A novel polypeptide encoded by the circRACK1 suppresses gastric cancer via cytoskeleton remodeling

A novel polypeptide encoded by the circRACK1 suppresses gastric cancer via cytoskeleton remodeling | 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 A novel polypeptide encoded by the circRACK1 suppresses gastric cancer via cytoskeleton remodeling Lei Qiao, Zhengyang Zhou, Yanan Cheng, Wen Pan, Yueting Han, Qihang Zhu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4522505/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Gastric cancer (GC), particularly prevalent in China, is associated with high rates of morbidity and mortality. This study focuses on the role and mechanistic pathways of a novel 127-amino acid peptide, circR-127aa, encoded by hsa_circ_0075402 (circRACK1) in GC cells, exploring its implications in cancer development. Circular RNAs (circRNAs), which vary in expression across different cancers, contribute to tumorigenesis via mechanisms such as miRNA sponging, protein binding, and influencing transcription and translation. Our research involved an extensive analysis of circRNAs ribo-seq data, complemented by mass spectrometry, Western blotting, and immunofluorescence to validate the encoding of circR-127aa by circRACK1. We explored the functional impact of circR-127aa, examining its effects on cell proliferation, apoptosis, and tumor formation in nude mice. A critical aspect of this study was investigating the interaction between circR-127aa and Vimentin, a significant player in cytoskeleton remodeling and cellular mobility during epithelial-mesenchymal transition (EMT). The findings reveal that the circRACK1-encoded peptide functions as a tumor suppressor, facilitating Vimentin ubiquitination, highlighting its potential as a novel therapeutic target and biomarker in GC treatment. In conclusion, this research unveils a groundbreaking role of the circRACK1-encoded peptide, circR-127aa, in GC, emphasizing its tumor-suppressing function through the ubiquitination of Vimentin. This discovery enhances our understanding of GC progression and presents circR-127aa as a valuable candidate for therapeutic strategies and as a biomarker in GC treatment. Gastric cancer circRACK1 Vimentin cytoskeleton Epithelial-mesenchymal transition (EMT） Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Gastric cancer, ranking as the fifth most prevalent cancer globally, is the third leading cause of cancer-related deaths. Particularly in China[1], this malignancy presents a formidable challenge due to its complex metastatic routes encompassing direct infiltration, hematogenous spread, transcoelomic dissemination, and lymphatic metastasis. Compounded by the frequent late-stage diagnosis, the overall 5-year survival rate in China falls below 30%[2, 3], underscoring the urgency for novel insights into gastric cancer's molecular mechanisms and effective early detection and intervention strategies. Circular RNAs (circRNAs) have emerged as a distinct class of molecules, characterized by their covalently closed-loop structures, absence of 5'-3' polarity, and lack of a polyadenylated tail[4, 5]. Their unique configuration endows them with remarkable stability, making them abundant across various species[6]. Although the full spectrum of their functions is not entirely understood, recent evidence points to their significant roles in cell proliferation[7], apoptosis[8], migration and invasion[9, 10], thereby implicating them in various cancer types. In gastric cancer, the role of circRNAs and their underlying mechanisms remain largely unexplored, presenting a frontier for in-depth research. RACK1 (Receptor for Activated C Kinase 1) is a multifunctional scaffold protein integral to numerous cellular processes[11]. Its involvement in growth, differentiation, invasion, and migration, and its dysregulation in various tumors, highlights its importance in cancer biology[12]. Interestingly, RACK1 exhibits both tumor-promoting and tumor-suppressing activities, depending on the cancer type and cellular context[13-17]. This duality adds complexity to understanding its role in gastric cancer. In this study, we identified a novel circular RNA, circRACK1, originating from the RACK1 gene, in gastric cancer cell lines. This circRNA was found to be down-regulated in these cells. More intriguingly, we discovered its potential to encode a novel peptide, circR-127aa, which appears to play a significant role in modulating vimentin ubiquitination during the epithelial-mesenchymal transition (EMT), a key process in cytoskeleton remodeling and cell mobility. Our research thus adds a new dimension to understanding circRACK1's involvement in gastric cancer and positions circR-127aa as a potential biomarker and therapeutic target. Methods Cell Lines and Culture Conditions: Our study utilized the human gastric epithelial cell line GES-1 and various human gastric cancer cell lines (HGC27, MKN-28, AGS, MKN-45, MGC-803, BGC-823), all sourced from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. GES-1 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS), while the gastric cancer cell lines were cultured in RPMI 1640 medium also enriched with 10% FBS. Standard incubation conditions involved a temperature of 37°C and an atmosphere of 5% CO 2 . Transfection, Oligonucleotides, and Plasmids: Custom oligonucleotides and plasmids were designed to manipulate the expression of circRACK1 and Vimentin. Targeted siRNAs against circRACK1 were synthesized, alongside overexpression constructs for circRACK1-3×Flag and Vimentin. These were transfected into cells using Lipofectamine™ 2000, following manufacturer protocols. Stable cell lines were generated via lentiviral infection, followed by a 2-week selection in puromycin. The sequences for siRNA and the control group are detailed in Table 1. RNA Isolation and RT-qPCR: Total RNA extraction from gastric cell lines was performed using TRIzol reagent, followed by reverse transcription into cDNA. Quantitative real-time PCR was conducted to assess the expression levels of target RNAs, with GAPDH serving as an internal control. The 2 -ΔΔCt method was employed for data analysis. The sequences of all primers used in the experiments are listed in Table 2 Protein Studies: Western Blotting and Coimmunoprecipitation (Co-IP): Protein extraction utilized SDS-lysis buffer, followed by concentration determination using a BCA Protein Assay kit. Proteins were separated via SDS-PAGE, transferred to PVDF membranes, and probed with primary and secondary antibodies for visualization. For Co-IP, cell lysates were incubated with primary antibodies and Protein A/G beads, followed by immunoprecipitation and analysis via Western blotting. The antibody information used for the Western Blot (WB) experiment is presented in Table 3. The details of the antibodies employed in the Co-immunoprecipitation (CO-IP) experiment can be found in Table 4. Immunofluorescence Assay: Cells were fixed, permeabilized, and blocked prior to incubation with primary antibodies. Following washes, fluorescently labeled secondary antibodies were applied. Nuclei were stained with DAPI, and fluorescence microscopy was utilized for visualization. the antibody information for the Immunofluorescence (IF) experiment is located in Table 5." Additional Methods: The methods section also detailed procedures for mass spectrometry analysis, ubiquitination assays, cell wound healing and transwell assays, cell proliferation assays (including CCK8, colony-forming, and EdU assays), flow cytometry analysis for apoptosis, HUVEC tube formation assay, immunohistochemistry, and animal experiments. Each of these techniques was carefully executed to ensure the reliability and validity of our findings. Statistical Analysis: Data were presented as mean ± SEM and analyzed using two-tailed paired or non-paired t-tests as appropriate, with P values <0.05 considered statistically significant. Results The expression of circRACK1 is downregulated in GC cells. Recent advancements in our understanding of circRNAs, particularly their role in protein translation via CAP-independent mechanisms, have not only expanded our knowledge of these molecules but also highlighted their potential in disease diagnosis and treatment. Current research indicates that circRNA translation is tightly regulated under stress conditions, possibly serving as a compensatory cellular mechanism. Notably, hypoxia, a hallmark of the tumor microenvironment and a key feature of solid tumors, may influence circRNA expression. In this context, we investigated whether circRNA expression in gastric cancer (GC) cells alters under hypoxic conditions. We cultured HGC27 cells, which were in the logarithmic growth phase, in a hypoxic incubator (5% CO 2 , <1% O 2 ) for 36 hours. Post-cultivation, we lysed the cells and conducted Ribo-seq detection. Our circular heatmap analysis revealed a notable decrease in the expression of a novel circRNA, designated as novel_circ_000494, under these hypoxic conditions (see Fig. 1A). Further investigation in the Circbase database identified this circRNA as hsa_circ_0075402, located on chromosome 5:181241492-181242345. Its host gene is RACK1 (ENSG00000204628). Subsequent Sanger sequencing analysis showed that this circRNA, which we have named circRACK1, forms a closed-loop structure through the reverse splicing of two exons from the RACK1 gene (exon1: 181242345-181242174, exon2: 181241639-181241492), as illustrated in（Fig. 1B）. To confirm the circular structure of circRACK1, we designed convergent and divergent primers for amplifying circRACK1 and linear RACK1, respectively. Our results, depicted in Fig. 1C, demonstrate that circRACK1 is exclusively amplified from cDNA, confirming its nature as a back-spliced product of the host mRNA. Additionally, circRACK1 exhibited significantly higher resistance to RNase R degradation compared to its linear mRNA counterpart (Fig. 1D-E). Actinomycin D assays further confirmed the enhanced stability of circRACK1, showing a markedly longer half-life than the linear RACK1 transcript (Fig. 1F-G). RT-qPCR detection in common GC cells revealed a reduction in circRACK1 expression. Particularly, AGS and HGC27 cell lines, showing the lowest circRACK1 levels, were selected for further study (Fig. 1H). Upon re-examination under hypoxic conditions, we observed an additional decrease in circRACK1 expression levels (Fig. 1I-J). CircRACK1 inhibits malignant phenotypes of gastric cancer cells To delve into the functional importance of circRACK1 in GC cells, we strategically designed siRNAs targeting circRACK1's specific binding sites and introduced overexpression plasmids into HGC27 and AGS cells. This intervention notably impacted the expression levels of circRACK1, while the mRNA and protein levels of its host gene, RACK1, remained largely unaffected, as illustrated in Fig(S1 A-F). Our research extended to evaluating the effect of circRACK1 on the proliferation of GC cells. EdU incorporation analysis further corroborated these findings, showing increased EdU assimilation in cells with diminished circRACK1 expression, and the opposite in those with heightened expression (Fig 2A-B, S2A-B). The results from colony formation assays were in line with these observations. Cells overexpressing circRACK1 formed fewer and smaller colonies, whereas inhibition of circRACK1 resulted in a significant increase in colony numbers, underscoring its suppressive role in GC cell proliferation (Figure S3A).The role of circRACK1 in apoptosis was elucidated through flow cytometry, employing ANEXIN V-PI. The data revealed that circRACK1 knockdown markedly diminished apoptosis in GC cells, while its overexpression promoted apoptotic processes (Fig S2E-F).Our investigation further spanned the influence of circRACK1 on GC cell migration and angiogenesis. Transwell assays demonstrated an increased migration of cells in the circRACK1 knockdown groups and a decrease in the overexpressed groups (Fig 2C, S2C-D). Scratch assays reinforced these findings, indicating that high levels of circRACK1 slow down wound healing, whereas its knockdown accelerates this process (Figure S3B-C). These outcomes suggest a significant inhibitory effect of circRACK1 on the migratory capabilities of GC cells. Moreover, the angiogenic potential of GC cells, assessed through their impact on HUVEC-induced migration and tube formation, was enhanced in circRACK1 knockdown cells, while overexpression of circRACK1 exhibited the opposite effect (Figure 2D). CircR-127aa encoded by circRACK1 inhibits gastric cancer cell malignancy In exploring the coding potential of circRACK1, our search in the CIRCRNADb and TransCirc databases revealed multiple internal ribosome entry sites (IRES) and open reading frames (ORFs) within circRACK1, hinting at its possible coding function. To test the translatability of circRACK1, we analyzed its putative ORF using ORF Finder. We identified a potential overlapping ORF (384nt) within circRACK1, capable of encoding a peptide of 127 amino acids in length (Fig 3A). We cloned the circRACK1 sequence into a circRNA expression vector (circRACK1-3xFLAG), with a FLAG tag positioned before the stop codon to detect successful circularization. Immunoblotting suggested that circRACK1 might encode a new protein of approximately 15kDa. To verify this protein's origin from circRACK1, we engineered plasmids with FLAG-tagged start codon mutations (ATG-ATT) and empty vector controls. The unique peptide fragment formed at the junction site of circRACK1 is illustrated in Fig 3B. Post-transfection of these plasmids into HGC27 and AGS cells, followed by Western Blot analysis after 48 hours, FLAG was detected in cells overexpressing circRACK1 but not in those with the vector or circRACK1-mut plasmid (Fig 3C). Given that part of circRACK1-encoded circR-127aa sequence overlaps with the host gene RACK1, we considered the possibility of RACK1 polyclonal antibody binding to circR-127aa. Using an anti-RACK1 antibody, we detected circR-127aa, observing increased expression levels following overexpression of the OE plasmid, while the expression of host gene RACK1 remained relatively unchanged (Fig 3D). Immunofluorescence experiments further confirmed the presence of circR-127aa in HGC27 and AGS cells, predominantly localized in the cytoplasm and enriched on the cell membrane (Fig 3E).To ascertain whether the biological impact of circRACK1 on gastric cancer cells is mediated through itself or its translation products, we re-examined common tumor phenotypes using circRACK1 overexpression plasmids, circRACK1-mut, and control plasmids. Gastric cancer cells transfected with circRACK1-mut plasmids showed no significant changes in proliferation, migration, and scratch healing capabilities compared to control group plasmids. However, cells transfected with circRACK1 overexpression plasmids exhibited an inhibitory effect on these phenotypes (Fig S4 A-D). CircR-127aa promotes ubiquitination and degradation of vimentin To elucidate the biological mechanism behind circR-127aa's influence on gastric cancer behavior, we conducted immunoprecipitation experiments to identify proteins potentially interacting with circR-127aa. The immunoprecipitated products were subjected to gel electrophoresis and stained with Coomassie Brilliant Blue to accentuate the distinct bands between the Vector and OE groups. These bands were then analyzed through mass spectrometry, indicating potential interactions of circR-127aa with Vimentin and ACTIN (Fig 3F, S5A). Further immunoprecipitation assays validated these interactions (Fig 3G, H, S6B). We also performed immunofluorescence co-localization experiments, observing that circR-127aa co-localizes with Vimentin and ACTIN, particularly on the cell membrane (Fig S5C-D). To explore the regulatory mechanism of circR-127aa on Vimentin expression in gastric cancer cells, we conducted RT-qPCR and Western Blotting experiments. These revealed that Vimentin expression did not significantly change in the gastric cancer cell line with overexpressed or knocked-down circRACK1 compared to the control group (Fig S6A,-B). However, Western Blotting showed a decrease in Vimentin expression following circRACK1 overexpression and an increase following circRACK1 knockdown (Fig4A-B). We further treated OE and shRNA group cells with cycloheximide (CHX) to inhibit new protein synthesis in gastric cancer cells. Results indicated a significant reduction in Vimentin expression in the OE group compared to the ShRNA group (Fig 4C). After treatment with the proteasome inhibitor MG132, Vimentin expression levels in the OE group's two cell lines recovered, suggesting that circR-127aa might influence Vimentin expression through ubiquitin-mediated proteasomal degradation (Fig 4D). In HEK293T cells transfected with HA-ubiquitin and Vimentin overexpression plasmids, circRACK1, or Vector plasmids for 48 hours, exogenous ubiquitin detection revealed increased ubiquitination modifications in the OE group compared to the Vector group (Fig 4E). Lastly, immunoprecipitation experiments on two stable cell lines showed that circRACK1 knockdown decreased Vimentin ubiquitination, while its overexpression increased Vimentin ubiquitination (Fig 4F) Modulatory Effects of the HIF-1α/circRACK1/vimentin Axis on Gastric Cancer Cell Behavior In the HGC27 gastric cancer cell line, circRACK1 overexpression (OE) led to an increase in E-cadherin levels and a decrease in N-cadherin, β-catenin, MMP9, and Snail, indicative of reduced invasive capacity. This modulation was reversed by concurrent HIF-1α overexpression (HIF-1α OE), which suggests a regulatory mechanism where HIF-1α enhances cancer cell invasion and migration by suppressing circRACK1 (Fig5A,S6A). Colony formation and invasion assays further supported the role of circRACK1 in altering cellular behaviors. The cells with circRACK1 OE displayed a diminished ability to form colonies and invade through the matrix. This was notably counteracted by co-overexpression with vimentin (OE+Vim), placing vimentin as a key effector in circRACK1-driven functions (Fig5B-C,S6B-C). Immunofluorescence assays demonstrated that circRACK1 silencing (sh-circR) results in significant cytoskeletal reorganization, characterized by increased pseudopodia, suggesting a promotion of cellular motility, as opposed to the more stable architecture observed in the OE group (Fig5D,S6D). Western blot analysis across different genetic backgrounds established that circRACK1 knockdown and vimentin co-overexpression increased the levels of mesenchymal markers while decreasing the epithelial marker E-cadherin, aligning with the molecular signature of EMT (Fig5E,S6E). Collectively, these results illuminate the comprehensive role of circRACK1 and its peptide product, CircR-127aa, in modulating gastric cancer cell behavior through EMT and cytoskeletal dynamics, potentially orchestrated by vimentin interactions. Knockdown of circRACK1 promotes gastric cancer growth and metastasis in vivo To further validate the influence of circRACK1 on the proliferation of gastric cancer cells in vivo, we utilized stable cell lines with circRACK1 knockdown (experimental group) and corresponding control cell lines (control group) to establish subcutaneous tumors in nude mice. Regular tumor volume measurements indicated that circRACK1 knockdown enhances subcutaneous tumor growth (Fig6A，C, S7C). live animal imaging showed larger tumors in the circRACK1 knockdown group (Fig6B). Subsequent H&E staining and immunohistochemical analyses of the tumors indicated significantly increased expression of Ki-67, Vimentin, MMP9, and CD31 in the experimental group compared to the control group. This suggests enhanced proliferation and invasion capacities of tumor cells and increased microvascular density when circRACK1 is knocked down. Lower expression of Caspase 3 in the experimental group implied reduced apoptosis in tumor cells (FigS7F). Additionally, F-ACTIN staining within the tissues showed increased fluorescence intensity in mouse tumors with circRACK1 knockdown (Fig 6D).During the live imaging and pathological examination of the subcutaneously implanted tumors in the nude mice, we observed more instances of intrathoracic and abdominal metastases in the experimental group than in the control group (S7D,E). We injected the stable cell lines with circRACK1 knockdown (experimental group) and the corresponding control cell lines (control group) into the tail vein of mice to create a distant metastasis model. After four weeks, we conducted regular small animal live imaging and CT scans for distant metastases monitoring. Starting from the fifth week post-model establishment, more distant metastases were observed in the experimental group compared to the control group (Fig6E, S7G). Upon sacrificing and dissecting the mice at eight weeks post-modeling, we found that the experimental group exhibited more and larger lung metastases (Fig6F-G). Additionally, we examined the livers of the mice and noted metastatic lesions in the liver of some mice in the experimental group. We selected typical hepatic metastatic lesions for observation and documentation (Fig S7H). In the concluding section of our study, we present a detailed schematic diagram illustrating the role of the peptide circR-127aa, encoded by circRACK1, in gastric cancer (GC). This diagram emphasizes the tumor-suppressing function of circR-127aa, particularly highlighting its involvement in the ubiquitination process of Vimentin, thereby elucidating its significance in the pathophysiology of GC（Fig6H）. Discussion Our research has identified a novel circRNA, circRACK1, originating from the RACK1 gene. This discovery is pivotal in understanding gastric cancer biology. Notably, we observed a significant downregulation of circRACK1 in gastric cancer cells, particularly under hypoxic conditions. The unique circular structure of circRACK1, formed via exon splicing, comprises a 320-nucleotide sequence. This finding enriches the existing literature on RACK1's tumor-suppressive functions in gastric cancer, including its role in inhibiting cell proliferation and the epithelial-mesenchymal transition (EMT) through various signaling pathways[18, 19]. Hypoxia, prevalent in the tumor microenvironment of solid tumors[20, 21], induces the upregulation of HIF-1α, a factor known to accelerate metastasis gastric cancer[22]. Under hypoxic conditions, the expression of RACK1 and consequently circRACK1 is reduced, promoting invasion and angiogenesis via the HIF-1α and VEGF pathways[23]. This highlights the critical need for further investigation into circRACK1's function in gastric cancer, particularly considering environmental factors like hypoxia. Compared to linear host gene products, proteins encoded by circRNAs often possess unique and independent biological functions[24, 25].Our findings extend beyond the sequence of circRACK1 to its encoded protein, circR-127aa. This peptide exerts tumor-suppressive effects by interacting with vimentin/actin proteins and promoting vimentin ubiquitination, crucial for cytoskeleton remodeling. These interactions inhibit gastric cancer cell proliferation, migration, and invasion, while promoting apoptosis. Thus, circR-127aa plays a significant role in modulating the biological functions of gastric cancer. Vimentin ubiquitination, a crucial post-translational modification, regulates its signaling pathway, pivotal in controlling gastric cancer cell malignancy [26, 27]. Our findings suggest that the dynamic balance of vimentin ubiquitination and deubiquitination offers a complex regulatory mechanism, potentially exploitable in gastric cancer therapy. Mass spectrometry analysis confirmed circR-127aa's interaction with Vimentin and Actin, key proteins in cytoskeletal remodeling and tumor cell metastasis[28, 29],elucidating the underlying mechanisms of tumor malignancy and metastasis in gastric cancer. Despite our significant findings, this study has limitations. The insights into circRACK1's role in gastric cancer require further validation through extensive in vivo studies and analysis of clinical samples. Additionally, while we explored the interactions of circR-127aa with vimentin and actin, a comprehensive understanding of its network within gastric cancer cells is yet to be achieved. Future research should investigate these interactions in greater detail and assess circR-127aa's impact on patient prognosis and treatment response. Also, considering the complexity of the tumor microenvironment, the role and expression of circRACK1 and circR-127aa under varying conditions warrant further investigation. In conclusion, our study introduces circR-127aa as a novel peptide encoded by circRACK1, with significant implications as a diagnostic marker and therapeutic target in gastric cancer. Our findings pave the way for future clinical studies to fully elucidate the therapeutic potential and clinical utility of circR-127aa in gastric cancer management. Declarations Acknowledgements Not applicable. Funding Information This work was funded by Tianjin Key Medical Discipline(Specialty) Construction Project（TJYXZDXK-009A） Conflict of Interest The authors have no conflict of interest. Ethics Statement Approval of the research protocol by an Institutional Reviewer Board. N/A. Informed Consent. N/A. Registry and the Registration No. of the study/trial. N/A. Animal Studies. The operational process was executed within the approve of the Institutional Animal Care and Research Advisory Committee of Tianjin Medical University Cancer Institute and Hospital. References H. Sung, J. Ferlay, R.L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F. Bray, Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries, CA: a cancer journal for clinicians, 71 (2021) 209-249. H. Zeng, W. Chen, R. Zheng, S. Zhang, J.S. Ji, X. Zou, C. Xia, K. Sun, Z. Yang, H. Li, N. Wang, R. Han, S. Liu, H. Li, H. Mu, Y. He, Y. Xu, Z. Fu, Y. Zhou, J. Jiang, Y. Yang, J. Chen, K. Wei, D. Fan, J. Wang, F. Fu, D. Zhao, G. Song, J. Chen, C. Jiang, X. Zhou, X. Gu, F. Jin, Q. Li, Y. Li, T. Wu, C. Yan, J. Dong, Z. Hua, P. Baade, F. Bray, A. Jemal, X.Q. Yu, J. He, Changing cancer survival in China during 2003-15: a pooled analysis of 17 population-based cancer registries, The Lancet. Global health, 6 (2018) e555-e567. W. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X.Q. Yu, J. He, Cancer statistics in China, 2015, CA: a cancer journal for clinicians, 66 (2016) 115-132. L.S. Kristensen, M.S. Andersen, L.V.W. Stagsted, K.K. Ebbesen, T.B. Hansen, J. Kjems, The biogenesis, biology and characterization of circular RNAs, Nature reviews. Genetics, 20 (2019) 675-691. L.L. Chen, The expanding regulatory mechanisms and cellular functions of circular RNAs, Nature reviews. Molecular cell biology, 21 (2020) 475-490. J. Salzman, R.E. Chen, M.N. Olsen, P.L. Wang, P.O. Brown, Cell-type specific features of circular RNA expression, PLoS genetics, 9 (2013) e1003777. H. Pan, T. Li, Y. Jiang, C. Pan, Y. Ding, Z. Huang, H. Yu, D. Kong, Overexpression of Circular RNA ciRS-7 Abrogates the Tumor Suppressive Effect of miR-7 on Gastric Cancer via PTEN/PI3K/AKT Signaling Pathway, Journal of cellular biochemistry, 119 (2018) 440-446. Y. Enuka, M. Lauriola, M.E. Feldman, A. Sas-Chen, I. Ulitsky, Y. Yarden, Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor, Nucleic acids research, 44 (2016) 1370-1383. M. Jie, Y. Wu, M. Gao, X. Li, C. Liu, Q. Ouyang, Q. Tang, C. Shan, Y. Lv, K. Zhang, Q. Dai, Y. Chen, S. Zeng, C. Li, L. Wang, F. He, C. Hu, S. Yang, CircMRPS35 suppresses gastric cancer progression via recruiting KAT7 to govern histone modification, Molecular cancer, 19 (2020) 56. J. Meng, S. Chen, J.X. Han, B. Qian, X.R. Wang, W.L. Zhong, Y. Qin, H. Zhang, W.F. Gao, Y.Y. Lei, W. Yang, L. Yang, C. Zhang, H.J. Liu, Y.R. Liu, H.G. Zhou, T. Sun, C. Yang, Twist1 Regulates Vimentin through Cul2 Circular RNA to Promote EMT in Hepatocellular Carcinoma, Cancer research, 78 (2018) 4150-4162. D.R. Adams, D. Ron, P.A. Kiely, RACK1, A multifaceted scaffolding protein: Structure and function, Cell communication and signaling : CCS, 9 (2011) 22. D. Duff, A. Long, Roles for RACK1 in cancer cell migration and invasion, Cellular signalling, 35 (2017) 250-255. Y. Ruan, L. Sun, Y. Hao, L. Wang, J. Xu, W. Zhang, J. Xie, L. Guo, L. Zhou, X. Yun, H. Zhu, A. Shen, J. Gu, Ribosomal RACK1 promotes chemoresistance and growth in human hepatocellular carcinoma, The Journal of clinical investigation, 122 (2012) 2554-2566. C. Shen, H. Hua, L. Gu, S. Cao, H. Cai, X. Yao, X. Chen, Overexpression of RACK1 Predicts Poor Prognosis in Melanoma, Journal of Cancer, 11 (2020) 795-803. X. Li, Y. Xiao, S. Fan, M. Xiao, X. Wang, X. Chen, C. Li, G. Zong, G. Zhou, C. Wan, RACK1 overexpression associates with pancreatic ductal adenocarcinoma growth and poor prognosis, Experimental and molecular pathology, 101 (2016) 176-186. L. Chen, L. Min, X. Wang, J. Zhao, H. Chen, J. Qin, W. Chen, Z. Shen, Z. Tang, Q. Gan, Y. Ruan, Y. Sun, X. Qin, J. Gu, Loss of RACK1 Promotes Metastasis of Gastric Cancer by Inducing a miR-302c/IL8 Signaling Loop, Cancer research, 75 (2015) 3832-3841. Z. Yu, X. Jiang, L. Qin, H. Deng, J. Wang, W. Ren, H. Li, L. Zhao, H. Liu, H. Yan, W. Shi, Q. Wang, C. Luo, B. Long, H. Zhou, H. Sun, Z. Jiao, A novel UBE2T inhibitor suppresses Wnt/β-catenin signaling hyperactivation and gastric cancer progression by blocking RACK1 ubiquitination, Oncogene, 40 (2021) 1027-1042. J.J. Li, D. Xie, RACK1, a versatile hub in cancer, Oncogene, 34 (2015) 1890-1898. C. Liu, L. Ren, Y. Wang, Y. Liu, J. Xiao, The interaction between RACK1 and WEE1 regulates the growth of gastric cancer cell line HGC27, Oncology letters, 14 (2017) 4784-4792. H. Choudhry, A.L. Harris, Advances in Hypoxia-Inducible Factor Biology, Cell metabolism, 27 (2018) 281-298. Z. Chen, F. Han, Y. Du, H. Shi, W. Zhou, Hypoxic microenvironment in cancer: molecular mechanisms and therapeutic interventions, Signal transduction and targeted therapy, 8 (2023) 70. X. Wang, X. Che, Y. Yu, Y. Cheng, M. Bai, Z. Yang, Q. Guo, X. Xie, D. Li, M. Guo, K. Hou, W. Guo, X. Qu, L. Cao, Hypoxia-autophagy axis induces VEGFA by peritoneal mesothelial cells to promote gastric cancer peritoneal metastasis through an integrin α5-fibronectin pathway, Journal of experimental & clinical cancer research : CR, 39 (2020) 221. S.J. Yang, Y.S. Park, J.H. Cho, B. Moon, H.J. An, J.Y. Lee, Z. Xie, Y. Wang, D. Pocalyko, D.C. Lee, H.A. Sohn, M. Kang, J.Y. Kim, E. Kim, K.C. Park, J.A. Kim, Y.I. Yeom, Regulation of hypoxia responses by flavin adenine dinucleotide-dependent modulation of HIF-1α protein stability, The EMBO journal, 36 (2017) 1011-1028. J.N. Vo, M. Cieslik, Y. Zhang, S. Shukla, L. Xiao, Y. Zhang, Y.M. Wu, S.M. Dhanasekaran, C.G. Engelke, X. Cao, D.R. Robinson, A.I. Nesvizhskii, A.M. Chinnaiyan, The Landscape of Circular RNA in Cancer, Cell, 176 (2019) 869-881.e813. J. Guarnerio, M. Bezzi, J.C. Jeong, S.V. Paffenholz, K. Berry, M.M. Naldini, F. Lo-Coco, Y. Tay, A.H. Beck, P.P. Pandolfi, Oncogenic Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal Translocations, Cell, 165 (2016) 289-302. Q. Li, M.S. Deng, R.T. Wang, H. Luo, Y.Y. Luo, D.D. Zhang, K.J. Chen, X.F. Cao, G.M. Yang, T.M. Zhao, B. Xu, C.X. Xu, J.M. Wang, PD-L1 upregulation promotes drug-induced pulmonary fibrosis by inhibiting vimentin degradation, Pharmacological research, 187 (2023) 106636. W. Shao, J. Li, Q. Piao, X. Yao, M. Li, S. Wang, Z. Song, Y. Sun, L. Zheng, G. Wang, L. Liu, C. Yu, Y. Huang, Y. Bao, L. Sun, FRMD3 inhibits the growth and metastasis of breast cancer through the ubiquitination-mediated degradation of vimentin and subsequent impairment of focal adhesion, Cell death & disease, 14 (2023) 13. Y. Hashimoto, D.J. Kim, J.C. Adams, The roles of fascins in health and disease, The Journal of pathology, 224 (2011) 289-300. S. Lin, M.D. Taylor, P.K. Singh, S. Yang, How does fascin promote cancer metastasis?, The FEBS journal, 288 (2021) 1434-1446. Additional Declarations No competing interests reported. Supplementary Files DetailedInformationofAntibodiesUsedintheStudy.docx S1New.tif S2New.tif S3New.tif S4New.tif S5New.tif S6New.tif S7New.tif SequencesofPrimersandsiRNAsUsedintheStudy.docx Supplementaryfigurelegends.docx Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4522505","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":313410535,"identity":"643c93ce-0955-4f02-a9b4-9e6bc87d1936","order_by":0,"name":"Lei Qiao","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Qiao","suffix":""},{"id":313410536,"identity":"c7a8d800-0eb5-47b0-ae9c-6a5290f7b383","order_by":1,"name":"Zhengyang Zhou","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhengyang","middleName":"","lastName":"Zhou","suffix":""},{"id":313410537,"identity":"3c8c17d8-88bc-45bf-9871-2052a7f5dea8","order_by":2,"name":"Yanan Cheng","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanan","middleName":"","lastName":"Cheng","suffix":""},{"id":313410538,"identity":"7004bc87-6f6a-4600-b2ad-4e844ff84350","order_by":3,"name":"Wen Pan","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Pan","suffix":""},{"id":313410539,"identity":"26c54e2f-69de-4aa3-a7c9-46ffa20d8b80","order_by":4,"name":"Yueting Han","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yueting","middleName":"","lastName":"Han","suffix":""},{"id":313410541,"identity":"e2d3b454-cde3-4770-bd03-4937de2ae81b","order_by":5,"name":"Qihang Zhu","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qihang","middleName":"","lastName":"Zhu","suffix":""},{"id":313410543,"identity":"92bf7627-5ac4-43fc-a5c7-a5211138c025","order_by":6,"name":"Haiyang Zhang","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haiyang","middleName":"","lastName":"Zhang","suffix":""},{"id":313410544,"identity":"c4de863e-a64e-470c-848c-bf2702ddcf8f","order_by":7,"name":"Yi Ba","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYBACAyA+8IGBDUjxkKDl4IwEUrUw8yQwkKDFnP2M4WHbH3zRBsfPHmD4UbGNsBbLnhyDwzkJbLkbzuQlMPacuU2Eww7AtNzgMWBmbCNGy/k3BoctSNNyA2gLA0laLGc8KzjYk8aWO/NMjsFBovxizp+8+cMPm2O5fcfPGD74UUGEFgYGDlDUHAMzDxCjHgjYHwCJGiIVj4JRMApGwYgEAC5dP3+pvt5iAAAAAElFTkSuQmCC","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Ba","suffix":""}],"badges":[],"createdAt":"2024-06-03 14:37:30","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4522505/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4522505/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58706083,"identity":"2a77a345-f363-4fac-8079-547f0a1e8e91","added_by":"auto","created_at":"2024-06-20 05:21:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":736004,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of circRACK1 in gastric cancer cells under hypoxia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Circular heatmap from Ribo-seq showing downregulated expression of novel_circ_075402, identified as circRACK1, in HGC27 cells exposed to hypoxia.\u003c/p\u003e\n\u003cp\u003eB. Schematic representation of circRACK1 genesis through back-splicing of RACK1 gene exons, as verified by Sanger sequencing.\u003c/p\u003e\n\u003cp\u003eC. PCR analysis distinguishes circular circRACK1 presence in cDNA versus gDNA, confirming its circular configuration.\u003c/p\u003e\n\u003cp\u003eD-E. RNase R resistance assay of circRACK1 compared to linear RACK1 mRNA in HGC27 (D) and AGS (E) cells, demonstrating enhanced stability of circRACK1.\u003c/p\u003e\n\u003cp\u003eF-G. Comparative decay profiles of circRACK1 and linear RACK1 mRNA in HGC27 (F) and AGS (G) cells over 24 hours, establishing the longer half-life of circRACK1.\u003c/p\u003e\n\u003cp\u003eH. Baseline expression of circRACK1 across various GC cell lines, with AGS and HGC27 showing notably lower levels.\u003c/p\u003e\n\u003cp\u003eI. Western blot analysis indicating reduced circRACK1 protein abundance in AGS and HGC27 cells under hypoxic conditions.\u003c/p\u003e\n\u003cp\u003eJ. RT-qPCR assessment showing a hypoxia-induced decrease in circRACK1 expression in AGS and HGC27 cells.\u003c/p\u003e\n\u003cp\u003eAll data were representative of at least three biological replicates and shown as mean±SD. Statistical significance was determined by 2-tailed Student’s t-test. *p \u0026lt; 0.05, **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Fig1New.png","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/e80d9ec5a5c7aae5b6ca407a.png"},{"id":58706567,"identity":"fb4bef94-609f-410d-9b3b-dbdffd86cbd7","added_by":"auto","created_at":"2024-06-20 05:29:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9087133,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional analyses of circRACK1's role in GC cell proliferation and metastatic behavior.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-B. EdU incorporation assays revealing lower DNA synthesis in cells overexpressing circRACK1 and higher synthesis with reduced circRACK1 levels.\u003c/p\u003e\n\u003cp\u003eC. Transwell assays demonstrating inhibited cell migration upon circRACK1 overexpression, and enhanced migration with circRACK1 knockdown.\u003c/p\u003e\n\u003cp\u003eD Assessment of HUVEC migration and tube formation, indicating circRACK1 knockdown promotes angiogenesis, while its overexpression has an inhibitory effect.\u003c/p\u003e","description":"","filename":"Fig2New.png","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/bcaa64a3083907924963a4dd.png"},{"id":58706082,"identity":"cbc644c4-abdb-41c9-87d2-f5b8f513a097","added_by":"auto","created_at":"2024-06-20 05:21:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2272083,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircRACK1 exhibits coding potential and its translated product, circR-127aa, influences GC cell behaviors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. ORF Finder analysis depicting the potential overlapping ORF within circRACK1, capable of encoding a 127 amino acid peptide.\u003c/p\u003e\n\u003cp\u003eB. Schematic representation of circRACK1-3xFLAG construct, with a unique peptide sequence at the junction site, indicating a translation product.\u003c/p\u003e\n\u003cp\u003eC. Western blot (WB) detection of the FLAG tag in HGC27 and AGS cells transfected with circRACK1, circRACK1-mut, and empty vector, demonstrating the translation of circRACK1 but not its mutated form or from the control vector.\u003c/p\u003e\n\u003cp\u003eD. WB analysis using an anti-RACK1 antibody to identify the circRACK1-encoded peptide, circR-127aa, confirming its overexpression does not significantly alter host gene RACK1 expression.\u003c/p\u003e\n\u003cp\u003eE. Immunofluorescence assays showing the subcellular localization of the circR-127aa peptide in the cytoplasm and on the cell membrane of HGC27 and AGS cells.\u003c/p\u003e\n\u003cp\u003eF. Protein bands obtained from immunoprecipitation of circR-127aa were visualized with Coomassie Brilliant Blue staining, and bands of interest were further analyzed by mass spectrometry.\u003c/p\u003e\n\u003cp\u003eG-H. Confirmation of circR-127aa interactions with Vimentin and ACTIN through immunoprecipitation assays.\u003c/p\u003e","description":"","filename":"Fig3New.png","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/0197f2152e2ed99d2640da89.png"},{"id":58706087,"identity":"2d768cc0-2c18-4357-9460-318e1768e474","added_by":"auto","created_at":"2024-06-20 05:21:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1137539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of circR-127aa on Vimentin expression and its post-translational regulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-B. Western blot analyses showing changes in Vimentin protein levels in gastric cancer cells with circRACK1 overexpression or knockdown.\u003c/p\u003e\n\u003cp\u003eC. Examination of Vimentin expression in the presence of cycloheximide (CHX), which inhibits new protein synthesis, indicating reduced Vimentin levels in the OE group compared to the shRNA group.\u003c/p\u003e\n\u003cp\u003eD. The effect of proteasome inhibition by MG132 on Vimentin expression, suggesting that circR-127aa may regulate Vimentin stability through the proteasomal pathway.\u003c/p\u003e\n\u003cp\u003eE. Ubiquitination assays in HEK293T cells transfected with relevant plasmids, demonstrating enhanced ubiquitination of proteins in the OE group.\u003c/p\u003e\n\u003cp\u003eF. Immunoprecipitation experiments indicating that ubiquitination levels of Vimentin are modulated by the expression status of circRACK1, with an increase upon overexpression and decrease upon knockdown.\u003c/p\u003e","description":"","filename":"Fig4New.png","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/2784c5a4457255e16bda7f58.png"},{"id":58706568,"identity":"16e2e65e-97c0-4ddc-8fb3-fba1070be223","added_by":"auto","created_at":"2024-06-20 05:29:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4039799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of circR-127aa on the cytoskeletal architecture and EMT-related protein expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.Overexpression of circRACK1 curtails EMT protein markers, an effect reversed by HIF-1α co-expression, highlighting HIF-1α's regulatory influence over circRACK1-modulated EMT in gastric cancer cells.\u003c/p\u003e\n\u003cp\u003eB-C. circRACK1 overexpression reduces proliferation and migration, effects reversed by Vimentin co-expression, highlighting Vimentin's role in modulating these cellular functions.\u003c/p\u003e\n\u003cp\u003eD. Phalloidin staining of F-ACTIN in HGC27 cell lines, showing alterations in fluorescence intensity and filament density corresponding to changes in circRACK1 expression levels, with knockdown leading to increased, and overexpression leading to decreased F-ACTIN representation.\u003c/p\u003e\n\u003cp\u003eE. Western blot analysis of EMT marker proteins in gastric cancer cell lines with transient or stable circRACK1 expression modulation.\u003c/p\u003e","description":"","filename":"Fig5New.png","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/7755f7b63ccb2fe9f7ae9c90.png"},{"id":58706088,"identity":"ec299846-6a49-4225-8216-7ca32affc7d3","added_by":"auto","created_at":"2024-06-20 05:21:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4448405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo effects of circRACK1 knockdown on gastric cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA．Graph illustrating the accelerated growth of subcutaneous tumors in nude mice injected with circRACK1 knockdown cells compared to control.\u003c/p\u003e\n\u003cp\u003eB. Imaging data showcasing larger tumor sizes in the knockdown group versus control.\u003c/p\u003e\n\u003cp\u003eC. Measurement data supporting the increased tumor volumes post circRACK1 suppression.\u003c/p\u003e\n\u003cp\u003eD. F-ACTIN staining indicating increased cytoskeletal activity in tumors from the circRACK1 knockdown group.\u003c/p\u003e\n\u003cp\u003eE. Imaging data revealing a higher frequency and volume of distant metastases in the knockdown group.\u003c/p\u003e\n\u003cp\u003eF-G. Post-mortem examination data showing a greater incidence and size of lung metastases in mice with circRACK1 knockdown.\u003c/p\u003e\n\u003cp\u003eH. Functional and Mechanistic Insights of circR-127aa Mediated Regulation in Gastric Cancer.\u003c/p\u003e","description":"","filename":"Fig6New1.png","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/54045326b5292670de8d868f.png"},{"id":58831807,"identity":"c11f100c-461f-45a1-8e60-6c8bb47654ad","added_by":"auto","created_at":"2024-06-21 17:42:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26253673,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/993cd013-2871-42ed-8afe-51d62c664551.pdf"},{"id":58706085,"identity":"d3fccb0d-7d2b-4425-b418-94285fe36c52","added_by":"auto","created_at":"2024-06-20 05:21:15","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":18552,"visible":true,"origin":"","legend":"","description":"","filename":"DetailedInformationofAntibodiesUsedintheStudy.docx","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/8152c55d78f0e68207784e10.docx"},{"id":58706091,"identity":"2466d64b-3561-4514-9a80-92a7dab526d5","added_by":"auto","created_at":"2024-06-20 05:21:16","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":2451848,"visible":true,"origin":"","legend":"","description":"","filename":"S1New.tif","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/3e22ed8fefd3b0557f33aa5e.tif"},{"id":58706089,"identity":"6fa543e8-ad9e-4001-9f8a-985aa001c27f","added_by":"auto","created_at":"2024-06-20 05:21:16","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":3646452,"visible":true,"origin":"","legend":"","description":"","filename":"S2New.tif","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/7f3558ebcdc95503b3ec406a.tif"},{"id":58706096,"identity":"6ed0ace2-7979-416d-8969-084b6c0f2255","added_by":"auto","created_at":"2024-06-20 05:21:17","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":27477920,"visible":true,"origin":"","legend":"","description":"","filename":"S3New.tif","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/8faf4f6510a9112d5d6ef87b.tif"},{"id":58706093,"identity":"8f4ebfc3-67cd-41b3-b9fc-4db94ae555d4","added_by":"auto","created_at":"2024-06-20 05:21:16","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":24521056,"visible":true,"origin":"","legend":"","description":"","filename":"S4New.tif","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/af42d059cf228461a026a6cd.tif"},{"id":58706095,"identity":"4e01d585-aa0f-4c9f-9b96-71f47da97a46","added_by":"auto","created_at":"2024-06-20 05:21:16","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":14171040,"visible":true,"origin":"","legend":"","description":"","filename":"S5New.tif","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/e175f7e5d102175340f97b65.tif"},{"id":58706097,"identity":"f67a7398-0d55-4308-84a1-e6a12997b983","added_by":"auto","created_at":"2024-06-20 05:21:17","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":15110388,"visible":true,"origin":"","legend":"","description":"","filename":"S6New.tif","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/bab154fbfc490af69fb051a8.tif"},{"id":58706094,"identity":"ef05b00e-3188-41fe-ab7f-c49f182d8a6f","added_by":"auto","created_at":"2024-06-20 05:21:16","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":27977696,"visible":true,"origin":"","legend":"","description":"","filename":"S7New.tif","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/ed8ca78f7051b2e5a7a50d22.tif"},{"id":58706092,"identity":"17d80e5e-dc12-4e9b-b332-fab4ab43b93d","added_by":"auto","created_at":"2024-06-20 05:21:16","extension":"docx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":16207,"visible":true,"origin":"","legend":"","description":"","filename":"SequencesofPrimersandsiRNAsUsedintheStudy.docx","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/9531fd4898013a6bb2221ea8.docx"},{"id":58706090,"identity":"50630695-1973-4f16-a808-07a18888754f","added_by":"auto","created_at":"2024-06-20 05:21:16","extension":"docx","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":10712916,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigurelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-4522505/v1/daa2e49f4c2681fdc25b994f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel polypeptide encoded by the circRACK1 suppresses gastric cancer via cytoskeleton remodeling","fulltext":[{"header":"Background","content":"\u003cp\u003eGastric cancer, ranking as the fifth most prevalent cancer globally, is the third leading cause of cancer-related deaths. Particularly in China[1], this malignancy presents a formidable challenge due to its complex metastatic routes encompassing direct infiltration, hematogenous spread, transcoelomic dissemination, and lymphatic metastasis. Compounded by the frequent late-stage diagnosis, the overall 5-year survival rate in China falls below 30%[2, 3], underscoring the urgency for novel insights into gastric cancer\u0026apos;s molecular mechanisms and effective early detection and intervention strategies.\u003c/p\u003e\n\u003cp\u003eCircular RNAs (circRNAs) have emerged as a distinct class of molecules, characterized by their covalently closed-loop structures, absence of 5\u0026apos;-3\u0026apos; polarity, and lack of a polyadenylated tail[4, 5]. Their unique configuration endows them with remarkable stability, making them abundant across various species[6]. Although the full spectrum of their functions is not entirely understood, recent evidence points to their significant roles in cell proliferation[7], apoptosis[8], migration and invasion[9, 10], thereby implicating them in various cancer types. In gastric cancer, the role of circRNAs and their underlying mechanisms remain largely unexplored, presenting a frontier for in-depth research.\u003c/p\u003e\n\u003cp\u003eRACK1 (Receptor for Activated C Kinase 1) is a multifunctional scaffold protein integral to numerous cellular processes[11]. Its involvement in growth, differentiation, invasion, and migration, and its dysregulation in various tumors, highlights its importance in cancer biology[12]. Interestingly, RACK1 exhibits both tumor-promoting and tumor-suppressing activities, depending on the cancer type and cellular context[13-17]. This duality adds complexity to understanding its role in gastric cancer.\u003c/p\u003e\n\u003cp\u003eIn this study, we identified a novel circular RNA, circRACK1, originating from the RACK1 gene, in gastric cancer cell lines. This circRNA was found to be down-regulated in these cells. More intriguingly, we discovered its potential to encode a novel peptide, circR-127aa, which appears to play a significant role in modulating vimentin ubiquitination during the epithelial-mesenchymal transition (EMT), a key process in cytoskeleton remodeling and cell mobility. Our research thus adds a new dimension to understanding circRACK1\u0026apos;s involvement in gastric cancer and positions circR-127aa as a potential biomarker and therapeutic target.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell Lines and Culture Conditions:\u003c/strong\u003e Our study utilized the human gastric epithelial cell line GES-1 and various human gastric cancer cell lines (HGC27, MKN-28, AGS, MKN-45, MGC-803, BGC-823), all sourced from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. GES-1 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS), while the gastric cancer cell lines were cultured in RPMI 1640 medium also enriched with 10% FBS. Standard incubation conditions involved a temperature of 37\u0026deg;C and an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransfection, Oligonucleotides, and Plasmids:\u0026nbsp;\u003c/strong\u003eCustom oligonucleotides and plasmids were designed to manipulate the expression of circRACK1 and Vimentin. Targeted siRNAs against circRACK1 were synthesized, alongside overexpression constructs for circRACK1-3\u0026times;Flag and Vimentin. These were transfected into cells using Lipofectamine\u0026trade; 2000, following manufacturer protocols. Stable cell lines were generated via lentiviral infection, followed by a 2-week selection in puromycin. The sequences for siRNA and the control group are detailed in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Isolation and RT-qPCR:\u003c/strong\u003e Total RNA extraction from gastric cell lines was performed using TRIzol reagent, followed by reverse transcription into cDNA. Quantitative real-time PCR was conducted to assess the expression levels of target RNAs, with GAPDH serving as an internal control. The 2\u003csup\u003e-\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method was employed for data analysis. The sequences of all primers used in the experiments are listed in Table 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein Studies:\u0026nbsp;\u003c/strong\u003eWestern Blotting and Coimmunoprecipitation (Co-IP): Protein extraction utilized SDS-lysis buffer, followed by concentration determination using a BCA Protein Assay kit. Proteins were separated via SDS-PAGE, transferred to PVDF membranes, and probed with primary and secondary antibodies for visualization. For Co-IP, cell lysates were incubated with primary antibodies and Protein A/G beads, followed by immunoprecipitation and analysis via Western blotting. The antibody information used for the Western Blot (WB) experiment is presented in Table 3. The details of the antibodies employed in the Co-immunoprecipitation (CO-IP) experiment can be found in Table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence Assay:\u0026nbsp;\u003c/strong\u003eCells were fixed, permeabilized, and blocked prior to incubation with primary antibodies. Following washes, fluorescently labeled secondary antibodies were applied. Nuclei were stained with DAPI, and fluorescence microscopy was utilized for visualization. the antibody information for the Immunofluorescence (IF) experiment is located in Table 5.\u0026quot;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Methods:\u003c/strong\u003e The methods section also detailed procedures for mass spectrometry analysis, ubiquitination assays, cell wound healing and transwell assays, cell proliferation assays (including CCK8, colony-forming, and EdU assays), flow cytometry analysis for apoptosis, HUVEC tube formation assay, immunohistochemistry, and animal experiments. Each of these techniques was carefully executed to ensure the reliability and validity of our findings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis:\u0026nbsp;\u003c/strong\u003eData were presented as mean \u0026plusmn; SEM and analyzed using two-tailed paired or non-paired t-tests as appropriate, with P values \u0026lt;0.05 considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe expression of circRACK1 is downregulated in GC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecent advancements in our understanding of circRNAs, particularly their role in protein translation via CAP-independent mechanisms, have not only expanded our knowledge of these molecules but also highlighted their potential in disease diagnosis and treatment. Current research indicates that circRNA translation is tightly regulated under stress conditions, possibly serving as a compensatory cellular mechanism. Notably, hypoxia, a hallmark of the tumor microenvironment and a key feature of solid tumors, may influence circRNA expression. In this context, we investigated whether circRNA expression in gastric cancer (GC) cells alters under hypoxic conditions.\u003c/p\u003e\n\u003cp\u003eWe cultured HGC27 cells, which were in the logarithmic growth phase, in a hypoxic incubator (5% CO\u003csub\u003e2\u003c/sub\u003e, \u0026lt;1% O\u003csub\u003e2\u003c/sub\u003e) for 36 hours. Post-cultivation, we lysed the cells and conducted Ribo-seq detection. Our circular heatmap analysis revealed a notable decrease in the expression of a novel circRNA, designated as novel_circ_000494, under these hypoxic conditions (see Fig. 1A). Further investigation in the Circbase database identified this circRNA as hsa_circ_0075402, located on chromosome 5:181241492-181242345. Its host gene is RACK1 (ENSG00000204628). Subsequent Sanger sequencing analysis showed that this circRNA, which we have named circRACK1, forms a closed-loop structure through the reverse splicing of two exons from the RACK1 gene (exon1: 181242345-181242174, exon2: 181241639-181241492), as illustrated in（Fig. 1B）.\u003c/p\u003e\n\u003cp\u003eTo confirm the circular structure of circRACK1, we designed convergent and divergent primers for amplifying circRACK1 and linear RACK1, respectively. Our results, depicted in Fig. 1C, demonstrate that circRACK1 is exclusively amplified from cDNA, confirming its nature as a back-spliced product of the host mRNA. Additionally, circRACK1 exhibited significantly higher resistance to RNase R degradation compared to its linear mRNA counterpart (Fig. 1D-E). Actinomycin D assays further confirmed the enhanced stability of circRACK1, showing a markedly longer half-life than the linear RACK1 transcript (Fig. 1F-G).\u003c/p\u003e\n\u003cp\u003eRT-qPCR detection in common GC cells revealed a reduction in circRACK1 expression. Particularly, AGS and HGC27 cell lines, showing the lowest circRACK1 levels, were selected for further study (Fig. 1H). Upon re-examination under hypoxic conditions, we observed an additional decrease in circRACK1 expression levels (Fig. 1I-J).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCircRACK1 inhibits malignant phenotypes of gastric cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delve into the functional importance of circRACK1 in GC cells, we strategically designed siRNAs targeting circRACK1\u0026apos;s specific binding sites and introduced overexpression plasmids into HGC27 and AGS cells. This intervention notably impacted the expression levels of circRACK1, while the mRNA and protein levels of its host gene, RACK1, remained largely unaffected, as illustrated in Fig(S1 A-F).\u003c/p\u003e\n\u003cp\u003eOur research extended to evaluating the effect of circRACK1 on the proliferation of GC cells. EdU incorporation analysis further corroborated these findings, showing increased EdU assimilation in cells with diminished circRACK1 expression, and the opposite in those with heightened expression (Fig 2A-B, S2A-B). The results from colony formation assays were in line with these observations. Cells overexpressing circRACK1 formed fewer and smaller colonies, whereas inhibition of circRACK1 resulted in a significant increase in colony numbers, underscoring its suppressive role in GC cell proliferation (Figure S3A).The role of circRACK1 in apoptosis was elucidated through flow cytometry, employing ANEXIN V-PI. The data revealed that circRACK1 knockdown markedly diminished apoptosis in GC cells, while its overexpression promoted apoptotic processes (Fig S2E-F).Our investigation further spanned the influence of circRACK1 on GC cell migration and angiogenesis. Transwell assays demonstrated an increased migration of cells in the circRACK1 knockdown groups and a decrease in the overexpressed groups (Fig 2C, S2C-D). Scratch assays reinforced these findings, indicating that high levels of circRACK1 slow down wound healing, whereas its knockdown accelerates this process (Figure S3B-C). These outcomes suggest a significant inhibitory effect of circRACK1 on the migratory capabilities of GC cells. Moreover, the angiogenic potential of GC cells, assessed through their impact on HUVEC-induced migration and tube formation, was enhanced in circRACK1 knockdown cells, while overexpression of circRACK1 exhibited the opposite effect (Figure 2D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCircR-127aa encoded by circRACK1 inhibits gastric cancer cell malignancy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn exploring the coding potential of circRACK1, our search in the CIRCRNADb and TransCirc databases revealed multiple internal ribosome entry sites (IRES) and open reading frames (ORFs) within circRACK1, hinting at its possible coding function. To test the translatability of circRACK1, we analyzed its putative ORF using ORF Finder. We identified a potential overlapping ORF (384nt) within circRACK1, capable of encoding a peptide of 127 amino acids in length (Fig 3A). We cloned the circRACK1 sequence into a circRNA expression vector (circRACK1-3xFLAG), with a FLAG tag positioned before the stop codon to detect successful circularization. Immunoblotting suggested that circRACK1 might encode a new protein of approximately 15kDa. To verify this protein\u0026apos;s origin from circRACK1, we engineered plasmids with FLAG-tagged start codon mutations (ATG-ATT) and empty vector controls. The unique peptide fragment formed at the junction site of circRACK1 is illustrated in Fig 3B. Post-transfection of these plasmids into HGC27 and AGS cells, followed by Western Blot analysis after 48 hours, FLAG was detected in cells overexpressing circRACK1 but not in those with the vector or circRACK1-mut plasmid (Fig 3C). Given that part of circRACK1-encoded circR-127aa sequence overlaps with the host gene RACK1, we considered the possibility of RACK1 polyclonal antibody binding to circR-127aa. Using an anti-RACK1 antibody, we detected circR-127aa, observing increased expression levels following overexpression of the OE plasmid, while the expression of host gene RACK1 remained relatively unchanged (Fig 3D). Immunofluorescence experiments further confirmed the presence of circR-127aa in HGC27 and AGS cells, predominantly localized in the cytoplasm and enriched on the cell membrane (Fig 3E).To ascertain whether the biological impact of circRACK1 on gastric cancer cells is mediated through itself or its translation products, we re-examined common tumor phenotypes using circRACK1 overexpression plasmids, circRACK1-mut, and control plasmids. Gastric cancer cells transfected with circRACK1-mut plasmids showed no significant changes in proliferation, migration, and scratch healing capabilities compared to control group plasmids. However, cells transfected with circRACK1 overexpression plasmids exhibited an inhibitory effect on these phenotypes (Fig S4 A-D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCircR-127aa promotes ubiquitination and degradation of vimentin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the biological mechanism behind circR-127aa\u0026apos;s influence on gastric cancer behavior, we conducted immunoprecipitation experiments to identify proteins potentially interacting with circR-127aa. The immunoprecipitated products were subjected to gel electrophoresis and stained with Coomassie Brilliant Blue to accentuate the distinct bands between the Vector and OE groups. These bands were then analyzed through mass spectrometry, indicating potential interactions of circR-127aa with Vimentin and ACTIN (Fig 3F, S5A). Further immunoprecipitation assays validated these interactions (Fig 3G, H, S6B).\u003c/p\u003e\n\u003cp\u003eWe also performed immunofluorescence co-localization experiments, observing that circR-127aa co-localizes with Vimentin and ACTIN, particularly on the cell membrane\u0026nbsp;(Fig S5C-D). To explore the regulatory mechanism of circR-127aa on Vimentin expression in gastric cancer cells, we conducted RT-qPCR and Western Blotting experiments. These revealed that Vimentin expression did not significantly change in the gastric cancer cell line with overexpressed or knocked-down circRACK1 compared to the control group\u0026nbsp;(Fig S6A,-B).\u0026nbsp;However, Western Blotting showed a decrease in Vimentin expression following circRACK1 overexpression and an increase following circRACK1 knockdown (Fig4A-B).\u003c/p\u003e\n\u003cp\u003eWe further treated OE and shRNA group cells with cycloheximide (CHX) to inhibit new protein synthesis in gastric cancer cells. Results indicated a significant reduction in Vimentin expression in the OE group compared to the ShRNA group (Fig 4C). After treatment with the proteasome inhibitor MG132, Vimentin expression levels in the OE group\u0026apos;s two cell lines recovered, suggesting that circR-127aa might influence Vimentin expression through ubiquitin-mediated proteasomal degradation (Fig 4D). In HEK293T cells transfected with HA-ubiquitin and Vimentin overexpression plasmids, circRACK1, or Vector plasmids for 48 hours, exogenous ubiquitin detection revealed increased ubiquitination modifications in the OE group compared to the Vector group (Fig 4E). Lastly, immunoprecipitation experiments on two stable cell lines showed that circRACK1 knockdown decreased Vimentin ubiquitination, while its overexpression increased Vimentin ubiquitination (Fig 4F)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModulatory Effects of the HIF-1\u0026alpha;/circRACK1/vimentin Axis on Gastric Cancer Cell Behavior\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the HGC27 gastric cancer cell line, circRACK1 overexpression (OE) led to an increase in E-cadherin levels and a decrease in N-cadherin, \u0026beta;-catenin, MMP9, and Snail, indicative of reduced invasive capacity. This modulation was reversed by concurrent HIF-1\u0026alpha; overexpression (HIF-1\u0026alpha; OE), which suggests a regulatory mechanism where HIF-1\u0026alpha; enhances cancer cell invasion and migration by suppressing circRACK1 (Fig5A,S6A). Colony formation and invasion assays further supported the role of circRACK1 in altering cellular behaviors. The cells with circRACK1 OE displayed a diminished ability to form colonies and invade through the matrix. This was notably counteracted by co-overexpression with vimentin (OE+Vim), placing vimentin as a key effector in circRACK1-driven functions (Fig5B-C,S6B-C). Immunofluorescence assays demonstrated that circRACK1 silencing (sh-circR) results in significant cytoskeletal reorganization, characterized by increased pseudopodia, suggesting a promotion of cellular motility, as opposed to the more stable architecture observed in the OE group (Fig5D,S6D). Western blot analysis across different genetic backgrounds established that circRACK1 knockdown and vimentin co-overexpression increased the levels of mesenchymal markers while decreasing the epithelial marker E-cadherin, aligning with the molecular signature of EMT (Fig5E,S6E). Collectively, these results illuminate the comprehensive role of circRACK1 and its peptide product, CircR-127aa, in modulating gastric cancer cell behavior through EMT and cytoskeletal dynamics, potentially orchestrated by vimentin interactions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of circRACK1 promotes gastric cancer growth and metastasis in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate the influence of circRACK1 on the proliferation of gastric cancer cells in vivo, we utilized stable cell lines with circRACK1 knockdown (experimental group) and corresponding control cell lines (control group) to establish subcutaneous tumors in nude mice. Regular tumor volume measurements indicated that circRACK1 knockdown enhances subcutaneous tumor growth (Fig6A，C, S7C). live animal imaging showed larger tumors in the circRACK1 knockdown group (Fig6B). Subsequent H\u0026amp;E staining and immunohistochemical analyses of the tumors indicated significantly increased expression of Ki-67, Vimentin, MMP9, and CD31 in the experimental group compared to the control group. This suggests enhanced proliferation and invasion capacities of tumor cells and increased microvascular density when circRACK1 is knocked down. Lower expression of Caspase 3 in the experimental group implied reduced apoptosis in tumor cells (FigS7F). Additionally, F-ACTIN staining within the tissues showed increased fluorescence intensity in mouse tumors with circRACK1 knockdown (Fig 6D).During the live imaging and pathological examination of the subcutaneously implanted tumors in the nude mice, we observed more instances of intrathoracic and abdominal metastases in the experimental group than in the control group (S7D,E). We injected the stable cell lines with circRACK1 knockdown (experimental group) and the corresponding control cell lines (control group) into the tail vein of mice to create a distant metastasis model. After four weeks, we conducted regular small animal live imaging and CT scans for distant metastases monitoring. Starting from the fifth week post-model establishment, more distant metastases were observed in the experimental group compared to the control group (Fig6E, S7G). Upon sacrificing and dissecting the mice at eight weeks post-modeling, we found that the experimental group exhibited more and larger lung metastases (Fig6F-G).\u003c/p\u003e\n\u003cp\u003eAdditionally, we examined the livers of the mice and noted metastatic lesions in the liver of some mice in the experimental group. We selected typical hepatic metastatic lesions for observation and documentation (Fig S7H).\u003c/p\u003e\n\u003cp\u003eIn the concluding section of our study, we present a detailed schematic diagram illustrating the role of the peptide circR-127aa, encoded by circRACK1, in gastric cancer (GC). This diagram emphasizes the tumor-suppressing function of circR-127aa, particularly highlighting its involvement in the ubiquitination process of Vimentin, thereby elucidating its significance in the pathophysiology of GC（Fig6H）.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur research has identified a novel circRNA, circRACK1, originating from the RACK1 gene. This discovery is pivotal in understanding gastric cancer biology. Notably, we observed a significant downregulation of circRACK1 in gastric cancer cells, particularly under hypoxic conditions. The unique circular structure of circRACK1, formed via exon splicing, comprises a 320-nucleotide sequence. This finding enriches the existing literature on RACK1\u0026apos;s tumor-suppressive functions in gastric cancer, including its role in inhibiting cell proliferation and the epithelial-mesenchymal transition (EMT) through various signaling pathways[18, 19].\u003c/p\u003e\n\u003cp\u003eHypoxia, prevalent in the tumor microenvironment of solid tumors[20, 21], induces the upregulation of HIF-1\u0026alpha;, a factor known to accelerate metastasis gastric cancer[22]. Under hypoxic conditions, the expression of RACK1 and consequently circRACK1 is reduced, promoting invasion and angiogenesis via the HIF-1\u0026alpha; and VEGF pathways[23]. This highlights the critical need for further investigation into circRACK1\u0026apos;s function in gastric cancer, particularly considering environmental factors like hypoxia.\u003c/p\u003e\n\u003cp\u003eCompared to linear host gene products, proteins encoded by circRNAs often possess unique and independent biological functions[24, 25].Our findings extend beyond the sequence of circRACK1 to its encoded protein, circR-127aa. This peptide exerts tumor-suppressive effects by interacting with vimentin/actin proteins and promoting vimentin ubiquitination, crucial for cytoskeleton remodeling. These interactions inhibit gastric cancer cell proliferation, migration, and invasion, while promoting apoptosis. Thus, circR-127aa plays a significant role in modulating the biological functions of gastric cancer.\u003c/p\u003e\n\u003cp\u003eVimentin ubiquitination, a crucial post-translational modification, regulates its signaling pathway, pivotal in controlling gastric cancer cell malignancy [26, 27]. Our findings suggest that the dynamic balance of vimentin ubiquitination and deubiquitination offers a complex regulatory mechanism, potentially exploitable in gastric cancer therapy. Mass spectrometry analysis confirmed circR-127aa\u0026apos;s interaction with Vimentin and Actin, key proteins in cytoskeletal remodeling and tumor cell metastasis[28, 29],elucidating the underlying mechanisms of tumor malignancy and metastasis in gastric cancer.\u003c/p\u003e\n\u003cp\u003eDespite our significant findings, this study has limitations. The insights into circRACK1\u0026apos;s role in gastric cancer require further validation through extensive in vivo studies and analysis of clinical samples. Additionally, while we explored the interactions of circR-127aa with vimentin and actin, a comprehensive understanding of its network within gastric cancer cells is yet to be achieved. Future research should investigate these interactions in greater detail and assess circR-127aa\u0026apos;s impact on patient prognosis and treatment response. Also, considering the complexity of the tumor microenvironment, the role and expression of circRACK1 and circR-127aa under varying conditions warrant further investigation.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study introduces circR-127aa as a novel peptide encoded by circRACK1, with significant implications as a diagnostic marker and therapeutic target in gastric cancer. Our findings pave the way for future clinical studies to fully elucidate the therapeutic potential and clinical utility of circR-127aa in gastric cancer management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Tianjin Key Medical Discipline(Specialty) Construction Project（TJYXZDXK-009A）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproval of the research protocol by an Institutional Reviewer Board. N/A.\u003c/p\u003e\n\u003cp\u003eInformed Consent. N/A.\u003c/p\u003e\n\u003cp\u003eRegistry and the Registration No. of the study/trial. N/A.\u003c/p\u003e\n\u003cp\u003eAnimal Studies. The operational process was executed within the approve of the\u003c/p\u003e\n\u003cp\u003eInstitutional Animal Care and Research Advisory Committee of Tianjin Medical\u003c/p\u003e\n\u003cp\u003eUniversity Cancer Institute and Hospital.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eH. Sung, J. Ferlay, R.L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F. Bray, Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries, CA: a cancer journal for clinicians, 71 (2021) 209-249.\u003c/li\u003e\n\u003cli\u003eH. Zeng, W. Chen, R. Zheng, S. Zhang, J.S. Ji, X. Zou, C. Xia, K. Sun, Z. Yang, H. Li, N. Wang, R. Han, S. Liu, H. Li, H. Mu, Y. He, Y. Xu, Z. Fu, Y. Zhou, J. Jiang, Y. Yang, J. Chen, K. Wei, D. Fan, J. Wang, F. Fu, D. Zhao, G. Song, J. Chen, C. Jiang, X. Zhou, X. Gu, F. Jin, Q. Li, Y. Li, T. Wu, C. Yan, J. Dong, Z. Hua, P. Baade, F. Bray, A. Jemal, X.Q. Yu, J. He, Changing cancer survival in China during 2003-15: a pooled analysis of 17 population-based cancer registries, The Lancet. Global health, 6 (2018) e555-e567.\u003c/li\u003e\n\u003cli\u003eW. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X.Q. Yu, J. He, Cancer statistics in China, 2015, CA: a cancer journal for clinicians, 66 (2016) 115-132.\u003c/li\u003e\n\u003cli\u003eL.S. Kristensen, M.S. Andersen, L.V.W. Stagsted, K.K. Ebbesen, T.B. Hansen, J. Kjems, The biogenesis, biology and characterization of circular RNAs, Nature reviews. Genetics, 20 (2019) 675-691.\u003c/li\u003e\n\u003cli\u003eL.L. Chen, The expanding regulatory mechanisms and cellular functions of circular RNAs, Nature reviews. Molecular cell biology, 21 (2020) 475-490.\u003c/li\u003e\n\u003cli\u003eJ. Salzman, R.E. Chen, M.N. Olsen, P.L. Wang, P.O. Brown, Cell-type specific features of circular RNA expression, PLoS genetics, 9 (2013) e1003777.\u003c/li\u003e\n\u003cli\u003eH. Pan, T. Li, Y. Jiang, C. Pan, Y. Ding, Z. Huang, H. Yu, D. Kong, Overexpression of Circular RNA ciRS-7 Abrogates the Tumor Suppressive Effect of miR-7 on Gastric Cancer via PTEN/PI3K/AKT Signaling Pathway, Journal of cellular biochemistry, 119 (2018) 440-446.\u003c/li\u003e\n\u003cli\u003eY. Enuka, M. Lauriola, M.E. Feldman, A. Sas-Chen, I. Ulitsky, Y. Yarden, Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor, Nucleic acids research, 44 (2016) 1370-1383.\u003c/li\u003e\n\u003cli\u003eM. Jie, Y. Wu, M. Gao, X. Li, C. Liu, Q. Ouyang, Q. Tang, C. Shan, Y. Lv, K. Zhang, Q. Dai, Y. Chen, S. Zeng, C. Li, L. Wang, F. He, C. Hu, S. Yang, CircMRPS35 suppresses gastric cancer progression via recruiting KAT7 to govern histone modification, Molecular cancer, 19 (2020) 56.\u003c/li\u003e\n\u003cli\u003e J. Meng, S. Chen, J.X. Han, B. Qian, X.R. Wang, W.L. Zhong, Y. Qin, H. Zhang, W.F. Gao, Y.Y. Lei, W. Yang, L. Yang, C. Zhang, H.J. Liu, Y.R. Liu, H.G. Zhou, T. Sun, C. Yang, Twist1 Regulates Vimentin through Cul2 Circular RNA to Promote EMT in Hepatocellular Carcinoma, Cancer research, 78 (2018) 4150-4162.\u003c/li\u003e\n\u003cli\u003e D.R. Adams, D. Ron, P.A. Kiely, RACK1, A multifaceted scaffolding protein: Structure and function, Cell communication and signaling : CCS, 9 (2011) 22.\u003c/li\u003e\n\u003cli\u003e D. Duff, A. Long, Roles for RACK1 in cancer cell migration and invasion, Cellular signalling, 35 (2017) 250-255.\u003c/li\u003e\n\u003cli\u003e Y. Ruan, L. Sun, Y. Hao, L. Wang, J. Xu, W. Zhang, J. Xie, L. Guo, L. Zhou, X. Yun, H. Zhu, A. Shen, J. Gu, Ribosomal RACK1 promotes chemoresistance and growth in human hepatocellular carcinoma, The Journal of clinical investigation, 122 (2012) 2554-2566.\u003c/li\u003e\n\u003cli\u003e C. Shen, H. Hua, L. Gu, S. Cao, H. Cai, X. Yao, X. Chen, Overexpression of RACK1 Predicts Poor Prognosis in Melanoma, Journal of Cancer, 11 (2020) 795-803.\u003c/li\u003e\n\u003cli\u003e X. Li, Y. Xiao, S. Fan, M. Xiao, X. Wang, X. Chen, C. Li, G. Zong, G. Zhou, C. Wan, RACK1 overexpression associates with pancreatic ductal adenocarcinoma growth and poor prognosis, Experimental and molecular pathology, 101 (2016) 176-186.\u003c/li\u003e\n\u003cli\u003e L. Chen, L. Min, X. Wang, J. Zhao, H. Chen, J. Qin, W. Chen, Z. Shen, Z. Tang, Q. Gan, Y. Ruan, Y. Sun, X. Qin, J. Gu, Loss of RACK1 Promotes Metastasis of Gastric Cancer by Inducing a miR-302c/IL8 Signaling Loop, Cancer research, 75 (2015) 3832-3841.\u003c/li\u003e\n\u003cli\u003e Z. Yu, X. Jiang, L. Qin, H. Deng, J. Wang, W. Ren, H. Li, L. Zhao, H. Liu, H. Yan, W. Shi, Q. Wang, C. Luo, B. Long, H. Zhou, H. Sun, Z. Jiao, A novel UBE2T inhibitor suppresses Wnt/\u0026beta;-catenin signaling hyperactivation and gastric cancer progression by blocking RACK1 ubiquitination, Oncogene, 40 (2021) 1027-1042.\u003c/li\u003e\n\u003cli\u003e J.J. Li, D. Xie, RACK1, a versatile hub in cancer, Oncogene, 34 (2015) 1890-1898.\u003c/li\u003e\n\u003cli\u003e C. Liu, L. Ren, Y. Wang, Y. Liu, J. Xiao, The interaction between RACK1 and WEE1 regulates the growth of gastric cancer cell line HGC27, Oncology letters, 14 (2017) 4784-4792.\u003c/li\u003e\n\u003cli\u003e H. Choudhry, A.L. Harris, Advances in Hypoxia-Inducible Factor Biology, Cell metabolism, 27 (2018) 281-298.\u003c/li\u003e\n\u003cli\u003e Z. Chen, F. Han, Y. Du, H. Shi, W. Zhou, Hypoxic microenvironment in cancer: molecular mechanisms and therapeutic interventions, Signal transduction and targeted therapy, 8 (2023) 70.\u003c/li\u003e\n\u003cli\u003e X. Wang, X. Che, Y. Yu, Y. Cheng, M. Bai, Z. Yang, Q. Guo, X. Xie, D. Li, M. Guo, K. Hou, W. Guo, X. Qu, L. Cao, Hypoxia-autophagy axis induces VEGFA by peritoneal mesothelial cells to promote gastric cancer peritoneal metastasis through an integrin \u0026alpha;5-fibronectin pathway, Journal of experimental \u0026amp; clinical cancer research : CR, 39 (2020) 221.\u003c/li\u003e\n\u003cli\u003e S.J. Yang, Y.S. Park, J.H. Cho, B. Moon, H.J. An, J.Y. Lee, Z. Xie, Y. Wang, D. Pocalyko, D.C. Lee, H.A. Sohn, M. Kang, J.Y. Kim, E. Kim, K.C. Park, J.A. Kim, Y.I. Yeom, Regulation of hypoxia responses by flavin adenine dinucleotide-dependent modulation of HIF-1\u0026alpha; protein stability, The EMBO journal, 36 (2017) 1011-1028.\u003c/li\u003e\n\u003cli\u003e J.N. Vo, M. Cieslik, Y. Zhang, S. Shukla, L. Xiao, Y. Zhang, Y.M. Wu, S.M. Dhanasekaran, C.G. Engelke, X. Cao, D.R. Robinson, A.I. Nesvizhskii, A.M. Chinnaiyan, The Landscape of Circular RNA in Cancer, Cell, 176 (2019) 869-881.e813.\u003c/li\u003e\n\u003cli\u003e J. Guarnerio, M. Bezzi, J.C. Jeong, S.V. Paffenholz, K. Berry, M.M. Naldini, F. Lo-Coco, Y. Tay, A.H. Beck, P.P. Pandolfi, Oncogenic Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal Translocations, Cell, 165 (2016) 289-302.\u003c/li\u003e\n\u003cli\u003e Q. Li, M.S. Deng, R.T. Wang, H. Luo, Y.Y. Luo, D.D. Zhang, K.J. Chen, X.F. Cao, G.M. Yang, T.M. Zhao, B. Xu, C.X. Xu, J.M. Wang, PD-L1 upregulation promotes drug-induced pulmonary fibrosis by inhibiting vimentin degradation, Pharmacological research, 187 (2023) 106636.\u003c/li\u003e\n\u003cli\u003e W. Shao, J. Li, Q. Piao, X. Yao, M. Li, S. Wang, Z. Song, Y. Sun, L. Zheng, G. Wang, L. Liu, C. Yu, Y. Huang, Y. Bao, L. Sun, FRMD3 inhibits the growth and metastasis of breast cancer through the ubiquitination-mediated degradation of vimentin and subsequent impairment of focal adhesion, Cell death \u0026amp; disease, 14 (2023) 13.\u003c/li\u003e\n\u003cli\u003e Y. Hashimoto, D.J. Kim, J.C. Adams, The roles of fascins in health and disease, The Journal of pathology, 224 (2011) 289-300.\u003c/li\u003e\n\u003cli\u003e S. Lin, M.D. Taylor, P.K. Singh, S. Yang, How does fascin promote cancer metastasis?, The FEBS journal, 288 (2021) 1434-1446.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gastric cancer, circRACK1, Vimentin, cytoskeleton, Epithelial-mesenchymal transition (EMT）","lastPublishedDoi":"10.21203/rs.3.rs-4522505/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4522505/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGastric cancer (GC), particularly prevalent in China, is associated with high rates of morbidity and mortality. This study focuses on the role and mechanistic pathways of a novel 127-amino acid peptide, circR-127aa, encoded by hsa_circ_0075402 (circRACK1) in GC cells, exploring its implications in cancer development. Circular RNAs (circRNAs), which vary in expression across different cancers, contribute to tumorigenesis via mechanisms such as miRNA sponging, protein binding, and influencing transcription and translation.\u003c/p\u003e\n\u003cp\u003eOur research involved an extensive analysis of circRNAs ribo-seq data, complemented by mass spectrometry, Western blotting, and immunofluorescence to validate the encoding of circR-127aa by circRACK1. We explored the functional impact of circR-127aa, examining its effects on cell proliferation, apoptosis, and tumor formation in nude mice. A critical aspect of this study was investigating the interaction between circR-127aa and Vimentin, a significant player in cytoskeleton remodeling and cellular mobility during epithelial-mesenchymal transition (EMT). The findings reveal that the circRACK1-encoded peptide functions as a tumor suppressor, facilitating Vimentin ubiquitination, highlighting its potential as a novel therapeutic target and biomarker in GC treatment.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this research unveils a groundbreaking role of the circRACK1-encoded peptide, circR-127aa, in GC, emphasizing its tumor-suppressing function through the ubiquitination of Vimentin. This discovery enhances our understanding of GC progression and presents circR-127aa as a valuable candidate for therapeutic strategies and as a biomarker in GC treatment.\u003c/p\u003e","manuscriptTitle":"A novel polypeptide encoded by the circRACK1 suppresses gastric cancer via cytoskeleton remodeling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-20 05:21:10","doi":"10.21203/rs.3.rs-4522505/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3725e0dc-f019-49b6-9cd6-42a3ed9c247e","owner":[],"postedDate":"June 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-21T17:38:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-20 05:21:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4522505","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4522505","identity":"rs-4522505","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}