RBIS regulates ribosome biogenesis to affect progression in lung adenocarcinoma

Abstract

Background: Increased ribosome biogenesis is required for tumor growth. In this study, we investigated the function and underlying molecular mechanism of ribosome biogenesis factor (RBIS) in the progression of non-small cell lung cancer (NSCLC).

Methods: In our study, we conducted a comprehensive analysis to identify key genes implicated in ribosome biogenesis by leveraging a Gene Set Enrichment Analysis (GSEA) dataset. Subsequently, we performed a comparative analysis of gene expression profiles by utilizing data from the Gene Expression Omnibus (GEO) datasets to ascertain differentially expressed genes (DEGs) between cancerous and adjacent non-cancerous tissues. Through the intersection of gene sets derived from GSEA and GEO, we identified a cohort of ribosome-associated genes that might exert a substantial influence on the progression of lung adenocarcinoma. Following an extensive literature review, we have identified the RBIS gene as an interesting candidate for further investigation. To elucidate the in vitro functional role of RBIS, several assays was employed, including the Transwell migration and invasion assay, wound healing assay, Cell Counting Kit-8 (CCK-8) proliferation assay, and colony formation assay. Subcutaneous and tail vein injection-based lung metastasis xenograft tumor models were used in evaluating the tumorigenic potential, growth, and metastatic spread of lung cancer cells. Flow cytometry analysis was employed to investigate cell cycle distribution and apoptotic rates. Additionally, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was utilized to quantify the mRNA expression levels of genes. To comprehensively assess the translational efficiency of nascent proteins, we employed polysome profiling analysis to provide insights into the cellular translational landscape. Furthermore, we quantified global protein synthesis using a fluorescence-based assay to measure protein synthesis rates. The immunofluorescence technology was utilized to study the subcellular reorganization of the nucleolus. We conducted co-immunoprecipitation (Co-IP) assays followed by Western blot analysis to identify potential proteins interacted with RBIS. The half maximal inhibitory concentration (IC50) was used for evaluating the chemosensitivity of lung cancer cells to gemcitabine. Additionally, the colony formation assay was employed to assess the survival and proliferative capacity post-treatment of gemcitabine.

Results: The database analysis showed that RBIS was upregulated in lung adenocarcinoma, and its high expression was associated with poor prognosis; Knockdown of RBIS significantly inhibited NSCLC cell migration, invasion and proliferation in vitro and xenograft tumor growth and metastasis in vivo. Additionally, knockdown of RBIS led to G0/G1 phase arrest and significantly increased apoptosis in lung adenocarcinoma cells. Mechanistically, downregulation of RBIS significantly decreased the expression of 47S ribosomal RNA (rRNA), a component associated with ribosome assembly. Polysome profiling analysis indicated that RBIS knockdown affected protein translation efficiency, and global protein synthesis assay further verified that RBIS knockdown inhibited synthesis of newborn proteins. Additionally, the ribosomal biogenesis-targeting drugs CX-5461 and the loss of RBIS exhibited synergistic effects in inhibiting cell cycle progression and inducing apoptosis. Furthermore, the ribosomal maturation factor GNL2 was identified as the key downstream regulator of RBIS in ribosome biogenesis. Notably, knockdown of RBIS substantially increased the sensitivity of lung adenocarcinoma cells to the chemotherapeutic drug gemcitabine, highlighting its l role in chemotherapy.

Conclusions: Collectively, these studies suggested the close involvement of RBIS in the progression of lung adenocarcinoma, providing new insights for targeted therapeutic interventions involving ribosomes.

Keywords: Gemcitabine sensitivity; Lung adenocarcinoma; RBIS; Ribosome biogenesis.

Fig. 1

Expression of RBIS in clinical lung cancer samples and its correlation with prognosis. A, B Expression of RBIS in different cancer types from the TIMER and ULCAN databases. C Expression of RBIS in the GSE102287, GSE30219 and ULCAN-TCGA datasets. D, E Expression of RBIS in different pathological stages and lymph node metastasis stages of LUAD in the ULCAN database. F Analysis of the correlation between the RBIS and the prognosis of lung adenocarcinoma patients using the Kaplan, GEPIA and TIMER databases. G mRNA expression of RBIS in 60 clinical lung cancer samples. H Statistical analysis revealed high expression of RBIS in 55.7% of the lung cancer tissues

Fig. 2

Significant inhibition of cell migration and invasion in lung adenocarcinoma cells with RBIS knockdown. A mRNA expression levels of RBIS in lung cancer cell lines. B Knockdown of RBIS expression in A549 and PC9 cells via transient transfection of RBIS siRNA. C, D Transwell assay assessing the effects of RBIS knockdown on the migration and invasion abilities of lung adenocarcinoma cells. E–G Wound healing assay to detect the migration ability of lung adenocarcinoma cells. All experiments were conducted with three replicates

Fig. 3

Significant inhibition of cell growth in lung adenocarcinoma cells with RBIS knockdown. A CCK-8 assay showing the viability of A549, H1299 and PC9 lung adenocarcinoma cells with transient RBIS knockdown. B Colony formation assay examining the clonogenic potential of A549, H1299 and PC9 lung adenocarcinoma cells with transient RBIS knockdown. C Knockdown efficiency of RBIS in A549, H1299 and PC9 lung adenocarcinoma cells with shRNA. D, E CCK-8 and colony formation assays were conducted in A549, H1299 and PC9 lung adenocarcinoma cells with stable RBIS knockdown. All experiments were conducted with three technical replicates

Fig. 4

Knockdown of RBIS inhibits in vivo tumor growth and metastasis of lung cancer cells. A–C A549 cells stably expressing shNC or shRBIS were injected subcutaneously into 6-week-old BALB/c female nude mice (n = 20). The tumor images (A), tumor weights (B), and tumor volumes (C) are presented 31 days postinjection. D–F A549 cells stably expressing shNC or shRBIS were injected into the tail vein of 6-week-old male NOD-SCID mice (n = 6). Representative images of lung metastasis (D) and the incidence of lung metastasis (E) and H&E (F) results are shown. The Mann–Whitney test was used to assess differences in tumor weight and tumor volume among the different experimental treatment groups. The Chi-square test was used to evaluate the incidence of lung metastasis.  All experiments were conducted with three replicates

Fig. 5

RBIS knockdown alters the DNA content distribution in lung adenocarcinoma cells. A–C Flow cytometry was used to detect changes in the DNA content distribution in the lung adenocarcinoma cell lines A549, H1299 and PC9 with RBIS knockdown. D EdU staining was conducted to detect DNA synthesis. E Detection of expression changes by Western blot in cell cycle-related protein markers in lung cancer cells after RBIS knockdown. All experiments were conducted with three technical replicates

Fig. 6

RBIS knockdown significantly promotes the cell apoptosis rate. A–C Flow cytometry was used to detect the cell apoptosis rate in the lung adenocarcinoma cell lines A549, H1299 and PC9 with RBIS knockdown. D Changes in the expression of apoptotic related protein markers determined by Western blot in lung cancer cells with RBIS knockdown. E, F Flow cytometry were used to assess the apoptosis indicators in the subcutaneous tumors formed from A549 cells stably expressing shNC or shRBIS. All experiments was conducted with three replicates

Fig. 7

RBIS deficiency alters ribosome biogenesis. A, B Evaluation of RBIS knockdown efficacy by qPCR (A) and IF (B). C Expression of 47S rRNA in A549 cells. D Protein translation activity evaluated by polysome profiling analysis. E A global protein synthesis assay kit was used to evaluate the global protein synthesis status. F Nucleolin immunofluorescence staining was used to analyze changes in the nucleolus. G Following the addition of 75 µM of the ribosome assembly inhibitor CX-5461 to A549 cells stably expressing shNC or shRBIS, Western blot analysis was conducted to detect the expression of CDK2 and MCL-1. H Co-IP assay in H293T cell. I, J Western blot was conducted to identify the interaction between RBIS and GNL2 in H293T (I) and A549 cell (J). K GNL2 expression was detected by Western blot in A549 cell stably over-expressing PCDH or RBIS. All experiments were conducted with three technical replicates except experiments related to IP.

Fig. 8

RBIS knockdown significantly increases the sensitivity of lung adenocarcinoma cells to gemcitabine. A IC50 of gemcitabine in A549 and H1299 lung adenocarcinoma cells with RBIS knockdown. B, C Effects of different concentrations of gemcitabine on the clonogenic potential of A549 and H1299 lung adenocarcinoma cells with RBIS knockdown. All experiments were conducted with three replicates