It is worth noting that this study also has certain limitations, such as: Only the TCGA database contains PFS information, and the relationship between AP004608.1 gene expression and PFS, thus it could not be verified in the GEO database. In addition, in this study, through GO and KEGG enrichment analysis, the role of AP004608.1 gene expression in PCa was preliminarily discussed. However, the AP004608.1 gene modification (such as methylation) and the potential mechanism linking The correlation analysis of AP004608.1 expression and clinical indicators. The correlation analysis of AP004608.1 expression and Age, M stage, T stage, and N stage. Forest plot of AP004608.1 expression with overall prognosis in PCa patients from TCGA and GEO datasets.

Conclusion
We examined the sequencing data of a large sample of PCa using bioinformatics tools, and exposed the potential therapeutic and prognostic relevance of the AP004608.1 in PCa. Low AP004608.1 expression predicts favorable prognosis in PCa patients. Hence, AP004608.1 could act as a promising biomarker in PCa patients. Our study provided hints and a foundation for further research into the gene's biological function and mechanism of action.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ supplementary material.

Author contributions
Author contribution: XXand WL designed the study and interpreted data; WL, RZ and BS analyzed the data and wrote the manuscript; RZ, BS, XJ and YC collected the data; WL, RZ, BS, XJ and YC analyzed the data. XX and WL revised the manuscript. All authors contributed to the article and approved the submitted version.

Funding
This study was supported by the National Natural Science Foundation of China (Nos. 81703542) and the Natural Science Foundation of Nanjing University of Chinese Medicine (NZY81703542).