C8ORF59 regulates ribosome biogenesis to affect progression in lung adenocarcinoma

C8ORF59 regulates ribosome biogenesis to affect progression in lung adenocarcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article C8ORF59 regulates ribosome biogenesis to affect progression in lung adenocarcinoma Hongyu Pan, Li Liao, Siwei Xu, Yujian Xu, Wenjun Chai, Xiaoli Liu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4182106/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Dec, 2024 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Elevated ribosome biogenesis was required by tumor growth. In this study, we initially screened a set of key genes related to ribosome biogenesis from the GSEA dataset. Then, we obtained differentially expressed gene sets between cancer tissues and adjacent non-cancerous tissues from the GSE datasets. By intersecting these gene sets, we identified potential genes that may play a significant role in the progression of lung adenocarcinoma. Subsequently, through extensive literature review, we finally identified the gene Chromosome 8 Open Reading Frame 59 (C8ORF59) as an interesting candidate. Our research findings demonstrated that the knockdown of C8ORF59 significantly inhibits the migration, invasion potential, cell growth, and clonogenicity of lung adenocarcinoma cells. Additionally, apoptosis assays revealed a significant increase in apoptosis, including both early and late stages, in lung adenocarcinoma cells upon C8ORF59 knockdown. Cell cycle analysis showed that C8ORF59 knockdown arrests cells predominantly in the G0/G1 phase, indicating inhibited cell proliferation. Moreover, knocking down C8ORF59 significantly inhibits the in vivo growth of lung cancer cells. Mechanistically, downregulation of C8ORF59 significantly decreases the expression of 47S rRNA, a component associated with ribosome assembly, ribosome proteins Fibrillarin (FBL) and Ribosomal Protein L3 (RPL3). Additionally, ribosomal biogenesis targeting drugs CX-5461 and C8ORF59 loss generate synergistic effects on key proteins regulating cell cycle and apoptosis. Knocking down C8ORF59 also substantially enhanced the sensitivity of lung adenocarcinoma cells to the chemotherapeutic drug gemcitabine, suggesting a potential association between C8ORF59 and drug resistance. Collectively, these studies suggest the close involvement of C8ORF59 in the progression of lung adenocarcinoma, providing new insights for its therapeutic intervention. Lung adenocarcinoma C8ORF59 Ribosomes biogenesis Gemcitabine sensitivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction 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 ]. Pathologically, lung cancer is categorized into two subtypes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC comprises approximately 85% of all lung cancer cases, with squamous cell carcinoma (LUSC) representing 25–30%, adenocarcinoma (LUAD) representing approximately 40% of NSCLC cases, which is the most common subtype in NSCLC [ 2 ]. Currently, the vast majority of patients are usually diagnosed at advanced stages or have metastasis, palliative chemotherapy is the primary treatment approach for advanced non-small cell lung cancer, though immune checkpoint inhibitors and targeted therapy can further improve treatment outcomes and serve as consolidation treatment options after radiation and chemotherapy, they will not replace chemotherapy in future [ 3 ]. However, the emergence of drug resistance in many LUAD cases has led to final treatment failures, emphasizing the urgent need to uncover novel therapeutic targets that drive LUAD progression and chemotherapy resistance. The ribosome, a molecular machine responsible for protein synthesis, consists of ribosomal RNA (rRNA) and ribosomal proteins. Eukaryotic cells harbor an 80S ribosome composed of a small 40S subunit and a large 60S subunit. The 40S subunit comprises the 18S rRNA and 33 distinct ribosomal proteins (RPS), while the 60S subunit contains the 28S, 5.8S, and 5S rRNA in addition to 47 ribosomal proteins (RPL) [ 4 ]. Under the drive of RNA polymerase I, ribosomal DNA (rDNA) is transcribed into 47S pre-rRNA (also known as 45S pre-rRNA), encoding 18S rRNA, 5.8S rRNA, and 28S rRNA [ 5 ]. Increasing evidence suggests that perturbations in human ribosome biogenesis can lead to various diseases, including cancer [ 6 ]. Notably, dysregulated ribosome biogenesis frequently occurs in tumor cells to sustain their rapid growth and high protein synthesis efficiency [ 7 ]. Significant progress has been made in both research and clinical treatment of tumors by targeting ribosome biogenesis. Several clinically approved drugs, including cisplatin, oxaliplatin, doxorubicin, mitoxantrone, and bleomycin, exert their therapeutic effects, at least in part, by inhibiting ribosome synthesis at the rRNA transcription level, while 5-fluorouracil and vincristine inhibit ribosome synthesis at the rRNA processing level [ 8 ]. These findings highlight the critical role of ribosome biogenesis in tumor progression and emphasize the importance of identifying additional regulators of ribosome biogenesis as potential therapeutic targets for cancer treatment. In recent years, with the rapid development of next-generation sequencing technologies, high-throughput sequencing has emerged as an important tool for identifying tumor-related genes. In this study, we performed a bioinformatics analysis of differentially expressed genes from the lung cancer datasets GSE30219 and GSE102287, as well as ribosome biogenesis factors from the Gene Set Enrichment Analysis (GSEA) dataset, to identify key regulators of ribosome biogenesis potentially involved in lung cancer progression. Through comprehensive bioinformatics analysis and literature research, we identified C8ORF59 as a promising candidate gene. C8ORF59, located on chromosome 8, is a vertebrate-specific ribosome synthesis factor. A systematic nucleolar screening study has identified that C8ORF59 may be involved in the pre-rRNA processing [ 9 ]; Genome-wide RNAi Screening suggested its involvement in the biogenesis of the ribosomal 40S small subunit, and functional clustering analysis of molecules involved in 40S subunit synthesis revealed their participation in various biological processes and components, including ribosome biogenesis, ribosomal components, NPC transport, proteasome, transcription, translation, signal transduction, metabolism, and alternative splicing regulation [ 10 ]. The above two studies suggest that C8ORF59 may be related to the assembly of ribosomes, however, to date, there have been no reports investigating the functional role and mechanisms of C8ORF59 in relation to cancer. In this study, we demonstrated for the first time that C8ORF59 is upregulated in lung cancer tissues. Knockdown of C8ORF59 significantly inhibited the in vitro growth, migration, and invasion potential of lung adenocarcinoma cells. Moreover, C8ORF59 depletion enhanced the inhibitory effect of the ribosome biogenesis-targeting drug CX-5461 on cell growth, as well as increased the sensitivity of lung adenocarcinoma cells to the chemotherapy drug gemcitabine. These findings identify C8ORF59 as a potential therapeutic target for lung adenocarcinoma and provide new insights into the development of combined gene and chemotherapy treatment strategies. Results C8ORF59 is overexpressed in lung adenocarcinoma tissues and correlates with poor prognosis Analysis of the mRNA expression level of C8ORF59 via databases TIMER, ULCAN and GEO datasets GSE102287, GSE30219 showed a significantly higher expression of C8ORF59 in lung adenocarcinoma (LUAD) tissues compared to normal tissues (Fig. 1 A-C), yet no significant correlation was observed between its expression and the clinical pathological stage or metastasis of lung adenocarcinoma (Fig. 1 D-E). Further analysis utilizing Kaplan, GEPIA and TIMER's survival analysis modules showed that patients with high C8ORF59 expression had worse prognosis (Fig. 1 F). These findings suggest that C8ORF59 may be associated with the malignant phenotype of lung adenocarcinoma. To validate the the above database analysis, we further analyzed the expression of C8ORF59 in lung cancer clinical samples collected in our laboratory. In agreement with the results from database analysis, mRNA level of C8ORF59 in 60 cases of lung cancer tumor tissues was significantly higher than that in paired non-tumor tissues (Fig. 1 G), and cases with C8ORF59 overexpression account for 55.7% (Fig. 1 H). These results suggest a close correlation between C8ORF59 and the clinical progression and malignant phenotype of lung cancer. Knocking down C8ORF59 significantly inhibits the migration and invasion potential 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 C8ORF59. Firstly, expression levels of C8ORF59 in lung cancer cell lines were detected and siRNA was used to knock down its expression in A549 and PC9 cells (Fig. 2 A-B). Results from transwell assays indicate that knocking down C8ORF59 significantly inhibited the in vitro migration and invasion potential as well as wound healing abilities of lung cancer cells (Fig. 2 C-G). These results suggest that C8ORF59 may be a mediator of the metastatic properties of lung adenocarcinoma cells. Knockdown of C8ORF59 significantly suppresses the growth of lung adenocarcinoma cells To further assess whether C8ORF59 affects the in vitro growth of lung adenocarcinoma cells, we transfected lung adenocarcinoma cells including A549, H1299, and PC9 transiently with siRNA to suppress the expression of C8ORF59 (Fig. 3 A). Results from CCK-8 assay showed that lung adenocarcinoma cells with transient knockdown of C8ORF59 demonstrated significantly reduced cell viability (Fig. 3 B). Colony formation assays revealed fewer colonies formed when C8ORF59 was knocked down in lung adenocarcinoma cells (Fig. 3 C). Additionally, we established stable cell lines expressing shNC and shC8ORF59. Results from CCK-8 and colony formation assays were consistent between cells with stable and transient knockdown of C8ORF59 (Fig. 3 D-F). Taken to gather, these studies suggest a close relationship between C8ORF59 and the growth of lung cancer cells. Knocking down C8ORF59 induces significant cell cycle arrest Cell proliferation is closely related to cell growth. To understand how C8ORF59 affect cell growth, cell cycle analysis was conducted to observe if any changes occurred to the DNA content distribution in cells in which C8ORF59 was knocked down. The results showed significant cell cycle arrest at G0/G1 in A549, H1299, and PC9 cells with C8ORF59 knock down (Fig. 4 A-C). The MYC Proto-Oncogene, BHLH Transcription Factor (C-MYC) gene regulates the transition of cells from G1 to S phase [ 11 ]. Protein p21 can inhibit the activity of cyclinE-Cyclin Dependent Kinase 2 (CDK2), thus causing the cell cycle to stall in the G1 phase [ 12 ]. Thus, we further observed the expression levels of key proteins that regulate the transition of cells from G1 phase to S phase, and found that upon knockdown of C8ORF59, protein expressions of c-myc, p-p70s6k, p-CDK2 decreased significantly, while p21 protein expression increased significantly (Fig. 4 D). These results suggest that C8ORF59 might affect lung adenocarcinoma cell growth by affecting cell proliferation. Knockdown of C8ORF59 significantly induces apoptosis in lung cancer Cells Cell cycle arrest usually triggers apoptosis of cancer cells. Annexin V binds to the early apoptotic cell membrane to mark early apoptotic cells, while cell nuclear dye 7-AAD can enter late apoptotic or necrotic cells to stain DNA and mark late apoptotic or necrotic cells. Using AnexinV PE-7AAD to double stain cells to observe whether knocking down C8ORF59 can induce apoptosis. Results show that loss of C8ORF59 significantly promoted apoptosis in lung adenocarcinoma cells, including both early and late apoptosis (Fig. 5 A-C). Additionally, immunoblotting assays using antibodies of anti/pro-apoptotic cell markers were conducted to confirm the above results. We found that upon knockdown of C8ORF59 in lung cancer cells, the expression of Myeloid Cell Leukemia Sequence 1 (MCL-1), an anti-apoptotic cell marker, significantly decreased, while the expression of apoptosis-promoting proteins BH3 Interacting Domain Death Agonist (BID), BCL2 Antagonist/Killer (BAK), and BCL2 Binding Component 3 (Puma) significantly increased (Fig. 5 D). These findings suggest that growth inhibition caused by C8ORF59 deficiency could be associated with induced cell apoptosis. Significant reduction of 47S rRNA is observed in lung adenocarcinoma cells expressing low levels of C8ORF59 The assembly of ribosomes mainly involves the transcription and processing of rRNA, the transcription and translation of ribosomal proteins (RPs), and the regulation of ribosomal assembly factors (AFs). Among them, rRNA transcription is the rate limiting step of ribosomal biogenesis [ 13 ]; Additionaly, C8ORF59 may be a regulatory factor related to pre-rRNA processing [ 9 ]. Therefore, we detected the expression of 47S pre-rRNA in cells where C8ORF59 was knocked down and found a significant decrease in 47S rRNA expression in A549 cells (Fig. 6 A). RPs and Afs are indispensable parts for ribosome assembly, thus we collected proteins from control group and C8ORF59 knockdown cells and detected the protein expression of key RPs and AFs using immunoblotting assay. FBL, a highly conserved nucleolar methyltransferase responsible for rRNA and protein methylation [ 14 ], immunoblotting assay showed a significant decrease in the expression of FBL upon C8ORF59 knockdown (Fig. 6 B). It has been reported that RPL3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles, and the final maturation of pre-40S ribosome small subunits depends on functional integrity of RPL3 [ 15 ], our results demonstraed a significant decrease in RPL3 expression (Fig. 6 B). CX-5461 is a drug targetting ribosome biogenesis through inhibiting rDNA transcription, thus inhibiting HGSOC cell growth [ 16 ]. To explore whether ribosome biogenesis mediated growth regulation of C8ORF59 on lung cancer cells, CX-5461 was added to lung cancer cells stable expressing shNC and shC8ORF59, the inhibitory effects of C8ORF59 knockdown on CDK2 and MCL-1 was further enhanced upon adding CX-5461 48 hours later, results display synergistic effects of C8ORF59 deficiency and ribosomal biogenesis inhibition on key proteins of cell cycle and cell growth. Taken together, these results indicate that C8ORF59 might regulate the growth of lung cancer cells through, at least in part, impacting ribosome assembly. Knockdown of C8ORF59 enhances the drug sensitivity of lung adenocarcinoma cells to Gemcitabine The ribosomes are the site for all protein synthesis in the cell and therefore crucial for cell survival. Some studies have shown that ribosomal biogenesis is significantly related to radiotherapy resistance and chemotherapy resistance in cancer [ 17 ]. Gemcitabine, a cytidine nucleoside derivative, which undergo phosphorylation of deoxycytidine nucleoside kinase within cells to form diphosphate and triphosphate nucleosides, are incorporated into cells to interfere with normal DNA synthesis, causing DNA breakage and ultimately leading to cell death, they mainly kill 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 knocking down C8ORF59 caused cell cycle arrest at G0/G1, and significant decrease in the proportion of S phase cells, so we want to know whether loss of function of C8ORF59 and gemcitabine have synergistic effect on growth inhibition. Consequently, different concentrations of gemcitabine was added into lung cancer cells stable expressing shNC and shC8ORF59, results showed that the IC50 value of gemcitabine significantly decreased in lung cancer cells with C8ORF59 knockdown (Fig. 7 A). Moreover, similar results were obtained from colony formation assays and exhibiting a significant dose-dependent manner (Fig. 7 B-C). These experimental results imply that C8ORF59 may be related to gemcitabine resistance in lung cancer cells. Knocking down C8ORF59 inhibits in vivo tumor growth of lung cancer cells To verify whether C8ORF59 can affect tumor growth in vivo, we subcutaneously inoculated cells with stable expression of shC8ORF59 and control cells shNC, and tumor growth was monitored two weeks after injection until the tumor size reaches the ethical limit. Results showed that knocking down C8ORF59 significantly inhibited tumor growth in vivo (Fig. 8 A-C). We dissected the subcutaneous tumors, digested part of the subcutaneous tumors into cell suspensions and used flow cytometry to detect the apoptosis rate. Results show a substantial apoptosis proportion in the C8ORF59 knockdown group (Fig. 8 D-E). These results suggest that C8ORF59 is closely related to the in vivo growth of lung adenocarcinoma cells. Discussion Studies have showed elevated expression of 47S pre-rRNA has been observed in primary colorectal cancer tissues compared to normal colonic mucosa [ 18 ]; Overexpression of ribosomal protein RPL15 in circulating tumor cells promotes multi-organ metastasis and selectively enhances the translation of other ribosomal proteins and cell cycle regulators [ 19 ]. Targeting ribosome biogenesis by inhibiting the ribosomal small subunit protein RPS6 can suppress NSCLC cell growth by affecting cell cycle regulation [ 20 ], indicating that ribosome related factors play important roles in tumors. C8ORF59 is a ribosomal biogenic factor preliminarily indicated by GSEA, however, there is very limited research on this gene currently, with only two studies suggesting its potential involvement in ribosome assembly through systematic screening [ 9 , 10 ]. The specific function and mechanisms of C8ORF59 in ribosome assembly have not been directly and extensively investigated. In our study, we have revealed the preliminary function and mechanisms of C8ORF59 in ribosome biogenesis, LUAD progression, and chemotherapy resistance for the first time. CX-5461, an RNA polymerase I inhibitor, can inhibit ribosome biogenesis, leading to growth, migration, and DNA damage induction in pancreatic ductal adenocarcinoma cells [ 21 ]. Silencing of the rRNA metabolism-associated cancer gene EXOSC8 reduces levels of nucleolar proteins and proliferation markers, as well as rRNA/DNA and global protein synthesis, inhibiting colorectal cancer cell proliferation [ 22 ]. These studies suggest that ribosome stress causes a series of disruptions in cellular biology. In our study, we found that the downregulation of C8ORF59 results in decreased cell migration and invasion potential, inhibited cell growth, cell cycle arrest, and increased apoptotic ratio. Then, we investigated the molecular mechanisms of C8ORF59 in regulating LUAD progression. Transcription of rRNA is a rate-limiting step in ribosome biogenesis [ 13 ]. In our study, we observed a significant decrease in the transcriptional expression levels of 47S pre-rRNA in A549 cells with C8ORF59 knockdown. Studies show in the ribosome, rRNA plays a central role in structure and function, providing binding sites for ribosomal proteins. During the processing of pre-rRNA, ribosome assembly factors and ribosomal proteins assemble in a coordinated manner onto pre-rRNA, forming pre-ribosomal particles [ 23 ], suggesting that the abnormal expression of pre-rRNA may be associated with the co-expression abnormalities of ribosomal proteins. Another study involving TCGA data analysis also revealed a 30% median increase in ribosomal protein expression levels in tumor tissues compared to normal tissues. Furthermore, many ribosomal proteins exhibit distinct dysregulation patterns in specific tumors. For example, RPL21L1 and RPS27L are upregulated in breast and thyroid cancers, while RPL21 is downregulated [ 24 ], indicate the diversity of ribosomal protein expression patterns in cancer progression. Our present research found that FBL, which possesses rRNA methyltransferase activity, and RPL3, a structural protein of the ribosome large subunit, significantly decreased upon C8ORF59 knockdown. However, we cannot exclude the possibility that other untested ribosomal proteins may be regulated by C8ORF59. Treatment with CX-5461 to inhibit ribosome biogenesis enhanced the growth-inhibitory effect of C8ORF59 loss in lung cancer cells. In human tumors, ribosomal proteins and related ribosome biogenesis factors mediate radioresistance and chemoresistance in various cancers, including pancreatic cancer, lung cancer and glioma [ 25 – 27 ]. 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 [ 28 ]. However, resistance is a major obstacle to the benefit of cancer patients. Our study found that downregulation of C8ORF59 increased the sensitivity of lung cancer cells to gemcitabine. Considering that downregulation of C8ORF59 also causes changes in ribosome biogenesis, we believe that ribosome biogenesis may mediate, at least partially, the increased sensitivity of lung cancer cells to gemcitabine caused by C8ORF59 knockdown. Conclusion In summary, we have identified C8ORF59 as a potential oncogenic factor and a therapeutic target in LUAD. Mechanistically, C8ORF59 affects the cell cycle and apoptosis of lung cancer cells through its involvement in ribosome biogenesis, ultimately impacting LUAD progression. Furthermore, we have found that inhibiting C8ORF59 can enhance tumor sensitivity to anti-Gemcitabine therapy. Materials and Methods Clinical Samples: The clinical samples used in this study were obtained from Huashan Hospital, affiliated with Fudan University. Written informed consent was obtained from all patients, and the study was approved by the ethics committee of Huashan Hospital. Cell Culture The human lung adenocarcinoma cell lines, A549, H1299, and PC9, were obtained from the ATCC cell repository. A549 cells were cultured in Ham's F-12K medium, while H1299, and PC9 cells were cultured in RPMI-1640 medium. All media were supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Cell Growth Cell suspensions of lung adenocarcinoma cells were adjusted to a concentration of 800 cells/200 µl and seeded in 96-well cell culture plates with triplicate wells per group. After 24 hours, 10 µl of CCK-8 reagent was added to 90 µl of cell culture medium. After incubation for 2 hours, the absorbance at 450 nm was measured daily for 6 consecutive days to generate growth curves. Colony formation Assay Cells were seeded at a density of 500 cells per well in 6-well plates and cultured for 10–14 days. The colonies were fixed with methanol for 30 minutes and stained with 0.1% crystal violet for 30 minutes. After washing and air-drying, the colonies were counted. Migration and Invasion Assays Migration and invasion assays were performed using chambers purchased from Corning. The chambers were coated without FBS in the upper compartment with cell suspensions adjusted to 2.5×10^5 cells/ml for migration assays and 5×10^5 cells/ml for invasion assays. The lower compartment was filled with complete medium containing 10% FBS. The chambers were then incubated in a cell culture incubator for 16–20 hours, followed by removal of non-migrating or non-invading cells from the upper surface of the membrane with a cotton swab. The cells on the lower surface were fixed with methanol for 30 minutes, stained with 0.1% crystal violet for 15–20 minutes, washed, air-dried, and photographed under a microscope. Nine fields were randomly selected for counting in each chamber. RNA Extraction and qPCR Total RNA was extracted using RNAiso Plus (#9109) purchased from TAKARA. Reverse transcription was performed using the reverse transcription kit from Novogene. The obtained cDNA was diluted 10-fold, and qPCR was performed using SYBR Green from Yisheng Biotechnology. The primer sequence for C8ORF59 was synthesized by Genewiz. Cell apoptosis detection Cell inoculation was performed on a 6-well plate until reaching 80–90% confluency. The cell supernatant and adherent cells were collected and washed once with pre-chilled PBS. Then, 5 µl of PE and 7AAD from the apoptosis detection kit were added separately for dual staining of the cells. Blank control, PE single staining control, and 7AAD single staining control groups were set up. After 10 minutes of light-protected staining, the apoptosis rate was detected using a flow cytometer within 1 hour. Cell cycle analysis Cell cycle analysis was conducted by collecting adherent cells from a 6 cm cell culture dish, washing them twice with PBS, and fixing them in 70% ethanol at -20°C for at least 4 hours or overnight. After centrifugation and removal of the supernatant, the cells were washed once with PBS and stained with 500 µl of PI staining solution for 10 minutes. The DNA content of the cells was analyzed using a flow cytometer. Protein extraction and Western Blot Cells from the 6-well plate were washed three times with pre-chilled PBS, and 200 µl of protein lysis buffer (T-per) containing protease and phosphatase inhibitors was added. The cells were scraped and thoroughly lysed on ice for 30 minutes. After centrifugation at 12,000 rpm for 15–20 minutes, the supernatant containing the desired protein was collected. The protein concentration was measured using a BCA protein quantification kit, and samples from different groups were adjusted to the same protein concentration. Protein samples were treated with loading buffer and boiled at 100°C for 10 minutes before storage at -80°C. The following antibodies were used in this study. Cell transfection The siRNA targeting C8ORF59 is a commercial product purchased from Jin Weizhi. The cells were placed on a 6-well cell culture plate and transiently transfected into lung cancer cells using Lippofectamine 2000 24 hours later. The solution was changed 6 hours later, and subsequent experiments were conducted 48 hours after transfection. The siRNA sequence was shown in Supplementary Table 1. Plasmid construction and lentivirus packaging The shRNA sequence of C8ORF59 was synthesized by Jinweizhi Company and constructed into the PLKO.1 plasmid. The successfully constructed PLKO.1-C8ORF59-shRNA was transfected with lentivirus packaging envelope plasmids pMD2.G and psPAX2 in a ratio of 8:6:2 ug into 10 cm dishes of 293T cells using TenfectDNA transfection reagent. After 48 hours, the cell culture supernatant was collected, and cell debris was removed by filtering through a 0.45 µm membrane. The virus liquid was stored at 4°C for up to 1 week or at -80°C for longer periods. Drug Experiments In the stable cell lines expressing shNC and shC8ORF59, 0 µM and 75 µM of the ribosome-targeting drug CX-5461 were added, respectively. After incubating for 48 hours, cells were collected and protein was extracted. Protein concentration was measured using the BCA assay, and protein samples were adjusted to equal concentrations and mixed with protein loading buffer. The mixture was heated at 100°C for 10 minutes for subsequent Western blot experiments. In the gemcitabine IC50 experiment, A549 and H1299 cells stably expressing shNC and shC8ORF59 were seeded in a 96-well plate at a density of 4,000 cells per well. The cells were treated with different concentrations of gemcitabine (0, 0.00316, 0.01, 0.0316, 0.1, 0.316, 1, 3.16, 10, 31.6 µM) in 10 replicates for each concentration, and incubated for 72 hours. The absorbance at 450 nm (OD450) was measured, and the OD450 value of the blank well was subtracted from the OD450 value of each well. Then, the OD450 value of the experimental group was subtracted from the OD450 value of the control group to obtain the values for IC50 plotting. In the gemcitabine impact on colony formation experiment, A549 and H1299 cells stably expressing shNC and shC8ORF59 were seeded in 6-well plates at a density of 2,500 cells per well. Four different concentrations of gemcitabine (0, 0.1, 0.2, 0.3 µM) were added to triplicate wells for each concentration, and the cells were incubated until colonies formed. Crystal violet staining was performed, and the colonies were counted after imaging. Database analysis The expression level of C8ORF59 in lung cancer databases was analyzed using public databases TIMER ( http://cistrome.org/timer ), ULCAN ( https://ualcan.path.uab.edu/analysis.html ), and 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 of C8ORF59 with lung adenocarcinoma prognosis was analyzed using Kaplan, GEPIA, and TIMER. Statistical analysis Statistical analysis of the data in the article was performed using GraphPad Prism 9. The differences between two groups were analyzed using paired or unpaired Student's t-test. Survival analysis was conducted using the Log-rank Cox method, and data were presented as mean ± SEM. P-value less than 0.05 was considered statistically significant. Declarations Acknowledgments We are thankful to the TCGA, TIMER and GEPIA for providing the data analyzed in this study. Funding: The work in the Yan laboratory is supported, in whole or in part, by grants from the National Natural Science Foundation of China (No. 81972173; No. 82273371) and the Science and Technology Commission of Shanghai Municipality (No.22140901400). Authors’ contributions MXY and QL were responsible for the design of the study and wrote the manuscript; HYP and LL participated in the functional analysis and Western blot; SWX and YJX performed bioinformatic analysis and qPCR; WJC was responsible for the animal experiment; XLL contributed to conduct the stable cell lines; JL, YC and LS were incharge of drug sensitivity experiment; All authors reviewed the manuscript before submission and approved the final manuscript. Availability of data and materials All data generated or analyzed during this study are included either in this article or in the supplementary information files. Ethics approval and consent to participate The study was approved by the Ethics Committee of Fudan University (Shanghai, China). Consent for publication All authors agree on publication of the results of the present manuscript. Competing interests The authors declare that they have no competing interest. 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Eukaryotic ribosome assembly, transport and quality control . Nat Struct Mol Biol 24(9), 689-699 (2017). Guimaraes JC and Zavolan M. Patterns of ribosomal protein expression specify normal and malignant human cells . Genome Biol 17(1), 236 (2016). Wei F, Ding L, Wei Z, Zhang Y, Li Y, Qinghua L, et al. Ribosomal protein L34 promotes the proliferation, invasion and metastasis of pancreatic cancer cells . Oncotarget 7(51), 85259-85272 (2016). Yang HJ, Youn H, Seong KM, Jin YW, Kim J, and Youn B. Phosphorylation of ribosomal protein S3 and antiapoptotic TRAF2 protein mediates radioresistance in non-small cell lung cancer cells . J Biol Chem 288(5), 2965-75 (2013). Ko CY, Lin CH, Chuang JY, Chang WC, and Hsu TI. MDM2 Degrades Deacetylated Nucleolin Through Ubiquitination to Promote Glioma Stem-Like Cell Enrichment for Chemotherapeutic Resistance . Mol Neurobiol 55(4), 3211-3223 (2018). Mlak R, Krawczyk P, Ciesielka M, Kozioł P, Homa I, Powrózek T, et al. The relationship between RRM1 gene polymorphisms and effectiveness of gemcitabine-based first-line chemotherapy in advanced NSCLC patient . Clin Transl Oncol 18(9), 915-24 (2016). Supplementary Files SupplementaryTable1.xlsx Cite Share Download PDF Status: Published Journal Publication published 25 Dec, 2024 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 27 Apr, 2024 Reviewers invited by journal 23 Apr, 2024 Editor assigned by journal 29 Mar, 2024 First submitted to journal 28 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4182106","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":294483302,"identity":"383d4eeb-2c9a-4847-adb3-adc2fc93fd0d","order_by":0,"name":"Hongyu Pan","email":"","orcid":"","institution":"Fudan University Shanghai Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"Pan","suffix":""},{"id":294483303,"identity":"d51e28be-d85b-4d91-9eb5-26e7758230ff","order_by":1,"name":"Li Liao","email":"","orcid":"","institution":"Guangzhou Medical University Second Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Liao","suffix":""},{"id":294483304,"identity":"df48c024-2312-43a0-ad8c-bbad10d8ebf3","order_by":2,"name":"Siwei Xu","email":"","orcid":"","institution":"Fudan University Shanghai Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Siwei","middleName":"","lastName":"Xu","suffix":""},{"id":294483305,"identity":"3d105c83-f476-41d7-95c2-803bd7931269","order_by":3,"name":"Yujian Xu","email":"","orcid":"","institution":"Fudan University Shanghai Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Yujian","middleName":"","lastName":"Xu","suffix":""},{"id":294483306,"identity":"f30e3f01-792f-4c04-bf22-d5bfd9625636","order_by":4,"name":"Wenjun Chai","email":"","orcid":"","institution":"Fudan University Shanghai Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Wenjun","middleName":"","lastName":"Chai","suffix":""},{"id":294483307,"identity":"c248a047-2252-449d-b144-283ea389cbaf","order_by":5,"name":"Xiaoli Liu","email":"","orcid":"","institution":"Fudan University Shanghai Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Liu","suffix":""},{"id":294483308,"identity":"65daf6c2-0c3c-4313-beb8-d95ec445df11","order_by":6,"name":"Jing Li","email":"","orcid":"","institution":"Fudan University Shanghai Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Li","suffix":""},{"id":294483309,"identity":"daafd188-d47a-4769-b86c-287d453a4b30","order_by":7,"name":"Yue Cao","email":"","orcid":"","institution":"Fudan University Shanghai Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Cao","suffix":""},{"id":294483310,"identity":"b678c094-91aa-40f8-a142-2266480f8fb5","order_by":8,"name":"Lei Sun","email":"","orcid":"","institution":"Fudan University Shanghai Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Sun","suffix":""},{"id":294483311,"identity":"d3f83da1-c7c1-4bf6-8c2e-facb1ce51c52","order_by":9,"name":"Qian Liu","email":"","orcid":"","institution":"Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine: Shanghai Jiaotong University School of Medicine Xinhua Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Liu","suffix":""},{"id":294483312,"identity":"79b50969-33a5-488e-9ba7-afaa09713ff5","order_by":10,"name":"Mingxia Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDACZhBhAGYyPkiosCFNC7PBgzNppFnIJvmw7RBhZbrtvIdf8xTYMRgcP3usIoHtAAN/e3cCXi1mh/nSLGcYJDMYnMlLu5HAc4dB4szZDQS08JgZfDBgZjA4kGN2I0HiGYOBRC4RWhIM6hkMzr8xK0gwOEyUFuMHH0Aqb+SYMSQkEKfFjHGGwXEGyRtvjCUSDqTxEPbL+TPGn3n+VDPwnc8x/Pjzn40cf3svfi1AwCYBJOoXHIDweAgpBwHmDyBSvoEYtaNgFIyCUTAiAQAdJEhyELGr3QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4301-5793","institution":"Fudan University Shanghai Cancer Center Fudan University Cancer Institute","correspondingAuthor":true,"prefix":"","firstName":"Mingxia","middleName":"","lastName":"Yan","suffix":""}],"badges":[],"createdAt":"2024-03-28 12:03:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4182106/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4182106/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-024-05886-1","type":"published","date":"2024-12-25T15:57:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55400699,"identity":"6913a0ec-585a-47ed-9758-d147e56687c3","added_by":"auto","created_at":"2024-04-26 18:46:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4288675,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of C8ORF59 clinical lung cancer samples and its correlation with prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-B. Expression of C8ORF59 in different cancer types from the TIMER and ULCAN databases. C. Expression of C8ORF59 in GSE102287, GSE30219 and ULCAN-TCGA databases. D-E. Expression of C8ORF59 in different pathological stages and lymph node metastasis stages of LUAD in the ULCAN database. F. Analysis of the correlation between C8ORF59 and the prognosis of lung adenocarcinoma patients using Kaplan, GEPIA and TIMER databases. G. mRNA expression of C8ORF59 in 60 cases of clinical lung cancer samples. H. Statistical analysis revealed high expression of C8ORF59 in 55.7% of lung cancer tissues.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4182106/v1/da2e9a2bbb6bab8df1e82346.jpg"},{"id":55400700,"identity":"42607384-4b77-4afe-94e4-0eb85e38878a","added_by":"auto","created_at":"2024-04-26 18:46:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2173582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSignificant inhibition of cell migration and invasion in lung adenocarcinoma cells with C8ORF59 knockdown.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. mRNA expression levels of C8ORF59 in lung cancer cell lines. B. Knockdown of C8ORF59 expression in A549 and PC9 cells using transient transfection of C8ORF59 siRNA. C-D. Transwell assay assessing the effect of C8ORF59 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.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4182106/v1/bbebbb01f14cb83b253a141f.jpg"},{"id":55400973,"identity":"20034e57-61d7-4f40-8deb-d60d11b26201","added_by":"auto","created_at":"2024-04-26 18:54:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1906015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSignificant inhibition of cell growth in lung adenocarcinoma cells with C8ORF59 knockdown.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Knockdown efficiency of C8ORF59 in lung adenocarcinoma cells A549,\u003c/p\u003e\n\u003cp\u003eH1299 and PC9 with siRNA. B. CCK-8 assay detecting cell viability in lung adenocarcinoma cells A549, H1299 and PC9 with C8ORF59 transient knockdown. C. Colony formation assay examining the clonogenic potential of lung adenocarcinoma cells A549, H1299 and PC9 with C8ORF59 transient knockdown. D. Knockdown efficiency of C8ORF59 in lung adenocarcinoma cells A549, H1299 and PC9 with shRNA. E-F. CCK-8 and colony formation assay was conducted in lung adenocarcinoma cells A549, H1299 and PC9 with C8ORF59 stable knockdown.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4182106/v1/2fbf91597225b9a91006aecc.jpg"},{"id":55400701,"identity":"d1e75cd7-0ab0-4399-9457-4a5c9bc4ad4c","added_by":"auto","created_at":"2024-04-26 18:46:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":819045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC8ORF59 knockdown alters DNA content distribution in lung adenocarcinoma cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-C. Flow cytometry was used to detect changes in DNA content distribution in lung adenocarcinoma cell lines A549, H1299 and PC9 with C8ORF59 knockdown. D. Detecting the expression changes of cell cycle related protein markers in lung cancer cells using Western blot after knocking down C8ORF59.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4182106/v1/a66ef224884396c1a4d113ff.jpg"},{"id":55400702,"identity":"a409227b-3580-4f43-838e-d8660f07b0ff","added_by":"auto","created_at":"2024-04-26 18:46:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":790076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC8ORF59 knockdown significantly promotes cell apoptosis rate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-C. Flow cytometry used to detect cell apoptosis rate in lung adenocarcinoma cell lines A549, H1299 and PC9 with C8ORF59 knockdown. D. The expression changes of apoptotic related protein markers in lung cancer cells using Western blot after knocking down C8ORF59.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4182106/v1/fcfaaa639e6839468492c943.jpg"},{"id":55400706,"identity":"7364793e-f02a-4fce-8eca-f147543fce7d","added_by":"auto","created_at":"2024-04-26 18:46:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":406032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC8ORF59 deficiency alters ribosome assembly\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Evaluation of C8ORF59 knockdown efficacy and expression of 47S rRNA in A549 cells. B. Western blot analysis of the FBL and RPL3 expression in A549 cells stably expressing shNC and shC8ORF59. C. Following the addition of 75uM of the ribosome assembly inhibitor CX-5461 to A549 cells stably expressing shNC and shC8ORF59, Western blot analysis was conducted to detect the expression of CDK2 and MCL-1.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4182106/v1/f996cb73b02449057b4d3870.jpg"},{"id":55400704,"identity":"b7c6a826-5cd6-4763-bd15-b8539a429fa1","added_by":"auto","created_at":"2024-04-26 18:46:47","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1307033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC8ORF59 knockdown significantly increases cell sensitivity to gemcitabine in lung adenocarcinoma cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. IC50 detection of gemcitabine in lung adenocarcinoma cells A549 and H1299 with C8ORF59 knockdown. B-C. The effect of different concentrations of gemcitabine on the clonogenic potential of lung adenocarcinoma cells A549 and H1299 with C8ORF59 knockdown.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4182106/v1/cd96447dea6031c061a1d1e0.jpg"},{"id":55400974,"identity":"b58012c1-4c33-4541-a237-d98812423ef0","added_by":"auto","created_at":"2024-04-26 18:54:47","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":574911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnocking down C8ORF59 inhibits in vivo tumor growth of lung cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-C. A549 cells stably expressing shNC and shC8ORF59 were injected subcutaneously into 6-week-old BALB/c female nude mice (n=20). Tumor images (A), tumor weight (B), and tumor volume (C) are presented 31 days post-injection, after mice were sacrificed and xenograft tumors removed D-G. Flow cytometry (D-E), immunohistochemistry (F-G), were utilized to assess cell cycle and apoptosis indicators in subcutaneous tumors formed from A549 cells with stable expression of shNC and shC8ORF59.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4182106/v1/95ad6e926d261521263e5d27.jpg"},{"id":72640400,"identity":"29e70c8a-77c5-47ca-8dc0-2775b0e1eb9b","added_by":"auto","created_at":"2024-12-30 16:05:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13091282,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4182106/v1/2f302f41-488e-49ce-a0c3-591ff26e7cba.pdf"},{"id":55400707,"identity":"cc0e632c-9036-4a8c-9dc0-540f9a189bad","added_by":"auto","created_at":"2024-04-26 18:46:48","extension":"xlsx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":9578,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4182106/v1/e8e63e5089b44bb61b3e7a14.xlsx"}],"financialInterests":"","formattedTitle":"C8ORF59 regulates ribosome biogenesis to affect progression\n\nin lung adenocarcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer poses a significant global health burden, with approximately 19.3\u0026nbsp;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 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Pathologically, lung cancer is categorized into two subtypes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC comprises approximately 85% of all lung cancer cases, with squamous cell carcinoma (LUSC) representing 25\u0026ndash;30%, adenocarcinoma (LUAD) representing approximately 40% of NSCLC cases, which is the most common subtype in NSCLC [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Currently, the vast majority of patients are usually diagnosed at advanced stages or have metastasis, palliative chemotherapy is the primary treatment approach for advanced non-small cell lung cancer, though immune checkpoint inhibitors and targeted therapy can further improve treatment outcomes and serve as consolidation treatment options after radiation and chemotherapy, they will not replace chemotherapy in future [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the emergence of drug resistance in many LUAD cases has led to final treatment failures, emphasizing the urgent need to uncover novel therapeutic targets that drive LUAD progression and chemotherapy resistance.\u003c/p\u003e \u003cp\u003eThe ribosome, a molecular machine responsible for protein synthesis, consists of ribosomal RNA (rRNA) and ribosomal proteins. Eukaryotic cells harbor an 80S ribosome composed of a small 40S subunit and a large 60S subunit. The 40S subunit comprises the 18S rRNA and 33 distinct ribosomal proteins (RPS), while the 60S subunit contains the 28S, 5.8S, and 5S rRNA in addition to 47 ribosomal proteins (RPL) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Under the drive of RNA polymerase I, ribosomal DNA (rDNA) is transcribed into 47S pre-rRNA (also known as 45S pre-rRNA), encoding 18S rRNA, 5.8S rRNA, and 28S rRNA [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Increasing evidence suggests that perturbations in human ribosome biogenesis can lead to various diseases, including cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Notably, dysregulated ribosome biogenesis frequently occurs in tumor cells to sustain their rapid growth and high protein synthesis efficiency [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Significant progress has been made in both research and clinical treatment of tumors by targeting ribosome biogenesis. Several clinically approved drugs, including cisplatin, oxaliplatin, doxorubicin, mitoxantrone, and bleomycin, exert their therapeutic effects, at least in part, by inhibiting ribosome synthesis at the rRNA transcription level, while 5-fluorouracil and vincristine inhibit ribosome synthesis at the rRNA processing level [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These findings highlight the critical role of ribosome biogenesis in tumor progression and emphasize the importance of identifying additional regulators of ribosome biogenesis as potential therapeutic targets for cancer treatment.\u003c/p\u003e \u003cp\u003eIn recent years, with the rapid development of next-generation sequencing technologies, high-throughput sequencing has emerged as an important tool for identifying tumor-related genes. In this study, we performed a bioinformatics analysis of differentially expressed genes from the lung cancer datasets GSE30219 and GSE102287, as well as ribosome biogenesis factors from the Gene Set Enrichment Analysis (GSEA) dataset, to identify key regulators of ribosome biogenesis potentially involved in lung cancer progression. Through comprehensive bioinformatics analysis and literature research, we identified C8ORF59 as a promising candidate gene. C8ORF59, located on chromosome 8, is a vertebrate-specific ribosome synthesis factor. A systematic nucleolar screening study has identified that C8ORF59 may be involved in the pre-rRNA processing [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]; Genome-wide RNAi Screening suggested its involvement in the biogenesis of the ribosomal 40S small subunit, and functional clustering analysis of molecules involved in 40S subunit synthesis revealed their participation in various biological processes and components, including ribosome biogenesis, ribosomal components, NPC transport, proteasome, transcription, translation, signal transduction, metabolism, and alternative splicing regulation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The above two studies suggest that C8ORF59 may be related to the assembly of ribosomes, however, to date, there have been no reports investigating the functional role and mechanisms of C8ORF59 in relation to cancer.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated for the first time that C8ORF59 is upregulated in lung cancer tissues. Knockdown of C8ORF59 significantly inhibited the in vitro growth, migration, and invasion potential of lung adenocarcinoma cells. Moreover, C8ORF59 depletion enhanced the inhibitory effect of the ribosome biogenesis-targeting drug CX-5461 on cell growth, as well as increased the sensitivity of lung adenocarcinoma cells to the chemotherapy drug gemcitabine. These findings identify C8ORF59 as a potential therapeutic target for lung adenocarcinoma and provide new insights into the development of combined gene and chemotherapy treatment strategies.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eC8ORF59 is overexpressed in lung adenocarcinoma tissues and correlates with poor prognosis\u003c/h2\u003e \u003cp\u003eAnalysis of the mRNA expression level of C8ORF59 via databases TIMER, ULCAN and GEO datasets GSE102287, GSE30219 showed a significantly higher expression of C8ORF59 in lung adenocarcinoma (LUAD) tissues compared to normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C), yet no significant correlation was observed between its expression and the clinical pathological stage or metastasis of lung adenocarcinoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). Further analysis utilizing Kaplan, GEPIA and TIMER's survival analysis modules showed that patients with high C8ORF59 expression had worse prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These findings suggest that C8ORF59 may be associated with the malignant phenotype of lung adenocarcinoma.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate the the above database analysis, we further analyzed the expression of C8ORF59 in lung cancer clinical samples collected in our laboratory. In agreement with the results from database analysis, mRNA level of C8ORF59 in 60 cases of lung cancer tumor tissues was significantly higher than that in paired non-tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), and cases with C8ORF59 overexpression account for 55.7% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). These results suggest a close correlation between C8ORF59 and the clinical progression and malignant phenotype of lung cancer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKnocking down C8ORF59 significantly inhibits the migration and invasion potential of lung adenocarcinoma cells in vitro\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs 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 C8ORF59. Firstly, expression levels of C8ORF59 in lung cancer cell lines were detected and siRNA was used to knock down its expression in A549 and PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Results from transwell assays indicate that knocking down C8ORF59 significantly inhibited the in vitro migration and invasion potential as well as wound healing abilities of lung cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-G). These results suggest that C8ORF59 may be a mediator of the metastatic properties of lung adenocarcinoma cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of C8ORF59 significantly suppresses the growth of lung adenocarcinoma cells\u003c/h2\u003e \u003cp\u003eTo further assess whether C8ORF59 affects the in vitro growth of lung adenocarcinoma cells, we transfected lung adenocarcinoma cells including A549, H1299, and PC9 transiently with siRNA to suppress the expression of C8ORF59 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Results from CCK-8 assay showed that lung adenocarcinoma cells with transient knockdown of C8ORF59 demonstrated significantly reduced cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Colony formation assays revealed fewer colonies formed when C8ORF59 was knocked down in lung adenocarcinoma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Additionally, we established stable cell lines expressing shNC and shC8ORF59. Results from CCK-8 and colony formation assays were consistent between cells with stable and transient knockdown of C8ORF59 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). Taken to gather, these studies suggest a close relationship between C8ORF59 and the growth of lung cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eKnocking down C8ORF59 induces significant cell cycle arrest\u003c/h2\u003e \u003cp\u003eCell proliferation is closely related to cell growth. To understand how C8ORF59 affect cell growth, cell cycle analysis was conducted to observe if any changes occurred to the DNA content distribution in cells in which C8ORF59 was knocked down. The results showed significant cell cycle arrest at G0/G1 in A549, H1299, and PC9 cells with C8ORF59 knock down (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). The MYC Proto-Oncogene, BHLH Transcription Factor (C-MYC) gene regulates the transition of cells from G1 to S phase [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Protein p21 can inhibit the activity of cyclinE-Cyclin Dependent Kinase 2 (CDK2), thus causing the cell cycle to stall in the G1 phase [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thus, we further observed the expression levels of key proteins that regulate the transition of cells from G1 phase to S phase, and found that upon knockdown of C8ORF59, protein expressions of c-myc, p-p70s6k, p-CDK2 decreased significantly, while p21 protein expression increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results suggest that C8ORF59 might affect lung adenocarcinoma cell growth by affecting cell proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of C8ORF59 significantly induces apoptosis in lung cancer Cells\u003c/h2\u003e \u003cp\u003eCell cycle arrest usually triggers apoptosis of cancer cells. Annexin V binds to the early apoptotic cell membrane to mark early apoptotic cells, while cell nuclear dye 7-AAD can enter late apoptotic or necrotic cells to stain DNA and mark late apoptotic or necrotic cells. Using AnexinV PE-7AAD to double stain cells to observe whether knocking down C8ORF59 can induce apoptosis. Results show that loss of C8ORF59 significantly promoted apoptosis in lung adenocarcinoma cells, including both early and late apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Additionally, immunoblotting assays using antibodies of anti/pro-apoptotic cell markers were conducted to confirm the above results. We found that upon knockdown of C8ORF59 in lung cancer cells, the expression of Myeloid Cell Leukemia Sequence 1 (MCL-1), an anti-apoptotic cell marker, significantly decreased, while the expression of apoptosis-promoting proteins BH3 Interacting Domain Death Agonist (BID), BCL2 Antagonist/Killer (BAK), and BCL2 Binding Component 3 (Puma) significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These findings suggest that growth inhibition caused by C8ORF59 deficiency could be associated with induced cell apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSignificant reduction of 47S rRNA is observed in lung adenocarcinoma cells expressing low levels of C8ORF59\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe assembly of ribosomes mainly involves the transcription and processing of rRNA, the transcription and translation of ribosomal proteins (RPs), and the regulation of ribosomal assembly factors (AFs). Among them, rRNA transcription is the rate limiting step of ribosomal biogenesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; Additionaly, C8ORF59 may be a regulatory factor related to pre-rRNA processing [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, we detected the expression of 47S pre-rRNA in cells where C8ORF59 was knocked down and found a significant decrease in 47S rRNA expression in A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). RPs and Afs are indispensable parts for ribosome assembly, thus we collected proteins from control group and C8ORF59 knockdown cells and detected the protein expression of key RPs and AFs using immunoblotting assay. FBL, a highly conserved nucleolar methyltransferase responsible for rRNA and protein methylation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], immunoblotting assay showed a significant decrease in the expression of FBL upon C8ORF59 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). It has been reported that RPL3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles, and the final maturation of pre-40S ribosome small subunits depends on functional integrity of RPL3 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], our results demonstraed a significant decrease in RPL3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). CX-5461 is a drug targetting ribosome biogenesis through inhibiting rDNA transcription, thus inhibiting HGSOC cell growth [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To explore whether ribosome biogenesis mediated growth regulation of C8ORF59 on lung cancer cells, CX-5461 was added to lung cancer cells stable expressing shNC and shC8ORF59, the inhibitory effects of C8ORF59 knockdown on CDK2 and MCL-1 was further enhanced upon adding CX-5461 48 hours later, results display synergistic effects of C8ORF59 deficiency and ribosomal biogenesis inhibition on key proteins of cell cycle and cell growth. Taken together, these results indicate that C8ORF59 might regulate the growth of lung cancer cells through, at least in part, impacting ribosome assembly.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of C8ORF59 enhances the drug sensitivity of lung adenocarcinoma cells to Gemcitabine\u003c/h2\u003e \u003cp\u003eThe ribosomes are the site for all protein synthesis in the cell and therefore crucial for cell survival. Some studies have shown that ribosomal biogenesis is significantly related to radiotherapy resistance and chemotherapy resistance in cancer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Gemcitabine, a cytidine nucleoside derivative, which undergo phosphorylation of deoxycytidine nucleoside kinase within cells to form diphosphate and triphosphate nucleosides, are incorporated into cells to interfere with normal DNA synthesis, causing DNA breakage and ultimately leading to cell death, they mainly kill 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 knocking down C8ORF59 caused cell cycle arrest at G0/G1, and significant decrease in the proportion of S phase cells, so we want to know whether loss of function of C8ORF59 and gemcitabine have synergistic effect on growth inhibition. Consequently, different concentrations of gemcitabine was added into lung cancer cells stable expressing shNC and shC8ORF59, results showed that the IC50 value of gemcitabine significantly decreased in lung cancer cells with C8ORF59 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Moreover, similar results were obtained from colony formation assays and exhibiting a significant dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). These experimental results imply that C8ORF59 may be related to gemcitabine resistance in lung cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eKnocking down C8ORF59 inhibits in vivo tumor growth of lung cancer cells\u003c/h2\u003e \u003cp\u003eTo verify whether C8ORF59 can affect tumor growth in vivo, we subcutaneously inoculated cells with stable expression of shC8ORF59 and control cells shNC, and tumor growth was monitored two weeks after injection until the tumor size reaches the ethical limit. Results showed that knocking down C8ORF59 significantly inhibited tumor growth in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-C). We dissected the subcutaneous tumors, digested part of the subcutaneous tumors into cell suspensions and used flow cytometry to detect the apoptosis rate. Results show a substantial apoptosis proportion in the C8ORF59 knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-E). These results suggest that C8ORF59 is closely related to the in vivo growth of lung adenocarcinoma cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudies have showed elevated expression of 47S pre-rRNA has been observed in primary colorectal cancer tissues compared to normal colonic mucosa [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]; Overexpression of ribosomal protein RPL15 in circulating tumor cells promotes multi-organ metastasis and selectively enhances the translation of other ribosomal proteins and cell cycle regulators [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Targeting ribosome biogenesis by inhibiting the ribosomal small subunit protein RPS6 can suppress NSCLC cell growth by affecting cell cycle regulation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], indicating that ribosome related factors play important roles in tumors. C8ORF59 is a ribosomal biogenic factor preliminarily indicated by GSEA, however, there is very limited research on this gene currently, with only two studies suggesting its potential involvement in ribosome assembly through systematic screening [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The specific function and mechanisms of C8ORF59 in ribosome assembly have not been directly and extensively investigated. In our study, we have revealed the preliminary function and mechanisms of C8ORF59 in ribosome biogenesis, LUAD progression, and chemotherapy resistance for the first time.\u003c/p\u003e \u003cp\u003eCX-5461, an RNA polymerase I inhibitor, can inhibit ribosome biogenesis, leading to growth, migration, and DNA damage induction in pancreatic ductal adenocarcinoma cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Silencing of the rRNA metabolism-associated cancer gene EXOSC8 reduces levels of nucleolar proteins and proliferation markers, as well as rRNA/DNA and global protein synthesis, inhibiting colorectal cancer cell proliferation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These studies suggest that ribosome stress causes a series of disruptions in cellular biology. In our study, we found that the downregulation of C8ORF59 results in decreased cell migration and invasion potential, inhibited cell growth, cell cycle arrest, and increased apoptotic ratio.\u003c/p\u003e \u003cp\u003eThen, we investigated the molecular mechanisms of C8ORF59 in regulating LUAD progression. Transcription of rRNA is a rate-limiting step in ribosome biogenesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In our study, we observed a significant decrease in the transcriptional expression levels of 47S pre-rRNA in A549 cells with C8ORF59 knockdown. Studies show in the ribosome, rRNA plays a central role in structure and function, providing binding sites for ribosomal proteins. During the processing of pre-rRNA, ribosome assembly factors and ribosomal proteins assemble in a coordinated manner onto pre-rRNA, forming pre-ribosomal particles [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], suggesting that the abnormal expression of pre-rRNA may be associated with the co-expression abnormalities of ribosomal proteins. Another study involving TCGA data analysis also revealed a 30% median increase in ribosomal protein expression levels in tumor tissues compared to normal tissues. Furthermore, many ribosomal proteins exhibit distinct dysregulation patterns in specific tumors. For example, RPL21L1 and RPS27L are upregulated in breast and thyroid cancers, while RPL21 is downregulated [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], indicate the diversity of ribosomal protein expression patterns in cancer progression. Our present research found that FBL, which possesses rRNA methyltransferase activity, and RPL3, a structural protein of the ribosome large subunit, significantly decreased upon C8ORF59 knockdown. However, we cannot exclude the possibility that other untested ribosomal proteins may be regulated by C8ORF59. Treatment with CX-5461 to inhibit ribosome biogenesis enhanced the growth-inhibitory effect of C8ORF59 loss in lung cancer cells.\u003c/p\u003e \u003cp\u003eIn human tumors, ribosomal proteins and related ribosome biogenesis factors mediate radioresistance and chemoresistance in various cancers, including pancreatic cancer, lung cancer and glioma [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, resistance is a major obstacle to the benefit of cancer patients. Our study found that downregulation of C8ORF59 increased the sensitivity of lung cancer cells to gemcitabine. Considering that downregulation of C8ORF59 also causes changes in ribosome biogenesis, we believe that ribosome biogenesis may mediate, at least partially, the increased sensitivity of lung cancer cells to gemcitabine caused by C8ORF59 knockdown.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we have identified C8ORF59 as a potential oncogenic factor and a therapeutic target in LUAD. Mechanistically, C8ORF59 affects the cell cycle and apoptosis of lung cancer cells through its involvement in ribosome biogenesis, ultimately impacting LUAD progression. Furthermore, we have found that inhibiting C8ORF59 can enhance tumor sensitivity to anti-Gemcitabine therapy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eClinical Samples:\u003c/h2\u003e \u003cp\u003eThe clinical samples used in this study were obtained from Huashan Hospital, affiliated with Fudan University. Written informed consent was obtained from all patients, and the study was approved by the ethics committee of Huashan Hospital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eThe human lung adenocarcinoma cell lines, A549, H1299, and PC9, were obtained from the ATCC cell repository. A549 cells were cultured in Ham's F-12K medium, while H1299, and PC9 cells were cultured in RPMI-1640 medium. All media were supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCell Growth\u003c/h2\u003e \u003cp\u003eCell suspensions of lung adenocarcinoma cells were adjusted to a concentration of 800 cells/200 \u0026micro;l and seeded in 96-well cell culture plates with triplicate wells per group. After 24 hours, 10 \u0026micro;l of CCK-8 reagent was added to 90 \u0026micro;l of cell culture medium. After incubation for 2 hours, the absorbance at 450 nm was measured daily for 6 consecutive days to generate growth curves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eColony formation Assay\u003c/h2\u003e \u003cp\u003eCells were seeded at a density of 500 cells per well in 6-well plates and cultured for 10\u0026ndash;14 days. The colonies were fixed with methanol for 30 minutes and stained with 0.1% crystal violet for 30 minutes. After washing and air-drying, the colonies were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMigration and Invasion Assays\u003c/h2\u003e \u003cp\u003eMigration and invasion assays were performed using chambers purchased from Corning. The chambers were coated without FBS in the upper compartment with cell suspensions adjusted to 2.5\u0026times;10^5 cells/ml for migration assays and 5\u0026times;10^5 cells/ml for invasion assays. The lower compartment was filled with complete medium containing 10% FBS. The chambers were then incubated in a cell culture incubator for 16\u0026ndash;20 hours, followed by removal of non-migrating or non-invading cells from the upper surface of the membrane with a cotton swab. The cells on the lower surface were fixed with methanol for 30 minutes, stained with 0.1% crystal violet for 15\u0026ndash;20 minutes, washed, air-dried, and photographed under a microscope. Nine fields were randomly selected for counting in each chamber.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA Extraction and qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using RNAiso Plus (#9109) purchased from TAKARA. Reverse transcription was performed using the reverse transcription kit from Novogene. The obtained cDNA was diluted 10-fold, and qPCR was performed using SYBR Green from Yisheng Biotechnology. The primer sequence for C8ORF59 was synthesized by Genewiz.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCell apoptosis detection\u003c/h2\u003e \u003cp\u003eCell inoculation was performed on a 6-well plate until reaching 80\u0026ndash;90% confluency. The cell supernatant and adherent cells were collected and washed once with pre-chilled PBS. Then, 5 \u0026micro;l of PE and 7AAD from the apoptosis detection kit were added separately for dual staining of the cells. Blank control, PE single staining control, and 7AAD single staining control groups were set up. After 10 minutes of light-protected staining, the apoptosis rate was detected using a flow cytometer within 1 hour.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003eCell cycle analysis was conducted by collecting adherent cells from a 6 cm cell culture dish, washing them twice with PBS, and fixing them in 70% ethanol at -20\u0026deg;C for at least 4 hours or overnight. After centrifugation and removal of the supernatant, the cells were washed once with PBS and stained with 500 \u0026micro;l of PI staining solution for 10 minutes. The DNA content of the cells was analyzed using a flow cytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and Western Blot\u003c/h2\u003e \u003cp\u003eCells from the 6-well plate were washed three times with pre-chilled PBS, and 200 \u0026micro;l of protein lysis buffer (T-per) containing protease and phosphatase inhibitors was added. The cells were scraped and thoroughly lysed on ice for 30 minutes. After centrifugation at 12,000 rpm for 15\u0026ndash;20 minutes, the supernatant containing the desired protein was collected. The protein concentration was measured using a BCA protein quantification kit, and samples from different groups were adjusted to the same protein concentration. Protein samples were treated with loading buffer and boiled at 100\u0026deg;C for 10 minutes before storage at -80\u0026deg;C. The following antibodies were used in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection\u003c/h2\u003e \u003cp\u003eThe siRNA targeting C8ORF59 is a commercial product purchased from Jin Weizhi. The cells were placed on a 6-well cell culture plate and transiently transfected into lung cancer cells using Lippofectamine 2000 24 hours later. The solution was changed 6 hours later, and subsequent experiments were conducted 48 hours after transfection. The siRNA sequence was shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction and lentivirus packaging\u003c/h2\u003e \u003cp\u003eThe shRNA sequence of C8ORF59 was synthesized by Jinweizhi Company and constructed into the PLKO.1 plasmid. The successfully constructed PLKO.1-C8ORF59-shRNA was transfected with lentivirus packaging envelope plasmids pMD2.G and psPAX2 in a ratio of 8:6:2 ug into 10 cm dishes of 293T cells using TenfectDNA transfection reagent. After 48 hours, the cell culture supernatant was collected, and cell debris was removed by filtering through a 0.45 \u0026micro;m membrane. The virus liquid was stored at 4\u0026deg;C for up to 1 week or at -80\u0026deg;C for longer periods.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eDrug Experiments\u003c/h2\u003e \u003cp\u003eIn the stable cell lines expressing shNC and shC8ORF59, 0 \u0026micro;M and 75 \u0026micro;M of the ribosome-targeting drug CX-5461 were added, respectively. After incubating for 48 hours, cells were collected and protein was extracted. Protein concentration was measured using the BCA assay, and protein samples were adjusted to equal concentrations and mixed with protein loading buffer. The mixture was heated at 100\u0026deg;C for 10 minutes for subsequent Western blot experiments. In the gemcitabine IC50 experiment, A549 and H1299 cells stably expressing shNC and shC8ORF59 were seeded in a 96-well plate at a density of 4,000 cells per well. The cells were treated with different concentrations of gemcitabine (0, 0.00316, 0.01, 0.0316, 0.1, 0.316, 1, 3.16, 10, 31.6 \u0026micro;M) in 10 replicates for each concentration, and incubated for 72 hours. The absorbance at 450 nm (OD450) was measured, and the OD450 value of the blank well was subtracted from the OD450 value of each well. Then, the OD450 value of the experimental group was subtracted from the OD450 value of the control group to obtain the values for IC50 plotting. In the gemcitabine impact on colony formation experiment, A549 and H1299 cells stably expressing shNC and shC8ORF59 were seeded in 6-well plates at a density of 2,500 cells per well. Four different concentrations of gemcitabine (0, 0.1, 0.2, 0.3 \u0026micro;M) were added to triplicate wells for each concentration, and the cells were incubated until colonies formed. Crystal violet staining was performed, and the colonies were counted after imaging.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eDatabase analysis\u003c/h2\u003e \u003cp\u003eThe expression level of C8ORF59 in lung cancer databases was analyzed using public databases TIMER (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cistrome.org/timer\u003c/span\u003e\u003cspan address=\"http://cistrome.org/timer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), ULCAN (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ualcan.path.uab.edu/analysis.html\u003c/span\u003e\u003cspan address=\"https://ualcan.path.uab.edu/analysis.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and GEO datasets GSE102287 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE102287\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE102287\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and GSE30219 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30219\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30219\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The correlation of C8ORF59 with lung adenocarcinoma prognosis was analyzed using Kaplan, GEPIA, and TIMER.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of the data in the article was performed using GraphPad Prism 9. The differences between two groups were analyzed using paired or unpaired Student's t-test. Survival analysis was conducted using the Log-rank Cox method, and data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. P-value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are thankful to the TCGA, TIMER and GEPIA for providing the data analyzed in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work in the Yan laboratory is supported, in whole or in part, by grants from the National Natural Science Foundation of China (No. 81972173; No. 82273371) and the Science and Technology Commission of Shanghai Municipality (No.22140901400).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMXY and QL were responsible for the design of the study and wrote the manuscript; HYP and LL participated in the functional analysis and Western blot; SWX and YJX performed bioinformatic analysis and qPCR; WJC was responsible for the animal experiment; XLL contributed to conduct the stable cell lines; JL, YC and LS were incharge of drug sensitivity experiment; All authors reviewed the manuscript before submission and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included either in this\u003c/p\u003e\n\u003cp\u003earticle or in the supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of Fudan University (Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree on publication of the results of the present manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 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Phosphorylation of ribosomal protein S3 and antiapoptotic TRAF2 protein mediates radioresistance in non-small cell lung cancer cells\u003cem\u003e.\u003c/em\u003e J Biol Chem 288(5), 2965-75 (2013).\u003c/li\u003e\n\u003cli\u003eKo CY, Lin CH, Chuang JY, Chang WC, and Hsu TI. MDM2 Degrades Deacetylated Nucleolin Through Ubiquitination to Promote Glioma Stem-Like Cell Enrichment for Chemotherapeutic Resistance\u003cem\u003e.\u003c/em\u003e Mol Neurobiol 55(4), 3211-3223 (2018).\u003c/li\u003e\n\u003cli\u003eMlak R, Krawczyk P, Ciesielka M, Kozioł P, Homa I, Powr\u0026oacute;zek T, et al. The relationship between RRM1 gene polymorphisms and effectiveness of gemcitabine-based first-line chemotherapy in advanced NSCLC patient\u003cem\u003e.\u003c/em\u003e Clin Transl Oncol 18(9), 915-24 (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lung adenocarcinoma, C8ORF59, Ribosomes biogenesis, Gemcitabine sensitivity","lastPublishedDoi":"10.21203/rs.3.rs-4182106/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4182106/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eElevated ribosome biogenesis was required by tumor growth. In this study, we initially screened a set of key genes related to ribosome biogenesis from the GSEA dataset. Then, we obtained differentially expressed gene sets between cancer tissues and adjacent non-cancerous tissues from the GSE datasets. By intersecting these gene sets, we identified potential genes that may play a significant role in the progression of lung adenocarcinoma. Subsequently, through extensive literature review, we finally identified the gene Chromosome 8 Open Reading Frame 59 (C8ORF59) as an interesting candidate. Our research findings demonstrated that the knockdown of C8ORF59 significantly inhibits the migration, invasion potential, cell growth, and clonogenicity of lung adenocarcinoma cells. Additionally, apoptosis assays revealed a significant increase in apoptosis, including both early and late stages, in lung adenocarcinoma cells upon C8ORF59 knockdown. Cell cycle analysis showed that C8ORF59 knockdown arrests cells predominantly in the G0/G1 phase, indicating inhibited cell proliferation. Moreover, knocking down C8ORF59 significantly inhibits the in vivo growth of lung cancer cells. Mechanistically, downregulation of C8ORF59 significantly decreases the expression of 47S rRNA, a component associated with ribosome assembly, ribosome proteins Fibrillarin (FBL) and Ribosomal Protein L3 (RPL3). Additionally, ribosomal biogenesis targeting drugs CX-5461 and C8ORF59 loss generate synergistic effects on key proteins regulating cell cycle and apoptosis. Knocking down C8ORF59 also substantially enhanced the sensitivity of lung adenocarcinoma cells to the chemotherapeutic drug gemcitabine, suggesting a potential association between C8ORF59 and drug resistance. Collectively, these studies suggest the close involvement of C8ORF59 in the progression of lung adenocarcinoma, providing new insights for its therapeutic intervention.\u003c/p\u003e","manuscriptTitle":"C8ORF59 regulates ribosome biogenesis to affect progression\nin lung adenocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-26 18:46:42","doi":"10.21203/rs.3.rs-4182106/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-27T06:13:06+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-23T10:13:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-29T06:24:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2024-03-28T08:02:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"99880863-e25e-4599-a829-d23d6db145a1","owner":[],"postedDate":"April 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-30T15:59:14+00:00","versionOfRecord":{"articleIdentity":"rs-4182106","link":"https://doi.org/10.1186/s12967-024-05886-1","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2024-12-25 15:57:05","publishedOnDateReadable":"December 25th, 2024"},"versionCreatedAt":"2024-04-26 18:46:42","video":"","vorDoi":"10.1186/s12967-024-05886-1","vorDoiUrl":"https://doi.org/10.1186/s12967-024-05886-1","workflowStages":[]},"version":"v1","identity":"rs-4182106","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4182106","identity":"rs-4182106","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}