Holliday junction-recognizing protein is a potential predictive and prognostic biomarker for kidney renal clear cell carcinoma

doi:10.12122/j.issn.1673-4254.2024.12.10

Abstract

Abstract:

Objective To investigate the role of Holliday cross-recognition protein (HJURP) in tumorigenesis, progression, and immunotherapy responses. Methods Bioinformatics approaches were used to analyze the expression level of HJURP in various cancers and its association with prognosis, clinical stage, and immune cell infiltration using TCGA, GTEx, SangerBox and TIMER 2.0 databases. LinkedOmics database was employed to investigate HJURP-related genes and their potential functions in kidney renal clear cell carcinoma (KIRC). The expression of HJURP in KIRC samples was examined with immunohistochemistry, Western blotting and qRT-PCR, and the effect of HJURP silencing on cell proliferation and migration was tested in cultured KIRC cells. Results HJURP was highly expressed in 26 cancers with negative correlations with the patients' survival outcomes in 5 cancers including KIRC (P<0.05). HJURP expression levels was strongly correlated with clinical stages and immune cell infiltration in the tumors. In KIRC, HJURP expression was significantly elevated (P<0.0001) and showed a positive correlation with TNM stage (P<0.05), overall stage (P<0.01) and immune cell infiltration. Gene Ontology (GO) functional analysis showed that HJURP is predominantly enriched in biological processes such as biological regulation and metabolic processes. Concerning cellular components, HJURP is primarily localized to the cell membrane and nucleus. In terms of molecular functions, it is chiefly enriched in activities related to protein binding and ion binding. HJURP was highly expressed in both clinical KIRC tissues and KIRC cell lines (P<0.001). In cultured KIRC cells, silencing of HJURP significantly inhibited cell proliferation and migration abilities. Conclusion HJURP may serves as an indicator of prognosis and immunotherapy response of KIRC, and its high expression enhances malignant behaviors of KIRC cells.

Key words: Holliday junction-recognition protein, pan-cancer, kidney renal clear cell carcinoma, proliferation, migration

Huahua ZHANG, Qingyin TA, Yun FENG, Jiming HAN. Holliday junction-recognizing protein is a potential predictive and prognostic biomarker for kidney renal clear cell carcinoma[J]. Journal of Southern Medical University, 2024, 44(12): 2347-2358.

Figures/Tables 7

Fig.1 Expression of Holliday junction-recognizing protein (HJURP) in pan-cancer. A: Expression of HJURP in different human tumors based on TCGA database. B: HJURP expression in 26 human tumors analyzed by integrating normal tissue data from GTEx database and TCGA tumor tissue. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Normal group.

Fig.2 Survival forest map for prognostic analysis of HJURP expression in pan-cancer. OS: Overall survival; DSS: Disease-specific survival; DFI: Disease-free interval; PFI: Progression-free interval.

Fig.3 Associations between HJURP expression and clinicopathological characteristics of pan-cancer. A: T grade. B: N grade. C: M status. D: clinical stage. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs T1, N0, M0 or Stage I.

Fig.5 Correlation of HJURP expression with clinicopathological characteristics and prognosis of kidney renal clear cell carcinoma (KIRC). A: T grade. B: N grade. C: M status. D: clinical stage. E: HR (95% CI) for OS. F: HR (95% CI) for DSS. G: HR (95% CI) for DFI. H: HR (95% CI) for PFI. *P<0.05, **P<0.01, ***P<0.001 vs T1, N0, M0, or Stage I.

Fig.6 Analysis of HJURP-related genes in KIRC and the GO function. A: Volcanic plot of HJURP-related genes in KIRC. B: Heat map of genes positively associated with HJURP in KIRC. C: Heat map of genes negatively associated with HJURP in KIRC. D: GO function analysis of HJURP in KIRC.

Fig.7 Expression of HJURP in KIRC and validation of the efficiency of HJURP interference. A: HJURP in adjacent and KIRC tissues detected by immunohistochemistry (×100). B: Differential expression of HJURP in KIRC verified by Western blotting. C: Expression of HJURP mRNA in KIRC cell lines detected by qRT-PCR. D, E: Transfection efficiency of si-HJURP sequences assessed by Western blotting (D) and qRT-PCR (E). ***P<0.001 vs HK-2 or siNC group.

Fig.8 Silencing of HJURP suppresses proliferation and migration of KIRC cells in vitro. A:CCK-8 assays for cell viability. B: Clone formation test to detect the changes of cell clone formation ability. C, D: Flow cytometry for analyzing cell cycle distribution in 786-O and Caki-1 cells (Mean±SD, n=3). E: Changes in cell cycle-related proteins after silencing HJURP. F: Scratch test for assessing cell migration ability (×100). G: Transwell assays for assessing cell migration (×200). H: Western blotting for detecting cell migration-related protein expressions including E-cadherin, N-cadherin, MMP2 and vimentin. **P<0.01, ***P<0.001 vs siNC group.

References 31

1	Zheng RS, Chen R, Han BF, et al. Cancer incidence and mortality in China, 2022[J]. Zhonghua Zhong Liu Za Zhi, 2024, 46(3): 221-31.
2	Ansell SM, Lesokhin AM, Borrello I, et al. PD-1 blockade with nivolumab in relapsed or refractory Hodgkin's lymphoma[J]. N Engl J Med, 2015, 372(4): 311-9.
3	Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma[J]. N Engl J Med, 2015, 373(1): 23-34.
4	Robert C, Ribas A, Wolchok JD, et al. Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase 1 trial[J]. Lancet, 2014, 384(9948): 1109-17.
5	Brahmer JR, Tykodi SS, Chow LQ, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer[J]. N Engl J Med, 2012, 366(26): 2455-65.
6	Weinstein JN, Collisson EA, Mills GB, et al. The Cancer Genome Atlas Pan-Cancer analysis project[J]. Nat Genet, 2013, 45(10): 1113-20.
7	Kato T, Sato N, Hayama S, et al. Activation of Holliday junction recognizing protein involved in the chromosomal stability and immortality of cancer cells[J]. Cancer Res, 2007, 67(18): 8544-53.
8	Barnhart MC, Kuich PH, Stellfox ME, et al. HJURP is a CENP-a chromatin assembly factor sufficient to form a functional de novo kinetochore[J]. J Cell Biol, 2011, 194(2): 229-43.
9	Wei Y, Ouyang GL, Yao WX, et al. Knockdown of HJURP inhibits non-small cell lung cancer cell proliferation, migration, and invasion by repressing Wnt/β-catenin signaling[J]. Eur Rev Med Pharmacol Sci, 2019, 23(9): 3847-56.
10	Kang DH, Woo J, Kim H, et al. Prognostic relevance of HJURP expression in patients with surgically resected colorectal cancer[J]. Int J Mol Sci, 2020, 21(21): 7928.
11	Wang CJ, Li X, Shi P, et al. Holliday junction recognition protein promotes pancreatic cancer growth and metastasis via modulation of the MDM2/p53 signaling[J]. Cell Death Dis, 2020, 11(5): 386.
12	Cao R, Wang G, Qian KY, et al. Silencing of HJURP induces dysregulation of cell cycle and ROS metabolism in bladder cancer cells via PPARγ-SIRT1 feedback loop[J]. J Cancer, 2017, 8(12): 2282-95.
13	Wang L, Qu JL, Liang Y, et al. Identification and validation of key genes with prognostic value in non-small-cell lung cancer via integrated bioinformatics analysis[J]. Thorac Cancer, 2020, 11(4): 851-66.
14	Gu Y, Li J, Guo DL, et al. Identification of 13 key genes correlated with progression and prognosis in hepatocellular carcinoma by weighted gene co-expression network analysis[J]. Front Genet, 2020, 11: 153.
15	Hu Z, Huang G, Sadanandam A, et al. The expression level of HJURP has an independent prognostic impact and predicts the sensitivity to radiotherapy in breast cancer[J]. Breast Cancer Res, 2010, 12(2): R18.
16	Fu FQ, Zhang Y, Gao ZD, et al. Development and validation of a five-gene model to predict postoperative brain metastasis in operable lung adenocarcinoma[J]. Int J Cancer, 2020, 147(2): 584-92.
17	Shen WT, Song ZG, Zhong X, et al. Sangerbox: a comprehensive, interaction-friendly clinical bioinformatics analysis platform[J]. Imeta, 2022, 1(3): e36.
18	Li TW, Fan JY, Wang BB, et al. TIMER: a web server for comprehensive analysis of tumor-infiltrating immune cells[J]. Cancer Res, 2017, 77(21): e108-10.
19	Yoshihara K, Shahmoradgoli M, Martínez E, et al. Inferring tumour purity and stromal and immune cell admixture from expression data[J]. Nat Commun, 2013, 4: 2612.
20	Telloni SM. Tumor staging and grading: a primer[J]. Methods Mol Biol, 2017, 1606: 1-17.
21	Vasaikar SV, Straub P, Wang J, et al. LinkedOmics: analyzing multi-omics data within and across 32 cancer types[J]. Nucleic Acids Res, 2018, 46(D1): D956-63.
22	Li L, Yuan Q, Chu YM, et al. Advances in holliday junction recognition protein (HJURP): structure, molecular functions, and roles in cancer[J]. Front Cell Dev Biol, 2023, 11: 1106638.
23	Chen TC, Zhou LF, Zhou Y, et al. HJURP promotes epithelial-to-mesenchymal transition via upregulating SPHK1 in hepatocellular carcinoma[J]. Int J Biol Sci, 2019, 15(6): 1139-47.
24	Yang Y, Yuan JY, Liu ZZ, et al. The expression, clinical relevance, and prognostic significance of HJURP in cholangiocarcinoma[J]. Front Oncol, 2022, 12: 972550.
25	Zhang F, Yuan DB, Song JK, et al. HJURP is a prognostic biomarker for clear cell renal cell carcinoma and is linked to immune infiltration[J]. Int Immunopharmacol, 2021, 99: 107899.
26	Hinshaw DC, Shevde LA. The tumor microenvironment innately modulates cancer progression[J]. Cancer Res, 2019, 79(18): 4557-66.
27	Luo DC, Liao SN, Liu Y, et al. Holliday cross-recognition protein HJURP: association with the tumor microenvironment in hepatocellular carcinoma and with patient prognosis[J]. Pathol Oncol Res, 2022, 28: 1610506.
28	Liu L, Zhang ZF, Xia XL, et al. KIF18B promotes breast cancer cell proliferation, migration and invasion by targeting TRIP13 and activating the Wnt/β‑catenin signaling pathway[J]. Oncol Lett, 2022, 23(4): 112.
29	Xie JL, Wang B, Luo WJ, et al. Upregulation of KIF18B facilitates malignant phenotype of esophageal squamous cell carcinoma by activating CDCA8/mTORC1 pathway[J]. J Clin Lab Anal, 2022, 36(10): e24633.
30	Koike Y, Yin CZ, Sato Y, et al. TPX2 is a prognostic marker and promotes cell proliferation in neuroblastoma[J]. Oncol Lett, 2022, 23(4): 136.
31	Li LZ, Jiang PC, Hu WM, et al. AURKB promotes bladder cancer progression by deregulating the p53 DNA damage response pathway via MAD2L2[J]. J Transl Med, 2024, 22(1): 295.

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