Comprehensive analysis of TLX2 in pan cancer as a prognostic and immunologic biomarker and validation in ovarian cancer

T cell leukemia homeobox 2 (TLX2) plays an important role in some tumors. Bioinformatics and experimental validation represent a useful way to explore the mechanisms and functions of TLX2 gene in the cancer disease process from a pan cancer perspective. TLX2 was aberrantly expressed in pan cancer and cell lines and correlated with clinical stage. High TLX2 expression was significantly associated with poor overall survival in COAD, KIRC, OC, and UCS. The greatest frequency of TLX2 alterations in pan cancer was amplification. Alterations of NXF2B, MSLNL, PCGF1, INO80B-WBP1, LBX2-AS1, MRPL53, LBX2, TTC31, WDR54, and WBP1 co-occurred in the TLX2 alteration group. PFS was significantly shorter in the TLX2-altered group (n = 6) compared to the TLX2-unaltered group (n = 400). Methylation levels of TLX2 were high in 17 tumors. TLX2 expression was associated with MSI in seven tumors and TMB in five tumors. TLX2 expression was associated with immune infiltration and immune checkpoint genes. TLX2 may be associated with some pathways and chemoresistance. We constructed a possible competing endogenous RNA (ceRNA) network of LINC01010/miR-146a-5p/TLX2 in OC. TLX2 expression was significantly upregulated in ovarian cancer cell lines compared to ovarian epithelial cell lines. Aberrant expression of TLX2 in pan cancer may promote tumorigenesis and progression through different mechanisms. TLX2 may represent an important therapeutic target for human cancers.


Correlation analysis of TLX2 and prognosis in pan cancer
We used univariate Cox regression and the package forestplot for the analyses.We analyzed overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS).The statistical method was Wilcox test.

Diagnostic value of TLX2 in pan cancer
We used the package pROC [1.18.0] to perform ROC analysis on the data and the results were visualized using package ggplot2 [3.3.6].The data processing method is log2 (value + 1).
If a specific tumor contains normal samples in TCGA, the data from TCGA were used.If the specific tumor has no normal samples in TCGA, data from TCGA + GTEx were used.

Correlation between TLX2 expression and stage, TMB, and MSI in pan cancer
We used the GEPIA 2 website (http:// gepia2.cancer-pku.cn/# index, accessed on 10 June 2022) to analyze the expression of TLX2 at different clinical stages in pan cancer 18 .We used the "Expression DIY" module to analyze the expression of a gene in different cancer stages using the box plot.
We obtained RNAseq data (level3) and corresponding clinical information from the TCGA database for 33 tumors.We obtained TMB and MSI from the literature 22,23 .The statistical method was Wilcox test.

Genomic alterations of TLX2 in pan cancer
The cBioPortal database (http:// www.cbiop ortal.org/, accessed on 10 June 2022) was used to analyze the genetic alterations of TLX2 in the TCGA pan cancer dataset 24 .We analyzed TLX2 genetic alterations and mutant sites based on the "Oncoprint", "Cancer Type Summary", and "Mutation" modules 18 .The statistical method was Logrank test.

Correlation of TLX2 expression with immune infiltration and immune checkpoint genes
We explored the association between TLX2 expression and cancer-associated fibroblast (CAF) infiltration using the "immune" module of TIMER2 (http:// timer.cistr ome.org/, accessed on 10 June 2022).Algorithms included Extended Multidimensional Immunome Characterization (EPIC), Microenvironmental Cell Population-Counter (MCP-counter), Cell Type Enrichment Analysis (XCELL) and Tumor Immune Dysfunction and Exclusion (TIDE) algorithms 18 .The data were analyzed using Spearman's correlation analysis.

TLX2-related functional enrichment analysis
We analyzed proteins for TLX2 interactions using the STRING website (version 11.5, https:// string-db.org/, accessed on 10 June 2022).The confidence score was 0.4 27 .We used "Similar Genes Detection" module of GEPIA2 to analyze the top 100 genes with similar expression patterns to TLX2 in pan cancer.We searched for genes with similar expression patterns to TLX2 genes in pan cancer.
We performed GO enrichment analysis and KEGG pathway analysis of TLX2 and interacting proteins using the DAVID website (https:// david.ncifc rf.gov/, accessed on 10 June 2022) 27 .

Drug sensitivity of TLX2 in pan cancer
We used the RNAactDrug database (http:// bio-bigda ta.hrbmu.edu.cn/ RNAac tDrug/ index.jsp, accessed on 10 June 2022) to analyze the drug sensitivity of TLX2 in pan cancer 18 .We selected the top 5 significantly positively correlated drugs and the top 5 significantly negatively correlated drugs based on Spearman's correlation coefficient 18 .

Single cell sequencing data analysis
We used the CancerSEA database (http:// biocc.hrbmu.edu.cn/ Cance rSEA/ home.jsp, accessed on 10 June 2022) on the server to explore the correlation between TLX2 expression and different tumor functional states.It had only data for GBM and RB.The plot showing the correlation between TLX2 expression and different functional status in GBM was obtained from the CancerSEA database.We obtained t-SNE plots of individual cells from the CancerSEA database.

CeRNA network construction
We focused on the mining of molecular markers in OC.To explore the possible competing endogenous RNA (ceRNA) network of TLX2 in OC, we used TarBase V.8 (https:// carol ina.imis.athena-innov ation.gr/ diana_ tools/ web/ index.php?r= tarba sev8% 2Find ex, accessed on 10 June 2022) to predict the binding of TLX2-binding miRNAs 17,28 .We screened miRNAs based on their expression in OC and correlation with OC prognosis.Next, we used LncBase Predicted v.2 (https:// carol ina.imis.athena-innov ation.gr/ diana_ tools/ web/ index.php?r= lncba sev2/ index-predi cted, accessed on 10 June 2022) to predict the lncRNAs that interact with miRNAs bound to lncRNAs.We screened lncRNAs based on their expression in OC and correlation with OC prognosis.
We used GSE103708 to analyze the expression of miR-146a-5p in normal ovarian and OC tissues 24,29 .The molecules were LINC01010 [ENSG00000236700.6] and TLX2.The statistical method was the t-test.

Statistical analysis
We performed statistical analysis using R programming language (version 3.6.3).P-values below 0.05 were considered statistically significant.

Abnormal expression of TLX2 in pan cancer and cell lines
As shown in Fig. 1, TLX2 expression was significantly upregulated in BLCA, BRCA, CESC, CHOL, DLBC, ESCA, HNSC, KIRC, LAML, LIHC, LUAD, LUSC, OC, PCPG, PRAD, STAD, UCEC, and UCS, and significantly downregulated in ACC, COAD, GBM, LGG, READ, and TGCT compared with normal tissues.Abnormal TLX2 expression in pan cancer may be associated with the development and progression of these tumors.As shown in Fig. S1, analysis of the CCLE dataset also showed differential expression of some cell lines.

TLX2 expression correlates with prognosis in pan cancer
As shown in Fig. 2A

TLX2 related pathways in pan cancer
We used GEPIA2 to obtain the top 100 genes similar to TLX2 and the STRING tool to obtain 55 genes that interacted with TLX2.We performed enrichment analysis on these 155 genes.The results showed 5 significantly related biology processes, including SRP-dependent cotranslational protein targeting to membrane, protein targeting to ER, cotranslational protein targeting to membrane, establishment of protein localization to endoplasmic reticulum, and nuclear-transcribed mRNA catabolic process, nonsense-mediated decay (Fig. 10A).The results showed five significantly related cell components, including cytosolic ribosome, ribosomal subunit, cytosolic part, ribosome, and cytosolic large ribosomal subunit (Fig. 10A).The results showed five significantly related molecular functions, including structural constituent of ribosome, rRNA binding, activating transcription factor binding, histone deacetylase activity, and sodium: chloride symporter activity (Fig. 10A).As illustrated in Fig. 10B, five pathways, including ribosomes, amphetamine addiction, cocaine addiction, alcoholism, and dopaminergic synapses, may be the main pathways through which TLX2 mediates tumorigenesis and progression.

Expression of TLX2 in single cells correlated with cancer function
As shown in Fig. 11A, TLX2 expression in GBM was positively correlated with differentiation and negatively correlated with apoptosis, cell cycle, DNA damage, DNA repair, EMT, hypoxia, invasion, and metastasis.TLX2 expression in RB was positively correlated with angiogenesis, differentiation, inflammation, metastasis, and stemness and negatively correlated with apoptosis, cell cycle, DNA damage, DNA repair, and EMT.As shown in Fig. 11B, the results showed the relationship between TLX2 expression and DNA repair, TLX2 expression and DNA damage, TLX2 expression and invasion, and TLX2 expression and EMT in GBM.T-SNE plots showed the expression profile of TLX2 in GBM single cells (Fig. 11C).These results suggest that TLX2 may play a role in the development of cancer progression.

CeRNA network of TLX2
We next examined the possible regulatory network of TLX2 in OC.The expression of LINC01010 was significantly upregulated in OC compared to normal tissues (Fig. 12A).The expression of miR-146a-5p was downregulated in OC compared with normal tissues (Fig. 12B).The expression of TLX2 was significantly upregulated in OC compared to normal tissues (Fig. 12C).High expression of LINC01010 in OC suggested a poorer prognosis (Fig. 12D).Low expression of miR-146a-5p in OC suggested a poorer prognosis (Fig. 12E).High expression of TLX2 in OC suggested a poorer prognosis (Fig. 12F).

Discussion
Pan cancer analysis provides insight into the molecular expression and genomic alterations of various cancers and helps to provide effective biomarkers for cancer diagnosis and treatment.TCGA includes data from 33 tumors and facilitates the discovery of differential expression of molecules and genomic alterations at the pan cancer level using multi-omics data analysis 33,34 .Endogenous TLX2 gene expression occurs in many non-neuroblastoma tissues, including the heart, bladder, brain, adrenal glands, and intestine 32 .However, there is a paucity of research on TLX2 in other cancer types.Therefore, in this study, we explored the possible role of TLX2 in pan cancer.In the present study, we found that TLX2 was aberrantly expressed in pan cancer.TLX2 was significantly upregulated in OC cell lines.
Aberrant abnormalities of TLX2 correlated with prognosis in LUSC 9 .Aberrant expression of TLX2 was associated with prognosis in uterine sarcoma 10 .In this study, we investigated the correlation between TLX2 expression  However, there are no relevant studies on TLX2 gene alterations in human cancers.Thus, it was decided to use the cBioPortal database to discover that amplification is the greatest frequency of TLX2 alterations in pan cancer.In pan cancer, NXF2B, MSLNL, PCGF1, INO80B-WBP1, LBX2-AS1, MRPL53, LBX2, TTC31, WDR54 and WBP1 were co-altered in the TLX2-altered group.PFS in the TLX2 altered group suggested a worse prognosis in OC.
In the realm of scientific research, it has conventionally been established that intensive promoter DNA methylation is closely linked to the repression of transcription 35 .Notably, distinct modifications in the DNA methylome have been observed in the oncogenesis of various types of cancer 36,37 .In this study, we found that TLX2 presented high methylation levels and expression levels in 12 tumors (BLCA, BRCA, CESC, CHOL, ESCA, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, PAAD, PRAD, THCA, and UCEC) and high methylation levels and low expression levels in 2 tumors (COAD and READ).The TERT promoter undergoes DNA hypermethylation, resulting in an upregulation of TERT expression in cancer 38 .AML exhibits elevated levels of methylation and expression of the AWT1 gene 39 .The FOXA2 gene demonstrates heightened methylation and expression during the development of endoderm 40 .DNA hypermethylation amplifies the expression of telomerase reverse transcriptase in human induced pluripotent stem cells 41 .Our discoveries unveil a previously unknown role of DNA methylation in regulating TLX2 gene expression.
In this study, we found that TLX2 expression correlated with stage, MSI, and TMB.High expression of TLX2 in four tumors was significantly associated with tumor stage.Expression of TLX2 was significantly associated with MSI in seven cancer types and TMB in five cancer types.Expression of TLX2 may affect the response of cancer patients to immune checkpoint therapy, which will help us to further explore the mechanisms of TLX2based immunotherapy in pan cancer.
CAF, as a major component of Tumor microenvironment (TME), plays an important role in tumorigenesis 42 .To date, the role of TLX2 in the human immune system is unclear.In this study, we investigated whether TLX2 in pan cancer was associated with immune infiltration and immune checkpoint genes.Our work elucidated the potential role of TLX2 in tumor immunity.
TLX2 is an orphan homeodomain transcription factor whose expression is a transcriptional target of PHOX2B in neural crest-derived cells 32 .TLX2 is a gene involved in pain transmission 43 .TLX2 homology domain can bind to 14-3-3eta signaling proteins 44 .In this study, we found that TLX2 was associated with ribosomes, amphetamine  addiction, cocaine addiction, alcoholism, and dopaminergic synapses in pan cancer.Ribosome biogenesis plays a crucial role in the process of cancer metastasis and resistance to treatment 45 .The administration of amphetamine may lead to modifications in immune function and cytokine expression, which are linked to the development of lymphoma 46 .The genotoxicity, oxidative stress, and inflammatory responses induced by crack cocaine are associated with the process of carcinogenesis 47 .Consumption of alcohol poses a risk factor for various major cancers 48 .Par-4, which was identified through a screening process for genes with heightened expression in prostate tumor cells undergoing apoptosis, mediates dopaminergic synaptic plasticity 49 .TLX2 may mediate tumorigenesis and progression through these pathways.
In the present study, CancerSEA had only data for GBM and RB.We found that TLX2 expression in GBM was significantly and positively correlated with differentiation and negatively correlated with apoptosis, cell cycle, DNA damage, DNA repair, EMT, hypoxia, invasion, and metastasis.However, the expression of TLX2 in OC single-cell sequencing is unclear and deserves further in-depth study.
However, there were some limitations of this study.First, this study explored the clinical significance and genomic variation of TLX2 aberrant expression in pan cancer based on public databases, and these may require real-world samples for validation in the future.Second, we explored the possible regulatory network and drug sensitivity of TLX2 in pan cancer, and the specific mechanisms of TLX2 in pan cancer development and progression need to be further investigated.Finally, we constructed a ceRNA network of LINC01010/miR-146a-5p/TLX2 in OC, and the results of these bioinformatics analyses need some experiments for validation.

Conclusion
TLX2 can be a candidate diagnostic and prognostic factor in a variety of cancers.TLX2 expression was associated with MSI, TMB, CAF infiltration, immune checkpoint genes, and drug sensitivity.The methylation levels of TLX2 exhibited high values in 17 tumors.The possible ceRNA network of LINC01010/miR-146a-5p/TLX2

Figure 5 .
Figure 5.The expression of TLX2 in pan cancer was correlated with tumor mutational burden (TMB).

Figure 6 .
Figure 6.The expression of TLX2 in pan cancer was correlated with microsatellite instability (MSI).

Figure 7 .
Figure 7. Genomic alterations of TLX2 in pan cancer.(A) Summary of TLX2 alterations in pan cancer.(B) Structural variants, mutations, and copy number alterations of TLX2.(C) Mutation types, number, and loci of TLX2 gene alterations.(D) Types of TLX2 alterations in pan cancer.(E) Top 10 genes co-altered with TLX2 alterations.(F) The group of TLX2 alterations in OC suggested poor prognosis.

Figure 10 .
Figure 10.Enrichment analysis of TLX2 and its co-expressed genes in pan cancer.(A) GO analysis.(B) KEGG analysis.

Figure 11 .
Figure 11.Expression pattern of TLX2 and its correlation with tumor functional status (single cell sequencing).(A) Correlation between TLX2 expression and different tumor functional states (*P < 0.05).(B) Correlation between TLX2 expression and different functional status in GBM (*P < 0.05).(C) Expression of TLX2 in GBM monocytes (T-SNE plots).

Figure 12 .
Figure 12.Differential expression of TLX2-mediated ceRNA network in OC and correlation with prognosis.(A) Differential expression of LINC01010 in OC tumor tissues and normal tissues.(B) Differential expression of miR-146a-5p in OC tumor tissues and normal tissues.(C) Differential expression of TLX2 in OC tumor tissues and normal tissues.(D) Prognostic value of LINC01010 in OC. (E) Prognostic value of miR-146a-5p in OC. (F) Prognostic value of TLX2 in OC.

Figure 13 .
Figure 13.Expression of TLX2 in human normal ovarian epithelial cells IOSE29, ovarian cancer cell lines SKOV3 and A2780.

Table 1 .
Drug sensitivity analysis of TLX2.