ORIGINAL ARTICLE

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AUNIP was a candidate marker for prognosis and immunology in pan-cancer

Xiaorong Guo11D . Ting Liu2 . Nan Li3 · Li Jin4

Received: 23 January 2025 / Accepted: 27 March 2025 / Published online: 17 May 2025 @ The Author(s) 2025

Abstract

AUNIP (Aurora kinase A[Aurora-A] and ninein-interacting protein), is a key factor regulating the end-state of DNA cleavage. It has been reported that AUNIP affects the progression of some tumors; however, the molecular functions involved in AUNIP remain unknown. We employed some databases, such as TCGA, GTEx, TIMER, GEPIA2, cBioportal, and GSCALite, to study AUNIP gene expression, prognosis, gene variation, and drug sensitivity. The relationship between AUNIP and clinicopathological information was explored using Wilcoxon test. The association between AUNIP and TMB, MSI, immunocyte infiltration, and immune checkpoints were analyzed using Spearman correlation analysis. We employed GSEA to research the functional mechanisms involved in AUNIP for pan-cancer. Moreover, we conducted immunohistochemistry (IHC) to investigate AUNIP difference expression between liver hepatocellular carcinoma (LIHC) and normal tissues. The Chisq test was used to study the correlation of AUNIP with clinical characteristics. AUNIP was highly expressed in majority of tumors and IHC analysis demonstrated that AUNIP expression was higher in LIHC than normal tissues. AUNIP overexpression had adverse outcomes in adrenocortical carcinoma (ACC), brain lower grade glioma (LGG), LIHC, mesothelioma (MESO), and sarcoma (SARC). Furthermore, high AUNIP expression led to unfavorable prognosis in LIHC. AUNIP was associated with T stage, N stage, and clinicopathological analysis in several cancers and AUNIP expression had a correlation with histologic grade in LIHC by IHC. Mutation analysis showed that AUNIP was the highest frequency of genetic changes in cholangiocarcinoma (CHOL). AUNIP was negatively associated with 30 small-molecule drugs that inhibit tumor development. AUNIP expression had association with TMB, MSI, immune cell infiltration, and immune checkpoints for various tumors. GSEA results suggested that AUNIP mainly participated in the cell cycle, DNA replication, mismatch repair, and homologous recombination.Pan-cancer study considered AUNIP as a potential prognostic marker and high latent diagnostic biomarker.

Keywords AUNIP . Pan-cancer . Prognostic . Immunity . Hepatocellular carcinoma

Abbreviations

ACC Adrenocortical carcinoma

BLCA Bladder urothelial carcinoma

BRCA Breast invasive carcinoma

CESC Cervical squamous cell carcinoma and endocer- vical adenocarcinoma

CHOL Cholangio carcinoma COAD Colon adenocarcinoma

Xiaorong Guo and Ting Liu is equal contribution to the manuscript.

☒ Li Jin jinli1089@126.com

Xiaorong Guo gxr802013@163.com

1 Department of Pathology, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Heilongjiang, China

2 Department of Pathology, Beijing Ditan Hospital, Capital Medical University, No. 8 Jing Shun East Street, Chaoyang District, Beijing 100015, China

3 Department of Pathology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin 150081, Heilongjiang, China

4 Cancer Center, Department of Pathology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, China

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DLBC

Lymphoid neoplasm diffuse large B-cell lymphoma

ESCA Esophageal carcinoma

GBM Glioblastoma multiforme

HNSC Head and neck squamous cell carcinoma

KICH Kidney chromophobe

KIRC

Kidney renal clear cell carcinoma

KIRP

Kidney renal papillary cell carcinoma

LAML

Acute myeloid leukemia

LGG Brain lower grade glioma

LIHC

Liver hepatocellular carcinoma

LUAD

Lung adenocarcinoma

LUSC

Lung squamous cell carcinoma

MESO

Mesothelioma

OV

Ovarian serous cystadenocarcinoma

PAAD

Pancreatic adenocarcinoma

PCPG

Pheochromocytoma and paraganglioma

PRAD Prostate adenocarcinoma

READ Rectum adenocarcinoma

SARC

Sarcoma

SKCM

Skin cutaneous melanoma

STAD

Stomach adenocarcinoma

TGCT

Testicular germ cell tumors

THCA Thyroid carcinoma

THYM

Thymoma

UCEC

Uterine corpus endometrial carcinoma

UCS

Uterine carcinosarcoma

UVM

Uveal melanoma

Introduction

Cancer is a serious disease that becomes a threat to mankind’s health. The number of deaths from cancer is increasing every year. Pan-cancer research has been prevalent in recent years, with the aim of integrating TCGA data based on different tumor types and platforms, while analyzing and interpreting these data. Our research relies on multi-omics database to explore differences between tumors, guiding tumor diagnosis, prognosis, and treatment selection (Zhang and Wang 2015; Yang et al. 2018).

AUNIP (Aurora kinase A and Ninein-interacting protein) is a centrosomal protein that interacts to promote the maintenance of Aurora-A and Ninein centrosome structures and the formation of spindles (Zhang and Wang 2015). AUNIP regulates the mitotic entry and mitotic spindle assembly by activating of Plk1 and Aurora-A. Yang et al. used bioinformatics to investigate the high expression of AUNIP in oral squamous cell carcinoma (OSCC), which is associated with tumor microenvironment, human papillomavirus infection, and cell cycle. Inhibition of AUNIP can inhibit OSCC cells’ proliferation, resulting in the G0/G1 phase arrest of OSCC cells. AUNIP overexpression

predicts bad prognosis in OSCC patients (Yang et al. 2019). However, there are few reports on pan-cancer research in AUNIP.

Our work used bioinformatics aspect to discuss the expression, prognosis, clinicopathological features, mutation, tumor mutation load (TMB), microsatellite instability (MSI), immune characteristics, and drug sensitivity of AUNIP from the viewpoint of pan-cancer, and comprehensively analyzed the characteristics and mechanism of AUNIP, providing new ideas for tumor treatment and prognosis.

Materials and methods

Differential expression of AUNIP mRNA for cancers and normal samples

TIMER2 database studied immune cells infiltration in different tumors, as well as the differential expression of 33 kinds of tumors and normal tissues from TCGA database (Li et al. 2020). Owing to the absence of normal samples in several tumors in TIMER database, we merged TCGA and GTEx to discuss the differential expression of AUNIP in 33 tumors and normal tissues.

Prognosis and diagnostic value of AUNIP

GEPIA2 database is an online platform in which survival significance maps of genes in pan-cancer can be obtained. According to the median value of AUNIP expression, AUNIP was divided into low-expression group and high- expression group and Kaplan-Meier was used to show prognostic differences of both groups (Tang et al. 2019). In addition, a receiver-operating characteristic (ROC) curve estimated the diagnose value for AUNIP using pROC in R.

Clinicopathological features

We acquired the expression data and clinicopathological parameters of 33 cancers from the TCGA database and utilized Wilcoxon test to investigate its correlation with clinicopathology, including T stage, N stage, and pathological stage.

Immunohistochemical staining

Forty-four cases of liver cancer and paracancerous tissue were collected from the Department of Pathology of the Zhejiang Provincial People’s Hospital. The study was authorized by the ethics committee of Zhejiang Provincial People’s Hospital (batch number: QT2025083), and all patients received written informed consent before surgery.

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Patients with a pathologic diagnosis of hepatocellular carcinoma who had not received any preoperative chemotherapy or radiotherapy were included in the study. Patients with other diagnosed malignancies were not included in the study. The slices were cut into 4 um thick. The sections were dewaxed, hydrated, and repaired by high-pressure antigen. The endogenous catalase activity was inactivated by 3% H2O2 at room temperature for 10 min. The non-specific antigen was blocked by 10% sheep serum after rinsed with PBS at 37 ℃ for 10 min, and rabbit anti-AUNIP polyclonal antibody (bs-15019R, 1:200, Bioss Company) was added at 4 ℃ overnight. The next day, the secondary antibody (Goat anti-Rabbit IgG, PV-6000, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) was added, and then developed color with DAB. Finally, the slices were observed in the microscope. The standard of expression strength is: 0 points without staining; light yellow is 1 point; light brown is 2 points; dark brown is 3 points. The scoring criteria for positive cells were: 0 points for ≤ 5%; 6% ~25% is 1 score; 26% ~ 50% is 2 points; 51% ~ 75% is 3 points; > 76% is 4 points. AUNIP expression is interpreted by the percentage of positive cells multiplied by the staining intensity. The degree of positive staining was defined: ≤7 is classified as low expression, and > 7 is classified as high expression.

Mutational analysis of AUNIP

cBioPortal studied the frequency of AUNIP gene change in various tumors (Cerami et al. 2012).

Correlative analysis of AUNIP expression with TMB and MSI

TMB is the total number of genetic coding errors, base substitution, gene insertion, or deletion errors detected in somatic cells from millions of bases, and it can effectively evaluate tumor mutation and neoantigen load and is related to immunotherapy response (Zhang et al. 2019; Chan et al. 2019). MSI is due to mismatch repair gene defects. Tumors with MSl molecular characteristics increase tumor antigen load due to high-frequency gene mutation, inducing killer T lymphocyte infiltration and corresponding immunosuppressive molecule high expression, and respond well to corresponding immunotherapy (Dudley and Le 2016). The interrelation of AUNIP expression with TMB and MSI in 33 tumors was discussed by Spearman analysis.

Association of AUNIP with immune cell infiltration and immune checkpoints

Cells and molecules of the tumor microenvironment (TME) can influence the efficiency of immunotherapy, so

research on TME is of great significance in immunotherapy. Tumor immune cell infiltration is closely linked to tumor progression in the TME. The relationship of AUNIP with 23 types of immune cell infiltration in different cancers was applied using ssGSEA algorithm in R language. In addition, the stromal score, immune score, and estimate score for different tumors were investigated using ESTIMATE algorithm. Immunotherapy with immune checkpoint inhibitors has initiated a new era of tumor treatment, and finding predictable biomarkers is a necessary pathway for achieving precise tumor immunotherapy. At present, the eight commonest immune checkpoints are PD-1, PD-L1, CTLA-4, PDCD1LG2, TIGIT, HAVCR2, SIGLEC15, and LAG3. We discussed the association of AUNIP with immune checkpoints through Spearman analysis.

Correlative analysis between AUNIP expression and drug sensitivity

GSCALite is an integrated platform for genomic, pharmacogenomic, and immunogenomic gene set cancer analysis. The CTRP dataset from the GSCALite database (http://bioinfo.life.hust.edu.cn/web/GSCALite/) was employed to explore the relationship between gene expression and drug sensitivity (Liu et al. 2022).

Gene set enrichment analysis of AUNIP

We conducted GSEA analysis between high AUNIP expression and low AUNIP expression according to the KEGG dataset in the MSigDB database.

Statistical analysis

Wilcoxon test was employed to study the difference expression between tumors and normal tissues. The relationship of AUNIP expression with clinicopathological features was applied by Chi-square test. We investigated the association of AUNIP with TME and immune checkpoints using Spearman correlation. P < 0.05 was considered statistically significant.

Results

Overexpression of AUNIP mRNA in various tumors

The TIMER database showed that compared with normal tissue, AUNIP had high expression in BLCA, BRCA, CESC, CHOL, COAD, ESCA, HNSC, LIHC, LUAD, LUSC, PAAD, READ, STAD, THCA, and UCEC, and low expres- sion in KICH, KIRC, KIRP, and PCPG (Fig. 1A). Because of no normal tissues in some tumors in the TIMER database,

AUNIP Expression Level(log2 TPM)

ACC.Tumor (n=79)

BLCA.Tumor (n=408)

BLCA.Normal (n=19)

BRCA.Tumor (n=1093)

BRCA.Normal (n=112)

BRCA-Basal.Tumor (n=190)

BRCA-Her2.Tumor (n=82)

BRCA-LumA. Tumor (n=564) BRCA-LumB.Tumor (n=217)

CESC.Tumor (n=304)

CESC.Normal (n=3)

CHOL.Tumor (n=36)

CHOL.Normal (n=9)

COAD.Tumor (n=457)

COAD.Normal (n=41)

DLBC.Tumor (n=48)

ESCA.Tumor (n=184)

ESCA.Normal (n=11)-

GBM.Tumor (n=153)

GBM.Normal (n=5)

HNSC.Tumor (n=520)

HNSC.Normal (n=44)

HNSC-HPV+.Tumor (n=97) HNSC-HPV -. Tumor (n=421)

KICH.Tumor (n=66)

KICH.Normal (n=25)

KIRC.Tumor (n=533)

KIRC.Normal (n=72)

KIRP.Tumor (n=290)

KIRP.Normal (n=32)

LAML.Tumor (n=173) LGG.Tumor (n=516)

LIHC.Tumor (n=371) LIHC.Normal (n=50)

LUAD.Tumor (n=515)

LUAD.Normal (n=59)

LUSC.Tumor (n=501)

LUSC.Normal (n=51)

MESO.Tumor (n=87)

OV.Tumor (n=303)

PAAD.Tumor (n=178)

PAAD.Normal (n=4) PCPG.Tumor (n=179) PCPG.Normal (n=3)

PRAD.Tumor (n=497) PRAD.Normal (n=52)

READ.Tumor (n=166)

READ.Normal (n=10)

SARC.Tumor (n=259)

SKCM.Tumor (n=103)

SKCM.Metastasis (n=368)

STAD.Tumor (n=415)

STAD.Normal (n=35) TGCT.Tumor (n=150)

THCA.Tumor (n=501)

THCA.Normal (n=59)

THYM.Tumor (n=120)

UCEC.Tumor (n=545)

UCEC.Normal (n=35)

UCS.Tumor (n=57)

UVM.Tumor (n=80)

0

N

1

.

-0

1

II

*

*


A

B

The expression of AUNIP Log2 (TPM+1)

0

2

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4

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ACC

ns

BLCA


BRCA


CESC


CHOL

COAD

DLBC

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ESCA


:

HNSC

GBM


KICH

ns

KIRC

KIRP

Ps

LAML

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PCPG

I

PRAD

ns

READ

SARC

SKCM


STAD

TGCT

THCA

THYM


UCEC


UCS

UVM

Tumor Normal

*P < 0.05)

Fig. 1 The expression of AUNIP in pan-cancer and normal tissues in TIMER database (A) and TCGA +GTEx (B) ( *** P < 0.001, ** P < 0.01,

TCGA was combined with GTEx to explore AUNIP expres- sion in tumors and corresponding normal samples. We dis- covered that AUNIP expression in BLCA, BRCA, CESC, CHOL, COAD, DLBC, ESCA, GBM, HNSC, LGG, LIHC, LUAD, LUSC, OV, PAAD, READ, SKCM, STAD, THCA, THYM, UCEC, and UCS was higher than in normal tissues, but lower in KIRC, LAML, PCPG, and TGCT (Fig. 1B).

Prognosis and diagnostic value of AUNIP

in pan-cancer

We analyzed the impact of AUNIP expression on survival in 33 tumors. Prognostic index included overall survival (OS) and disease-free survival (DFS). The findings dem- onstrated that the OS of low-expression AUNIP was bet- ter than that of high-expression AUNIP for ACC, KIRP, LAML, LGG, LIHC, LUAD, MESO, PRAD, SARC, and SKCM, (Fig. 2A). Regarding DFS, the DFS for low- expression AUNIP in ACC, KIRP, LGG, LIHC, MESO,

PAAD, PRAD, and SARC was better than that of high- expression AUNIP (Fig. 2B). Among them, the OS and DFS with low AUNIP expression for ACC, KIRP, LGG, LIHC, MESO, PRAD, and SARC were higher than those with high AUNIP expression. There was no significant difference of AUNIP expression in KIRP and PRAD, compared to their corresponding normal tissues. There- fore, AUNIP was related to OS and DFS in ACC, LGG, LIHC, MESO, and SARC. Furthermore, we evaluated the diagnostic value of AUNIP in tumors. The area under the receiver-operating characteristic curve (AUC) >0.7 is

considered certain accuracy, and AUC > 0.9 is considered higher accuracy (Mishra et al. 2023a). The results demon- strated that AUNIP has high diagnostic value in BLCA, BRCA, CESC, CHOL, COAD, ESCA, HNSC, LUAD, LUSC, PCPG, READ, STAD, UCEC, and certain diag- nostic values for KICH, KIRC, LIHC, PAAD, and SARC (Figure S1).

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log 10(HR)

1.0

Fig. 2 The survival hotmaps and Kaplan-Meier survival curve in pan-cancer using GEPIA2. A The OS of AUNIP in pan-cancer. B The DFS of AUNIP in pan-cancer

A

ENSG00000127423.10

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ACC

BLCA

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CHOL

COAD

DLBC

ESCA

GBM

HNSC

KICH

KIRC

KIRP

LAML

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LIHC

LUAD

LUSC

MESO

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PCPG

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READ

SARC

SKCM

STAD

TGCT

THCA

THYM

UCEC

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UVM

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Overall Survival

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HIỆN AUNIP TPM

LOW AUNIP TPM

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LOW AUNIP TPM

Lograr pro 6e-06

ogrank p=0.0013

Lagrank p=0. 0068

High AUNIP TPM

Logrank pu0.00052

POKRY-9 de

Pipinghy-2.6

nghghjejj now(=38

D(HR)=0.0044

HRphiphi-2.1

Logrank p=6.36-05

High AUNIP TPM

=

=

DOFR)-0.011

Highghij=2.2

DO-)=9.5e-05

-

Hecaghij=1.9

Percent survival

Percent survival

níhigh)= 141 nílow)=131

Percent survival

nghighj=53 now !: 53

DOHR-48-04

Percent survival

n(high)=254

now)=252

Percent survival

níhighje 180 n(om)=179

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Low TUNP TPM

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High AUNIP TPM

JNP TPM

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Logan p=0 013

Lograna pa6. 16-07

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HIỆN AUNIP TPM

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Lograra p0 043

=

Hk[high)=3.6

D(FG=1.Se-06

=

Highghi=1.5

Logrark p=0.006

DO-FOTO.014 nghighi=238

B

3

Percent survival

P(HR)=0.044 n(high)=130

HRpighj=1.3

Percent survival

P(PR)-0.006

Percent survival

Con()=236

ngow)= 130

Percent survival

nghighi-229

3

ngon)=239

Percent survival

nghighi=41 n(ou)=41

AN

now)-228

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LUAD

MESO

PRAD

SARC

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ENSG00000127423.10

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ACC

BLCA

BRCA

CESC

CHOL

COAD

DLBC

ESCA

GBM

HNSC

KICH

KIRC

KIRP

LAML

LGG

LIHC

LUAD

LUSC

MESO

OV

PAAD

PCPG

PRAD

READ

SARC

SKCM

STAD

TGCT

THCA

THYM

UCEC

UCS

UVM

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Disease Free Survival

Disease Free Survival

Disease Free Survival

Low AUNIP

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LOW

9

High AUNIP TPM

LOW AUNIP TPM

0

Logrank p=9.5e-06

High AUNIP TPM

Low AUNIP

Logrank p=0.0013

High AUNIP TPM

Logrank p=0.00014

High AUNIP TPM

Logrank p=0.0038

4

HR(high)=4

6

HR(high)=2.7

08

0.8

Percent survival

P(HR)=0.00028

P(HR)=0.002

HR(high)=1.8

HR(high)=1.6

n(high)=38

Percent survival

n(high)=141

now ?- 38

Percent survival

P(HR)=0.00017

ngow)=131

n(high)=254 n(low)=252

Percent survival

P(HR)=0.004

n(high)=180

0.6

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06

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Logrank p=0.0074

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HR(high)=2.2

Logrank p=0.017

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Logrank p=0.036

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Logrark p=0.044

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P(R)=0.0084 n(high)=41

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p(HR)=0.019 nghigh)=87 now)=87

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P(HR)=0.038 n(high)=244

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Percent survival

P(HR)=0.042

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níjon)=41

Percent survival

Percent survival

Mów)=235

Percent survival

n(high)= 130

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AUNIP was associated with clinicopathological features in some tumors

We downloaded clinicopathological data for 33 tumors. Regarding T staging, AUNIP expression was higher at T3 + T4 than at T1 + T2 in ACC, KIRP, LIHC, PRAD, and lower at T3+T4 in THCA (Fig. 3A). In ACC, HNSC, KICH, KIRC, KIRP, LUAD, LUSC, and PRAD, AUNIP was higher expressed in patients with N1&N2&N3 than in patients with N0, while in SKCM, AUNIP was higher expressed in patients with N0(Fig. 3B). In ACC, BLCA, HNSC, KIRP, LIHC, LUAD, UCEC, and UCS, AUNIP

expression increased with the increase of pathological staging, while in OV and SKCM, AUNIP expression decreased with the increase of pathological staging (Fig. 3C).

Gene variation of AUNIP in pan-cancer

Genetic alterations are a form of epigenetics. Genetic altera- tions of AUNIP in different tumors appear in the form of mutation, structural variant, amplification, deep deletion, and multiple alterations. Among them, CHOL has the high- est frequency of gene change, which is manifested as deep

Fig. 3 The correlation of AUNIP with clinical pathological features in some tumors. A The correlation of AUNIP with T stage in ACC, KIRP, LIHC, PRAD, and THCA. B The association between AUNIP and N stage in ACC, HNSC, KICH, KIRC, KIRP, LUAD, LUSC, PRAD, and SKCM. C The relationship of AUNIP with pathologi- cal stage in ACC, BLCA, HNSC, KIRP, LIHC, LUAD, OV, SKCM, UCEC, and UCS ( *** P< 0.001, ** P < 0.01, *P < 0.05)

A

4

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The expression of AUNIP Log2 (TPM+1)

The expression of AUNIP Log, (TPM=1)

The expression of AUNIP

S

The expression of AUNIP Log2 (TPM+1)

4

3

The expression of AUNIP

The expression of AUNIP

The expression of AUNIP

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The expression of ALINIP

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2.0

Log_ (TPM+1)

Log (TPM+1)

1.5

Log2 (TPM+1)

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Log (TPM+1)

N

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KIRP

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PRAD

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·

THAT2

T3&T4

T1872

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T stage

T stage

12

T4673

T1&12

T3414

Pathologic T stage

T stage

NO

NT

NO

NIANG

T stage

Pathologic N stage

NO

NIANIANO

Pathologic N stage

Pathologie N stage

C

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5

5

2

=

The expression of AUNIP Log_ (TPM+1)

. 6

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The expression of AUNIP Log_ (TPM+1)

The expression of AUNIP Log, (TPM+1)

The expression of AUNIP Logy (TPM+1)

3

The expression of AUNIP

3

The expression of AUNIP Log, (TPM+1)

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4

Log2 (TPM+1)

The expression of AUNIP To: Log, (TPM+1)

The expression of AUNIP Log(TPM+1)

2.0

2

2

2

4

2

Z

1

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1.0

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ACC

BLCA

1

HNSC

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KIRP

0

A

LIHC

0.5

KIRC

KIRP

LUAD

Stage IAStage # Stage il&Stage IV Pathologic stage

0

Stage I&Stage Il Stage IT&Stage IV Pathologic stage

Stage I&Stage E Stage HAStage IV Clinical stage

Stage I&Stage Il Stage I&Stage IV Pathologic stage

Stage I&Stage Ii Stage B&Stage IV Pathologic stage

NO

NIAN2

NO

Pathologic N stage

NIANG

NO

Pathologic N stage

NIAN2&N3

Pathologic N stage

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s-

1

5.01

5

=

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The expression of AUNIP Log_ (TPM+1)

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The expression of AUNIP Log (TPM+1)

The expression Of AUNIP Log2 (TPM+1)

6

S

The expression of AUNIP Log, (TPM+1)

4.5

3

Too. (TPM+1)

The expression of AUNIP

Log2 (TPM+1)

The expression of AUNIP

Log2 (TPM+1)

The expression of ALINIP Log, [TPM+1)

&

4.0

4

2

y

3.5

0

0

0

3

0

2

0

3.0

0

1

1

2

1

T

LUAD

OV

SKCM

UCEC

25

UCS

LUSC

PRAD

SKCM

0

Stage I&Sange Il Singe Ili&Stage IV Pathologic stage

Stage I&Stage Il Sage HAStage IV FIGO stage

0

0

Stage I&Stage Il Stage HISStage IV Pathologic stage

Stage ISStage Il Stage INAStage IV Clinical stage

Stage I&Stage Ii Stage II&Stage IV Clinical stage

0

NO

NTANGENS

NO

N1

Pathologic N stage

Pathologic N stage

NO

NIAN2ANS

Pathologic N stage

deletion, followed by PCPG, which is mainly manifested as deep deletion. The third genetic alteration is PAAD (muta- tion, amplification and deep deletion). No genetic alteration

Fig. 4 The gene alteration of AUNIP in pan-cancer

2.5%-

Mutation

Structural Variant

Amplification

Deep Deletion

Multiple Alterations

Alteration Frequency

2%-

1.5%

1%-

0.5%-

Structural variant data

Mutation data

CNA data

Cholangiocarcinoma (TCGA, PanCancer Atlas)

Pheochromocytoma and Paraganglioma (TCGA, PanCancer Atlas)

Pancreatic Adenocarcinoma (TCGA, PanCancer Atlas)

Uterine Corpus Endometrial Carcinoma (TCGA, PanCancer Atlas)

Sarcoma (TCGA, PanCancer Atlas)

Bladder Urothelial Carcinoma (TCGA, PanCancer Atlas)

Stomach Adenocarcinoma (TCGA, PanCancer Atlas)

Mesothelioma (TCGA, PanCancer Atlas)

Esophageal Adenocarcinoma (TCGA, PanCancer Atlas) Adrenocortical Carcinoma (TCGA, PanCancer Atlas)

Ovarian Serous Cystadenocarcinoma (TCGA, PanCancer Atlas) Cervical Squamous Cell Carcinoma (TCGA, PanCancer Atlas)

Lung Adenocarcinoma (TCGA, PanCancer Atlas) Prostate Adenocarcinoma (TCGA, PanCancer Atlas)

Skin Cutaneous Melanoma (TCGA, PanCancer Atlas) Testicular Germ Cell Tumors (TCGA, PanCancer Atlas)

Breast Invasive Carcinoma (TCGA, PanCancer Atlas)

Lung Squamous Cell Carcinoma (TCGA, PanCancer Atlas) Glioblastoma Multiforme (TCGA, PanCancer Atlas)

Kidney Renal Clear Cell Carcinoma (TCGA, PanCancer Atlas)

Kidney Renal Papillary Cell Carcinoma (TCGA, PanCancer Atlas)

Colorectal Adenocarcinoma (TCGA, PanCancer Atlas)

Diffuse Large B-Cell Lymphoma (TCGA, PanCancer Atlas) Liver Hepatocellular Carcinoma (TCGA, PanCancer Atlas)

Head and Neck Squamous Cell Carcinoma (TCGA, PanCancer Atlas)

Thyroid Carcinoma (TCGA, PanCancer Atlas)

Brain Lower Grade Glioma (TCGA, PanCancer Atlas)

Kidney Chromophobe (TCGA, PanCancer Atlas)

Thymoma (TCGA, PanCancer Atlas)

Acute Myeloid Leukemia (TCGA, PanCancer Atlas)

Uveal Melanoma (TCGA, PanCancer Atlas)

Uterine Carcinosarcoma (TCGA, PanCancer Atlas)

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of AUNIP was observed in DLBC, THCA, LGG, KICH, THYM, LAML, UVM, and UCS (Fig. 4).

TMB and MSI are two common potential indicators for tumor immunotherapy response. The findings suggested that AUNIP expression had positive correlation with TMB in ACC, BLCA, BRCA, COAD, HNSC, LGG, LUAD, LUSC, PAAD, PRAD, SARC, and STAD, and the correlation coef- ficient with STAD is the highest (Fig. 5A). AUNIP had posi- tive interrelation with MSI in BLCA, LUSC, MESO, SARC, and STAD, and negative association with MSI in THCA (Fig. 5B).

Association of AUNIP with immune cell infiltration and immune checkpoints in pan-cancer

We applied ssGSEA to investigate the interrelation between AUNIP and 23 kinds of immune cell infiltration in pan-cancer and found that AUNIP was positively linked with Th2 cells in 30 kinds of tumors, and the positive cor- relation coefficient was the highest. Among the remaining immune cells, AUNIP had a correlation with one or more immune cells in different cancers (Fig. 6A). For the ESTI- MATE algorithm, in ACC, CESC, COAD, ESCA, GBM, HNSC, LUAD, LUSC, OV, READ, SKCM, STAD, THCA, UCEC, and UCS, AUNIP expression had negative associa- tion with stromalscore, immunescore, and estimatescore. In BLCA, CHOL, DLBC, KICH, LAML, LGG, MESO,

PAAD, PCPG, PRAD, and SARC, AUNIP was not associ- ated with stromalscore, immune score, and estimatescore, and AUNIP was related to stromalscore, immunescore, and estimate score in the remaining 7 tumors(Fig. 6B). Immune checkpoint is the target of immunotherapy at this stage, and our results showed that AUNIP was positively or negatively associated with an immune checkpoint in all tumors except ACC, CESC, CHOL, MESO, OV, and UCS (Fig. 6C).

Drug sensitivity analysis

We found that AUNIP expression was negatively linked with 50% inhibitory concentration (IC50) values of 30 drugs based on the results of the CTRP dataset in GSCA. There was a strong negative correlation with IC50 of COL-3, dinaciclib, and docetaxel (Fig. 7). These findings indicated that AUNIP was significantly associated with different drug sensitivities in various tumor cell lines and may be a latent target for cancer therapy.

Gene functional enrichment of AUNIP in pan-cancer

The results of GSEA demonstrated that AUNIP was pri- marily participated in cell cycle, DNA replication, mis- match repair, and homologous recombination in most tumors (Fig. 8). In these tumors, AUNIP was mainly involved in the development of tumors through the above pathways.

Fig. 5 The relationship of AUNIP with TMB (A) and MSI (B) ( *** P< 0.001, ** P < 0.01, *P < 0.05)

A

ACC

B

UCS

UVM

BLCA

ACC

0.6

BRCA ***

UCS

UVM

BLCA **

0.4

BRCA

UCEC

CESC

UCEC

CESC

0.3

THYM

0.4

CHOL

THYM

CHOL

0.2

THCA

COAD ***

** THCA

0.

COAD

0.2

TGCT

DLBC

TGCT

0

DLBC

0.1

*** STAD

ESCA


STAD

-0.2

ESCA

SKCM

GBM

SKCM

-0.3

GBM

*** SARC

HNSC *

*** SARC

HNSC

READ

KICH

READ

KICH


PRAD

KIRC

PRAD

KIRC

PCPG

KIRP

PCPG

KIRP

*** PAAD

LAML

PAAD

LAML

OV

LGG ***

OV

LGG

MEŞQ

*** LUSC

*** LUAD

LIHC

* MESQ

** LUSC

LUAD

LIHC

TMB

MSI

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Fig. 6 The relationship between AUNIP and immunity in pan- cancer. A The relationship of AUNIP with immune cell infiltration using ssGSEA. B The association between AUNIP and stromalscore, immunescore, and estimatescore using ESTIMATE. C The correla- tion between AUNIP and immune checkpoints. (*P < 0.05)

A

LAG3

SIGLEC15

PDCD1LG2

C

TIGIT

HAVCR2

CTLA4

PD-1

17-Od

aDC

ACC

B cells

BLCA

CD8 T cells

BRCA

Cytotoxic cells

DC

CESC

Eosinophils

CHOL

IDC

Macrophages

COAD

Mast cells

p < 0.05

OLBC

Neutrophils

NK CD56bright cells

Cor

1.0

ESCA

NK CD56dim cells

NK cells

0.5

GBM

PDC

0.0

HNSC

T cells

T helper cells

-0.5

KICH

Tcm

-1.0

KIRC

Tem

TFH

KIRP

Tgd

LAMEL

Th1 cells

Th17 cells

LGG

Th2 cells

UHC

TReg

ACC BLCA

BRCA

CESC CHOL

COAD

DLBC

ESCA

GBM

HNSC

KICH

KIRC

KIRP

LAML

LGG

LIHC

LUAD

LUSC

MESO

OV

PAAD

PCPG

PRAD

READ

SARC

SKCM

STAD

TGCT

THCA THYM

UCEC

UCS

UVM

LUAD

LUSC

B

MESO

or

PAAD

StromalScore

¥

*

* p < 0.05

PCPG

PRAD

Cor

1.0

READ

SARC

ImmuneScore

0.5

SKCM

0.0

STAD

TGCT

ESTIMATEScore

*

-0.5

THCA

-1.0

THYM

ACC

BLCA

BRCA

CESC

CHOL

COAD

DLBC

ESCA

GBM HNSC

KICH

KIRC

KIRP

LAML

LGG

LIHC

LUAD

LUSC

MESO

OV

PAAD

PCPG

PRAD

READ

SARC SKCM

STAD

TGCT

THCA THYM

UCEC

UCS

UVM

UCEC

UCS

UVM

Cor

900 > d.

-1.0

S’O-

00

0.5

1.0

Fig. 7 The correlation between AUNIP expression and drug sensitivity using the CTRP dataset

Correlation between CTRP drug sensitivity and mRNA expression

Correlation

-0.4

-0.1

0.0

AUNIP

FDR

0.05

FDR

3-CI-AHPC

AZD7762

BI-2536

BRD-K66453893

BRD-K70511574

CD-437

COL-3

CR-1-31B

FQI-2

GSK461364

GW-843682X

KX2-391

ML311

NVP-231

PHA-793887

SB-225002

SB-743921

SR-II-138A

clofarabine

cytarabine hydrochloride

dinaciclib

docetaxel

leptomycin B

nakiterpiosin

narciclasine

pevonedistat

rigosertib

tivantinib

triazolothiadiazine

vincristine

0.001

0.0001

Drugs

Overexpression AUNIP was correlated with clinical information in LIHC and an independent prognostic gene for LIHC

IHC analysis demonstrated that AUNIP was overexpressed, compared to normal liver tissues (Fig. 9A,B). AUNIP

expression was linked with histologic grade, not corre- lated with age, gender, and pathologic stage (Table 1). Kaplan-Meier analysis suggested that the patients with high-expression group had worse prognosis (Fig. 9C).

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Fig. 8 The functional enrichment of AUNIP in pan-cancer using GSEA

ACC BLCA

BRCA

CESC

CHOL

COAD

DLBC

ESCA

GBM

HNSC

KICH

KIRC

KIRP

LAML

LGG

LIHC

LUAD

LUSC

MESO

OV

PAAD

PCPG

PRAD

READ

SARC

SKCM

NSE

TGCT

THCA

THYM

UCEC

UCS

UVM

KEGG_HOMOLOGOUS_RECOMBINATION

2

KEGG_MISMATCH_REPAIR

KEGG_CELL_CYCLE

1

KEGG_DNA_REPLICATION

0

KEGG_RIBOSOME

-1

KEGG_ECM_RECEPTOR_INTERACTION

-2

KEGG_MATURITY_ONSET_DIABETES_OF_THE_YOUNG

-3

KEGG_P53_SIGNALING_PATHWAY

KEGG_HEDGEHOG_SIGNALING_PATHWAY

KEGG_STEROID_BIOSYNTHESIS

Discussion

The global mortality burden is predominantly attributed to malignant neoplasms resulting from dysregulated cellular proliferation. Recent decades have witnessed remarkable progress in early disease detection methodologies, encompassing the conventional approaches including radiotherapy, surgical procedures, tailored therapeutic regimens, and chemotherapeutic interventions (Mishra et al. 2023a, 2023b, 2023c). However, cancer remains a major threat to human health. Therefore, it is important to find an effective biomarker to predict the development and prognosis of cancer. DNA double-strand breakage damage is the most severe form of damage, if not repaired in time or abnormal repair occurs, it will lead to a series of changes in the cell genome, directly lead to deactivation of tumor suppressor genes or overexpression of oncogenes, and eventually lead to cell cancer (Burma et al. 2006). The most critical factor affecting the selection of DNA double-strand break repair pathways is the state of the cut end of DNA, and AUNIP is a key factor regulating the state of DNA cleavage ends. AUNIP, a binding protein of protein kinase A and Ninein proteins, also known as AIBP, is a structurally specific DNA-binding protein that is localized to the 135 open-reading framework of chromosome 1. According to reports, it is highly expressed in various tumors (Ma et al. 2020). AUNIP is highly expressed in astrocytoma and other brain tumors, suggesting that AUNIP may play a role as oncogenic genes in the development of brain tumors (Lieu et al. 2010).

Our study analyzed the expression, clinical significance, prognosis, mutation, and immunity of AUNIP from the perspective of pan-cancer using a multi-omics system. It was found that AUNIP expression was increased significantly in most tumors compared to normal tissues, suggesting

that AUNIP may be a key gene in cancer development. We conducted IHC analysis to confirm the higher expression of AUNIP in LIHC, which was consistent with TCGA database. In addition, AUNIP with high expression in ACC, LGG, LIHC, MESO, and SARC had poorer OS and DFS than those of AUNIP with low expression, suggesting that the high expression of AUNIP in some tumors influenced patients’ prognosis. Furthermore, AUNIP expression was related to the T stage, N stage, and clinicopathological stage in some cancers, indicating that AUNIP may be a promising valuable diagnostic and prognostic marker in multiple tumors. IHC analysis indicated that AUNIP was linked with histologic grade in LIHC. Moreover, AUNIP expression was an independent prognostic index by univariate and multivariate regression in LIHC. We used cBioportal to study the frequency of AUNIP gene alteration in tumors. In CHOL, the frequency of genetic changes was the highest, with all deep deletions, followed by PCPG, with all deep deletions.

Immune cell infiltration is closely linked to cancer progression (Marcas and Walzer 2018). Recent studies have suggested that tumor progression is caused by an imbalance between the tumor’s immune state and the host’s immune response (Nabbi et al. 2019). We studied the relationship between AUNIP and immunocyte infiltration and observed that AUNIP expression was positively related to Th2 for most tumors, indicating that with the increase of AUNIP expression, Th2 concentration was up-regulated. Th2 cells are not conducive to the anti-tumor effect of cellular immunity. Th1/Th2 drift will protect the tumor from immune surveillance and immune attack, thus promoting the development and progression of tumors (Sharma et al. 2007). Furthermore, we applied the ESTIMATE algorithm to discuss the correlation between AUNIP and stromalscore, immunescore, and estimatescore in different tumors. In most

Fig. 9 The expression of AUNIP in LIHC (A) and Kaplan-Meier analysis of AUNIP in LIHC (B)

A

Normal tissues

Hepatocellular carcinoma

B

C

p = 2.8e-05

12

1.0

Low groups

High groups

10

0.9

IHC score

Survival probability

8

0.8

6

0.7

4

7

0.6

2

HR = 3.16 (1.59 - 6.28)

0.5

P = 0.001

Normal

LIHC

0

1

2

3

4

5

6

Time

tumors, AUNIP was negatively correlated with these three scores. The application in immune checkpoint inhibitors has elevated immunotherapy to a new level. Immunotherapy has been considered as an effective therapy for various advanced and invasive cancers (Morse et al. 2005; Zhou and Zhong 2004). At present, immunotherapy has been applied to a variety of tumors. Common immune checkpoints include PD-1, PD-L1, CTLA-4, PDCD1LG2, TIGIT, HAVCR2, SIGLEC15, and LAG3. In some tumors, AUNIP expression was positively related to immune checkpoint expression, suggesting that these patients with high AUNIP expression may benefit from immunotherapy. TMB and MSI are effective markers to predict the effect of immunotherapy. MSI-H’s tumor gene repair system is abnormal, and there may be more gene mutations, which are easily recognized by

T cells and may respond better to immunotherapy (Bateman 2021). The higher the TMB, the greater the probability that neoantigens expressed by the tumor will be identified by the immune system. Therefore, tumors with high TMB are more sensitive to immune therapy (Liu et al. 2019). Our study demonstrated that AUNIP had positive association with TMB and MSI in BLCA, SARC, and STAD, and patients with high AUNIP expression in these three types of tumors were more susceptible to immunotherapy.

The results of gene enrichment analysis showed that AUNIP caused tumors progression through the cell cycle, DNA replication, mismatch repair, and homologous recombination in most tumors. This is consistent with the literature reports (Lou et al. 2017) In addition, we also performed a correlative analysis between AUNIP and drug

Table 1 The correlation of AUNIP expression with clinical informa- tion in LIHC
CharacteristicsAUNIPXP
Low expressionHigh expression
Age0.250.72
≤ 551420
> 5555
Gender0.240.63
Female1015
Male910
Pathologic stage0.090.76
Stage I+ Stage II913
Stage III + Stage IV1012
Histologic grade4.540.03
G1+G2139
G3616

sensitivity, and we used a public database to predict sev- eral candidate targeted small-molecule drugs. We found that AUNIP was negatively related to IC50 values of 30 drugs, indicating that these drugs stop the progression of the tumor. This provides a novel insight into expanding the therapeutic selection of these targeted small-molecule drugs and developing new drugs specifically targeting AUNIP.

We performed IHC analysis to discuss AUNIP expression in LIHC and the findings demonstrated that AUNIP expression was up-regulated in LIHC. The patients with high-expression group had unfavorable prognosis. These findings suggested that overexpression AUNIP was correlated with the progress of LIHC development and prognosis.

In our work, the expression, prognosis, and characteristics of AUNIP were elucidated by pan-cancer analysis. However, there are some shortcomings in this study. The characteristics of AUNIP were analyzed through bioinformatics and only conducted IHC to verify the overexpression of AUNIP in LIHC. However, there was no biological experiment to verify it. Therefore, in the following studies, it needs more experiment to further validate the mechanism of effect of AUNIP in inducing tumors.

Acknowledgements The authors express our gratitude for the contribu- tions of TCGA, GTEx, TIMER, GEPIA2, cBioportal, and GSCALite databases.

Author contributions All authors participated in the design, methodology, data analysis, and manuscript review of the study; the contributions of XRG and TL are equal. NL provided experimental concepts and designs. XRG, TL, and LJ have made contributions in conceptualization, project management, writing review, and editing. All authors have read and approved the final manuscript.

Funding This study was granted from Zhejiang Province Traditional Chinese Medicine Technology Project (2023ZL249).

Availability of data and materials The data included in the research report are included in the article. Further inquiries can be made directly to the corresponding author.

Declarations

Conflict of interest The authors declare no competing interests.

Ethics approval and consent to participate The study has been per- formed in accordance with the Declaration of Helsinki and was approved by Institutional Research Ethics Committee of the Zheji- ang Provincial People’s Hospital, and written informed consent was obtained from all patients.

Consent for publication.

Not applicable.

Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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