Bioinformatic analysis of CCND2 expression in papillary thyroid carcinoma and its impact on immune infiltration

doi:10.12122/j.issn.1673-4254.2024.05.21

Abstract

Abstract:

Objective To investigate cyclin D2 (CCND2) expression in papillary thyroid carcinoma (PTC) and its association with the clinicopathological features. Methods The public databases TCGA, TIMER 2.0 and UALCAN were used to explore CCND2 expression level in PTC and adjacent tissues, and its diagnostic value for PTC was analyzed using ROC curves. GO enrichment analysis of CCND2-related differentially expressed genes (DEGs) in PTC was performed, and tumor immune infiltration of CCND2 in thyroid cancer was analyzed using TIMER database and CIBERSORT data source. RT-qPCR and Western blot were used to detect CCND2 expression in normal human thyroid cell line Nthy-ori-3-1 and human PTC cell lines TPC-1 and BCPAP. CCND2 expression was also detected in clinical specimens of PTC and adjacent tissues by immunohistochemistry, and its correlation with clinicopathological features of the patients were analyzed. Results Informatic analysis revealed significantly higher CCND2 mRNA expression in thyroid cancer than in the adjacent tissues (P<0.001) in close correlation with tumor stage, gender, age, pathological subtype, and lymph node involvement (P<0.05). ROC curve analysis showed that at the cutoff value of 4.983, the diagnostic sensitivity, specificity, and accuracy of CCND2 expression for PTC was 83.6%, 94.9%, and 78.5%, respectively. CCND2 expression was positively correlated with B cells, CD4⁺ T cells, and macrophages (P<0.001) and negatively with CD8⁺ T cells (P<0.01), and also correlated with memory B-cell infiltration, CD4⁺ T-cell memory activation, M2 macrophages, resting mast cells, and mast cell activation (P<0.05). RT-qPCR, Western blot and immunohistochemistry showed significantly higher CCND2 expression in the PTC cells than in Nthy-ori-3-1 cells (P<0.01) and also in clinical PTC tissues than in the adjacent tissues (P<0.05) in correlation with tumor size, lymph node metastasis and TNM stage (P<0.05). Conclusion CCND2 overexpression is closely correlated with tumor progression and immune cell infiltration in PTC patients..

Key words: papillary thyroid carcinoma, Cyclin D2, bioinformatics, tumor immune infiltration

Qinzhi WANG, Bing SONG, Shirui HAO, Zhiyuan XIAO, Lianhui JIN, Tong ZHENG, Fang CHAI. Bioinformatic analysis of CCND2 expression in papillary thyroid carcinoma and its impact on immune infiltration[J]. Journal of Southern Medical University, 2024, 44(5): 981-988.

Figures/Tables 8

Fig.1 Differential expressions of CCND2 and related genes in thyroid cancer. A: CCND2expression in different types of cancer investigated with the Tumor Immune Estimation Resource (TIMER) database. B: CCND2 expression in THCA was examined by using the UALCAN database; C: Analysis of CCND2 expression in THCA and adjacent normal tissues in the TCGA database; D: Analysis of CCND2 expression in THCA and paired adjacent normal tissues in TCGA database; E: Expression heatmap of differential gene related to CCND2. ***P<0.001

Fig.2 Correlation analysis of CCND2 expression with tumor stage (A), gender (B), lymph node metastasis (C), age (D) and tumor histology (E). *P<0.05, ***P<0.001 vs Normal group

Fig.3 ROC curve of CCND2 expression levels in tumor tissues for PTC diagnosis.

Fig.4 Functional enrichment analysis of differentially expressed genes. A: GO enrichment analysis; B: GO enrichment analysis

Fig.5 Relationship between expression levels of CCND2 and TIICs. A: Correlation analyses of CCND2 with the infiltration of different immune cells using the TIMER database; B: Difference in infiltration fractions of 22 immune cell subsets in high- and low-risk groups. Red, the high-risk group and blue, low-risk group. *P<0.05, **P<0.01 vs groups.

Fig.6 Expression of CCND2 in Nthy-ori-3-1, TPC-1, and BCPAP cells. A: RT-qPCR for detecting mRNA expression of CCNDD2. B: Western blot of CCND2 and β-tubulin (loading control). C: Relative expression level of CCND2/β-tubulin. Data are expressed as Mean±SD. **P<0.01.

Fig.7 Expression of CCND2 in PTC tissues and adjacent tissues detected by immunohistochemistry (Original magnification:×200). A: Normal tissue. B: PTC tissue. C: Expression levels calculated with average optical density (AOD). ***P<0.001.

Tab.1 Relationship between CCND2 expression and clinicopathological characteristics of PTC patients ［n (%)］

Clinicopathological parameters		n	CCND2 positive	χ²	P
Gender	Male	32	22 (68.75)	0.4622	0.4966
Gender	Female	84	63 (75.00)	0.4622	0.4966
Age (year)	<55	79	55 (69.62)	1.9627	0.1612
Age (year)	≥55	37	30 (81.08)	1.9627	0.1612
Tumor size (cm)	<1	76	50 (65.79)	4.6911	0.0303
Tumor size (cm)	≥1	40	35 (75.00)	4.6911	0.0303
Multifocal	Yes	40	26 (65.00)	2.1353	0.1439
Multifocal	No	76	59 (77.63)	2.1353	0.1439
Extraglandular invasion	Yes	13	11 (84.62)	0.9614	0.3268
Extraglandular invasion	No	103	74 (71.84)	0.9614	0.3268
Lymphatic metastasis	Yes	43	38 (88.37)	7.9520	0.0048
Lymphatic metastasis	No	73	47 (64.38)	7.9520	0.0048
TNM staging	I-II	100	70 (70.00)	12.1309	0.0005
TNM staging	III-IV	16	15 (93.75)	12.1309	0.0005

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