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.

Background

Cancer poses a significant global health burden, with approximately 19.3 million new cases reported annually. Lung cancer accounts for 11.4% of all cancer cases, and its mortality rate represents 18% of all cancer-related deaths, making it a leading cause of cancer-related fatalities [1]. Lung cancer is histologically categorized into two subtypes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The latter accounts for approximately 85% of all lung cancer cases. Within NSCLC, squamous cell carcinoma (LUSC) constitutes 25–30% of cases, while adenocarcinoma (LUAD) represents approximately 40% of all NSCLC cases, making it the most common subtype of NSCLC [2]. Currently, the vast majority of patients are usually diagnosed at advanced stages or have metastasis. In such advanced cases, palliative chemotherapy stands as the primary therapeutic strategy. However, drug resistance in many LUAD patients has led to final treatment failure, emphasizing the urgent need to uncover novel therapeutic targets that drive LUAD progression and chemotherapy resistance.

Increasing evidence suggests that perturbations in human ribosome biogenesis can lead to various diseases, including cancer [3]. Notably, dysregulated ribosome biogenesis is closely related to cancer cell growth, metastasis, and drug resistance [4,5,6], prompting us to identify additional regulators of ribosome biogenesis as potential therapeutic targets for cancer treatment. In this study, we performed a comprehensive bioinformatics analysis and identified RBIS as a candidate ribosomal biogenesis factor that may play a role in the progression of lung cancer. A systematic nucleolar screening study revealed that RBIS may be involved in pre-rRNA processing [7]. Genome-wide RNAi screening suggested involvement of RBIS in the biogenesis of the ribosomal 40S small subunit [8]. The above studies implicated that RBIS may play a role in the assembly of ribosomes. However, to date, comprehensive functional and mechanistic investigations of RBIS in cancer progression, particularly in LUAD, remain largely unexplored.

In this study, we demonstrated for the first time that RBIS was upregulated in lung cancer tissues. Knockdown of RBIS significantly inhibited the growth and metastasis potential of lung adenocarcinoma cells in vitro and in vivo. Flow cytometry assay demonstrated that loss of function of RBIS enhanced the cell cycle arrest and induced apoptosis. Mechanistically, RBIS is hypothesized to modulate ribosomal biogenesis, potentially through its interaction with GNL2, a potential ribosomal maturation factor. Moreover, knockdown of RBIS increased the sensitivity of lung adenocarcinoma cells to gemcitabine. These findings identify RBIS as an oncogenic regulator in the progression of lung adenocarcinoma and provide novel perspective on therapeutic strategies of targeting ribosomal biogenesis.

Materials and methods

Bioinformatics analysis

Differentially expressed genes (DEGs) between lung cancer tissue and adjacent tissues from GSE30219 and GSE102287 datasets were identified by Analyze-2R. The genes with a fold change > 1.6 were subsequently selected as differentially expressed gene set. Ribosome biogenesis genes were obtained from GSEA by searching for “ribosome biogenesis genes”. The expression levels of the RBIS gene were examined by utilizing public databases, including Tumor Immune Estimation Resource (TIMER, http://cistrome.org/timer) [9], Understanding ALterations in Cancer (ULCAN, https://ualcan.path.uab.edu/analysis.html), GEO datasets GSE102287 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE102287) and GSE30219 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30219). The correlation between RBIS and the prognosis of lung adenocarcinoma was analyzed from Kaplan–Meier analysis, Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/detail.php?gene=&clicktag=survival) [10], and TIMER.

Clinical samples

Sixty paired human lung cancer tissues and matched adjacent noncancerous tissues were obtained from Fudan University Shanghai Cancer Center. Written informed consent was obtained from all patients, and the study was approved by the ethics committee of Fudan University Shanghai Cancer Center (Certificate number: 050432-4-2108*).

Cell culture

The human lung adenocarcinoma cell lines A549, H1299, and PC9 were obtained from the American Type Culture Collection (ATCC) cell repository. A549 cells were cultured in Ham’s F-12K medium (Basal Media, L450KJ), while H1299 and PC9 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Basal Media, L220KJ). All medium were supplemented with 10% fetal bovine serum (FBS) (YeaSen, 40130ES76) and 1% penicillin–streptomycin (Basal Media, S110jv). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

RNA extraction and qPCR

Total RNA was extracted using RNAiso Plus (TAKARA, 9109). Subsequently, reverse transcription was performed using a reverse transcription kit (TAKARA, RR036A). For qPCR analysis, the obtained cDNA was diluted tenfold, and the reaction was performed using SYBR Green (YeaSen, 11202ES08). The primer sequence for RBIS was synthesized by Genewiz.

Migration and invasion assays

Migration and invasion assays were performed using 8-um pore chambers (Corning, 353097). Briefly, 5 × 10⁴ cells for migration assay and 1 × 10⁵ cells for invasion assay in 200 µl serum free culture medium were seeded in the upper chamber, culture medium containing 10% FBS was used as a chemoattractant in the lower chamber. The migrated or invaded cells were fixed with methanol for 30 min, then stained with 0.1% crystal violet for 15–20 min after 16–20 h incubation. Nine random fields were photographed under microscope (Olympus, IX73) at a magnification of 100×. Statistical analysis of the data was performed using GraphPad Prism 9.

Wound-healing assays

Cells were seeded into 6-well plates and allowed to reach approximately 90% confluence. The monolayers were then carefully scratched using a sterile 200 µl pipette tip. Subsequently, the cells were washed with phosphate-buffered saline (PBS) (Basal Media, B320KJ) and cultured in the appropriate cell culture medium supplemented with 1% FBS. Images of the wounded monolayers were captured at 0 h and specified time points following initiation of the wounds.

Cell survival assays

For cell viability assays, cells were seeded in 96-well plates (Corning, 3599) with triplicate wells per group and incubated overnight. Then cells were subjected to CCK8 reagent kit (YeaSen, 40203ES80). For colony formation assays, cells were seeded in 6-well plates (Corning, 3516) and cultured for 10–14 days. The survival colonies were fixed with methanol for 30 min and visualized by staining with 0.1% crystal violet (Beyotime, C0121) for 30 min. After washing and air-drying, the colonies were counted.

Cell transfection

The siRNAs were designed and purchased from RiboBio (Guangzhou, China). The cells were seeded on a 6-well plate and incubated overnight. When cells reached approximately 70% confluencer, a mixture of 120 nmol of siRNA and 5 µl of Lipofectamine 2000 (Invitrogen, 11668019) were incubated at room temperature for 20 min and added to cells. The siRNA sequences are shown in Supplementary Table 1.

Plasmid construction and lentivirus packaging

The shRNA sequence of RBIS was synthesized by Genewiz and inserted into the PLKO.1-TRC plasmid. To generate stable cell lines expressing shRNAs, HEK293T cells were transfected with lentivirus expression vector and packaging plasmids pMD2.G (Addgene, 12259) and psPAX2 (Addgene, 12260) using TenfectDNA transfection reagent (TEYE, FT19301). The cell culture supernatant was collected 48 h after transfection, filtered through a 0.45-μm membrane (Millipore, SLHPR33RB). Lentivirus was added to cells in the presence of 8 µg/ml of polybrene prior to drug selection with 2 mg/ml of puromycin for antibiotic selection of stable knockdown cells for 1 to 2 weeks.

In vivo tumor growth and metastasis assay

Female BALB/c nude mice, aged 6 to 8 weeks, were housed in specific pathogen-free grade standard experimental animal facilities. In the in vivo xenograft assays, 3 × 10⁶ A549 cells stably expressing shRBIS or the negative control were individually inoculated subcutaneously into the dorsal right flanks of the nude mice. Tumor size was measured every 3 days using calipers and calculated via the following formula: (length × width × width)/2. After 4 to 5 weeks, the mice were humanely euthanized, and their tumors were harvested and processed into single cells for subsequent detection of apoptosis. In the in vivo metastasis assays, 2 × 10⁶ A549 cells stably expressing shRBIS or the negative control were separately injected into the lateral tail veins of the NOD-SCID mice. Upon completion of 7 weeks, the mice were euthanized, and their lungs were collected during necropsy and then fixed in 10% neutral formalin. The fixed lung tissues were subsequently subjected to hematoxylin–eosin (H&E) staining and examined for any signs of metastasis.

Cell cycle analysis

Cells were collected from a 6-cm cell culture dish, washed cells twice with PBS, then fixed in 70% ethanol at − 20 °C overnight. After centrifugation and removal of the supernatant, the cells were washed once with PBS and stained with 500 µl of PI staining solution (BD, 550825) for 10 min. The DNA content of the cells was analyzed via a flow cytometer (Beckman coulter, CytoFlex S).

Protein extraction and Western blotting

Cells were washed with prechilled PBS triplicately, then lysed in buffer (Thermo, 78510) supplemented with protease inhibitors (YeaSen, 20123ES50) and phosphatase inhibitors (YeaSen, 20109ES20). Cells were scraped and thoroughly lysed on ice for 30 min. Lysates were centrifuged at 12,000 rpm for 15–20 min, and the supernatants were collected. Protein concentrations were measured using a BCA assay kit (Thermo Fisher Scientific, 23227), and samples were normalized to equivalent concentrations. Following the addition of 5× protein loading buffer (Yeasen, cat. no. 20315ES20), samples were denatured at 100 °C for 10 min and stored at − 80 °C. Proteins were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, 1620177). Membranes were blocked at room temperature for 2 h prior to incubation with primary antibodies. The primary antibodies used in this study were as follows: rabbit anti-Myc proto-oncogene, BHLH transcription factor (c-myc) (Proteintech, 10828-1-Ap, 1:2000), rabbit anti-p-p70s6k (Affinity, AF3228, 1:500), rabbit anti-p21 (Proteintech, 10355-1-ap, 1:1000), rabbit anti-p-cyclin dependent kinase 2 (p-CDK2) (Biodragon, BD-PP0592, 1:500), rabbit anti-CDK2 (Biodragon, BD-PT0832, 1:500), rabbit anti-myeloid cell leukemia 1 (mcl-1) (CST, 5453, 1:1000), and horseradish peroxidase (HRP)-conjugated β-actin (Proteintech, HRP-66009, 1:5000), and then incubated with the following secondary antibody: HRP-conjugated goat anti-rabbit antibody (CST, 7074, 1:5000). Bands were visualized by enhanced chemiluminescence (Share-Bio, sb-wb011), and densitometry measurements of the bands were acquired with Quantity One software (Bio-Rad).

EdU labeling

Cells were seeded into a 24-well plate containing cell culture slides. On the second day, the cells were incubated with 50 μM EdU (YeaSen, 40276ES60) for 2 h. Then cells were subjected to 4% neutral paraformaldehyde (PFA) for fixation at room temperature for 30 min. 2 mg/ml glycine solution was then added and incubated at room temperature for 5 min to neutralize residual fixative. Washed cells twice with 3% bovine serum albumin (BSA) (Basal Media, S475T7). Permeabilized cells with 0.5% Triton X-100 (Beyotime, P0096) and incubated at room temperature for 20 min. Washed cells twice with 3% BSA. Subsequently, the EdU reaction mixture was added to the culture plate, and incubated in the dark at room temperature for 30 min. Washed cells twice with 3% BSA. Slides were mounted using a mounting medium containing 4′,6-Diamidino-2′-phenylindole (DAPI) (Beyotime, P0131), and imaged using a Leica DMI6000 B optical microscope.

Cell apoptosis detection

The cells were inoculated in a 6-well plate. The supernatant and adherent cells were both collected when cells reached 80–90% confluency. Washed cells once with prechilled PBS. Then, added 5 µl of PE and 7AAD from the apoptosis detection kit (Vazyme, A213-02) to cells for dual staining of the cells. After a 10-min staining in the dark, apoptotic cell percentages were analyzed within 1 h using a flow cytometer (Beckman coulter, CytoFlex S).

Cellular immunofluorescence

Inoculated cells in a 24-well plate containing cell culture slides and allow them to adhere and grow naturally. Wash the cells three times with PBS the next day to remove any residual culture medium. The cells were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. Wash the cells three times with PBS to remove the fixative. Then, permeabilized cells with 0.5% Triton X-100 solution at room temperature for 15 min. The cells were then washed three times with PBS to remove the permeabilization agent. The nonspecific binding sites were blocked with 10% nonfat milk (Beyotime, P0216) and incubated for 30 min at room temperature. Anti-RBIS primary antibody (Bioss, BS 15298, 1:50), anti-nucleolin primary antibody (Proteintech, 10556-1-AP, 1:100) was added and incubated at 4 °C overnight. Washed cells with 1% PBST (PBS with 0.1% Tween-20) three times. The secondary antibody (Proteintech, SA00013-2, 1:100) was added in 3% nonfat milk and incubated for 1 h at room temperature. Finally, the slides were mounted with mounting medium containing DAPI. Images were captured via a laser confocal microscope (Leica, STELLARIS5).

Polysome profiling analysis

Human lung cancer cells were pretreated with cycloheximide (CHX, Selleck, S7418, 100 µg/ml) at 37 °C for 15 min. The subsequent lysis and component separation of the samples were operated by Siwega. Briefly, cells were collected and lysed on ice for 10 min. Centrifuged cells for 10 min at 13,000×g at 4 °C, the lysates were carefully loaded onto a pre-established 10–45% sucrose gradient and further ultracentrifuged at 4 °C for 3 h at 36,000 rpm. Samples were subjected to fractionate and monitored at a wavelength of 260 nm. A total of 18 fractions of equal volume were collected and their absorbances were measured.

Global protein synthesis detection

The Global Protein Synthesis Kit (Abcam, ab273286) was utilized to evaluate alterations in cellular protein translation efficiency. Initially, cells were plated in a 24-well plate containing cell culture slides. On the subsequent day, the cells were treated with protein label and incubated for 24 h. After incubation, cells were rinsed once with PBS and fixed with a fixative solution for 15 min at room temperature. After a single wash with the washing buffer, the cells were permeabilized for 10 min with a permeabilizing agent. Subsequently, 300 µl of the reaction reagent was added and incubated in the dark at room temperature for 30 min. The cells were then rinsed thrice with the washing buffer. Finally, cells were mounted using a mounting medium containing DAPI. The mounted cells are visualized and imaged using a confocal laser scanning microscope (Leica, STELLARIS5).

Drug experiments

For the CX-5461 treatment experiment, the cells expressing shNC or shRBIS, was treated with the ribosome-targeting drug CX-5461 (MCE, S2684) for 48 h, then protein was extracted as previously described. For the gemcitabine (MCE, HY-17026) IC50 experiment, A549 and H1299 cells stably expressing shNC or shRBIS were seeded in a 96-well plate, 4000 cells per well. The cells were treated with specified concentrations of gemcitabine (0, 0.00316, 0.01, 0.0316, 0.1, 0.316, 1, 3.16, 10, 31.6 μM) in 10 replicates and incubated for 72 h. Absorbance at 450 nm (OD450) was measured, with the blank well’s OD450 subtracted. The experimental group’s OD450 was then subtracted from the control group’s OD450 to derive values for IC50 plotting. To determine the impact of gemcitabine on colony formation, A549 and H1299 cells stably expressing shNC or shRBIS were seeded in 6-well plates, 2500 cells per well. Four different concentrations of gemcitabine (0, 0.1, 0.2, and 0.3 μM) in 3 replicates were added to cells, and incubated for 10–14 days until colonies formed. Colonies were visualized with 0.1% Crystal violet staining, then imaged and counted.

Immunoprecipitation assays and proteomics analysis

To explore the proteins that interact with RBIS, a combination of coimmunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC–MS/MS) was utilized to pinpoint the downstream effects mediated by RBIS conducted by Oebiotech. For Immunoprecipitation assay, lysis buffer with protease and phosphatase inhibitors (150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 2 mM MgCl₂, 1 mM EDTA, 10% glycerol, 0.5% NP-40) was added to cells and incubated on ice for 30 min, followed by centrifugation at 4 °C for 15 min at 12000 rpm. The protein extracts were incubated at 4 °C overnight with anti-Flag beads (Selleck, B23102). After incubation, the beads were extensively washed to remove nonspecifically bound proteins, dissolved in 2X protein loading buffer, and subjected to immunoblotting assays. For proteomics analysis, the online Chromatography separation was performed on the Easy nLC 1200 system (ThermoFisher, USA). DDA (data-dependent acquisition) mass spectrum techniques were used to acquire tandem MS data on a ThermoFisher Q Exactive mass spectrometer (ThermoFisher, USA) fitted with a Nano Flex ion source. The MS/MS data were analyzed for protein identification and quantification using Thermo Proteome Discoverer. Proteins with at least three unique peptides and a confidence level exceeding 95% were considered in subsequent analyses. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository [11, 12] with the dataset identifier PXD057090. The URL was http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD057090 and https://www.iprox.cn/page/project.html?id=IPX0010052000.

Statistical analysis

Statistical analysis of the data was performed using GraphPad Prism 9. Prior to t-test analysis, we employed an F-test to ascertain the variance homogeneity between the two datasets. Only those with equivalent variances proceeded to further analysis using either paired or unpaired Student’s t-tests. Survival analysis was conducted via the log-rank Cox method. The Mann–Whitney test was used to compare tumor weight and tumor volume between different experimental treatment groups. The data are presented as the means ± SEMs. P < 0.05 was considered statistically significant. *represents p < 0.05, **represents p < 0.01, ***represents p < 0.001, ****represents p < 0.0001.

Results

RBIS is overexpressed in lung adenocarcinoma tissues and is correlated with a poor prognosis

Ribosome biogenesis plays a crucial role in tumor progression. To identify potential ribosome biogenesis-related genes contributing to lung adenocarcinoma progression, we conducted a comprehensive analysis. Differentially expressed genes between lung cancer tissues and adjacent noncancerous tissues from the GSE datasets were obtained via the online Analyze-2R tool (Supplementary Tables 2, 3). Genes related to ribosome biogenesis were collected from the GSEA dataset (Supplementary Table 4). By intersecting these gene sets, we identified potential ribosome-related genes that might play a significant role in lung adenocarcinoma progression (Supplementary Fig. 1). Through an extensive literature review, we identified the RBIS gene as an interesting candidate. Analysis of the mRNA expression level of RBIS via the TIMER, ULCAN and GEO datasets (GSE102287 and GSE30219), revealed significantly higher expression of RBIS in LUAD tissues than that in normal tissues (Fig. 1A–C), yet no significant correlation was detected between its expression and the clinicopathological stage or metastasis of lung adenocarcinoma (Fig. 1D, E). Further analysis utilizing Kaplan, GEPIA and TIMER survival analysis modules revealed that patients with high RBIS expression had a worse prognosis (Fig. 1F). These findings suggested that RBIS might be associated with the malignant phenotype of lung adenocarcinoma.

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

To validate the above database analysis, we further analyzed the expression of RBIS in lung cancer clinical samples collected in our laboratory. Consistent with the findings from the database analysis, the mRNA level of RBIS in 60 cases of lung cancer tumor tissues was significantly higher than that in paired non-tumor tissues (Fig. 1G), and 55.7% of lung cancer tumor tissues showed RBIS overexpression (Fig. 1H). These results suggested a close correlation between the RBIS and the clinical progression and malignant phenotype of lung cancer.

Knockdown of RBIS significantly inhibits the migration, invasion potential and growth of lung adenocarcinoma cells in vitro

As metastasis is a major cause of death in patients with lung cancer, we continued to examine the in vitro migration and invasion potential of lung adenocarcinoma cells following the knockdown of RBIS. We first detected the expression levels of RBIS in lung cancer cell lines, and used transient transfection with siRNA to inhibit its expression in A549, H1299 and PC9 cells (Fig. 2A, B). Transwell assays indicated that knocking down RBIS significantly inhibited the in vitro migration and invasion potential as well as the wound healing ability of lung cancer cells (Fig. 2C–G). These results suggested that RBIS might be a mediator of the metastatic properties of lung adenocarcinoma cells.

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

To further assess whether RBIS affects the in vitro growth of lung adenocarcinoma cells, we next transiently knocked down endogenous RBIS in A549, H1299, and PC9 cells using siRNA. The results of the CCK-8 assay indicated that transient knockdown of RBIS in lung adenocarcinoma cells led to a significant reduction in cell viability (Fig. 3A). Colony formation assays revealed that knockdown of RBIS resulted in a decrease in the formation of colonies in lung cancer cells (Fig. 3B). Additionally, we established stable cell lines expressing shNC or shRBIS. The results of the CCK-8 and colony formation assays were consistent between RBIS stable expressing and transient knockdown cells (Fig. 3C–E). Taken together, these results suggest that low expression of RBIS inhibits lung cancer cell migration, invasion potential in vitro.

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

Knockdown of RBIS inhibits tumor growth and metastasis of lung cancer cells in vivo

To verify whether RBIS could affect tumor growth in vivo, we subcutaneously inoculated cells stably expressing shRBIS or shNC control cells, and tumor growth was monitored 2 weeks after injection until the tumor size reached the ethical limit. The results revealed that knocking down RBIS significantly inhibited tumor growth in vivo (Fig. 4A–C). To further verify whether RBIS affected the in vivo metastasis of lung cancer cells, we established stable cell lines expressing shNC or shRBIS in A549 cells and established a NOD-SCID mice lung metastasis model through tail vein injection (6 male mice per group). After 7 weeks, the animals were anesthetized with carbon dioxide and sacrificed for lung dissection. The results showed that knockdown of RBIS significantly reduced the lung metastasis rate (Fig. 4D–F). These results suggested that knockdown of RBIS obviously inhibited xenograft tumor growth and metastasis of lung adenocarcinoma cells in vivo.

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

Knockdown of RBIS induces significant cell cycle arrest and apoptosis

To understand how RBIS affected cell growth, we conducted cell cycle analysis to observe changes in DNA content distribution after RBIS knockdown. The results revealed that cells were significantly arrested at G0/G1 in A549, H1299, and PC9 cells after RBIS knockdown (Fig. 5A–C). We employed the EdU labeling method to further validate the impact of RBIS knockdown on DNA synthesis. The results showed that the proportion of EdU-positive cells was significantly reduced in A549, H1299, and PC9 cells with RBIS knockdown, suggesting that the suppression of RBIS markedly inhibited DNA synthesis (Fig. 5D). Based on the observed changes in the cell cycle, we examined whether the expression of cell cycle-related protein markers had changed. Studies have demonstrated that the c-myc gene plays a pivotal role in regulating the transition of cells from the G1 phase to the S phase [13]. Additionally, the p21 protein has been shown to inhibit the activity of CDK2, thereby inducing cell cycle arrest in the G1 phase [14]. Studies also show that the protein p70s6k is essential for G1 progression [15, 16]. Therefore, we further detected the protein expression levels of c-myc, p-p70s6k, CDK2 and p21, the results showed that the protein expression of c-myc, p-p70s6k, and p-CDK2 decreased significantly, whereas the protein expression of p21 increased significantly (Fig. 5E). These results suggested that RBIS affected cell cycle of lung adenocarcinoma cells.

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

Cell cycle arrest usually triggers the apoptosis of cancer cells. Annexin V PE-7AAD was used to double-stain the cells to observe whether knocking down RBIS could induce apoptosis. The results revealed that the loss of RBIS significantly promoted both early and late apoptosis in lung adenocarcinoma cells (Fig. 6A–C). Additionally, immunoblotting assays using antibodies against antiapoptotic/proapoptotic cell markers were conducted to confirm the above results. We found that upon knockdown of RBIS in lung cancer cells, the expression of MCL-1, an antiapoptotic cell marker, significantly decreased (Fig. 6D). In order to further confirm whether the cell apoptosis in the subcutaneous transplanted tumor was regulated by RBIS. We isolated and digested the subcutaneous tumors into cell suspensions, then detected the apoptotic rate by flow cytometry, the results revealed a substantial proportion of apoptotic cells in the RBIS knockdown group (Fig. 6E, F). In vitro and in vivo results showed that the growth inhibition caused by RBIS deficiency may be related to induced cellular apoptosis.

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

Impaired ribosome biogenesis is observed in lung adenocarcinoma cells expressing low levels of RBIS

Notably, dysregulated ribosome biogenesis frequently occurs in tumor cells to sustain their rapid growth and high protein synthesis efficiency [17]. Therefore, we further explore whether the effects of RBIS on the progression of lung adenocarcinoma was mediated through its regulation of ribosome biogenesis. A study has found that RBIS may be a regulatory factor related to pre-rRNA processing [7]. The pre-rRNA processing is an important rate-limiting step in ribosome biogenesis [18]. In order to further clarify the mechanism by which RBIS affects cell cycle and apoptosis is related to ribosome biogenesis, we first examined the transcription of rRNA. Our qPCR results showed a significant decrease of 47S rRNA expression in A549 cells following RBIS knockdown (Fig. 7A–C). The ribosome is a molecular machine responsible for protein synthesis [19], Therefore, we further investigated whether RBIS affects the efficiency of protein translation within cells. Polysome profiling analysis results showed that knockdown of RBIS increased the ratio of polysomes to nonpolysomes (Fig. 7D). A study posits that elevated heavy/light polysome ratio may indicate either (i) an increase in translation initiation, leading to a greater number of ribosomes being incorporated into polysomes and suggesting an upregulation of protein synthesis, or (ii) a downregulation of protein synthesis due to reduced elongation rates, which can result in prolonged occupancy and stalling of polysomes on mRNA [20]. Given that these conditions are anticipated to exert opposing influences on the rate of mRNA translation, we directly measured protein synthesis using the global protein synthesis assay kit. Our results indicated that RBIS knockdown led to a reduction in overall protein synthesis (Fig. 7E). As the nucleolus are the hub for ribosome biogenesis [21], we subsequently detected nucleolin expression using immunofluorescence staining analysis. As shown in Fig. 7F, the nucleolus number in A549 cells with RBIS knockdown exhibited a decreasing trend, suggesting the occurrence of nucleolar stress (Fig. 7F). CX-5461 is a drug that targets ribosome biogenesis through the inhibition of rDNA transcription, thus inhibiting HGSOC cell growth [22]. To further verify ribosome biogenesis mediated growth regulation of RBIS in lung cancer cells, we added CX-5461 to lung cancer cells stably expressing shNC or shRBIS and detected protein expression of key biomarkers involved in cell cycle and cell apoptosis. The results showed that the combined use of rRNA synthesis inhibitors could enhance the suppressive effect of RBIS knockdown on CDK2 and MCL-1 expression in tumor cells (Fig. 7G), indicating that RBIS might regulate the growth of lung cancer cells at least in part through impacting ribosome assembly.

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.

Given that the molecular mechanism through which RBIS influences ribosome biogenesis in eukaryotic cells remains elusive, our subsequent objective is to identify potential regulatory factors that mediate the role of RBIS in ribosome biogenesis. We performed IP assays using anti-Flag beads (Fig. 7H), followed by LC–MS/MS analysis. The results showed that total 18 proteins that specifically interacted with RBIS were identified according to the cut-off value of identified unique peptide number over 3 and high confidence (Supplementary Table 5). Experimental validation of co-IP revealed a potential interaction between RBIS and GNL2 in both HEK293T and A549 cells (Fig. 7I, J), with the capacity to modulate the expression of GNL2 (Fig. 7K). Study has found that GNL2 is implicated in the maturation of the 60S ribosomal subunit, which is a component of 80S ribosome and the regulation of global protein synthesis [23]. Combining literature and our research findings, we posited that the impact on the 60S ribosomal large subunit via interaction of RBIS with GNL2 might also influence the characteristics of the 80S ribosome, ultimately impacting ribosome biogenesis and protein synthesis. Another study has demonstrated that the loss of GNL2 is associated with a failure to exit the cell cycle. Additionally, GNL2 loss is found to induce the stabilization of p53 and subsequent p53-mediated apoptosis [24]. Therefore, we hypothesized that the perturbation of cell cycle and the upregulation of apoptosis induced by the loss of RBIS may be orchestrated through the interplay between RBIS and GNL2.

Knockdown of RBIS enhances the drug sensitivity of lung adenocarcinoma cells to gemcitabine

Some studies have shown that ribosomal biogenesis is significantly related to radiotherapy resistance and chemotherapy resistance in cancer [5]. Gemcitabine mainly kills cells in the DNA synthesis phase (S phase) and can also block the transition from G1 phase to S phase. In our study, we found that knockdown of RBIS caused cell cycle arrest at the G0/G1 phase and a significant decrease in the proportion of cells in S phase. To clarify the combined therapeutic effect of RBIS deficiency with gemcitabine treatment, indicated concentrations of gemcitabine were added to lung cancer cells stably expressing shNC or shRBIS, and the results showed that the IC50 value of gemcitabine significantly decreased in lung cancer cells with RBIS knockdown (Fig. 8A). Moreover, similar results were obtained in the colony formation assays, which revealed a significant dose-dependent effect (Fig. 8B, C). These experimental results suggested that RBIS may play a significant role in mediating gemcitabine resistance in lung cancer cells.

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

Discussion

Studies have shown that the expression of 47S pre-rRNA is higher in primary colorectal cancer tissues than in normal colonic mucosa [25]. Another study shows that the overexpression of the ribosomal protein RPL15 in circulating tumor cells promotes multiorgan metastasis and selectively enhances the translation of other ribosomal proteins and cell cycle regulators [26]. Targeting ribosome biogenesis by inhibiting the ribosomal small subunit protein RPS6 can suppress NSCLC cell growth by affecting cell cycle regulation [27], indicating that ribosome-related factors play important roles in tumors. RBIS is a ribosomal biogenic factor preliminarily indicated by GSEA. However, researches on this gene are very limited, with only two studies suggesting its potential involvement in ribosome assembly through systematic screening [7, 8]. The specific function and mechanisms of RBIS in ribosome assembly have not been extensively investigated. In our study, for the first time, we revealed the preliminary functions of RBIS in LUAD progression through its role in ribosome biogenesis.

In our study, we found that the downregulation of RBIS resulted in decreased cell migration and invasion potential, inhibited cell growth. Mechanistically, we observed a significant decrease in the transcription levels of 47S pre-rRNA in A549 cells with RBIS knockdown. A study found that silencing of the rRNA metabolism-associated cancer gene EXOSC8 reduces the levels of nucleolar proteins and proliferation markers, as well as rRNA/DNA and global protein synthesis, thereby inhibiting colorectal cancer cell proliferation [28]. Transcription of rRNA is a rate-limiting step in ribosome biogenesis [18]. These studies suggest that ribosome stress causes a series of disruptions in cellular biology. A study found that CX-5461 treatment leads to the induction of growth, migration, and DNA damage in pancreatic ductal adenocarcinoma cells [29]. In our study, we found that CX-5461 treatment enhanced the growth-inhibitory effect of RBIS loss in lung cancer cells, further verified that ribosome biogenesis is closely related to the growth of tumor cells. Therefore, we hypothesized that the suppressive effects of RBIS on the growth of lung cancer cells was mediated by compromised ribosome biogenesis, at least to a significant extent.

CO-IP assay identified that RBIS could interacted with GNL2. GNL2 was reported to play a role in the maturation of 60S ribosome subunit and global protein synthesis [23]. Moreover, our study on polysome profiling and global protein synthesis experiment showed impaired protein translation activity upon RBIS knockdown. Taken together, we speculated that RBIS regulated ribosome biogenesis by interacting with GNL2, leaded to alterations in the overall protein translational efficiency and ultimately influencing the progression of lung cancer.

In human tumors, ribosomal proteins and related ribosome biogenesis factors mediate radioresistance and chemoresistance in various cancers, including pancreatic cancer, lung cancer and glioma [30,31,32]. While targeted therapy and immunotherapy are becoming increasingly important in the treatment of advanced NSCLC, platinum-based chemotherapy in combination with gemcitabine remains a commonly used first-line treatment for advanced NSCLC patients [33]. However, resistance is a major obstacle to the benefit of cancer patients. Our study revealed that downregulation of RBIS increased the sensitivity of lung cancer cells to gemcitabine. Considering that downregulation of RBIS also causes changes in ribosome biogenesis, we believe that ribosome biogenesis may at least partially mediate the increased sensitivity of lung cancer cells to gemcitabine caused by RBIS knockdown.

Conclusion

In summary, we identified RBIS as a potential oncogenic factor and a therapeutic target in LUAD. Mechanistically, RBIS may interacted with GNL2 and therefore involved in ribosome biogenesis, ultimately impacting LUAD progression. Furthermore, we found that inhibition of RBIS can increase tumor sensitivity to anti-gemcitabine therapy.