Comprehensive analysis of prognostic value and immune infiltration of Regulator of Chromosome Condensation 2 in lung adenocarcinoma

Abstract

Background: Lung adenocarcinoma (LUAD) incidence and mortality take the leading place of most malignancies. Previous studies have revealed the regulator of chromosome condensation 1 (RCC1) family members played an essential role during tumorigenesis. However, its biological functions in LUAD still need further investigation. Methods: Several databases were applied to explore potential effects of RCC1 family members on LUAD, such as Oncomine, GEPIA, and cBioPortal. Real-time PCR and immunohistochemistry were used to verify the expression of RCC2 in stage I LUAD. H1975 and A549 were selected to explore the biological function of RCC2 in cellular malignant phenotype. Results: The expressions of RCC1 and RCC2 showed marked differences in malignant tissue compared to lung tissue. The higher the expression levels of RCC1 or RCC2 in LUAD patients, the shorter their overall survival (OS). In normal lung tissues, RCC1 expression was highly enriched in alveolar cells and endothelial cells. Compare with RCC1, RCC2 expression in normal lung tissue was significantly enriched in macrophages, B cells and granulocytes. Additionally, RCC2 expression level was correlated with multiple immune cell infiltration in LUAD. Moreover, the mutation or different sCNA status of RCC2 exerted influence on multiple immune cell infiltration distribution. We found that the upregulation of RCC1 and RCC2 were obviously related to TP53 mutation. GSEA analysis revealed that RCC2 was involved in the process of DNA replication, nucleotide excision repair and cell cycle, which might affect tumor progression through P53 signaling pathway. We further elucidated that downregulation of RCC2 could dramatically repress the migration and invasion of LUAD cells. Conclusions: The study demonstrated that RCC1 and RCC2 expression were markedly increased in early-stage of LUAD. Patients with high expression of RCC1 or RCC2 had a worse prognosis. Based on our analysis, RCC1 and RCC2 might exert influence on LUAD process through DNA replication, nucleotide excision repair and cell cycle, as well as cells migration and invasion. Different from RCC1, RCC2 also involved in immune infiltration. These analyses provided a novel insight into the identification of diagnostic biomarker.

Keywords: RCC1; RCC2; TP53; immune infiltration; lung adenocarcinoma.

Figure 1

The mRNA level of distinct RCC1 superfamily members in LUAD tissues and normal lung tissues. (A) The transcription levels of RCC1 superfamily in different cancer types (Oncomine): upregulated (red) and downregulated (blue). The following criteria were used: p value < 0.05; fold change absolute value < 2, gene rank: 10%. (B) Expression of RCC1 superfamily members in LUAD tissues and normal lung tissues (UALCAN). (C) Correlations between RCC1 and RCC2 expression and tumor stage of LUAD patients (UALCAN). (D) Relationship between RCC1 or RCC2 mRNA level and LUAD patient with TP53 mutation (UALCAN). (E) The protein expression level of RCC1 in LUAD tissues and normal lung tissues (HPA). (F) The protein expression level of RCC2 in LUAD tissues and normal lung tissues (HPA). (ns means p > 0.05; **** means p < 0.0001)

Figure 2

The survival analysis of LUAD patients with different expression level of RCC1 and RCC2. (A) Higher expression level of RCC1or RCC2 was significantly associated with shorter overall survival (OS) in LUAD patients (GEPIA). (B) Different expression of RCC1 or RCC2 exerted impact on LUAD patients' overall survival (OS), first-progression survival (PF) and post-progression survival (PPS) (Kaplan-Meier Plotter).

Figure 3

The univariate and multivariate Cox regression analysis of RCC1 and RCC2 in LUAD patients. (A) Univariate Cox regression analysis of RCC1 in LUAD patients. (B) Univariate Cox regression analysis of RCC2 in LUAD patients. (C) Multivariate Cox regression analysis of RCC2 in LUAD patients.

Figure 4

The alteration frequency and prognostic analysis of RCC1 and RCC2 in LUAD were derived from cBioPortal. (A) Summary of RCC1 and RCC2 alteration in LUAD patients. (B) Using the cBioPortal database, we assessed the RCC1 and RCC2 genetic alteration frequency of LUAD samples in TCGA database. (C) The overall survival (OS) between LUAD patients with/without RCC1 or RCC2 alteration was assessed by Kaplan-Meier.

Figure 5

Correlation between RCC1 and RCC2 expression and immune cell infiltration. (A-B) The distribution of RCC1 and RCC2 expression in normal lung tissues was assessed by the single cell RNA analysis in the HPA database. (C-D) The RNA profiles of RCC1 and RCC2 in human immune cells was evaluated by HPA database. (E) Correlation between multiple types of immune cell infiltration and RCC2 expression were detected by TIMER database. The result was adjusted by purity. (F) The TIMER database was used to assess the effect of RCC2 mutation on immune cell infiltration. (G) Comparing immune infiltration distribution based on the sCNA status of RCC2 across TCGA cancer types (TIMER).

Figure 6

Interaction network and enrichment analysis of RCC1 and RCC2 in LUAD patients. (A) Co-expression network of RCC1 and RCC2 in LUAD (GeneMANIA). (B) The top five functional categories were evaluated by Metascape analysis. (C) Co-relationship among the top five enrich terms, color by cluster ID (Metascape). (D) Protein-protein interaction network of RCC1 and RCC2 and their co-expression genes (STRING). (E) The prediction of transcription factor targets for RCC1, RCC2 and their related genes (Metascape). (F) The mechanism of RCC2 influencing LUAD progression was assessed by GSEA.

Figure 7

Verification of RCC2 expression in stage Ⅰ of LUAD patients. (A) Comparison of RCC2 expression between LUAD tissues and normal lung tissues by GEO database (GSE31548). (B) Verifying the RCC2 differential expression in LUAD patients by RT-PCR (32 LUAD patients). (C-D) RCC2 shows significantly high expression level in LUAD tumor tissue compared to adjacent tissues, as determined by immunohistochemical assay (58 patients, p < 0.05). (**** means p < 0.0001)

Figure 8

Down-regulation of RCC2 suppressed the migration and invasion abilities of A549 and H1975. (A-B) Migration distance of sh-RCC2 cells were significantly shorter than sh-NC cells. (** means p < 0.01, **** means p < 0.0001) (C-D) Transwell assay was used to evaluate the migration and invasion ability difference between sh-NC cells and sh-RCC2 cells. (** means p < 0.01, *** means p < 0.001, **** means p < 0.0001).