The tumor promoter role and molecular mechanism of C8orf76/CALB2 axis in clear cell renal cell carcinoma

The tumor promoter role and molecular mechanism of C8orf76/CALB2 axis in clear cell renal cell carcinoma | 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 The tumor promoter role and molecular mechanism of C8orf76/CALB2 axis in clear cell renal cell carcinoma Yuxiao Li, Xinyang Niu, Pengju Liu, Xueyou Ma, Jiazhu Sun, Suyuelin Huang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7305276/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background: Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system, with clear cell renal cell carcinoma (ccRCC) accounting for 90% of all RCC subtypes. Chromosome 8 open reading frame 76 (C8orf76) is significantly upregulated in various tumor types and has been involved in tumor cell proliferation, migration, invasion, and is associated with poor prognosis. However, the expression profile and molecular mechanisms of C8orf76 in ccRCC have not been fully elucidated, and further investigations are required to clarify these aspects. Methods: We systematically investigated the mechanism of action of C8orf76 in ccRCC through in vitro biological function experiments. Cellular function assessments were performed, including CCK-8 assay, colony formation assay, flow cytometry, SA-β-gal staining, Transwell chamber assay, and wound healing assay. Additionally, combined with a subcutaneous xenograft mouse model and an in vivo imaging system, we studied the phenotypic changes following C8orf76 knockdown. Potential downstream targets of C8orf76 were screened via RNA-sequencing and bioinformatics analysis. Additionally, we utilized The Cancer Genome Atlas (TCGA) database to analyze the expression patterns of C8orf76 and CALB2 in ccRCC, as well as their correlations with clinical prognosis. Results: Both C8orf76 and CALB2 are highly expressed in ccRCC and correlate with poor prognosis. Knockdown of C8orf76 significantly inhibits the proliferation and migration of ccRCC cells both in vivo and in vitro. Specifically, Knockdown of C8orf76 downregulates the transcriptional level of CALB2, leading to G1-phase cell cycle arrest, enhanced cellular senescence, and subsequent suppression of ccRCC proliferation and migration. Furthermore, ectopic overexpression of CALB2 can partially reverse these effects. Dual-luciferase reporter assay confirms that C8orf76 directly binds to the promoter region of CALB2. Similarly, CALB2 knockdown also induces tumor cell cycle arrest and cellular senescence, accompanied by inhibited proliferation and migration of ccRCC. Notably, the aforementioned phenomena are partially rescued following further knockdown of CDKN2A. Conclusions: C8orf76 is highly expressed in clear cell renal cell carcinoma and correlates with poor prognosis. C8orf76 directly binds to the CALB2 promoter, thereby promoting CALB2 transcription and downstream biological behaviors. Inhibition of the C8orf76/CALB2 axis induces G1-phase cell cycle arrest and activates cellular senescence signaling pathways, which in turn suppresses the proliferation and migration of ccRCC. ccRCC C8orf76 CALB2 Cell senescence Cell cycle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Renal cell carcinoma (RCC) is a common malignant tumor of the urinary system, accounting for about 90% of renal malignancies and constituting about 3% of adult malignant tumors globally[ 1 , 2 ]. In 2022, there were over 430,000 new RCC cases and 150,000 deaths worldwide[ 3 ]. Notably, the incidence rate among younger populations (< 50 years old) is on the rise[ 4 ]. Clear cell renal cell carcinoma (ccRCC), the most prevalent subtype of RCC, originates from the proximal tubules of nephrons. It is characterized by a highly aggressive phenotype and poor prognosis[ 5 , 6 ]. Surgical treatment is the main strategy for ccRCC[ 7 ]. However, due to the lack of effective early diagnostic measures, ccRCC often presents inconspicuously, with nearly 30% of patients diagnosed with metastatic disease at initial presentation[ 8 ]. Metastatic tumor patients frequently develop treatment resistance and disease progression[ 9 , 10 ]. Therefore, it is necessary to further investigate the molecular mechanisms of the occurrence, development, and metastasis of ccRCC. Such exploration may be the key to assisting clinical early diagnosis, identifying new therapeutic targets, and developing personalized treatment strategies. C8orf76 (chromosome 8 open reading frame 76) is a novel nuclear protein-encoding gene located on chromosome 8q24.13–24.3, which encodes a 380-aa protein. It was first discovered in gastric cancer. C8orf76 could directly interact with the oncogenic long non-coding RNA (lncRNA) dual specificity phosphatase 5 pseudogene 1 (DUSP5P1), triggering its transcriptional upregulation adn subsequent activation of the downstream mitogen-activated protein kinase (MAPK) signaling pathway[ 11 ]. Studies have also shown that C8orf76 participates in ferroptosis regulation by transcriptionally activating SLC7A11, thus promoting the progression of liver cancer[ 12 ]. The protein encoded by the calbindin 2 ( CALB2 ) gene is a member of the troponin C superfamily, primarily regulating intracellular calcium levels and exerting biological functions such as signal transduction and calcium ion buffering. CALB2 is also associated with metastasis and drug resistance in multiple tumors[ 13 – 15 ]. For example, CALB2 can activate the TRPV2-Ca²⁺-ERK1/2 signaling pathway to induce metastasis in hepatocellular carcinoma[ 16 ]. However, the specific roles and molecular mechanisms of C8orf76 and CALB2 in clear cell renal cell carcinoma have not yet been thoroughly investigated. The concept of cellular senescence was first proposed in the 1960s[ 17 ], referring to a stable and persistent state of cell cycle arrest. This state is associated with upregulation of cyclin-dependent kinase inhibitors (CKIs), such as p16INK4a and p21WAF1/CIP1, chronic DNA damage responses, and a hyper-secretory state of the senescence-associated secretory phenotype (SASP)[ 18 ]. Among these, p16INK4a (p16) is a critically important cell cycle inhibitor. It can suppress the activity of CDK4/6, prevent the phosphorylation of retinoblastoma protein (RB), and thereby lead to G1 phase arrest in the cell cycle, promoting the occurrence of cellular senescence[ 19 ]. This strong association with cellular senescence has made p16 a priority for research[ 20 ]. In tumor progression, cellular senescence exhibits a dual role: cell entry into senescence can act as an effective barrier against tumorigenesis[ 21 ], while under certain conditions, persistently senescent cells can also acquire pro-tumorigenic properties[ 22 , 23 ]. Preclinical evidence suggests that senescent cancer cells exhibit enhanced immunogenic properties, offering a novel avenue for therapeutic modulation of anti-cancer immunity[ 24 ].Therefore, in-depth research on cellular senescence and tumorigenesis may serve as a new tool to break through the barriers of cancer therapy and tackle the challenges of early tumor diagnosis. Materials and methods Cell Culture HEK-293T, HK-2, A-498, Caki-1, 769-P and 786-O cell lines were obtained from the Chinese Academy of Sciences Shanghai Cell Bank (Shanghai, China). HEK-293T cells were cultured in DMEM medium (Procell, Wuhan, China) supplemented with 10% fetal bovine serum (Biologic Industries, Israel) and 1% penicillin/streptomycin (Servicebio, Wuhan, China). A-498 cells were cultured in MEM medium (Procell, Wuhan, China) supplemented with 10% fetal bovine serum (Biologic Industries, Israel) and 1% penicillin/streptomycin (Servicebio, Wuhan, China). HK-2, Caki-1, 786-O and 769-P cells were cultured in RPMI 1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Biologic Industries, Israel) and 1% penicillin/streptomycin (Servicebio, Wuhan, China). All cell lines are regularly tested for mycoplasma contamination. All cell lines were cultured in a humidified atmosphere at 37°C with 5% CO 2 . Western Blot Protein concentrations were measured utilizing a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, China). An equivalent quantity of protein (30 µg) was subjected to SDS-PAGE gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After a blocking step with 5% non-fat dried milk in Tris-buffered saline containing Tween 20 (TBST) for 1.5 h at room temperature (RT), the membranes were subjected to primary antibodies specific to each target protein, followed by overnight incubation at 4°C. The next day, the membranes were washed three times with TBST before being treated with horseradish peroxidase-conjugated secondary antibodies (1:4000, Catalog No: GB23301, GB23303; Servicebio, Wuhan, China) for 2 h at RT. Protein bands were identified using an enhanced chemiluminescence (ECL) detection system. β-Actin was used as an internal control. Supplementary Table 1 lists the utilized primary antibodies. Quantitative Real-time PCR Total RNA was extracted from cell samples and stored at -80°C using RNAiso Plus Reagent (Takara), ensuring the genomic DNA contamination was removed, according to the manufacturer’s instructions. RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the cDNA Reverse Transcription Kit (Vazyme, China) following the protocol. Quantitative real-time PCR ( qPCR) was performed with SYBR qPCR Mix (Vazyme, China) on a Biorad CFX96 Detection System. The qPCR data were analyzed using the ΔΔCT method for relative quantification, normalizing to the β-actin. Data were analyzed using GraphPad Prism software (GraphPad Software10.0). Supplementary Table 2 lists the utilized primer sequences. Cell Counting Kit-8 (CCK-8) Assay Seed cells at an appropriate density (800 ~ 1000 cells/well) in a 96-well plate with 100 µL of cell culture medium per well. Incubate the plate in a humidified CO 2 incubator (e.g., 37°C, 5% CO 2 ) for 24 hours to allow cell attachment. Addition of CCK-8 Solution (Fude Bio, Hangzhou, China): Add 10 µL of CCK-8 solution to each well of the plate. Put the plate to the incubator and incubate for 1–2 hours. Measure the absorbance at 450 nm using a microplate reader. Calculate the cell viability by comparing the absorbance values of the test wells to those of the control wells. Colony Formation Assay Cell proliferation ability was detected by clonal formation assay [ 25 ]. Seed 800–1000 cells per well in a 6-well plate. Incubate the cells in a humidified incubator at 37°C with 5% CO 2 for approximately 1–3 weeks, changing the medium every 5 days and monitoring cell growth. Once visible colonies have formed, terminate the culture. Wash the cells with PBS, then fix with 4% polyoxymethylene for 30 minutes. After washing again, stain with crystal violet for 30 minutes. Count the number of colonies under a microscope for later analysis. Colonies are typically defined as groups of 50 or more cells. Plating Efficiency = (Number of Colonies / Number of Cells Seeded) × 100%. Wound Healing Assay Cells were seeded into 6-well plates and were cultured to confluency. Wounds were generated by scratching cell layer with 200 µL plastic pipette tips. The final images were taken and the gap distances of migrating cells were measured using optical microscope. Transwell Cell Migration Assay Cell migration assay was performed with transwell chamber (Corning, USA) according to the standard method. 600 µL of complete media was added to the lower chamber, and 200 µL of medium containing 1 × 10 4 cells in serum-free RPMI-1640 was put in the upper chamber (8-mm pore size, Corning). After being cultured for 24 h, cells were fixed in 4% paraformaldehyde and stained with crystal violet. Five fields per well were randomly selected and images were acquired by a optical microscope. Senescence Associated β-galactosidase (SA-β-gal) Staining The Senescence Associated β-Galactosidase Staining Kit were purchased from Beyotime (Shanghai, China). 786-O and 769-P cells were cultured in 6-well plates. After washing with PBS and fixation, dispense the working fluid according to the protocol. Incubate overnight at 37°C. The SA-β-gal positve cells were obtained via optical microscope and quantified using Image J software (version 1.48v). Cell Cycle Flow Cytometry The cell cycle flow cytometry was performed using the Cell Cycle Staining Kit (MultiSciences, Hangzhou, China). Data were analyzed using the FlowJo 10.8.1 and Modfit LT 5.0 software. Plasmids Transfection Seed cells in a 6-well plate at a density that allows for 60–70% confluence on the day of transfection. Incubate the cells in an incubator at 37°C with 5% CO 2 . For each well, dilute 1 µg of plasmid DNA and Lipofectamine 3000 (Thermofisher, USA) or JetPRIME transfection agents (Polyplus, USA) in buffer according to each protocol. Lipofectamine 3000 needs to be diluted before mixing with DNA. Incubate for 20 minutes at room temperature to allow the formation of transfection complexes. While the transfection complexes are forming, replace the medium in the cell culture wells with 1500 µL of fresh medium without serum. Add the transfection complexes dropwise to the cells and gently rock the plate to ensure even distribution. Return the plate to the incubator for 8 hours to allow for transfection. After the incubation period, remove the transfection medium and replace it with 1 mL of complete growth medium (containing serum and antibiotics) to support cell growth and expression of the transfected gene. Supplementary Table 3 presents the shRNA and siRNA sequences for knocking down C8orf76, CALB2 and CDKN2A. Lentiviral Construction Day 0: Seed 293T cells into 6-well plates at a density of 1.0*10^ 6 cells per well in 2 mL of lentivirus packaging medium. Incubate overnight at 37°C, 5% CO 2 to reach 70% confluence. Day 1: Prepare the transfection complexes by mixing 3 µg of the transfer vector and 9 µg of packaging plasmids with JetPRIME transfection agents in Opti-MEM medium according to the manufacturer's instructions. Incubate for 10–20 minutes at room temperature. Replace medium in each well with 1 mL of fresh lentivirus packaging medium. Add the lipid-DNA complex to each well, gently agitating the plate to distribute the mixture evenly. Incubate overnight at 37°C, 5% CO 2 . Day 2: Replace the medium containing the lipid-DNA complexes with 2 mL of pre-warmed lentivirus packaging medium. Return the plate to the incubator overnight. Collect the supernatant containing the virus and store at 4°C. Replace the collected medium with 2 mL of fresh lentivirus packaging medium. Day 3: Collect the second batch of supernatant. Combine this with the first collection and centrifuge at 2,000 rpm for 10 minutes at room temperature to remove cellular debris. Filter the supernatant through a 0.45 µm filter to remove any remaining debris. Aliquot the virus into cryovials and store at -80°C. Cell Infection GLI2 overexpression adenovirus containing the GLI2 coding sequence was obtained from Shanghai Genechem Co., Ltd. Cells were seeded for infection in a six-well plate, achieving 60% confluence by the next day. The reagent/medium containing adenovirus/lentivirus was removed from the − 80°C freezer and placed on ice. According to the instructions, a suitable volume of reagent/medium was administered to the cells in each well. Polybrene (Beijing Solarbio Science & Technology, China) was included at 8 µg/mL for lentiviral infection. After 24 h of infection, the medium was changed, and the cells were cultured in the incubator for another 24 h. Puromycin was added at an initial working concentration of 2 µg/mL to select stably infected clones, with the concentration gradually increased to 5 µg/mL by the fifth day. Cells were incubated for subsequent experiments. Dual-luciferase Reporter Gene Assay 293T cells were used to detect dual-luciferase according to Promega protocol as described in the literature [ 26 ]. Briefly, plate cells in 24-well plates at a density of 100,000 cells/well in appropriate medium. The next day, co-transfect cells with the firefly luciferase reporter plasmid (under the control of the promoter of interest) and the Renilla luciferase control plasmid using Lipofectamine 3000. Incubate cells at 37°C with 5% CO 2 overnight to allow for plasmid uptake and expression. Remove medium and wash cells with PBS. Lyse cells using 0.1 mL of Passive Lysis Buffer (PLB) per well. Aliquot lysates (5–20 µL) into duplicate wells of a white 96-well plate. Initiate bioluminescence by automatic injection of 0.1 mL of LAR II into the lysates. After a 1-minute delay, record the firefly luciferase emission signals using a microplate luminometer. Quench the firefly luciferase signal and activate Renilla luciferase by adding Stop & Glo® Reagent. Record the Renilla luciferase emission signals. Normalize the firefly luciferase activity to the Renilla luciferase activity to account for transfection efficiency and cell viability. Calculate the fold-change of transcriptional activity by comparing the normalized luciferase activity in the presence of the transcription factor to the vector control condition. Supplementary Table 4 lists the mutant plasmid sequence utilized in dual luciferase reporter gene experiments. Immumohistochemical (IHC) Staining Immumohistochemical staining was performed as described in the previous study [ 27 ]. Tumor tissue specimens were obtained from nude mice and fixed in 10% neutral buffered formalin for 24 h at RT to preserve cellular morphology and antigenicity. The fixed tissues were embedded in paraffin wax and sectioned into 4 µm thick slices using a microtome. The sections were mounted onto silane-coated slides and allowed to dry at 60°C on a hot plate. Paraffin was removed by incubating the slides in xylene, and the sections were rehydrated in a graded ethanol series and distilled water. Antigen retrieval was performed through heat treatment in sodium citrate buffer (pH 6.0) utilizing a water bath. The samples were incubated with a 3% hydrogen peroxide solution in distilled water for 10 min to inhibit endogenous peroxidase activity. Subsequently, sections were incubated with normal serum from the same species as the secondary antibody to diminish instances of non-specific binding. The diluted primary antibodies were applied to the sections and allowed to incubate at RT for 1 h. The slides were washed with TBST three times for 5 min each to eliminate unbound primary antibodies. Secondary antibodies were administered and incubated for 30 min at RT. The antigen-antibody complex was visualized using a chromogenic substrate, DAB, resulting in a brown color reaction at the antigen site. The sections were counterstained with hematoxylin for 1 min to yield a blue nuclear contrast. Tissue sections were systematically dehydrated using a graded ethanol series, thereafter mounted in an aqueous-based medium, and sealed with precisely cut glass coverslips. The slides were examined under a light microscope to visualize the specific antigen localization within the tissue sections. Statistical analysis All in vitro experimental procedures conducted were repeated a minimum of three times to ensure reproducibility. For the in vivo studies, each experimental group was comprised of five BALB/c-nu mice. The survival data were assessed utilizing the Kaplan-Meier method, with the log-rank test. The statistical processing of the data was carried out employing Prism 10.0 software by GraphPad. The results are depicted as the mean ± standard deviation (SD). Both unpaired Student's t-tests for parametric data and nonparametric tests were implemented to determine p -values. In detail, the normality of the data distribution was initially assessed using the normal-test function available in Prism 10.0, ensuring the applicability of the parametric t-test. For datasets that deviated from a normal distribution, non-parametric tests were employed. When the F test in the parametric t-test indicated statistical significance at a p -value less than 0.05, Welch’s correction was applied to adjust the data. A p -value threshold of less than 0.05 was set to define statistical significance. Results without statistical significance were denoted as ns, while significant differences were indicated with asterisks: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. Results C8orf76 up-regulation in ccRCC and its association with prognosis. The TIMER 2.0 database was employed to assess C8orf76 expression levels across pan-cancer, and the results indicated its high expression in these settings (Fig. 1 A). Both paired (Fig. 1 C) and unpaired (Fig. 1 B) analyses of KIRC-TCGA revealed elevated C8orf76 expression in tumor samples. An independent dataset containing information on 47 clinical samples yielded the similar results (Fig. 1 D, Tab. S5). Consistently, C8orf76 protein levels were significantly higher in clinical tumor samples compared with adjacent normal tissues from 10 ccRCC patients (Fig. 1 E). We also performed immunohistochemical experiments on ccRCC specimens using tissue microarray technology, with results further validating the above conclusion (Fig. 1 F). Moreover, KIRC-TCGA analysis revealed that patients with higher C8orf76 expression had poorer OS, PFI, and DSS than those with lower expression (Fig. 1 G-I). C8orf76 also showed high diagnostic value for ccRCC, with an area under the curve (AUC) of 0.851 (Fig. 1 J), highlighting its specificity in ccRCC. Therefore, we can conclude that C8orf76 is upregulated in ccRCC and associated with a poor prognosis. C8orf76 knockdown inhibits the proliferation and migration of ccRCC both in vivo and in vitro . Western blot and qPCR analyses were performed in ccRCC cell lines (786-O, A-498, Caki-1, and 769-P). The results demonstrated that the mRNA and protein levels of C8orf76 were relatively elevated in the 786-O and 769-P cell lines (Fig. 2 A, 2 B). As shown in Figure. 2C-2E, transient transfection with siC8orf76 resulted in a reduction in C8orf76 expression levels in both 786-O and 769-P cells relative to the negative control (NC). To explore the in vivo effect of C8orf76 on tumor progression, 769-P cells infected with C8orf76 knockdown lentivirus were selected and subcutaneously inoculated into separate experimental groups. The results demonstrated that tumor volume and weight in the C8orf76 knockdown group were significantly decreased compared with the control group (Fig. 2 F). CCK-8 and transwell cell migration assays demonstrated that C8orf76 knockdown groups displayed decreased migratory capacity (Fig. 2 G, 2 H, 2 K). Additionally, wound healing and colony formation assays also revealed that C8orf76 knockdown groups exhibited significantly reduced relative cell viability and colony formation ability (Fig. 2 I, 2 J, 2 L). Collectively, these results can suggest that C8orf76 knockdown inhibits the proliferation and migration of ccRCC both in vivo and in vitro . Knockdown of C8orf76 induces cell cycle arrest and triggers cellular senescence in ccRCC. Both Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Reactome analyses on 786-O cells between the siNC and the siC8orf76 group revealed enrichment for the cell cycle and cellular senescence pathways (Fig. 3 A, 3 B). Cell Cycle Flow Cytometry indicated that after knocking down C8orf76 in 786-O and 769-P cell lines, G1 phase arrest occurred in the cell cycle (Fig. 3 C). After knocking down C8orf76, western blot analyses revealed an upregulation of p16INK4a, a key senescence-associated protein, and a downregulation of cyclin-dependent kinases (CDK4 and CDK6). These findings suggest that C8orf76 may mediate cellular senescence pathways. Concomitantly, reduced expression levels were observed for Lamin B1 and FOXM1—two critical proteins involved in cell cycle regulation and senescence—indicating suppressed cell cycle progression and enhanced cellular senescence (Fig. 3 D). To more intuitively demonstrate the correlation between C8orf76 and cellular senescence, we knocked down C8orf76 in 786-O and 769-P cells and conducted SA-β-Gal staining. The results revealed a significantly higher proportion of SA-β-Gal positive cells in the knockdown group compared to the control (Fig. 3 E). Therefore, we can assume that C8orf76 knockdown induces cell cycle arrest and triggers cellular senescence. CALB2, potentially downstream of C8orf76, is overexpressed in ccRCC and correlates with poor prognosis. We reviewed relevant literature[ 11 ] and identified five binding motifs of C8orf76. Meanwhile, by integrating RNA-seq results with the TCGA-KIRC database, we screened out CALB2 as a potential downstream target gene of C8orf76 (Fig. 4 A). RNA sequencing also revealed CALB2 downregulation following C8orf76 knockdown (Fig. 4 B, 4 C). Furthermore, both paired (Fig. 4 E) and unpaired (Fig. 4 D) analyses of KIRC-TCGA revealed elevated CALB2 expression in tumor samples. Therefore, we conducted further verification in ccRCC cell lines (786-O, A-498, Caki-1, and 769-P cells). Western blot and qPCR analyses also revealed that CALB2 exhibited relatively high expression in 786-O and 769-P cells (Fig. 4 F, 4 G). To prove from multiple perspectives, we collected clinical samples of ccRCC. As depicted in Fig. 4 H and 4 I, CALB2 exhibits elevated expression in ccRCC tumor samples. Consequently, it is reasonable to conclude that CALB2 is highly expressed in ccRCC. Moreover, KIRC-TCGA analysis showed that higher CALB2 expression was associated with worse OS, PFI, and DSS in patients (Fig. 4 J-L), and the AUC was 0.827 (Fig. 4 M), which suggested that high C8orf76 expression was associated with unfavorable clinical outcomes. Knockdown of CALB2 suppresses the proliferation and migration of ccRCC. We firstly knocked down C8orf76 in ccRCC cells and found that the expression level of CALB2 also decreased (Fig. 5 A- 5 B, 5 E- 5 F). CALB2 was then knocked down in 786-O and 769-P cells, and the knockdown efficiency was verified by qPCR and western blot analyses (Fig. 5 C- 5 D, 5 G- 5 H). We conducted subsequent experiments in the CALB2 knockdown cells. CCK-8 and colony formation assays were performed to assess relative cell viability and colony formation capacity, respectively (Fig. 5 I, 5 J, 5 M). Additionally, wound healing and transwell cell migration assays were utilized to assess the migratory ability of ccRCC (Fig. 5 K, 5 L, 5 N). Based on the aforementioned experiments, we can conclude that the knockdown of CALB2 in vitro results in a certain degree of inhibition of the proliferative and migratory capacities in ccRCC. CALB2 knockdown induces cell cycle arrest and elicits cellular senescence in ccRCC. Similarly, we employed Cell Cycle Flow Cytometry to determine the proportions of cells in different phases of the cell cycle. The results revealed that following CALB2 knockdown, the proportion of cells in the G1 phase increased significantly, indicating that the cell cycle had been arrested (Fig. 6 A). Meanwhile, we also assessed the expression levels of proteins associated with the cell cycle and cellular senescence at the protein level. When CALB2 was knocked down, the expression level of p16 increased significantly. Given that high p16 expression is known to inhibit CDK4 and CDK6[ 28 ], this result is consistent with the findings from our western blot analysis. Furthermore, the expressions of Lamin B1 and FOXM1 were also downregulated. Collectively, these results indicate disruption of the cell cycle and the occurrence of cellular senescence (Fig. 6 B). Furthermore, we directly observed the proportion of senescent cells through SA-β-Gal staining. The results indicate that cellular senescence is significantly exacerbated upon CALB2 knockdown. In summary, the knockdown of CALB2 exerts a profound impact on the biological behavior of ccRCC, with a particular propensity to induce cell cycle arrest and promote cellular senescence. C8orf76 directly regulates CALB2, thereby mediating downstream biological behaviors. From the preceding text, we can infer that CALB2 may act downstream of C8orf76 in biological processes. To further explore the interaction between C8orf76 and CALB2, we re-transfected the CALB2-OE plasmid into C8orf76-knockdown 786-O and 769-P cells. The results demonstrated that the phenotypes of clear cell renal cell carcinoma were all reversed to some extent. Transfection efficiency was validated at both the mRNA and protein levels (Fig. 7 A- 7 D). Following C8orf76 knockdown, we repeated the CCK-8 and colony formation assays, and the results revealed that cell proliferation activity was inhibited (Fig. 7 E, 7 F, 7 I). Moreover, further overexpression of CALB2 could partially restore this proliferation activity. Additionally, in both the wound healing assay and Transwell migration experiment, ccRCC cells with C8orf76 knockdown exhibited an increased relative residual scratch area accompanied by a decreased relative migration rate, indicating impaired cell migratory capacity. Notably, these alterations could also be partially mitigated by further overexpression of CALB2 (Fig. 7 G, 7 H, 7 J). Based on the above experimental findings, we subsequently performed a dual-luciferase reporter assay. Considering the three potential binding sites within the CALB2 promoter region, three sets of pGL3-basic plasmids harboring the mutant genes were constructed (Fig. 7 K). After co-transfecting the cells with C8orf76-OE plasmid, no significant change was observed in the relative luciferase activity of the MUT2 group. This indicates that C8orf76 directly binds to the CALB2 promoter via this specific site (Fig. 7 L). Thus, we can conclude that C8orf76 directly binds to the CALB2 promoter, and the C8orf76/CALB2 axis functions as an independent signaling axis to exert biological effects. CALB2 facilitates the proliferation and migration of ccRCC through the inhibition of cellular senescence pathways. As shown in Fig. 7 , C8orf76 directly binds to the promoter region of CALB2. The C8orf76/CALB2 axis acts as an independent signaling module that mediates cellular senescence pathways, thereby influencing the proliferation and migration capabilities of downstream tumor cells. Based on these observations, we conducted further in-depth experiments. Transfection of sh-CALB2 plasmid into 786-O and 769-P cells resulted in a significant increase in the proportion of cells arrested in the cell cycle G1 phase (Fig. 8 A, S1 A, S1 B), accompanied by altered expression of senescence-associated proteins (upregulation of p16INK4a and downregulation of CDK4/6, Lamin B1, and FOXM1) (Fig. 8 B, S1 D, S1 E). Additionally, an elevated percentage of senescent cells was observed (Fig. 8 C, S1 C). However, subsequent transfection with the sh-CDKN2A plasmid reversed all of these effects (Fig. 8 A- 8 C). Subsequently, we performed CCK-8 assays (Fig. 8 D), colony formation assays (Fig. 8 G), wound healing assays (Fig. 8 E, 8 F), and Transwell migration assays (Fig. 8 H), which allowed us to comprehensively evaluate the changes in the proliferation and migration abilities of tumor cells before and after plasmid transfection. The results indicated that co-transfection of sh-CALB2 and sh-CDKN2A plasmids reversed the inhibitory effects on cell proliferation and migration induced by sh-CALB2 transfection alone. Collectively, these findings suggest that CALB2 facilitates the proliferation and migration of ccRCC through the inhibition of cellular senescence pathways. Conclusion In this study, we demonstrated the critical role of C8orf76 in the progression of clear cell renal cell carcinoma. The high expression of C8orf76 in ccRCC is associated with poor prognosis. Knockdown of C8orf76 leads to G1-phase cell cycle arrest and enhanced cellular senescence, and significantly inhibits the proliferation and migration of ccRCC cells both in vitro and in vivo. Furthermore, bioinformatics data and laboratory research confirmed that CALB2 is also highly expressed in ccRCC cells and correlates with adverse prognosis. As an upstream transcription factor of CALB2, C8orf76 can bind to the promoter region of the CALB2 gene and promote the transcription of CALB2 mRNA. The C8orf76/CALB2 axis mediates the proliferation and migration of ccRCC by inhibiting cellular senescence signaling pathways. Discussion Despite continuous advancements in the research and treatment of clear cell renal cell carcinoma, numerous challenges persist. For localized tumors, surgical resection can achieve satisfactory outcomes[ 8 ]. However, approximately 40% of patients experience recurrence and distant metastasis each year after surgery[ 29 ]. For advanced/metastatic ccRCC, chemotherapy and radiotherapy show limited sensitivity[ 30 ], and immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs) remain the primary treatment options[ 31 – 33 ]. Nevertheless, patients with advanced ccRCC typically develop resistance to TKI therapy within 6 to 15 months[ 34 ]. Tumor progression also frequently occurs in patients treated with ICIs, necessitating subsequent further treatment[ 10 , 34 , 35 ]. In this study, we found that the expression level of C8orf76 in ccRCC is significantly higher than that in normal tissues. Moreover, C8orf76 promotes the progression of ccRCC by regulating downstream effectors and signaling pathways. C8orf76 is a novel nuclear protein-encoding gene first identified for its ability to bind specific lncRNAs in gastric cancer and promote tumor progression[ 11 ]. Additionally, C8orf76 also plays roles in the occurrence and development of multiple tumors[ 12 , 36 , 37 ]. However, current understanding of C8orf76 remains superficial. No large-scale studies have deeply explored its mechanism of action, and the potential role of C8orf76 in renal cancer lacks sufficient literature data support. Against this backdrop, we conducted a study and for the first time reported the biological status and mechanism of action of C8orf76 in clear cell renal cell carcinoma. We found that C8orf76 was highly expressed in ccRCC, and high expression of C8orf76 predicted poor prognosis. C8orf76 knockdown caused G1 phase arrest in the cell cycle, activated cellular senescence, and inhibited tumor cell proliferation and migration both in vivo and in vitro. These results indicate that C8orf76 mediates the progression of ccRCC through the cellular senescence signaling pathway, which warrants further investigation. By reviewing the literature[ 11 ] and integrating RNA-seq results, we screened out CALB2 as a potential interactor of C8orf76. CALB2 belongs to the calmodulin family, and its primary biological functions involve calcium homeostasis regulation and signal transduction modulation. CALB2 has been shown to promote the progression of various tumors[ 13 , 16 ]. However, its role in ccRCC remains poorly documented. This study found that CALB2 is upregulated in ccRCC, and that high expression of CALB2 is associated with poor prognostic outcomes. Similar to C8orf76, CALB2 knockdown induced G1 phase cell cycle arrest, cellular senescence, and inhibited the proliferation and migration of ccRCC cells. We further elucidated the functional relationship between C8orf76 and CALB2. Overexpression of CALB2 in C8orf76-knockdown ccRCC cell lines suppressed cell cycle arrest and cellular senescence, and significantly rescued the proliferative and migratory capacities of tumor cells both in vitro and in vivo. Additionally, dual-luciferase reporter assays confirmed that C8orf76 directly binds to the promoter region of CALB2 to promote its transcription. Thus, CALB2 acts as a downstream effector of C8orf76 to exert its biological functions. Cellular senescence refers to a unique state of cell cycle arrest, which has been widely discussed due to its dual roles in tumors[ 38 ]. Cyclin-dependent kinase inhibitors represent a critical component in the occurrence of cellular senescence, primarily functioning through the p53/p21 and p16/pRb pathways[ 39 ]. The p53 protein directly binds to the promoter region of CDKN1A to promote transcription of p21CIP1, thereby inducing cell cycle arrest and cellular senescence[ 40 , 41 ]. Similar findings have been reported in renal cell carcinoma[ 42 , 43 ]. On the other hand, the p16INK4a protein, encoded by the CDKN2A gene, shows low expression or loss in RCC[ 44 ]. In this study, we found that further knockdown of CDKN2A in stably CALB2-knockdown ccRCC cell lines significantly suppressed cell cycle arrest and cellular senescence, and partially restored the proliferative and migratory capacities of tumor cells. These results indicate that the C8orf76/CALB2 axis promotes the proliferation and migration of ccRCC by inhibiting the cellular senescence pathway, potentially involving the p16/pRb signaling cascade. Given the critical role of the cellular senescence pathway in cancer therapy, multiple related drugs have been developed clinically. For example, the selective CDK4/6-targeted agent palbociclib has achieved new progress in breast cancer treatment[ 45 ]. This study supports the development of inhibitors targeting the C8orf76/CALB2 axis to activate the downstream cellular senescence pathway and inhibit the progression of ccRCC. Notably, this research still has several limitations to address. Although we propose that the C8orf76/CALB2 axis exerts its function through the cellular senescence pathway, the specific mechanisms involved require more in-depth investigation. Additionally, the p53/p21 pathway also plays a significant role in ccRCC cell senescence. Whether there is a further relationship between the C8orf76/CALB2 axis and this pathway remains unexplored, which will be our future research direction. Declarations Ethics approval and consent to participate The operation of animals in our study were approved by the Medical Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine. Consent for publication Consent for publication was obtained from the participants. Availability of data and materials The data that support the findings of this study are available in the supplementary material of this article. Competing interests The authors declare that they have no competing interests. Funding This work was supported by grants from the National Natural Science Foundation of China (82273132, 82403933), the Medical and Health Research Project of Zhejiang Province (2024KY065), 'Leading Goose' Science and Technology Program of Zhejiang Province (2025C02071), China Postdoctoral Science Foundation Grant (2023M743022). Authors' contributions The conception and design of the study: Ben Liu, Dingheng Lu; The acquisition of data: Yuxiao Li, Xinyang Niu, Pengju Liu, Fenghao Zhang, Zhixiang Qi, Kai Yu; Analysis and interpretation of data: Xueyou Ma, Jiazhu Sun, Suyuelin Huang, Yuchen Shi, Xuan Shu; Drafting the article or revising it critically for important intellectual content: Yuxiao Li, Xinyang Niu, Dingheng Lu; Final approval of the version to be submitted: all authors. Acknowledgements We are grateful for the supports from Jie Fang in all animal experiments. Supporting Information Supporting Information is available from the corresponding author. References A. Bex, Y.A. Ghanem, L. Albiges, S. Bonn, R. Campi, U. Capitanio, S. Dabestani, M. Hora, T. Klatte, T. Kuusk, L. Lund, L. Marconi, C. Palumbo, G. Pignot, T. Powles, N. Schouten, M. Tran, A. Volpe, J. Bedke, European Association of Urology Guidelines on Renal Cell Carcinoma: The 2025 Update, Eur Urol 87(6) (2025) 683-696. Y. Wu, S. He, M. Cao, Y. 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Jager, Inhibiting CDK4/6 in Breast Cancer with Palbociclib, Ribociclib, and Abemaciclib: Similarities and Differences, Drugs 81(3) (2021) 317-331. Supplementary Files Supplementalmaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 16 Aug, 2025 Reviewers invited by journal 16 Aug, 2025 Editor assigned by journal 07 Aug, 2025 First submitted to journal 05 Aug, 2025 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-7305276","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":501220891,"identity":"592f9f6c-f337-49a4-baad-d4fec96401a3","order_by":0,"name":"Yuxiao Li","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuxiao","middleName":"","lastName":"Li","suffix":""},{"id":501220892,"identity":"f7d85f57-ba59-410b-9c41-73d4da070073","order_by":1,"name":"Xinyang Niu","email":"","orcid":"","institution":"Zhejiang University School of 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03:38:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7305276/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7305276/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89978693,"identity":"a71b0341-4757-4282-9f2f-34781eba5f53","added_by":"auto","created_at":"2025-08-27 06:15:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":701022,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC8orf76 up-regulation in ccRCC and its association with prognosis.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Differential C8orf76 expression in various types of tumor\u003c/p\u003e\n\u003cp\u003etissues and adjacent normal tissues was analyzed by TIMER 2.0 database. \u003cstrong\u003eB, C\u003c/strong\u003eC8orf76 was highly expressed in tumor tissues according to the TCGA-KIRC database. \u003cstrong\u003eD\u003c/strong\u003e C8orf76 mRNA levels in 47 ccRCC tissues and adjacent normal tissues (the control group: the mRNA level of normal tissues). \u003cstrong\u003eE\u003c/strong\u003e Western blot analyses were conducted to assess the protein levels of C8orf76 in tumor and adjacent tissues collected from 10 patients with ccRCC (N, normal; T, tumor). \u003cstrong\u003eF\u003c/strong\u003e Representative IHC images of C8orf76 in ccRCC tissues and adjacent normal tissues. \u003cstrong\u003eG-I\u003c/strong\u003e Overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS) were analyzed according to high or low expression of C8orf76, and the log-rank t test was used (*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001). \u003cstrong\u003eJ\u003c/strong\u003e ROC curve analysis demonstrated the expression of C8orf76 is specific to ccRCC.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7305276/v1/8b18f41d5de70e36fa413f2f.png"},{"id":89976873,"identity":"fb0fe48d-589b-4f67-b7c0-aa7315d0b008","added_by":"auto","created_at":"2025-08-27 06:07:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":614945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC8orf76 knockdown inhibits the proliferation and migration of ccRCC both \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. A, B \u003c/strong\u003eThe mRNA levels and protein expression of C8orf76 in multiple ccRCC cell lines. \u003cstrong\u003eC-E\u003c/strong\u003e In 786-O and 769-P cells, C8orf76 expression was knocked down at both the transcriptional and translational levels, and the knockdown efficiency was validated. \u003cstrong\u003eF\u003c/strong\u003e Representative images and tumor volume curves of subcutaneous tumors in nude mice after transfection with shNC and shC8orf76 plasmids (n = 5 per group). \u003cstrong\u003eG, H\u003c/strong\u003e The cell viability of 786-O and 769-P cells was assessed using CCK-8 assay. \u003cstrong\u003eI, J\u003c/strong\u003e The migratory capacity of 786-O and 769-P cells was assessed using wound healing assay. I\u003csub\u003e1\u003c/sub\u003e, J\u003csub\u003e1\u003c/sub\u003e: cell wound healing images; I\u003csub\u003e2\u003c/sub\u003e, J\u003csub\u003e2\u003c/sub\u003e: quantitative analysis images. \u003cstrong\u003eK\u003c/strong\u003e The colony formation ability of 786-O and 769-P cells was evaluated using colony formation assay. K\u003csub\u003e1\u003c/sub\u003e: colony formation images; K\u003csub\u003e2\u003c/sub\u003e: quantitative analysis images. \u003cstrong\u003eL\u003c/strong\u003e The migratory capacity of 786-O and 769-P cells was evaluated using transwell cell migration assay. L\u003csub\u003e1\u003c/sub\u003e: transwell cell migration images; L\u003csub\u003e2\u003c/sub\u003e: quantitative analysis images.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7305276/v1/5e8b2c1f9f69f6dcb5b9fd8e.png"},{"id":89976012,"identity":"55831755-bd60-4217-9606-98e0d9980033","added_by":"auto","created_at":"2025-08-27 05:59:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":506445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of C8orf76 induces cell cycle arrest and triggers cellular senescence in ccRCC. A, B \u003c/strong\u003eReactome and KEGG enrichment analyses on 786-O cells between the siNC control group and the siC8orf76 group. \u003cstrong\u003eC\u003c/strong\u003e Cell cycle analysis showed inhibition of C8orf76 resulted in G1 phase arrest of cell cycle. \u003cstrong\u003eD\u003c/strong\u003e Western blot analysis demonstrated that inhibiting C8orf76 led to the downregulation of CDK4, CDK6, Lamin B1, and FOXM1, as well as the upregulation of p16INK4a. \u003cstrong\u003eE\u003c/strong\u003e SA-β-gal staining revealed that inhibiting C8orf76 increased the number of senescent cells in both 786-O and 769-P cells. Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7305276/v1/d4c85d8d2378287fafb84140.png"},{"id":89976875,"identity":"269dfbf7-0a11-428f-9e08-30ecb824e259","added_by":"auto","created_at":"2025-08-27 06:07:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":511171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCALB2, potentially downstream of C8orf76, is overexpressed in ccRCC and correlates with poor prognosis.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Integrating RNA sequencing, TCGA database, and literature data, we identified the downstream target genes of C8orf76. \u003cstrong\u003eB, C\u003c/strong\u003eRNA-sequencing results showed that after knocking down C8orf76 in 786-O cells, the expression of CALB2 was upregulated. \u003cstrong\u003eD, E\u003c/strong\u003e CALB2 was highly expressed in ccRCC according to the TCGA-KIRC database. \u003cstrong\u003eF, G\u003c/strong\u003e The mRNA levels and protein expression of CALB2 in multiple ccRCC cell lines. \u003cstrong\u003eH\u003c/strong\u003e CALB2 mRNA levels in 47 ccRCC tissues and adjacent normal tissues (the control group: the mRNA level of normal tissues). \u003cstrong\u003eI\u003c/strong\u003e Western blot analyses were performed to evaluate CALB2 protein levels in tumor and adjacent tissues from 10 patients with ccRCC (N, normal; T, tumor). \u003cstrong\u003eJ-L\u003c/strong\u003e OS, PFI, and DSS were analyzed according to high or low expression of CALB2, and the log-rank t test was used (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001). \u003cstrong\u003eM\u003c/strong\u003e ROC curve analysis shown that the expression of CALB2 is specific to ccRCC.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7305276/v1/80ab8cf139c88d76820934dc.png"},{"id":89976011,"identity":"f3904081-2fd6-4899-8c0c-54cb70cb9eca","added_by":"auto","created_at":"2025-08-27 05:59:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":633064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of CALB2 suppresses the proliferation and migration of ccRCC. A-B, E-F \u003c/strong\u003eThe expression level changes of CALB2 after C8orf76 knockdown. \u003cstrong\u003eC-D, G-H \u003c/strong\u003eValidate the knockdown efficiency of CALB2 at mRNA and protein levels. \u003cstrong\u003eI, J, M\u003c/strong\u003e Cell viability and colony formation ability were evaluated in 786-O and 769-P cells utilizing CCK-8 assay and colony formation assay. M\u003csub\u003e1\u003c/sub\u003e: colony formation images; M\u003csub\u003e2\u003c/sub\u003e: quantitative analysis images. \u003cstrong\u003eK, L, N\u003c/strong\u003e The migratory capacity of 786-O and 769-P cells was assessed using wound healing assay and transwell cell migration assay. K\u003csub\u003e1\u003c/sub\u003e, L\u003csub\u003e1\u003c/sub\u003e: cell wound healing images; N\u003csub\u003e1\u003c/sub\u003e: transwell cell migration images; K\u003csub\u003e2\u003c/sub\u003e, L\u003csub\u003e2\u003c/sub\u003e, N\u003csub\u003e2\u003c/sub\u003e: quantitative analysis images.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7305276/v1/0b8425cd1ad27e5c3d169c53.png"},{"id":89976015,"identity":"cfb299c0-aa64-48dc-b446-0f214a53342d","added_by":"auto","created_at":"2025-08-27 05:59:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":386198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCALB2 knockdown induces cell cycle arrest and elicits cellular senescence in ccRCC. A \u003c/strong\u003eCell cycle analysis showed inhibition of CALB2 resulted in G1 phase arrest of cell cycle in 786-O and 769-P cells. \u003cstrong\u003eB\u003c/strong\u003e Western blot analysis demonstrated that inhibiting CALB2 led to the downregulation of CDK4, CDK6, Lamin B1, and FOXM1, as well as the upregulation of p16INK4a. \u003cstrong\u003eC\u003c/strong\u003e SA-β-gal staining revealed that inhibiting CALB2 increased the number of senescent cells in both 786-O and 769-P cells. Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7305276/v1/40ef1c0efc53f6d92d72baae.png"},{"id":89976013,"identity":"c9a28185-7669-4f37-85e6-70c91121494c","added_by":"auto","created_at":"2025-08-27 05:59:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":661837,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC8orf76 directly regulates CALB2, thereby mediating downstream biological behaviors.\u003c/strong\u003e \u003cstrong\u003eA-D\u003c/strong\u003e The alterations in mRNA and protein levels following the transfection of shC8orf76 and CALB2-OE plasmids into ccRCC. \u003cstrong\u003eE, F, I\u003c/strong\u003e CCK-8 and colony formation assay showed that CALB2 overexpression rescued the impaired cell proliferation and colony formation ability induced by C8orf76 inhibition. I\u003csub\u003e1\u003c/sub\u003e: colony formation images; I\u003csub\u003e2\u003c/sub\u003e: quantitative analysis images. \u003cstrong\u003eG, H, J\u003c/strong\u003e Wound healing assay and transwell cell cell migration experiment revealed that over-expression of CALB2 restored the attenuated migratory capacity. G\u003csub\u003e1\u003c/sub\u003e, H\u003csub\u003e1\u003c/sub\u003e: cell wound healing images; J\u003csub\u003e1\u003c/sub\u003e: transwell cell migration images; G\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003e, J\u003csub\u003e2\u003c/sub\u003e: quantitative analysis images. \u003cstrong\u003eK, L\u003c/strong\u003e Dual-luciferase reporter assays on binding site of the CALB2 promoter in 786-O and 769-P cell lines.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7305276/v1/00dab9a1af63e5594d1a4754.png"},{"id":89976028,"identity":"480eeacc-3464-4b82-805d-7e7aacd8868c","added_by":"auto","created_at":"2025-08-27 05:59:54","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":668928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCALB2 facilitates the proliferation and migration of ccRCC through the inhibition of cellular senescence pathways. A\u003c/strong\u003e After transfection with sh-CALB2 and sh-CDKN2A plasmids in ccRCC cells, the effects on cell cycle arrest can be observed. \u003cstrong\u003eB\u003c/strong\u003e The expression levels of cell cycle- and cellular senescence-related proteins after transfection with sh-CALB2 and sh-CDKN2A plasmids. \u003cstrong\u003eC\u003c/strong\u003e SA-β-gal staining showed that knocking down CDKN2A suppressed the cellular senescence phenotype induced by knocking down CALB2. Scale bar = 50 μm. \u003cstrong\u003eD, G\u003c/strong\u003e CCK-8 and colony formation assays revealed that transfection with the sh-CALB2 plasmid led to decreased cell proliferation and colony formation ability. However, co-transfection with sh-CDKN2A rescued these phenotypes. G\u003csub\u003e1\u003c/sub\u003e: colony formation images; G\u003csub\u003e2\u003c/sub\u003e: quantitative analysis images. \u003cstrong\u003eE, F, H\u003c/strong\u003e Wound healing and transwell cell cell migration assays demonstrated differential cell migration capacities among groups following transfection with respective plasmids. E\u003csub\u003e1\u003c/sub\u003e, F\u003csub\u003e1\u003c/sub\u003e: cell wound healing images; H\u003csub\u003e1\u003c/sub\u003e: transwell cell migration images; E\u003csub\u003e2\u003c/sub\u003e, F\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003e: quantitative analysis image.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7305276/v1/f900334f772212f7f17168fd.png"},{"id":89981008,"identity":"7e3a2c80-50be-4bbf-af1e-e995efdcf47b","added_by":"auto","created_at":"2025-08-27 06:24:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6063260,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7305276/v1/1a3741ac-9493-4fdc-9daf-34d239c5ea38.pdf"},{"id":89976007,"identity":"141f2e7b-7366-4cb3-8f4c-f90901d8dc86","added_by":"auto","created_at":"2025-08-27 05:59:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":202022,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7305276/v1/403571da113b2eb4ebe820ac.docx"}],"financialInterests":"","formattedTitle":"The tumor promoter role and molecular mechanism of C8orf76/CALB2 axis in clear cell renal cell carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRenal cell carcinoma (RCC) is a common malignant tumor of the urinary system, accounting for about 90% of renal malignancies and constituting about 3% of adult malignant tumors globally[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In 2022, there were over 430,000 new RCC cases and 150,000 deaths worldwide[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, the incidence rate among younger populations (\u0026lt;\u0026thinsp;50 years old) is on the rise[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Clear cell renal cell carcinoma (ccRCC), the most prevalent subtype of RCC, originates from the proximal tubules of nephrons. It is characterized by a highly aggressive phenotype and poor prognosis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Surgical treatment is the main strategy for ccRCC[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, due to the lack of effective early diagnostic measures, ccRCC often presents inconspicuously, with nearly 30% of patients diagnosed with metastatic disease at initial presentation[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Metastatic tumor patients frequently develop treatment resistance and disease progression[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, it is necessary to further investigate the molecular mechanisms of the occurrence, development, and metastasis of ccRCC. Such exploration may be the key to assisting clinical early diagnosis, identifying new therapeutic targets, and developing personalized treatment strategies.\u003c/p\u003e\u003cp\u003eC8orf76 (chromosome 8 open reading frame 76) is a novel nuclear protein-encoding gene located on chromosome 8q24.13\u0026ndash;24.3, which encodes a 380-aa protein. It was first discovered in gastric cancer. C8orf76 could directly interact with the oncogenic long non-coding RNA (lncRNA) dual specificity phosphatase 5 pseudogene 1 (DUSP5P1), triggering its transcriptional upregulation adn subsequent activation of the downstream mitogen-activated protein kinase (MAPK) signaling pathway[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Studies have also shown that C8orf76 participates in ferroptosis regulation by transcriptionally activating SLC7A11, thus promoting the progression of liver cancer[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The protein encoded by the calbindin 2 (\u003cem\u003eCALB2\u003c/em\u003e) gene is a member of the troponin C superfamily, primarily regulating intracellular calcium levels and exerting biological functions such as signal transduction and calcium ion buffering. CALB2 is also associated with metastasis and drug resistance in multiple tumors[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For example, CALB2 can activate the TRPV2-Ca\u0026sup2;⁺-ERK1/2 signaling pathway to induce metastasis in hepatocellular carcinoma[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the specific roles and molecular mechanisms of C8orf76 and CALB2 in clear cell renal cell carcinoma have not yet been thoroughly investigated.\u003c/p\u003e\u003cp\u003eThe concept of cellular senescence was first proposed in the 1960s[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], referring to a stable and persistent state of cell cycle arrest. This state is associated with upregulation of cyclin-dependent kinase inhibitors (CKIs), such as p16INK4a and p21WAF1/CIP1, chronic DNA damage responses, and a hyper-secretory state of the senescence-associated secretory phenotype (SASP)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Among these, p16INK4a (p16) is a critically important cell cycle inhibitor. It can suppress the activity of CDK4/6, prevent the phosphorylation of retinoblastoma protein (RB), and thereby lead to G1 phase arrest in the cell cycle, promoting the occurrence of cellular senescence[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This strong association with cellular senescence has made p16 a priority for research[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In tumor progression, cellular senescence exhibits a dual role: cell entry into senescence can act as an effective barrier against tumorigenesis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], while under certain conditions, persistently senescent cells can also acquire pro-tumorigenic properties[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Preclinical evidence suggests that senescent cancer cells exhibit enhanced immunogenic properties, offering a novel avenue for therapeutic modulation of anti-cancer immunity[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].Therefore, in-depth research on cellular senescence and tumorigenesis may serve as a new tool to break through the barriers of cancer therapy and tackle the challenges of early tumor diagnosis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eCell Culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHEK-293T, HK-2, A-498, Caki-1, 769-P and 786-O cell lines were obtained from the Chinese Academy of Sciences Shanghai Cell Bank (Shanghai, China). HEK-293T cells were cultured in DMEM medium (Procell, Wuhan, China) supplemented with 10% fetal bovine serum (Biologic Industries, Israel) and 1% penicillin/streptomycin (Servicebio, Wuhan, China). A-498 cells were cultured in MEM medium (Procell, Wuhan, China) supplemented with 10% fetal bovine serum (Biologic Industries, Israel) and 1% penicillin/streptomycin (Servicebio, Wuhan, China). HK-2, Caki-1, 786-O and 769-P cells were cultured in RPMI 1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Biologic Industries, Israel) and 1% penicillin/streptomycin (Servicebio, Wuhan, China). All cell lines are regularly tested for mycoplasma contamination. All cell lines were cultured in a humidified atmosphere at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern Blot\u003c/b\u003e\u003c/p\u003e\u003cp\u003eProtein concentrations were measured utilizing a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, China). An equivalent quantity of protein (30 \u0026micro;g) was subjected to SDS-PAGE gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After a blocking step with 5% non-fat dried milk in Tris-buffered saline containing Tween 20 (TBST) for 1.5 h at room temperature (RT), the membranes were subjected to primary antibodies specific to each target protein, followed by overnight incubation at 4\u0026deg;C. The next day, the membranes were washed three times with TBST before being treated with horseradish peroxidase-conjugated secondary antibodies (1:4000, Catalog No: GB23301, GB23303; Servicebio, Wuhan, China) for 2 h at RT. Protein bands were identified using an enhanced chemiluminescence (ECL) detection system. β-Actin was used as an internal control. Supplementary Table\u0026nbsp;1 lists the utilized primary antibodies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantitative Real-time PCR\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from cell samples and stored at -80\u0026deg;C using RNAiso Plus Reagent (Takara), ensuring the genomic DNA contamination was removed, according to the manufacturer\u0026rsquo;s instructions. RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from 1 \u0026micro;g of total RNA using the cDNA Reverse Transcription Kit (Vazyme, China) following the protocol. Quantitative real-time PCR \u003cb\u003e(\u003c/b\u003eqPCR) was performed with SYBR qPCR Mix (Vazyme, China) on a Biorad CFX96 Detection System. The qPCR data were analyzed using the ΔΔCT method for relative quantification, normalizing to the β-actin. Data were analyzed using GraphPad Prism software (GraphPad Software10.0). Supplementary Table\u0026nbsp;2 lists the utilized primer sequences.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Counting Kit-8 (CCK-8) Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeed cells at an appropriate density (800\u0026thinsp;~\u0026thinsp;1000 cells/well) in a 96-well plate with 100 \u0026micro;L of cell culture medium per well. Incubate the plate in a humidified CO\u003csub\u003e2\u003c/sub\u003e incubator (e.g., 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e) for 24 hours to allow cell attachment. Addition of CCK-8 Solution (Fude Bio, Hangzhou, China): Add 10 \u0026micro;L of CCK-8 solution to each well of the plate. Put the plate to the incubator and incubate for 1\u0026ndash;2 hours. Measure the absorbance at 450 nm using a microplate reader. Calculate the cell viability by comparing the absorbance values of the test wells to those of the control wells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eColony Formation Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell proliferation ability was detected by clonal formation assay [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Seed 800\u0026ndash;1000 cells per well in a 6-well plate. Incubate the cells in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for approximately 1\u0026ndash;3 weeks, changing the medium every 5 days and monitoring cell growth. Once visible colonies have formed, terminate the culture. Wash the cells with PBS, then fix with 4% polyoxymethylene for 30 minutes. After washing again, stain with crystal violet for 30 minutes. Count the number of colonies under a microscope for later analysis. Colonies are typically defined as groups of 50 or more cells. Plating Efficiency = (Number of Colonies / Number of Cells Seeded) \u0026times; 100%.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWound Healing Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were seeded into 6-well plates and were cultured to confluency. Wounds were generated by scratching cell layer with 200 \u0026micro;L plastic pipette tips. The final images were taken and the gap distances of migrating cells were measured using optical microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranswell Cell Migration Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell migration assay was performed with transwell chamber (Corning, USA) according to the standard method. 600 \u0026micro;L of complete media was added to the lower chamber, and 200 \u0026micro;L of medium containing 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells in serum-free RPMI-1640 was put in the upper chamber (8-mm pore size, Corning). After being cultured for 24 h, cells were fixed in 4% paraformaldehyde and stained with crystal violet. Five fields per well were randomly selected and images were acquired by a optical microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSenescence Associated β-galactosidase (SA-β-gal) Staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Senescence Associated β-Galactosidase Staining Kit were purchased from Beyotime (Shanghai, China). 786-O and 769-P cells were cultured in 6-well plates. After washing with PBS and fixation, dispense the working fluid according to the protocol. Incubate overnight at 37\u0026deg;C. The SA-β-gal positve cells were obtained via optical microscope and quantified using Image J software (version 1.48v).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Cycle Flow Cytometry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cell cycle flow cytometry was performed using the Cell Cycle Staining Kit (MultiSciences, Hangzhou, China). Data were analyzed using the FlowJo 10.8.1 and Modfit LT 5.0 software.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlasmids Transfection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeed cells in a 6-well plate at a density that allows for 60\u0026ndash;70% confluence on the day of transfection. Incubate the cells in an incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. For each well, dilute 1 \u0026micro;g of plasmid DNA and Lipofectamine 3000 (Thermofisher, USA) or JetPRIME transfection agents (Polyplus, USA) in buffer according to each protocol. Lipofectamine 3000 needs to be diluted before mixing with DNA. Incubate for 20 minutes at room temperature to allow the formation of transfection complexes. While the transfection complexes are forming, replace the medium in the cell culture wells with 1500 \u0026micro;L of fresh medium without serum. Add the transfection complexes dropwise to the cells and gently rock the plate to ensure even distribution. Return the plate to the incubator for 8 hours to allow for transfection. After the incubation period, remove the transfection medium and replace it with 1 mL of complete growth medium (containing serum and antibiotics) to support cell growth and expression of the transfected gene. Supplementary Table\u0026nbsp;3 presents the shRNA and siRNA sequences for knocking down C8orf76, CALB2 and CDKN2A.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLentiviral Construction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDay 0: Seed 293T cells into 6-well plates at a density of 1.0*10^\u003csup\u003e6\u003c/sup\u003e cells per well in 2 mL of lentivirus packaging medium. Incubate overnight at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e to reach 70% confluence. Day 1: Prepare the transfection complexes by mixing 3 \u0026micro;g of the transfer vector and 9 \u0026micro;g of packaging plasmids with JetPRIME transfection agents in Opti-MEM medium according to the manufacturer's instructions. Incubate for 10\u0026ndash;20 minutes at room temperature. Replace medium in each well with 1 mL of fresh lentivirus packaging medium. Add the lipid-DNA complex to each well, gently agitating the plate to distribute the mixture evenly. Incubate overnight at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. Day 2: Replace the medium containing the lipid-DNA complexes with 2 mL of pre-warmed lentivirus packaging medium. Return the plate to the incubator overnight. Collect the supernatant containing the virus and store at 4\u0026deg;C. Replace the collected medium with 2 mL of fresh lentivirus packaging medium. Day 3: Collect the second batch of supernatant. Combine this with the first collection and centrifuge at 2,000 rpm for 10 minutes at room temperature to remove cellular debris. Filter the supernatant through a 0.45 \u0026micro;m filter to remove any remaining debris. Aliquot the virus into cryovials and store at -80\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Infection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGLI2 overexpression adenovirus containing the GLI2 coding sequence was obtained from Shanghai Genechem Co., Ltd. Cells were seeded for infection in a six-well plate, achieving 60% confluence by the next day. The reagent/medium containing adenovirus/lentivirus was removed from the \u0026minus;\u0026thinsp;80\u0026deg;C freezer and placed on ice. According to the instructions, a suitable volume of reagent/medium was administered to the cells in each well. Polybrene (Beijing Solarbio Science \u0026amp; Technology, China) was included at 8 \u0026micro;g/mL for lentiviral infection. After 24 h of infection, the medium was changed, and the cells were cultured in the incubator for another 24 h. Puromycin was added at an initial working concentration of 2 \u0026micro;g/mL to select stably infected clones, with the concentration gradually increased to 5 \u0026micro;g/mL by the fifth day. Cells were incubated for subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDual-luciferase Reporter Gene Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003e293T cells were used to detect dual-luciferase according to Promega protocol as described in the literature [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, plate cells in 24-well plates at a density of 100,000 cells/well in appropriate medium. The next day, co-transfect cells with the firefly luciferase reporter plasmid (under the control of the promoter of interest) and the Renilla luciferase control plasmid using Lipofectamine 3000. Incubate cells at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e overnight to allow for plasmid uptake and expression. Remove medium and wash cells with PBS. Lyse cells using 0.1 mL of Passive Lysis Buffer (PLB) per well. Aliquot lysates (5\u0026ndash;20 \u0026micro;L) into duplicate wells of a white 96-well plate. Initiate bioluminescence by automatic injection of 0.1 mL of LAR II into the lysates. After a 1-minute delay, record the firefly luciferase emission signals using a microplate luminometer. Quench the firefly luciferase signal and activate Renilla luciferase by adding Stop \u0026amp; Glo\u0026reg; Reagent. Record the Renilla luciferase emission signals. Normalize the firefly luciferase activity to the Renilla luciferase activity to account for transfection efficiency and cell viability. Calculate the fold-change of transcriptional activity by comparing the normalized luciferase activity in the presence of the transcription factor to the vector control condition. Supplementary Table\u0026nbsp;4 lists the mutant plasmid sequence utilized in dual luciferase reporter gene experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmumohistochemical (IHC) Staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eImmumohistochemical staining was performed as described in the previous study [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Tumor tissue specimens were obtained from nude mice and fixed in 10% neutral buffered formalin for 24 h at RT to preserve cellular morphology and antigenicity. The fixed tissues were embedded in paraffin wax and sectioned into 4 \u0026micro;m thick slices using a microtome. The sections were mounted onto silane-coated slides and allowed to dry at 60\u0026deg;C on a hot plate. Paraffin was removed by incubating the slides in xylene, and the sections were rehydrated in a graded ethanol series and distilled water. Antigen retrieval was performed through heat treatment in sodium citrate buffer (pH 6.0) utilizing a water bath. The samples were incubated with a 3% hydrogen peroxide solution in distilled water for 10 min to inhibit endogenous peroxidase activity. Subsequently, sections were incubated with normal serum from the same species as the secondary antibody to diminish instances of non-specific binding. The diluted primary antibodies were applied to the sections and allowed to incubate at RT for 1 h. The slides were washed with TBST three times for 5 min each to eliminate unbound primary antibodies. Secondary antibodies were administered and incubated for 30 min at RT. The antigen-antibody complex was visualized using a chromogenic substrate, DAB, resulting in a brown color reaction at the antigen site. The sections were counterstained with hematoxylin for 1 min to yield a blue nuclear contrast. Tissue sections were systematically dehydrated using a graded ethanol series, thereafter mounted in an aqueous-based medium, and sealed with precisely cut glass coverslips. The slides were examined under a light microscope to visualize the specific antigen localization within the tissue sections.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll \u003cem\u003ein vitro\u003c/em\u003e experimental procedures conducted were repeated a minimum of three times to ensure reproducibility. For the \u003cem\u003ein vivo\u003c/em\u003e studies, each experimental group was comprised of five BALB/c-nu mice. The survival data were assessed utilizing the Kaplan-Meier method, with the log-rank test. The statistical processing of the data was carried out employing Prism 10.0 software by GraphPad. The results are depicted as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Both unpaired Student's t-tests for parametric data and nonparametric tests were implemented to determine \u003cem\u003ep\u003c/em\u003e-values. In detail, the normality of the data distribution was initially assessed using the normal-test function available in Prism 10.0, ensuring the applicability of the parametric t-test. For datasets that deviated from a normal distribution, non-parametric tests were employed. When the F test in the parametric t-test indicated statistical significance at a \u003cem\u003ep\u003c/em\u003e-value less than 0.05, Welch\u0026rsquo;s correction was applied to adjust the data. A \u003cem\u003ep\u003c/em\u003e-value threshold of less than 0.05 was set to define statistical significance. Results without statistical significance were denoted as ns, while significant differences were indicated with asterisks: * for \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and *** for \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eC8orf76 up-regulation in ccRCC and its association with prognosis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe TIMER 2.0 database was employed to assess C8orf76 expression levels across pan-cancer, and the results indicated its high expression in these settings (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Both paired (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and unpaired (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) analyses of KIRC-TCGA revealed elevated C8orf76 expression in tumor samples. An independent dataset containing information on 47 clinical samples yielded the similar results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Tab. S5). Consistently, C8orf76 protein levels were significantly higher in clinical tumor samples compared with adjacent normal tissues from 10 ccRCC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). We also performed immunohistochemical experiments on ccRCC specimens using tissue microarray technology, with results further validating the above conclusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Moreover, KIRC-TCGA analysis revealed that patients with higher C8orf76 expression had poorer OS, PFI, and DSS than those with lower expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I). C8orf76 also showed high diagnostic value for ccRCC, with an area under the curve (AUC) of 0.851 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ), highlighting its specificity in ccRCC. Therefore, we can conclude that C8orf76 is upregulated in ccRCC and associated with a poor prognosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eC8orf76 knockdown inhibits the proliferation and migration of ccRCC both\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eWestern blot and qPCR analyses were performed in ccRCC cell lines (786-O, A-498, Caki-1, and 769-P). The results demonstrated that the mRNA and protein levels of C8orf76 were relatively elevated in the 786-O and 769-P cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). As shown in Figure. 2C-2E, transient transfection with siC8orf76 resulted in a reduction in C8orf76 expression levels in both 786-O and 769-P cells relative to the negative control (NC). To explore the in \u003cem\u003evivo\u003c/em\u003e effect of C8orf76 on tumor progression, 769-P cells infected with C8orf76 knockdown lentivirus were selected and subcutaneously inoculated into separate experimental groups. The results demonstrated that tumor volume and weight in the C8orf76 knockdown group were significantly decreased compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). CCK-8 and transwell cell migration assays demonstrated that C8orf76 knockdown groups displayed decreased migratory capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Additionally, wound healing and colony formation assays also revealed that C8orf76 knockdown groups exhibited significantly reduced relative cell viability and colony formation ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). Collectively, these results can suggest that C8orf76 knockdown inhibits the proliferation and migration of ccRCC both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eKnockdown of C8orf76 induces cell cycle arrest and triggers cellular senescence in ccRCC.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBoth Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Reactome analyses on 786-O cells between the siNC and the siC8orf76 group revealed enrichment for the cell cycle and cellular senescence pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Cell Cycle Flow Cytometry indicated that after knocking down C8orf76 in 786-O and 769-P cell lines, G1 phase arrest occurred in the cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). After knocking down C8orf76, western blot analyses revealed an upregulation of p16INK4a, a key senescence-associated protein, and a downregulation of cyclin-dependent kinases (CDK4 and CDK6). These findings suggest that C8orf76 may mediate cellular senescence pathways. Concomitantly, reduced expression levels were observed for Lamin B1 and FOXM1\u0026mdash;two critical proteins involved in cell cycle regulation and senescence\u0026mdash;indicating suppressed cell cycle progression and enhanced cellular senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). To more intuitively demonstrate the correlation between C8orf76 and cellular senescence, we knocked down C8orf76 in 786-O and 769-P cells and conducted SA-β-Gal staining. The results revealed a significantly higher proportion of SA-β-Gal positive cells in the knockdown group compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Therefore, we can assume that C8orf76 knockdown induces cell cycle arrest and triggers cellular senescence.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCALB2, potentially downstream of C8orf76, is overexpressed in ccRCC and correlates with poor prognosis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe reviewed relevant literature[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and identified five binding motifs of C8orf76. Meanwhile, by integrating RNA-seq results with the TCGA-KIRC database, we screened out \u003cem\u003eCALB2\u003c/em\u003e as a potential downstream target gene of C8orf76 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). RNA sequencing also revealed CALB2 downregulation following C8orf76 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, both paired (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and unpaired (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) analyses of KIRC-TCGA revealed elevated CALB2 expression in tumor samples. Therefore, we conducted further verification in ccRCC cell lines (786-O, A-498, Caki-1, and 769-P cells). Western blot and qPCR analyses also revealed that CALB2 exhibited relatively high expression in 786-O and 769-P cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). To prove from multiple perspectives, we collected clinical samples of ccRCC. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, CALB2 exhibits elevated expression in ccRCC tumor samples. Consequently, it is reasonable to conclude that CALB2 is highly expressed in ccRCC. Moreover, KIRC-TCGA analysis showed that higher CALB2 expression was associated with worse OS, PFI, and DSS in patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-L), and the AUC was 0.827 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM), which suggested that high C8orf76 expression was associated with unfavorable clinical outcomes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eKnockdown of CALB2 suppresses the proliferation and migration of ccRCC.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe firstly knocked down C8orf76 in ccRCC cells and found that the expression level of CALB2 also decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). CALB2 was then knocked down in 786-O and 769-P cells, and the knockdown efficiency was verified by qPCR and western blot analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). We conducted subsequent experiments in the CALB2 knockdown cells. CCK-8 and colony formation assays were performed to assess relative cell viability and colony formation capacity, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). Additionally, wound healing and transwell cell migration assays were utilized to assess the migratory ability of ccRCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). Based on the aforementioned experiments, we can conclude that the knockdown of CALB2 in vitro results in a certain degree of inhibition of the proliferative and migratory capacities in ccRCC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCALB2 knockdown induces cell cycle arrest and elicits cellular senescence in ccRCC.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSimilarly, we employed Cell Cycle Flow Cytometry to determine the proportions of cells in different phases of the cell cycle. The results revealed that following CALB2 knockdown, the proportion of cells in the G1 phase increased significantly, indicating that the cell cycle had been arrested (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Meanwhile, we also assessed the expression levels of proteins associated with the cell cycle and cellular senescence at the protein level. When CALB2 was knocked down, the expression level of p16 increased significantly. Given that high p16 expression is known to inhibit CDK4 and CDK6[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], this result is consistent with the findings from our western blot analysis. Furthermore, the expressions of Lamin B1 and FOXM1 were also downregulated. Collectively, these results indicate disruption of the cell cycle and the occurrence of cellular senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, we directly observed the proportion of senescent cells through SA-β-Gal staining. The results indicate that cellular senescence is significantly exacerbated upon CALB2 knockdown. In summary, the knockdown of CALB2 exerts a profound impact on the biological behavior of ccRCC, with a particular propensity to induce cell cycle arrest and promote cellular senescence.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eC8orf76 directly regulates CALB2, thereby mediating downstream biological behaviors.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrom the preceding text, we can infer that CALB2 may act downstream of C8orf76 in biological processes. To further explore the interaction between C8orf76 and CALB2, we re-transfected the CALB2-OE plasmid into C8orf76-knockdown 786-O and 769-P cells. The results demonstrated that the phenotypes of clear cell renal cell carcinoma were all reversed to some extent. Transfection efficiency was validated at both the mRNA and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Following C8orf76 knockdown, we repeated the CCK-8 and colony formation assays, and the results revealed that cell proliferation activity was inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). Moreover, further overexpression of CALB2 could partially restore this proliferation activity. Additionally, in both the wound healing assay and Transwell migration experiment, ccRCC cells with C8orf76 knockdown exhibited an increased relative residual scratch area accompanied by a decreased relative migration rate, indicating impaired cell migratory capacity. Notably, these alterations could also be partially mitigated by further overexpression of CALB2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). Based on the above experimental findings, we subsequently performed a dual-luciferase reporter assay. Considering the three potential binding sites within the CALB2 promoter region, three sets of pGL3-basic plasmids harboring the mutant genes were constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK). After co-transfecting the cells with C8orf76-OE plasmid, no significant change was observed in the relative luciferase activity of the MUT2 group. This indicates that C8orf76 directly binds to the CALB2 promoter via this specific site (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL). Thus, we can conclude that C8orf76 directly binds to the CALB2 promoter, and the C8orf76/CALB2 axis functions as an independent signaling axis to exert biological effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCALB2 facilitates the proliferation and migration of ccRCC through the inhibition of cellular senescence pathways.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, C8orf76 directly binds to the promoter region of CALB2. The C8orf76/CALB2 axis acts as an independent signaling module that mediates cellular senescence pathways, thereby influencing the proliferation and migration capabilities of downstream tumor cells. Based on these observations, we conducted further in-depth experiments. Transfection of sh-CALB2 plasmid into 786-O and 769-P cells resulted in a significant increase in the proportion of cells arrested in the cell cycle G1 phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), accompanied by altered expression of senescence-associated proteins (upregulation of p16INK4a and downregulation of CDK4/6, Lamin B1, and FOXM1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE). Additionally, an elevated percentage of senescent cells was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). However, subsequent transfection with the sh-CDKN2A plasmid reversed all of these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Subsequently, we performed CCK-8 assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), colony formation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG), wound healing assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF), and Transwell migration assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH), which allowed us to comprehensively evaluate the changes in the proliferation and migration abilities of tumor cells before and after plasmid transfection. The results indicated that co-transfection of sh-CALB2 and sh-CDKN2A plasmids reversed the inhibitory effects on cell proliferation and migration induced by sh-CALB2 transfection alone. Collectively, these findings suggest that CALB2 facilitates the proliferation and migration of ccRCC through the inhibition of cellular senescence pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we demonstrated the critical role of C8orf76 in the progression of clear cell renal cell carcinoma. The high expression of C8orf76 in ccRCC is associated with poor prognosis. Knockdown of C8orf76 leads to G1-phase cell cycle arrest and enhanced cellular senescence, and significantly inhibits the proliferation and migration of ccRCC cells both in vitro and in vivo. Furthermore, bioinformatics data and laboratory research confirmed that CALB2 is also highly expressed in ccRCC cells and correlates with adverse prognosis. As an upstream transcription factor of CALB2, C8orf76 can bind to the promoter region of the \u003cem\u003eCALB2\u003c/em\u003e gene and promote the transcription of CALB2 mRNA. The C8orf76/CALB2 axis mediates the proliferation and migration of ccRCC by inhibiting cellular senescence signaling pathways.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite continuous advancements in the research and treatment of clear cell renal cell carcinoma, numerous challenges persist. For localized tumors, surgical resection can achieve satisfactory outcomes[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, approximately 40% of patients experience recurrence and distant metastasis each year after surgery[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For advanced/metastatic ccRCC, chemotherapy and radiotherapy show limited sensitivity[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs) remain the primary treatment options[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Nevertheless, patients with advanced ccRCC typically develop resistance to TKI therapy within 6 to 15 months[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Tumor progression also frequently occurs in patients treated with ICIs, necessitating subsequent further treatment[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, we found that the expression level of C8orf76 in ccRCC is significantly higher than that in normal tissues. Moreover, C8orf76 promotes the progression of ccRCC by regulating downstream effectors and signaling pathways.\u003c/p\u003e\u003cp\u003eC8orf76 is a novel nuclear protein-encoding gene first identified for its ability to bind specific lncRNAs in gastric cancer and promote tumor progression[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, C8orf76 also plays roles in the occurrence and development of multiple tumors[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, current understanding of C8orf76 remains superficial. No large-scale studies have deeply explored its mechanism of action, and the potential role of C8orf76 in renal cancer lacks sufficient literature data support. Against this backdrop, we conducted a study and for the first time reported the biological status and mechanism of action of C8orf76 in clear cell renal cell carcinoma. We found that C8orf76 was highly expressed in ccRCC, and high expression of C8orf76 predicted poor prognosis. C8orf76 knockdown caused G1 phase arrest in the cell cycle, activated cellular senescence, and inhibited tumor cell proliferation and migration both in vivo and in vitro. These results indicate that C8orf76 mediates the progression of ccRCC through the cellular senescence signaling pathway, which warrants further investigation.\u003c/p\u003e\u003cp\u003eBy reviewing the literature[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and integrating RNA-seq results, we screened out CALB2 as a potential interactor of C8orf76. CALB2 belongs to the calmodulin family, and its primary biological functions involve calcium homeostasis regulation and signal transduction modulation. CALB2 has been shown to promote the progression of various tumors[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, its role in ccRCC remains poorly documented. This study found that CALB2 is upregulated in ccRCC, and that high expression of CALB2 is associated with poor prognostic outcomes. Similar to C8orf76, CALB2 knockdown induced G1 phase cell cycle arrest, cellular senescence, and inhibited the proliferation and migration of ccRCC cells. We further elucidated the functional relationship between C8orf76 and CALB2. Overexpression of CALB2 in C8orf76-knockdown ccRCC cell lines suppressed cell cycle arrest and cellular senescence, and significantly rescued the proliferative and migratory capacities of tumor cells both in vitro and in vivo. Additionally, dual-luciferase reporter assays confirmed that C8orf76 directly binds to the promoter region of CALB2 to promote its transcription. Thus, CALB2 acts as a downstream effector of C8orf76 to exert its biological functions.\u003c/p\u003e\u003cp\u003eCellular senescence refers to a unique state of cell cycle arrest, which has been widely discussed due to its dual roles in tumors[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Cyclin-dependent kinase inhibitors represent a critical component in the occurrence of cellular senescence, primarily functioning through the p53/p21 and p16/pRb pathways[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The p53 protein directly binds to the promoter region of CDKN1A to promote transcription of p21CIP1, thereby inducing cell cycle arrest and cellular senescence[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Similar findings have been reported in renal cell carcinoma[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. On the other hand, the p16INK4a protein, encoded by the CDKN2A gene, shows low expression or loss in RCC[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In this study, we found that further knockdown of CDKN2A in stably CALB2-knockdown ccRCC cell lines significantly suppressed cell cycle arrest and cellular senescence, and partially restored the proliferative and migratory capacities of tumor cells. These results indicate that the C8orf76/CALB2 axis promotes the proliferation and migration of ccRCC by inhibiting the cellular senescence pathway, potentially involving the p16/pRb signaling cascade.\u003c/p\u003e\u003cp\u003eGiven the critical role of the cellular senescence pathway in cancer therapy, multiple related drugs have been developed clinically. For example, the selective CDK4/6-targeted agent palbociclib has achieved new progress in breast cancer treatment[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This study supports the development of inhibitors targeting the C8orf76/CALB2 axis to activate the downstream cellular senescence pathway and inhibit the progression of ccRCC. Notably, this research still has several limitations to address. Although we propose that the C8orf76/CALB2 axis exerts its function through the cellular senescence pathway, the specific mechanisms involved require more in-depth investigation. Additionally, the p53/p21 pathway also plays a significant role in ccRCC cell senescence. Whether there is a further relationship between the C8orf76/CALB2 axis and this pathway remains unexplored, which will be our future research direction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe operation of animals in our study were approved by the Medical Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent for publication was obtained from the participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available in the supplementary material of this article.\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 interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (82273132, 82403933), the Medical and Health Research Project of Zhejiang Province (2024KY065), \u0026apos;Leading Goose\u0026apos; Science and Technology Program of Zhejiang Province (2025C02071), China Postdoctoral Science Foundation Grant (2023M743022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe conception and design of the study: Ben Liu, Dingheng Lu; The acquisition of data: Yuxiao Li, Xinyang Niu, Pengju Liu, Fenghao Zhang, Zhixiang Qi, Kai Yu; Analysis and interpretation of data: Xueyou Ma, Jiazhu Sun, Suyuelin Huang, Yuchen Shi, Xuan Shu; Drafting the article or revising it critically for important intellectual content: Yuxiao Li, Xinyang Niu, Dingheng Lu; Final approval of the version to be submitted: all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful for the supports from Jie Fang in all animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting Information is available from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eA. 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Jager, Inhibiting CDK4/6 in Breast Cancer with Palbociclib, Ribociclib, and Abemaciclib: Similarities and Differences, Drugs 81(3) (2021) 317-331.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"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":"ccRCC, C8orf76, CALB2, Cell senescence, Cell cycle","lastPublishedDoi":"10.21203/rs.3.rs-7305276/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7305276/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eRenal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system, with clear cell renal cell carcinoma (ccRCC) accounting for 90% of all RCC subtypes. Chromosome 8 open reading frame 76 (C8orf76) is significantly upregulated in various tumor types and has been involved in tumor cell proliferation, migration, invasion, and is associated with poor prognosis. However, the expression profile and molecular mechanisms of C8orf76 in ccRCC have not been fully elucidated, and further investigations are required to clarify these aspects.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe systematically investigated the mechanism of action of C8orf76 in ccRCC through in vitro biological function experiments. Cellular function assessments were performed, including CCK-8 assay, colony formation assay, flow cytometry, SA-β-gal staining, Transwell chamber assay, and wound healing assay. Additionally, combined with a subcutaneous xenograft mouse model and an in vivo imaging system, we studied the phenotypic changes following C8orf76 knockdown. Potential downstream targets of C8orf76 were screened via RNA-sequencing and bioinformatics analysis. Additionally, we utilized The Cancer Genome Atlas (TCGA) database to analyze the expression patterns of C8orf76 and CALB2 in ccRCC, as well as their correlations with clinical prognosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eBoth C8orf76 and CALB2 are highly expressed in ccRCC and correlate with poor prognosis. Knockdown of C8orf76 significantly inhibits the proliferation and migration of ccRCC cells both in vivo and in vitro. Specifically, Knockdown of C8orf76 downregulates the transcriptional level of CALB2, leading to G1-phase cell cycle arrest, enhanced cellular senescence, and subsequent suppression of ccRCC proliferation and migration. Furthermore, ectopic overexpression of CALB2 can partially reverse these effects. Dual-luciferase reporter assay confirms that C8orf76 directly binds to the promoter region of CALB2. Similarly, CALB2 knockdown also induces tumor cell cycle arrest and cellular senescence, accompanied by inhibited proliferation and migration of ccRCC. Notably, the aforementioned phenomena are partially rescued following further knockdown of CDKN2A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e C8orf76 is highly expressed in clear cell renal cell carcinoma and correlates with poor prognosis. C8orf76 directly binds to the CALB2 promoter, thereby promoting CALB2 transcription and downstream biological behaviors. Inhibition of the C8orf76/CALB2 axis induces G1-phase cell cycle arrest and activates cellular senescence signaling pathways, which in turn suppresses the proliferation and migration of ccRCC.\u003c/p\u003e","manuscriptTitle":"The tumor promoter role and molecular mechanism of C8orf76/CALB2 axis in clear cell renal cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 05:59:48","doi":"10.21203/rs.3.rs-7305276/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-08-16T10:17:21+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-16T08:01:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-07T12:00:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2025-08-05T23:36:55+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":"9e30fc3f-7a08-4138-b1d3-e71b4b795475","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-08-27T05:59:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 05:59:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7305276","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7305276","identity":"rs-7305276","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}