LncRNA PAXIP1-AS1 is a Prognostic Biomarker and Correlated with Immune Inltrates in Ovarian Cancer

Background: The long non-coding RNA (LncRNA) PAXIP1 antisense RNA 1 (PAXIP1-AS1) was found to promote proliferation, migration, EMT, and apoptosis of ovarian cancer (OC) cells in OC cell lines, but the relationship between PAXIP1-AS1 expression and clinical characteristics, prognosis, and immune inltration of OC patients and its regulatory network are unclear. Methods: QRT-PCR, Kruskal-Wallis test, Wilcoxon sign-rank test, logistic regression, Kaplan-Meier method, Cox regression analysis, Gene set enrichment analysis (GSEA), and immuno-inltration analysis were used to evaluate the relationship between clinical characteristics and PAXIP1-AS1 expression, prognostic factors, and determine the signicant involvement of PAXIP1-AS1 in function. Results: Low PAXIP1-AS1 expression in OC was associated with age (P=0.045), histological grade (P=0.011), and lymphatic invasion (P=0.004). Low PAXIP1-AS1 expression predicted a poorer overall survival (OS) (HR: 0.71; 95% CI: 0.55–0.92; P=0.009), progression free interval (PFS) (HR: 1.776; 95% CI: 1.067–2.955; P=0.001) and disease specic survival (DSS) (HR: 0.67; 95% CI: 0.51–0.89; P=0.006). And PAXIP1-AS1 expression (HR: 0.711; 95% CI: 0.542-0.934; P=0.014) was independently correlated with PFS in OC patients. GSEA demonstrated that neutrophil degranulation, signaling by Interleukins, GPCR-ligand binding, G alpha I signaling events, VEGFAVEGFR-2 signaling pathway, naba secreted factors, Class A 1 Rhodopsin-Like Receptors, PI3K-Akt signaling pathway, and Focal Adhesion-PI3K-Akt-mTOR-signaling pathway were differentially enriched in PAXIP1-AS1 high expression phenotype. PAXIP1-AS1 may inhibit the function of aDC, B cells, CD8 T cells, Cytotoxic cells, DC, iDC, Macrophages, Mast cells, Neutrophils, NK CD56dim cells, T cells, TFH, Tgd, Th1 cells, Th2 cells and Treg. Conclusions: Low expression of PAXIP1-AS1 was signicantly associated with poor survival and immune inltration in OC. PAXIP1-AS1 could be a promising prognosis biomarker for OC.


Introduction
Ovarian cancer (OC) is one of the most deadly malignancies in the female reproductive system [1]. Nearly 295,000 women worldwide have been diagnosed with OC and 185,000 have died from the disease [2].
Over 70% of OC patients are diagnosed at an advanced stage (FIGO stage III or IV) due to nonspeci c symptoms in the early stages and the lack of effective screening methods [3]. The 5-year survival rate for stage III-IV patients is approximately 30% and for stage I patients is approximately 92% [4]. Despite considerable progress in some of these areas, the treatment of this tumor remains a major challenge in gynaecological oncology, with little change in long-term survival rates for HGSOC and several issues hindering progress in clinical outcomes.
Long noncoding RNAs (lncRNAs) comprise a class of RNA transcripts >200 nucleotides in length that act as key regulators of target gene expression in a variety of biological processes including chromatin modi cation, gene transcription, RNA splicing, RNA transport and translation [5]. Aberrant lncRNA expression may be critical for cancer development and progression, and lncRNA-mediated biology may be central in cancer development [6]. Unlike miRNAs and protein-encoding mRNAs, lncRNAs typically display restricted tissue-speci c and cancer-speci c expression patterns [7]. Furthermore, their expression is lower than that of protein-coding genes [8]. Given this lncRNA tissue speci city, they may be superior biomarkers to many current protein-coding biomarkers [9]. Therefore, screening for molecular markers associated with OC prognosis is important for the precise treatment of OC.
Changes in the expression of some lncRNAs have been reported in OC and in association with clinical characteristics [10,11]. Silencing of the lncRNA PAXIP1-AS1, a key mediator of cell death, was found to contribute to cell survival [12]. Aberrant expression of the lncRNA PAXIP1-AS1 was evident in glioma cells and tissues and was signi cantly associated with survival outcomes in glioma patients [13]. H3K27acinducible lncRNA PAXIP1-AS1 promotes cell proliferation, migration, EMT, and apoptosis by targeting the miR-6744-5p/PCBP2 axis in OC [14]. However, the relationship between PAXIP1-AS1 expression and clinical characteristics, prognosis, and immune in ltration of OC patients and its regulatory network has not been studied.
This study compared PAXIP1-AS1 expression differences between tumor tissues and normal samples based on The Cancer Genome Atlas (TCGA) database and OC RNA-seq data in GTEx, and assessed the correlation between PAXIP1-AS1 expression levels and clinical features of OC, and the prognostic value of PAXIP1-AS1 in OC. Genomic enrichment analysis (GSEA) was performed on the high and low PAXIP1-AS1 expression groups to reveal their possible functions. Correlation analysis between PAXIP1-AS1 expression and immune in ltration was performed to explore the potential mechanisms by which PAXIP1-AS1 regulates OC occurrence and development. This study provided a new direction for the individualized and precise treatment of OC.
The relationship between PAXIP1-AS1 and clinical characteristics Correlation analysis of gene expression with clinical characteristics was carried out according to the references [15,16]. Molecule: PAXIP1-AS1. Clinical variables: age, histological grade, and lymphatic invasion.

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Logistics analysis was carried out according to the references [15,16]

QRT-PCR
Tumor tissue and normal ovarian tissue samples were collected from 8 OC patients at the Obstetrics and Gynaecology Department of the A liated Hospital of Xuzhou Medical University. The study was approved by the Ethics Committee of the A liated Hospital of Xuzhou Medical University. All patients signed a written informed consent form. The clinical characteristics of the 8 OC patients were listed as shown in Table 1. PAXIP1-AS1 expression levels were identi ed in 8 OC tissue samples by qRT-PCR. The speci c steps were performed according to the reference [24]. The primer sequences used are shown in Table 2.    Figure 1A). The expression of PAXIP1-AS1 in OC were signi cantly lower than that in paired normal tissues (0.612±0.138 vs. 1.538±0.179, P<0.001) ( Figure 1B).
The characteristics of OC patients were shown in Table 4, clinical and gene expression data were collected from TCGA database. According to the mean value of relative PAXIP1-AS1 expression, the patients with OC were divided into high (n=190) and low (n=189) expression groups. PAXIP1-AS1 expression was associated with age (P=0.002), histological grade (P=0.007), and lymphatic invasion (P=0.007). As shown in Figure 2 and Table 5, PAXIP1-AS1 was signi cantly related to age (P=0.045), histological grade (P=0.011), and lymphatic invasion (P=0.004).  (Table 6). The results suggested that decreased expression of PAXIP1-AS1 level is associated with poor PFS. A nomogram was constructed to predict the 1-, 3-, and 5-year survival probability of OC patients by combining the expression level of PAXIP1-AS1 with clinical variables, as shown in Figure 5.

Discussion
LncRNAs have been implicated in the molecular mechanisms of carcinogenesis [25]. As regulators of the ow of genetic information interacting with epigenetic, transcriptional, and post-transcriptional pathways, lncRNAs promote tumor formation, progression, and metastasis in many human malignancies [26].
Understanding the speci c molecular events that underpin OC tumorigenesis can lead to early detection and improved outcomes. LOXL1-AS1 expression correlates with poor clinical outcome in EOC patients and can be used as an independent prognostic indicator as well as a new diagnostic biomarker [27].
LINC00472 may be a potential tumor suppressor in OS by interacting with miR-300 and FOXO1 [28]. High plasma levels of lncRNA ROR can be used as a potential biomarker for the diagnosis of OC [29].
Therefore, it is important to study lncRNAs as new prognosis OC biomarkers and therapeutic targets in the future. Overexpression of PAXIP1-AS1 advances glioma development by recruiting the transcription factor ETS1 to increase KIF14 expression [13]. PAXIP1-AS1 may modulate smooth muscle cell function by affecting multiple IPAH-speci c transcriptional programs [30]. Based on GSEA, PAXIP1-AS1 was found to be involved in the pathways including neutrophil degranulation, signaling by Interleukins, GPCR-ligand binding, G alpha I signaling events, VEGFAVEGFR-2 signaling pathway, naba secreted factors, Class A 1 Rhodopsin-Like Receptors, PI3K-Akt signaling pathway, and Focal Adhesion-PI3K-Akt-mTOR-signaling pathway.
Immune in ltration of OC is currently a hot topic and understanding of immune in ltration will facilitate the development of immunotherapy for OC. The results of this study showed a modest relationship between PAXIP1-AS1 expression and immune cells in OC. These correlations may suggest that PAXIP1- Although there are some limitations, this is the rst study to explore the relationship between PAXIP1-AS1 and OC. This study was mainly based on bioinformatic analysis and could be further strengthened by experimental studies. The mechanism of PAXIP1-AS1-mediated ovarian carcinogenesis needs to be further investigated.
Conclusions PAXIP1-AS1 was lowly expressed in OC relative to normal tissue and related to poor OS, PFS, and DSS. PAXIP1-AS1 might participate in the development of OC by pathways including neutrophil degranulation, signaling by Interleukins, GPCR-ligand binding, G alpha I signaling events, VEGFAVEGFR-2 signaling pathway, naba secreted factors, Class A 1 Rhodopsin-Like Receptors, PI3K-Akt signaling pathway, and Focal Adhesion-PI3K-Akt-mTOR-signaling pathway. PAXIP1-AS1 expression was associated with immune in ltrating cells. This study partly revealed the role of PAXIP1-AS1 in OC, providing a potential prognosis biomarker for OC.