COPB2 promotes hepatocellular carcinoma progression through regulation of YAP1 nuclear translocation

COPB2 promotes hepatocellular carcinoma progression through regulation of YAP1 nuclear translocation | 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 COPB2 promotes hepatocellular carcinoma progression through regulation of YAP1 nuclear translocation Biao Wu, Yumeng Wu, Yifei Liu, Hongjian Chen, Wenjing Zhao, Jibin Liu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4117273/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 The Hippo pathway has been shown to be upregulated in many cancer patients. Yes-associated protein 1 (YAP1), an oncogene and core factor of the Hippo pathway, plays a key role in tumorigenesis and progression. Although YAP1 is a vital oncogene in HCC progression, its nuclear localization prevents its consideration as a potential therapeutic target. Recently, studies have reported that COPB2 also plays a critical role in HCC development, but its mechanism of action is unclear. Here, we found that COPB2 affects the drug sensitivity of HCC cells to DDP by regulating YAP1 nuclear translocation and stability. More importantly, COPB2 combined with YAP1 expression was related to overall postoperative survival in HCC patients and was an independent prognostic factor. The established nomogram and artificial neural network models also highlighted the prognostic value of these two genes for HCC patients. In summary, our findings suggest that COPB2/YAP1 affects the drug sensitivity of HCC cells to DDP and that targeting COPB2/YAP1 may be a promising strategy for the precision treatment of HCC. COPB2 YAP1 HCC prognosis DDP Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer death worldwide in 2020 [1] . Most HCC patients in China have a background of hepatitis B virus infection and the resulting cirrhosis [2] . Many patients are already in the middle to late stages at diagnosis and have a poor prognosis. The 5-year survival rate for advanced HCC patients is currently less than 5% [3] . In addition to liver transplantation, radical hepatectomy and ablation are the most effective treatments for early-stage patients [4] . Transarterial chemoembolization (TACE) is the first-line treatment for patients with intermediate to advanced HCC in the United States, Asia, and Europe [5] . It is indicated for unresectable patients who have relapsed after surgery or who have limited disease and have satisfactory preserved liver function [6] . Despite increasing improvements in the management and perioperative care of HCC patients, recurrence and metastasis remain key challenges to long-term survival [7] . As a result, the study of HCC-related genes and molecular biological mechanisms is of great clinical significance in predicting the prognosis of HCC patients and identifying potential targets for HCC therapy. The Hippo signaling pathway is a major cancer suppression pathway and plays a key role in inhibiting the proliferation and survival of HCC cells [8] . The Hippo pathway was originally identified and named by screening for mutant tumor suppressors in Drosophila [9] . The main composition of the pathway include the serine threonine kinase module and the transcriptional module [10] . The Hippo pathway has significant regulatory functions for organ development, regeneration and stem cell biology [11] . Once activated, the Hippo pathway undergoes a series of kinase phosphorylation reactions that ultimately lead to the inability of the downstream transcription factor Yes-associated protein 1 (YAP1) phosphorylation to enter the nucleus to activate gene transcription [12] . In contrast, inactivation of the Hippo pathway in tumors results in very common YAP1 dephosphorylation, and dephosphorylated YAP1 entry into the nucleus induces cell proliferation and expression of anti-apoptotic genes [13] . There is growing evidence that YAP1 is an essential oncogene as a downstream target of the Hippo pathway [14] . Intranuclear YAP1 expression plays a vital role in predicting post-operative survival and tumor recurrence in patients with HCC [15] . In addition, YAP1 has prognostic implications for patients treated with TACE. Although the core ingredients of the Hippo pathway have been studied in detail, the upstream regulators remain unclear. The coatomer protein complex subunit beta 2 (COPB2) is one of the component subunits of the coatomer complex I(COPI), a nuclear protein containing 906 amino acid residues and a molecular weight of 102 kD [16] . COPB2 has a tryptophan-aspartate (WD) repeat sequence, which is associated with signal transduction, regulation of the cell cycle and apoptosis [17] . It was found that COPB2 interacts with ADP-ribosylation factor (ARF) GTPase-activating protein 2 (GAP2) and KKXX-containing substances, and the product of this reaction is a coating of COPI-coated vesicles that plays a crucial role in vesicle transport and Golgi budding between the endoplasmic reticulum and Golgi apparatus [18] . Wang et al. and Couzens et al. found that vesicle transport regulates many signaling pathways, including the Hippo pathway, through proteomic studies [19][20] . Vesicular transport can affect the stability of YAP1 by regulating its phosphorylation [21] . In addition, COPB2 is abnormally highly expressed in many tumor tissues, including colon cancer [22] , prostate cancer [23] and gallbladder cancer [24] . An et al. reported that COPB2 inhibits the progression of gastric cancer through the RTK pathway [25] . PU et al. also found that COPB2 promotes lung cancer progression by regulating YAP1 nuclear translocation [26] . In a recent report, COPB2 promotes HCC progression by regulating the EMT pathway [27] . These results indicate that COPB2 plays a significant role in cancer development. However, the correlation between COPB2 and YAP1 in HCC has not been investigated. Our study found that COPB2 combined with YAP1 predicted the prognosis of HCC patients and developed associated nomogram and artificial neural network (ANN) prediction models. In addition, we found that COPB2 reduced the drug sensitivity to DDP in HCC cells by regulating YAP1 nuclear translocation. This finding may be critical in the progression and metastasis of HCC. Understanding this pathway may help identify new methods for the treatment of HCC. Materials and methods Database analysis TCGA-LIHC gene expression data were downloaded from the TCGA data portal (https://tcga-data.nci.nih.gov/tcga/ ) and three gene expression profiles (GSE102079 , GSE121248 and GSE14520) as raw data. The Human Protein Atlas web (http://www.proteinatlas.org/) was used to obtain relevant immunohistochemical staining images of COPB2 and YAP1 in HCC tissues. WGCNA analysis Weighted gene co-expression network analysis (WGCNA) of GSE02079 and GSE121248 expression matrices using the R package "WGCNA". Patient and clinical samples We retrospectively analyzed two independent cohorts comprising HCC 214 patients. In the training group, 114 HCC patients from the Affiliated Hospital of Nantong University from 2012-2017 were randomly selected. Moreover, 100 HCC patients from the Affiliated Tumor Hospital of Nantong University from 2011-2016 were randomly selected as the validation group. Cases are selected according to the following criteria: no previous anti-cancer related treatment prior to surgery; pathological diagnosis of HCC; availability of resected tissue and follow-up data. Patients receive a physical examination, laboratory diagnosis, and imaging twice a year after surgery. Most patients undergo TACE after relapse. Overall survival (OS) was measured from the date of surgery to the date of death or last follow-up. For each patient, the following clinicopathological information was collected: age, gender, hepatitis B virus (HBV), presence of cirrhosis, serum alpha-fetoprotein (AFP) level, tumor number, tumor size, degree of tumor differentiation, vascular tumor thrombus and lymph node metastasis. The staging of tumors is defined according to the American Joint Committee on Cancer/International Union Against Cancer's Tumor Lymph Node Metastasis (TNM) classification system. Written informed consent was obtained from all participating patients for the use of this clinical information, and the project was approved by the ethics committees of both hospitals. Immunohistochemistry The tissue microarrays (TMA) sections are dewaxed in xylene and rehydrated through a graded alcohol series. Pressure cook the sections in alkaline buffer (pH=9.0) for 3 minutes. After cooling at room temperature, immerse in 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. The sections were then incubated with rabbit polyclonal anti-COPB2 antibody (1:1000, Abcam, Cambridge, UK), YAP1 monoclonal antibody (1:200, Abcam, Cambridge, UK) overnight at 4℃. After washing three times with PBS, the sections were incubated with secondary antibodies (1:4000, Abcam, Cambridge, UK) for 20 min at room temperature, and then diaminobenzidine (DAB) solution was applied. Finally, sections were re-stained with hematoxylin, dehydrated and fixed. All microarrays were scored independently by two experienced pathologists. COPB2 scoring criteria: The percentage of positive cells was evaluated on a scale of 0–3:0(0–25%),1 (26–50%), 2 (51–75%), or 3(76–100%). Classified as low expression group:0-1 point or high expression group; 2-3 points. YAP1 scoring criteria, with a percentage of positive nuclei greater than 10% recorded as high expression and less than 10% recorded as low expression. Western blot HCC cells were lysed in cell lysis buffer on ice (Beyotime, Shanghai, China). The protein concentration was determined using the bicinchoninic acid assay (Beyotime, Shanghai, China). Electrophoresis was then performed on SDS-PAGE gels and transferred to PVDF membranes. The following antibodies were used in this study: COPB2 (1:1000, rabbit, Abcam, Cambridge, UK), YAP1 (1:500, rabbit, Abcam, Cambridge, UK), pLATS1(1:500, rabbit, Cell Signaling Technology, Massachusetts, USA), pYAP1 (1:500, rabbit, Cell Signaling Technology, Massachusetts, USA), GAPDH (1:5000, mouse, Abcam, Cambridge, UK). Cell culture and vectors The human HCC cell lines Huh7 and SK-hep1 were purchased from the Academy of Sciences Committee of the China Cell Resource Centre (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle 92 medium (DMEM, Hyclone, Logan, Utah, USA) containing 10% fetal bovine serum (FBS, Gibco, Grand 93 Island, NY, USA) at 37°C in a humidified atmosphere of 5% CO2. HCC cells in the exponential growth phase were used for subsequent experiments. The COPB2 siRNA was designed by Polyplus (Sense: CCCAUUAUGUUAUGCAGAUTT; Antisense‐1: AUCUGCAUAACAUAAUGGGTT). YAP1/pcDNA3.1 was constructed and the sequence was examined by GENECHEM. For transient expression, plasmids were transfected with Lipofectamine 2000 (Invitrogen, Shanghai, China) for 24 hours, after which the cells were processed with the indicated reagents as described above. Cell Counting Kit-8 (CCK-8) assay The Huh7 and SK-hep1 cells were digested with 0.25% trypsin and diluted to a 5×10 4 cells/ml concentration. The cells were counted using a cell counter(Thermo Fisher Scientific, Massachusetts, USA) and the cell suspension was inoculated into 96-well plates at a density of 2000 cells/well. Add 10μL CCK-8 solution (NCM, Suzhou, China) to each well, and culture at 37℃ for 2 hours. Cell viability was determined by measuring the absorbance at 450nm. Clone formation assay Briefly, Huh7 and SK-hep1 cells (2000 cells/well) were inoculated in 6-well plates and incubated at 37℃. The medium was changed every three days and observed cell growth. A total of 14 days after inoculation of cells, colonies with a cell count of > 50 were observed under a microscope. Cells were washed with PBS and fixed in 4% paraformaldehyde (Biosharp, Hefei, China) for 20 minutes at room temperature. Fixed cells were stained with 1 ml of Crystal Violet Staining Solution (Beyotime, Shanghai, China) for 10 minutes, washed with PBS and air dried at room temperature. The cells were photographed and the number of clones visible was counted. Transwell assay Migration assay: Transwell chambers were placed in 24-well plates. 2 × 10 5 Huh7 and SK-hep1 cells starved in 200 μL of serum-free medium were added to the upper chamber, and 500 μL of medium with 10% serum was added to the lower chamber. After 24 hours of incubation at 37℃ and 5% CO2, the Transwell chambers were first fixed in paraformaldehyde for 30 minutes and then stained with Crystal Violet Staining Solution for 10 minutes. The cells were photographed and counted under the microscope, with three replicates per group. Invasion assay: Melt Matrigel at 4℃ overnight and dilute with pre-cooled serum-free DMEM medium. Add 100µL of diluted Matrigel to the upper chamber. The remaining steps are the same as those in the Transwell migration assay Immunofluorescence Huh7 and SK-hep1 cells were inoculated in 24-well plates for 24 hours. Cells were then fixed in 4% paraformaldehyde for 15 minutes and permeabilized for 10 minutes. After being closed with 5% BSA for 1 hour, the cells were incubated overnight at 4°C with YAP1 antibody (1:100, Abcam, Cambridge, UK) and then Alexa Fluor 594 anti-rabbit IgG secondary antibody (Invitrogen, Shanghai, China). Furthermore, cell nuclei were stained with DAPI (Thermo Fisher). The images were taken under a fluorescent microscope. Statistical analysis Differences in expression were analyzed by paired t-test. Clinicopathological characteristics were assessed using the chi-square test. P < 0.05 was considered a statistically significant difference. Statistical analyses were performed using the SPSS 26.0 statistical package (SPSS, Inc, Chicago, IL) and GraphPad Prism version 7.01 (GraphPad Software, Inc, La Jolla, CA). Nomogram was developed using the Regression Modelling Strategies package in R (version 3.0.2; R Package "rms" for nomogram establishment; www.r-project.org). ANN was built using SPSS Clementine version 12.0 software for Windows (IBM Corporation). Results Expression of COPB2 and YAP1 in HCC tissues We analyzed the two HCC datasets using WGCNA analysis and obtained 927 intersecting genes in the most relevant module of HCC, and then we used the two datasets with larger sample sizes in the HCC dataset to obtain 394 shared genes that were significantly different in the two datasets (Figure 1 A-C). Then we constructed Protein-Protein Interaction Networks (PPI) for these genes and performed Hub gene analysis on the network and found that YAP1 was in the Hub gene network (Figure 1 D). To explore the connection between COPB2 and YAP1 expression in HCC tissues, we first searched the database. Notably, we found that COPB2 and YAP1 have a high correlation in TCGA-LIHC (Figure 1 E), and in the human protein atlas database COPB2 was highly expressed in the cytoplasm, YAP1 was also highly expressed in the nucleus. When COPB2 was lowly expressed, YAP1 was also lowly expressed (Figure 1 F). It is worth noting that we discovered that the COPB2 expression in the cytoplasm is positively associated with YAP1 expression in the nucleus. To verify the findings in the database, we performed immunohistochemical staining of COPB2 and YAP1 on tissue microarrays of 214 specimens from two hospitals (Figure 1 G). We found that COPB2 was predominantly present in the cytoplasm and YAP1 was expressed in both the nucleus and cytoplasm, and we grouped samples for high and low expression based on the two antibody staining scores. Among the 114 HCC specimens in the training group, the COPB2 low expression rate was 43.9% (50/114), and high expression rate was 56.1% (64/114), YAP1 low expression rate was 42.1% (48/114), and YAP1 high expression rate was 57.9% (66/114). Similarly, among the 100 HCC specimens in the validation group, the low and high COPB2 expression rates were 43% (43/100) and 57% (57/100), respectively, and the low and high YAP1 expression rates were 33% (33/100) and 67% (67/100), respectively. Correlation of COPB2 and YAP1 expression with the clinicopathological characteristics of HCC patients As shown in Table 1, COPB2 expression was correlated with tumor size and TNM staging in both the training and validation groups (P<0.05). YAP1 expression related to the degree of tumor differentiation and TNM staging in both groups (P<0.05) (Table 2). Correlation of COPB2 and YAP1 expression with OS of HCC patients In the training and validation groups, we found that combined COPB2 and YAP1 expression was an independent prognostic factor for HCC patients in addition to the TNM staging by univariate and multivariate analyses, respectively (Table 3). Kaplan-Meier survival analysis showed that patients with lower COPB2 or YAP1 expression had a longer OS (Figure 2 A,B). Moreover, we found that patients with low expression of both COPB2 and YAP1 had longer OS than those with high COPB2 and YAP1expression (Figure 2 C). Nomogram and ANN model based on COPB2 and YAP1 expression Based on the nomogram, we scored several important clinicopathological features for each patient and the sum of the points obtained was used to predict the 3- and 5-year survival rates of HCC patients (Figure 3 A). A higher score predicts a worse prognosis for the patient. The model showed good accuracy in predicting OS of HCC after hepatectomy with a c-index of 0.673. Calibration plots show the model's good prediction of 3- and 5-year survival rates for HCC patients (Figure 3 B, C). Similarly, an ANN model related to COPB2 and YAP1 expression was developed based on several important risk factors (Figure 4 A). The proportion of OS importance accounted for by risk factors of TNM staging, degree of tumor differentiation, YAP1 expression, COPB2 expression, tumor number and tumor size in the ANN model was 0.23, 0.2168, 0.1823, 0.1338, 0.1288 and 0.1083 respectively (Figure 4 B). Correlation between COPB2 and YAP1 expression and OS in HCC patients treated with TACE after surgery TACE is the first-line treatment for intermediate to advanced hepatocellular carcinoma. 102 of 214 patients at both hospitals underwent postoperative TACE, and DDP was used in TACE at both hospitals. Kaplan-Meier survival analysis showed that among these patients, those with lower COPB2 and YAP1 expression had a longer OS (Figure 5). Multivariate analysis identified COPB2 combined with YAP1 expression, vascular tumor thrombus, and TNM staging as independent risk factors (Table 4). COPB2 mediates the drug sensitivity of HCC cells to DDP through the regulation of YAP1 Next, to demonstrate whether COPB2 and YAP1 affect the sensitivity of HCC cells to DDP, we first selected two cell lines, Huh7 and SK-hep1. We used siRNA to knock down COPB2 and plasmids to overexpress YAP1 in cells and Western blot to verify the efficiency of knockdown and overexpression (Figure 6 A). In the presence of DDP, through CCK8 and clone formation analysis, we discovered that knockdown of COPB2 significantly reduced the proliferation ability of HCC cells, while overexpression of YAP1 could reverse this phenomenon (Figure 6 B, C). Furthermore, we found that knockdown of COPB2 inhibited migration and invasion of HCC cells by transwell assay, while overexpression of YAP1 rescued the inhibitory effect of COPB2 knockdown (Figure 6 D, E). Knockdown of COPB2 promotes YAP1 out of the nucleus and affects its stability Ultimately, by immunofluorescence staining assays, we found that knockdown of COPB2 in Huh7 and SK-hep1 cells promote YAP1 out of the nucleus (Figure 7A). In addition, we found increased expression of pLATS1 and pYAP1 after knockdown of COPB2 (Figure 7B). Discussion HCC is one of the leading causes of cancer deaths worldwide and remains a major clinical challenge [28] . Despite increasingly sophisticated treatment modalities for HCC patients, the prognosis is still not promising [29] . Therefore, elucidating the molecular mechanisms underlying the pathogenesis of HCC and chemotherapy resistance, and exploring potential biomarkers, are essential to identify new targeted therapies and improve the prognosis of HCC patients. COPB2 has been reported to be responsible for the development of many human cancers, such as lung cancer [30] , colon cancer [31] and breast cancer [32] . However, studies on COPB2 in HCC are fewer and more limited. In this study, we found that COPB2 was associated with the Hippo signaling pathway and YAP1 and correlated with prognosis and drug sensitivity in HCC patients. There is growing evidence that YAP1 is becoming an appealing target for cancer treatment and contributing to our chemotherapy resistance insights [33] . It is worth noting that we obtained similar results in this study by TMA, survival analysis, and cellular experiments. In this study, we first obtained immunohistochemical profiles of COPB2 and YAP1 in HCC tissues through database, and after preliminary analysis, we discovered that the cytoplasmic expression of COPB2 was positively associated with the cytosolic expression of YAP1. To verify this phenomenon, we retrospectively analyzed TMA specimens from HCC patients who underwent surgical treatment at two hospitals and divided them into a training group and a validation group. By immunohistochemical staining of the TMA, we obtained similar results. By chi-square test, we found that COPB2 expression was significantly associated with tumor size and TNM staging in HCC patients, and YAP1 expression was significantly correlated with the degree of tumor differentiation and TNM staging in HCC patients. This result suggests that there is a correlation between COPB2 and YAP1 expression and the tumor load and staging of the patients. It is well known that the prognosis of HCC patients is strongly influenced by tumor load and TNM staging. To further explore whether combining these two genes was associated with OS in HCC patients, we found that both high expression of COPB2 and YAP1 and TNM staging were independent prognostic factors for patients undergoing radical hepatic resection for HCC by univariate and multivariate analyses. Kaplan-Meier survival analysis showed a shorter OS for patients with high expression of both COPB2 and YAP1 than for those with low expression. To further investigate the prognostic value of combined COPB2 and YAP1 expression on the clinical outcome of HCC patients, we generated a nomogram, which in turn predicted 3- or 5-year survival rates for HCC patients. In recent years, artificial neural network technology has been widely used in the field of medical diagnosis and prediction. It is a mathematical model that simulates the human brain's nervous system to synthesize complex information and has been shown to be superior to traditional discriminant analysis, and ANNs may be more accurate when multiple predictor variables involve multi-dimensional functions that interact with each other [34][35] . According to our model, YAP1 and COPB2 expression were second only to TNM staging and degree of differentiation in predicting overall survival in HCC patients. Further data mining of HCC patients in TMA revealed that a total of 102 patients had undergone at least one TACE treatment after surgery for tumor recurrence or consolidation, of which DDP was the commonly used drug in TACE. We also performed univariate and multivariate analyses to understand whether double positive COPB2 and YAP1 expression is a prognostic guide for HCC patients undergoing postoperative TACE treatment. Surprisingly, we discovered that COPB2 combined with YAP1 expression was also an independent risk factor for this group of patients, suggesting that they play an essential role in assessing the prognosis of HCC patients. Moreover, Kaplan-Meier survival analysis also indicated that patients with high expression of both COPB2 and YAP1 had the worst prognosis. In colon cancer, activation of YAP has been shown to be associated with DDP resistance [36] . Therefore, we speculate that the high expression of COBP2 and YAP1 may be related to the chemical sensitivity of HCC patients to DDP. Through in vitro experiments, we observed that HCC cells were more sensitive to DDP after COPB2 knockdown. However, overexpression of YAP1 on top of this partially reversed this phenomenon. YAP1 can regulate the sensitivity of cancer cells to drugs through a variety of mechanisms, such as the HIPPO-YAP1-TEAD signaling pathway, stem cell markers and increased expression of ABC transporter proteins, but all of these mechanisms must rely on YAP1 entering the nucleus in order to achieve [37][38][39] . Consistent with our speculation, upregulation of YAP1 increased the short- and long-term viability of tumor cells under DDP treatment. Subsequent immunofluorescence experiments revealed a significant increase in YAP1 in the cytoplasm of HCC cells following the knockdown of COPB2. Activation of the Hippo pathway will promote phosphorylation of YAP1, which is degraded upon binding to the cytoplasmic 14-3-3 protein and is unable to enter the nucleus, losing transcriptional activity [40] . Furthermore, we discovered that the expression of pYAP1 and pLATS1 was increased after the knockdown of COPB2 by western blot. There are several limitations to this study that warrant further discussion. Firstly, all patients in our study were from Asia and the results obtained need to be validated in other populations and larger cohorts. Second, the detailed mechanism by which COPB2 regulates chemotherapy sensitivity through modulation of YAP1 needs to be further investigated. In conclusion, we have identified increased COPB2/YAP1 expression as an independent risk factor in HCC patients and correlated with patient sensitivity to drugs. Inhibition of COPB2/YAP1 may be a promising new approach for the treatment of HCC. Declarations Acknowledgements: The study was supported by Cancer research center Nantong. Author Contributions: Biao Wu and Yumeng Wu contributed equally to this work and are co-first authors. BiaoWu made substantial contributions to conception and design of the work, in data analysis and drafting the manuscript. Yumeng Wu made substantial contributions to the interpretation of data. Xianlin Guo and Hongjian Chen conducted machine learning analysis. Yifei Liu, Wenjing Zhao and Yilang Wang took the patient's clinical data. Xudong Chen and Suqing Zhang made substantial contributions guiding and revising the work critically for important intellectual content and has been involved in drafting the manuscript, answering the referees ' comments and given final approval of the version to be published. All authors have read and approved the final manuscript. Funding : This study was supported by scientific research project of Nantong Health Commission (JC22022084, JC22022011) and Scientific Research Project of Nantong Health Commission (MS2022058) Ethical approval statement : The study was approved by the institutional review boards of the two participating hospitals. Data availability statement : The raw data supporting the conclusions of this article will be made available by the authors. Conflict of Interest : The authors declare that they have no competing interests. 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Zhong, BY; Yan, ZP; Sun, JH; et al.Prognostic Performance of Albumin-Bilirubin Grade With Artificial Intelligence for Hepatocellular Carcinoma Treated With Transarterial Chemoembolization Combined With Sorafenib.[J].Front Oncol.2020,10():525461. Li, K; Guo, J; Wu, Y; et al.Suppression of YAP by DDP disrupts colon tumor progression.[J].Oncol Rep.2018,39(5):2114-2126. Que, K; Tong, Y; Que, G; et al.Downregulation of miR-874-3p promotes chemotherapeutic resistance in colorectal cancer via inactivation of the Hippo signaling pathway.[J].Oncol Rep.2017,38(6):3376-3386. Zhao, W; Wu, M; Cui, L; et al.Limonin attenuates the stemness of cervical carcinoma cells by promoting YAP nuclear-cytoplasmic translocation.[J].Food Chem Toxicol.2019,125():621-628. Miyahara, K; Hirata, D; Miyakawa, T; yAP-1- and yAP-2-mediated, heat shock-induced transcriptional activation of the multidrug resistance ABC transporter genes in Saccharomyces cerevisiae.[J].Curr Genet.1996,29(2):103-5. Basu, S; Totty, NF; Irwin, MS; et al.Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis.[J].Mol Cell.2003,11(1):11-23. Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files tables.xlsx 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. 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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-4117273","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283144449,"identity":"b4096cb9-329c-4e33-a107-fa72e6c62299","order_by":0,"name":"Biao Wu","email":"","orcid":"","institution":"Affiliated Tumor Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Biao","middleName":"","lastName":"Wu","suffix":""},{"id":283144450,"identity":"a379e6ff-28f5-4161-9288-5a9b2054adbc","order_by":1,"name":"Yumeng Wu","email":"","orcid":"","institution":"Affiliated Tumor Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yumeng","middleName":"","lastName":"Wu","suffix":""},{"id":283144451,"identity":"d64863fc-8b2b-4c9d-b3e9-568241c0ac35","order_by":2,"name":"Yifei Liu","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yifei","middleName":"","lastName":"Liu","suffix":""},{"id":283144452,"identity":"f10b1313-a8e1-4c4e-a127-7642aa899820","order_by":3,"name":"Hongjian Chen","email":"","orcid":"","institution":"Affiliated Tumor Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Hongjian","middleName":"","lastName":"Chen","suffix":""},{"id":283144453,"identity":"21344130-2f82-411c-ada4-83c0f9d40ea4","order_by":4,"name":"Wenjing Zhao","email":"","orcid":"","institution":"Affiliated Tumor Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Zhao","suffix":""},{"id":283144454,"identity":"7ab771e1-12dc-49d6-807c-6c177f55def6","order_by":5,"name":"Jibin Liu","email":"","orcid":"","institution":"Affiliated Tumor Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Jibin","middleName":"","lastName":"Liu","suffix":""},{"id":283144455,"identity":"64c1c469-5703-425b-9bde-44253c485417","order_by":6,"name":"Xiubing Zhang","email":"","orcid":"","institution":"Nantong Second Peoples Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Xiubing","middleName":"","lastName":"Zhang","suffix":""},{"id":283144456,"identity":"13621ba5-51bb-478b-8a8e-5afb15bbb65b","order_by":7,"name":"Jian Xu","email":"","orcid":"","institution":"Nantong Second Peoples Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Xu","suffix":""},{"id":283144457,"identity":"2dc8f71a-3fc5-4677-b19d-ee84c742098d","order_by":8,"name":"Yilang Wang","email":"","orcid":"","institution":"Affiliated Maternity and Child Healthcare Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yilang","middleName":"","lastName":"Wang","suffix":""},{"id":283144458,"identity":"e8cb4c15-f926-4e8e-b505-1db3fffd5b1a","order_by":9,"name":"Xianlin Guo","email":"","orcid":"","institution":"Affiliated Tumor Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Xianlin","middleName":"","lastName":"Guo","suffix":""},{"id":283144459,"identity":"8016ca7c-b4b6-49d7-a15a-b4cfc22d891c","order_by":10,"name":"Xudong Chen","email":"","orcid":"","institution":"Affiliated Tumor Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Xudong","middleName":"","lastName":"Chen","suffix":""},{"id":283144460,"identity":"f6b26866-8d26-4854-8568-2cbe03f8fc1a","order_by":11,"name":"Suqing Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYHAC9h8fKmzk2NjbDxCvR3LGmTRjPp4zCcRrkeZtO5w4T8LBgDjl8v2HNxjOYDuc3ibBkMDwo2IbYS2MM9IKEj7wpOe2STceYOw5c5uwFmYJHoODMySsc9tkDiQwM7YRoYWN/4xhM48BczqbRIIBcVp4GHKMmXkSnBOI1yIhkVbGOONAmmEbMJAPEuUXYIhtY/j4z0Zevr394IMfFURoAQJEdBwgSj2KllEwCkbBKBgFWAEA5Mk5avrL+NgAAAAASUVORK5CYII=","orcid":"","institution":"Affiliated Tumor Hospital of Nantong University","correspondingAuthor":true,"prefix":"","firstName":"Suqing","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-03-17 13:44:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4117273/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4117273/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53500106,"identity":"a12f394e-f6a6-43d3-93c5-a1fb099ba32f","added_by":"auto","created_at":"2024-03-26 18:10:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3182398,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between COPB2 and YAP1 expression in HCC. (A, B) WGCNA analysis of HCC datasets GSE102079 and GSE21248. (C) There are a total of 927 intersecting genes of the red module of GSE102079 and the blue module of GSE21248 (left), and 394 of the intersecting gene are differentially expressed in both the TCGA-LIHC dataset and the GSE14520 dataset (right), |Log2FC|\u0026gt;1, P\u0026lt;0.05. (D) The 364 shared differential genes were further analyzed in STING for PPI construction using CytoHubba plug in Cytoscape for these PPI, and YAP1 was one of the key proteins. (E) Correlation analysis of YAP1 and COPB2 mRNA expression levels in the TCGA-LIHC dataset. (F) In The Human Protein Atlas liver cancer data, COPB2 was lowly expressed in the cytoplasm and YAP1 was lowly expressed in the nucleus in patient #2280. In patient #2279, COPP2 was highly expressed in the cytoplasm and YAP1 was highly expressed in the nucleus. (G) Expression of COPB2 and YAP1 in the Nantong HCC cohort TMA. In patient A, COPB2 was low expressed in the cytoplasm and YAP1 was low expressed in the nucleus, and in patient B, COPB2 was high expressed in the cytoplasm and YAP1 was high expressed in the nucleus.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4117273/v1/6fd94cb425ed077e6a0ccf72.png"},{"id":53500112,"identity":"51a1e276-48da-4a67-8e4a-c0b7d894a679","added_by":"auto","created_at":"2024-03-26 18:10:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":592578,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier survival analysis between COPB2/YAP1 expression and OS of HCC patients after surgery. (A) Patients with higher COPB2 expression have a shorter OS. (B) Patients with higher YAP1 expression have a shorter OS. (C) Patients with high expression of both COPB2 and YAP1 have the shortest OS.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4117273/v1/3b3b2f733b558b99a9d1d3e2.png"},{"id":53500107,"identity":"d45c1f83-d095-4d39-be5e-8f96ebe70b54","added_by":"auto","created_at":"2024-03-26 18:10:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":665773,"visible":true,"origin":"","legend":"\u003cp\u003eNomogram predicting the probability of survival at 3 and 5 years. (A) Nomogram of HCC patients after surgery based on COPB2 and YAP1 expression. (B-C) Good calibration for predicting survival at 3 and 5 years.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4117273/v1/895b86f19e53838d7151aaec.png"},{"id":53500103,"identity":"12954864-b680-49a3-9282-43a0bdafc9e6","added_by":"auto","created_at":"2024-03-26 18:10:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2255748,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic representation of an ANN for predicting OS after surgery in patients with HCC. (B) The importance of the variables in the ANN model.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4117273/v1/73205af44e64ebe4ccf5ab0d.png"},{"id":53501417,"identity":"c90f254d-66f4-4527-b2ef-ab416f675dad","added_by":"auto","created_at":"2024-03-26 18:18:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":620051,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier survival analysis between COPB2/YAP1 expression and OS of HCC patients receiving TACE after surgery. (A) Patients with higher COPB2 expression have a shorter OS. (B) Patients with higher YAP1 expression have a shorter OS. (C) Patients with high expression of both COPB2 and YAP1 have the shortest OS.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4117273/v1/a60bdcde73a53f20fd6b7366.png"},{"id":53500111,"identity":"c95be858-c1ed-416a-900f-f14393582ee1","added_by":"auto","created_at":"2024-03-26 18:10:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7854926,"visible":true,"origin":"","legend":"\u003cp\u003eCOPB2 mediates the drug sensitivity of HCC cells to DDP (5μg/ml) through the regulation of YAP1. (A) Huh7 and SK-hep1 were transfected with si-RNA to knock down COPB2 and plasmids to overexpress YAP1, and western blot was used to detect the efficiency. (B) Cell proliferation was detected by CCK-8 assay at different time points. (C) Representative picture and quantification of colony formation. (D-E) Representative picture and quantification of transwell assay. All data are displayed as mean ± standard deviation (SD). ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure61.png","url":"https://assets-eu.researchsquare.com/files/rs-4117273/v1/3a277232237c9ad47af6d10b.png"},{"id":53500115,"identity":"6c4ec28c-f729-4cee-8582-e36d6841e730","added_by":"auto","created_at":"2024-03-26 18:10:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1914129,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of COPB2 promotes YAP1 out of the nucleus and affects its stability. (A) Representative images of immunofluorescent staining for YAP1 distribution. (B) Representative pictures of western blotting analysis of pLATS1 and pYAP1 in Huh7 and SK-hep1 cells transfected with si-NC and si-COPB2.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4117273/v1/843857e289d59c6d492d3b86.png"},{"id":54059689,"identity":"25346e34-b897-4325-b203-4894da3e42b6","added_by":"auto","created_at":"2024-04-04 03:29:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4081728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4117273/v1/9925a137-5dda-4e46-84e6-e9eb72296c85.pdf"},{"id":53500101,"identity":"4d16f3d0-5e84-466f-a29b-a04f1d6ff643","added_by":"auto","created_at":"2024-03-26 18:10:31","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19208,"visible":true,"origin":"","legend":"","description":"","filename":"tables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4117273/v1/06582dab886229ade155455c.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"COPB2 promotes hepatocellular carcinoma progression through regulation of YAP1 nuclear translocation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer death worldwide in 2020\u003csup\u003e[1]\u003c/sup\u003e. Most HCC patients in China have a background of hepatitis B virus infection and the resulting cirrhosis\u003csup\u003e[2]\u003c/sup\u003e. Many patients are already in the middle to late stages at diagnosis and have a poor prognosis. The 5-year survival rate for advanced HCC patients is currently less than 5%\u003csup\u003e[3]\u003c/sup\u003e. In addition to liver transplantation, radical hepatectomy and ablation are the most effective treatments for early-stage patients\u003csup\u003e[4]\u003c/sup\u003e. Transarterial chemoembolization (TACE) is the first-line treatment for patients with intermediate to advanced HCC in the United States, Asia, and Europe\u003csup\u003e[5]\u003c/sup\u003e. It is indicated for unresectable patients who have relapsed after surgery or who have limited disease and have satisfactory preserved liver function\u003csup\u003e[6]\u003c/sup\u003e. Despite increasing improvements in the management and perioperative care of HCC patients, recurrence and metastasis remain key challenges to long-term survival\u003csup\u003e[7]\u003c/sup\u003e. As a result, the study of HCC-related genes and molecular biological mechanisms is of great clinical significance in predicting the prognosis of HCC patients and identifying potential targets for HCC therapy.\u003c/p\u003e\n\u003cp\u003eThe Hippo signaling pathway is a major cancer suppression pathway and plays a key role in inhibiting the proliferation and survival of HCC cells\u003csup\u003e[8]\u003c/sup\u003e. The Hippo pathway was originally identified and named by screening for mutant tumor suppressors in Drosophila\u003csup\u003e[9]\u003c/sup\u003e. The main composition of the pathway include the serine threonine kinase module and the transcriptional module\u003csup\u003e[10]\u003c/sup\u003e. The Hippo pathway has significant regulatory functions for organ development, regeneration and stem cell biology\u003csup\u003e[11]\u003c/sup\u003e. Once activated, the Hippo pathway undergoes a series of kinase phosphorylation reactions that ultimately lead to the inability of the downstream transcription factor Yes-associated protein 1 (YAP1) phosphorylation to enter the nucleus to activate gene transcription\u003csup\u003e[12]\u003c/sup\u003e. In contrast, inactivation of the Hippo pathway in tumors results in very common YAP1 dephosphorylation, and dephosphorylated YAP1 entry into the nucleus induces cell proliferation and expression of anti-apoptotic genes\u003csup\u003e[13]\u003c/sup\u003e. There is growing evidence that YAP1 is an essential oncogene as a downstream target of the Hippo pathway\u003csup\u003e[14]\u003c/sup\u003e. Intranuclear YAP1 expression plays a vital role in predicting post-operative survival and tumor recurrence in patients with HCC\u003csup\u003e[15]\u003c/sup\u003e. In addition, YAP1 has prognostic implications for patients treated with TACE. Although the core ingredients of the Hippo pathway have been studied in detail, the upstream regulators remain unclear.\u003c/p\u003e\n\u003cp\u003eThe coatomer protein complex subunit beta 2 (COPB2) is one of the component subunits of the coatomer complex I(COPI), a nuclear protein containing 906 amino acid residues and a molecular weight of 102 kD\u003csup\u003e[16]\u003c/sup\u003e. COPB2 has a tryptophan-aspartate (WD) repeat sequence, which is associated with signal transduction, regulation of the cell cycle and apoptosis\u003csup\u003e[17]\u003c/sup\u003e. It was found that COPB2 interacts with ADP-ribosylation factor (ARF) GTPase-activating protein 2 (GAP2) and KKXX-containing substances, and the product of this reaction is a coating of COPI-coated vesicles that plays a crucial role in vesicle transport and Golgi budding between the endoplasmic reticulum and Golgi apparatus\u003csup\u003e[18]\u003c/sup\u003e. Wang et al. and Couzens et al. found that vesicle transport regulates many signaling pathways, including the Hippo pathway, through proteomic studies\u003csup\u003e[19][20]\u003c/sup\u003e. Vesicular transport can affect the stability of YAP1 by regulating its phosphorylation\u003csup\u003e[21]\u003c/sup\u003e. In addition, COPB2 is abnormally highly expressed in many tumor tissues, including colon cancer\u003csup\u003e[22]\u003c/sup\u003e, prostate cancer\u003csup\u003e[23]\u003c/sup\u003e and gallbladder cancer\u003csup\u003e[24]\u003c/sup\u003e. An et al. reported that COPB2 inhibits the progression of gastric cancer through the RTK pathway\u003csup\u003e[25]\u003c/sup\u003e. PU et al. also found that COPB2 promotes lung cancer progression by regulating YAP1 nuclear translocation\u003csup\u003e[26]\u003c/sup\u003e. In a recent report, COPB2 promotes HCC progression by regulating the EMT pathway\u003csup\u003e[27]\u003c/sup\u003e. These results indicate that COPB2 plays a significant role in cancer development. However, the correlation between COPB2 and YAP1 in HCC has not been investigated.\u003c/p\u003e\n\u003cp\u003eOur study found that COPB2 combined with YAP1 predicted the prognosis of HCC patients and developed associated nomogram and artificial neural network (ANN) prediction models. In addition, we found that COPB2 reduced the drug sensitivity to DDP in HCC cells by regulating YAP1 nuclear translocation. This finding may be critical in the progression and metastasis of HCC. Understanding this pathway may help identify new methods for the treatment of HCC.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eDatabase analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTCGA-LIHC gene expression data were downloaded from the TCGA data portal (https://tcga-data.nci.nih.gov/tcga/ ) and three gene expression profiles (GSE102079 , GSE121248 and GSE14520) as raw data. The Human Protein Atlas web (http://www.proteinatlas.org/) was used to obtain relevant immunohistochemical staining images of COPB2 and YAP1 in HCC tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWGCNA analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWeighted gene co-expression network analysis (WGCNA) of GSE02079 and GSE121248 expression matrices using the R package \u0026quot;WGCNA\u0026quot;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient and clinical samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe retrospectively analyzed two independent cohorts comprising HCC 214 patients. In the training group, 114 HCC patients from the Affiliated Hospital of Nantong University from 2012-2017 were randomly selected. Moreover, 100 HCC patients from the Affiliated Tumor Hospital of Nantong University from 2011-2016 were randomly selected as the validation group. Cases are selected according to the following criteria: no previous anti-cancer related treatment prior to surgery; pathological diagnosis of HCC; availability of resected tissue and follow-up data. Patients receive a physical examination, laboratory diagnosis, and imaging twice a year after surgery. Most patients undergo TACE after relapse. Overall survival (OS) was measured from the date of surgery to the date of death or last follow-up. For each patient, the following clinicopathological information was collected: age, gender, hepatitis B virus (HBV), presence of cirrhosis, serum alpha-fetoprotein (AFP) level, tumor number, tumor size, degree of tumor differentiation, vascular tumor thrombus and lymph node metastasis. The staging of tumors is defined according to the American Joint Committee on Cancer/International Union Against Cancer\u0026apos;s Tumor Lymph Node Metastasis (TNM) classification system. Written informed consent was obtained from all participating patients for the use of this clinical information, and the project was approved by the ethics committees of both hospitals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissue microarrays (TMA) sections are dewaxed in xylene and rehydrated through a graded alcohol series. Pressure cook the sections in alkaline buffer (pH=9.0) for 3 minutes. After cooling at room temperature, immerse in 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. The sections were then incubated with rabbit polyclonal anti-COPB2 antibody (1:1000, Abcam, Cambridge, UK), YAP1 monoclonal antibody (1:200, Abcam, Cambridge, UK) overnight at 4℃. After washing three times with PBS, the sections were incubated with secondary antibodies (1:4000, Abcam, Cambridge, UK) for 20 min at room temperature, and then diaminobenzidine (DAB) solution was applied. Finally, sections were re-stained with hematoxylin, dehydrated and fixed. All microarrays were scored independently by two experienced pathologists. COPB2 scoring criteria: The percentage of positive cells was evaluated on a scale of 0\u0026ndash;3:0(0\u0026ndash;25%),1 (26\u0026ndash;50%), 2 (51\u0026ndash;75%), or 3(76\u0026ndash;100%). Classified as low expression group:0-1 point or high expression group; 2-3 points. YAP1 scoring criteria, with a percentage of positive nuclei greater than 10% recorded as high expression and less than 10% recorded as low expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHCC cells were lysed in cell lysis buffer on ice (Beyotime, Shanghai, China). The protein concentration was determined using the bicinchoninic acid assay (Beyotime, Shanghai, China). Electrophoresis was then performed on SDS-PAGE gels and transferred to PVDF membranes. The following antibodies were used in this study: COPB2 (1:1000, rabbit, Abcam, Cambridge, UK), YAP1 (1:500, rabbit, Abcam, Cambridge, UK), pLATS1(1:500, rabbit, Cell Signaling Technology, Massachusetts, USA), pYAP1 (1:500, rabbit, Cell Signaling Technology, Massachusetts, USA), GAPDH (1:5000, mouse, Abcam, Cambridge, UK).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and vectors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human HCC cell lines Huh7 and SK-hep1 were purchased from the Academy of Sciences Committee of the China Cell Resource Centre (Shanghai, China). The cells were cultured in Dulbecco\u0026apos;s modified Eagle 92 medium (DMEM, Hyclone, Logan, Utah, USA) containing 10% fetal bovine serum (FBS, Gibco, Grand 93 Island, NY, USA) at 37\u0026deg;C in a humidified atmosphere of 5% CO2. HCC cells in the exponential growth phase were used for subsequent experiments. The COPB2 siRNA was designed by Polyplus (Sense: CCCAUUAUGUUAUGCAGAUTT; Antisense‐1: AUCUGCAUAACAUAAUGGGTT). YAP1/pcDNA3.1 was constructed and the sequence was examined by GENECHEM. For transient expression, plasmids were transfected with Lipofectamine 2000 (Invitrogen, Shanghai, China) for 24 hours, after which the cells were processed with the indicated reagents as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Counting Kit-8 (CCK-8) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Huh7 and SK-hep1 cells were digested with 0.25% trypsin and diluted to a 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/ml concentration. The cells were counted using a cell counter(Thermo Fisher Scientific, Massachusetts, USA) and the cell suspension was inoculated into 96-well plates at a density of 2000 cells/well. Add 10\u0026mu;L CCK-8 solution (NCM, Suzhou, China) to each well, and culture at 37℃ for 2 hours. Cell viability was determined by measuring the absorbance at 450nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClone formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBriefly, Huh7 and SK-hep1 cells (2000 cells/well) were inoculated in 6-well plates and incubated at 37℃. The medium was changed every three days and observed cell growth. A total of 14 days after inoculation of cells, colonies with a cell count of \u0026gt; 50 were observed under a microscope. Cells were washed with PBS and fixed in 4% paraformaldehyde (Biosharp, Hefei, China) for 20 minutes at room temperature. Fixed cells were stained with 1 ml of Crystal Violet Staining Solution (Beyotime, Shanghai, China) for 10 minutes, washed with PBS and air dried at room temperature. The cells were photographed and the number of clones visible was counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMigration assay: Transwell chambers were placed in 24-well plates. 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e Huh7 and SK-hep1 cells starved in 200 \u0026mu;L of serum-free medium were added to the upper chamber, and 500 \u0026mu;L of medium with 10% serum was added to the lower chamber. After 24 hours of incubation at 37℃ and 5% CO2, the Transwell chambers were first fixed in paraformaldehyde for 30 minutes and then stained with Crystal Violet Staining Solution for 10 minutes. The cells were photographed and counted under the microscope, with three replicates per group.\u003c/p\u003e\n\u003cp\u003eInvasion assay: Melt Matrigel at 4℃ overnight and dilute with pre-cooled serum-free DMEM medium. Add 100\u0026micro;L of diluted Matrigel to the upper chamber. The remaining steps are the same as those in the Transwell migration assay\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuh7 and SK-hep1 cells were inoculated in 24-well plates for 24 hours. Cells were then fixed in 4% paraformaldehyde for 15 minutes and permeabilized for 10 minutes. After being closed with 5% BSA for 1 hour, the cells were incubated overnight at 4\u0026deg;C with YAP1 antibody (1:100, Abcam, Cambridge, UK) and then Alexa Fluor 594 anti-rabbit IgG secondary antibody (Invitrogen, Shanghai, China). Furthermore, cell nuclei were stained with DAPI (Thermo Fisher). The images were taken under a fluorescent microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferences in expression were analyzed by paired t-test. Clinicopathological characteristics were assessed using the chi-square test. P \u0026lt; 0.05 was considered a statistically significant difference. Statistical analyses were performed using the SPSS 26.0 statistical package (SPSS, Inc, Chicago, IL) and GraphPad Prism version 7.01 (GraphPad Software, Inc, La Jolla, CA). Nomogram was developed using the Regression Modelling Strategies package in R (version 3.0.2; R Package \u0026quot;rms\u0026quot; for nomogram establishment; www.r-project.org). ANN was built using SPSS Clementine version 12.0 software for Windows (IBM Corporation).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExpression of COPB2 and YAP1 in HCC tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe analyzed the two HCC datasets using WGCNA analysis and obtained 927 intersecting genes in the most relevant module of HCC, and then we used the two datasets with larger sample sizes in the HCC dataset to obtain 394 shared genes that were significantly different in the two datasets (Figure 1 A-C). Then we constructed Protein-Protein Interaction Networks (PPI) for these genes and performed Hub gene analysis on the network and found that YAP1 was in the Hub gene network (Figure 1 D). To explore the connection between COPB2 and YAP1 expression in HCC tissues, we first searched the database. Notably, we found that COPB2 and YAP1 have a high correlation in TCGA-LIHC (Figure 1 E), and in the human protein atlas database COPB2 was highly expressed in the cytoplasm, YAP1 was also highly expressed in the nucleus. When COPB2 was lowly expressed, YAP1 was also lowly expressed (Figure 1 F). It is worth noting that we discovered that the COPB2 expression in the cytoplasm is positively associated with YAP1 expression in the nucleus. To verify the findings in the database, we performed immunohistochemical staining of COPB2 and YAP1 on tissue microarrays of 214 specimens from two hospitals (Figure 1 G). We found that COPB2 was predominantly present in the cytoplasm and YAP1 was expressed in both the nucleus and cytoplasm, and we grouped samples for high and low expression based on the two antibody staining scores. Among the 114 HCC specimens in the training group, the COPB2 low expression rate was 43.9% (50/114), and high expression rate was 56.1% (64/114), YAP1 low expression rate was 42.1% (48/114), and YAP1 high expression rate was 57.9% (66/114). Similarly, among the 100 HCC specimens in the validation group, the low and high COPB2 expression rates were 43% (43/100) and 57% (57/100), respectively, and the low and high YAP1 expression rates were 33% (33/100) and 67% (67/100), respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation of COPB2 and YAP1 expression with the clinicopathological characteristics of HCC patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 1, COPB2 expression was correlated with tumor size and TNM staging in both the training and validation groups (P\u0026lt;0.05). YAP1 expression related to the degree of tumor differentiation and TNM staging in both groups (P\u0026lt;0.05) (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation of COPB2 and YAP1 expression with OS of HCC patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the training and validation groups, we found that combined COPB2 and YAP1 expression was an independent prognostic factor for HCC patients in addition to the TNM staging by univariate and multivariate analyses, respectively (Table 3). Kaplan-Meier survival analysis showed that patients with lower COPB2 or YAP1 expression had a longer OS (Figure 2 A,B). Moreover, we found that patients with low expression of both COPB2 and YAP1 had longer OS than those with high COPB2 and YAP1expression (Figure 2 C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNomogram and ANN model based on COPB2 and YAP1 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the nomogram, we scored several important clinicopathological features for each patient and the sum of the points obtained was used to predict the 3- and 5-year survival rates of HCC patients (Figure 3 A). A higher score predicts a worse prognosis for the patient. The model showed good accuracy in predicting OS of HCC after hepatectomy with a c-index of 0.673. Calibration plots show the model\u0026apos;s good prediction of 3- and 5-year survival rates for HCC patients (Figure 3 B, C).\u003c/p\u003e\n\u003cp\u003eSimilarly, an ANN model related to COPB2 and YAP1 expression was developed based on several important risk factors (Figure 4 A). The proportion of OS importance accounted for by risk factors of TNM staging, degree of tumor differentiation, YAP1 expression, COPB2 expression, tumor number and tumor size in the ANN model was 0.23, 0.2168, 0.1823, 0.1338, 0.1288 and 0.1083 respectively (Figure 4 B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation between COPB2 and YAP1 expression and OS in HCC patients treated with TACE after surgery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTACE is the first-line treatment for intermediate to advanced hepatocellular carcinoma. 102 of 214 patients at both hospitals underwent postoperative TACE, and DDP was used in TACE at both hospitals. Kaplan-Meier survival analysis showed that among these patients, those with lower COPB2 and YAP1 expression had a longer OS (Figure 5). Multivariate analysis identified COPB2 combined with YAP1 expression, vascular tumor thrombus, and TNM staging as independent risk factors (Table 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOPB2 mediates the drug sensitivity of HCC cells to DDP through the regulation of YAP1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, to demonstrate whether COPB2 and YAP1 affect the sensitivity of HCC cells to DDP, we first selected two cell lines, Huh7 and SK-hep1. We used siRNA to knock down COPB2 and plasmids to overexpress YAP1 in cells and Western blot to verify the efficiency of knockdown and overexpression (Figure 6 A). In the presence of DDP, through CCK8 and clone formation analysis, we discovered that knockdown of COPB2 significantly reduced the proliferation ability of HCC cells, while overexpression of YAP1 could reverse this phenomenon (Figure 6 B, C). Furthermore, we found that knockdown of COPB2 inhibited migration and invasion of HCC cells by transwell assay, while overexpression of YAP1 rescued the inhibitory effect of COPB2 knockdown (Figure 6 D, E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of COPB2 promotes YAP1 out of the nucleus and affects its stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUltimately, by immunofluorescence staining assays, we found that knockdown of COPB2 in Huh7 and SK-hep1 cells promote YAP1 out of the nucleus (Figure 7A). In addition, we found increased expression of pLATS1 and pYAP1 after knockdown of COPB2 (Figure 7B).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHCC is one of the leading causes of cancer deaths worldwide and remains a major clinical challenge\u003csup\u003e[28]\u003c/sup\u003e. Despite increasingly sophisticated treatment modalities for HCC patients, the prognosis is still not promising\u003csup\u003e[29]\u003c/sup\u003e. Therefore, elucidating the molecular mechanisms underlying the pathogenesis of HCC and chemotherapy resistance, and exploring potential biomarkers, are essential to identify new targeted therapies and improve the prognosis of HCC patients. COPB2 has been reported to be responsible for the development of many human cancers, such as lung cancer\u003csup\u003e[30]\u003c/sup\u003e, colon cancer\u003csup\u003e[31]\u003c/sup\u003e and breast cancer\u003csup\u003e[32]\u003c/sup\u003e. However, studies on COPB2 in HCC are fewer and more limited. In this study, we found that COPB2 was associated with the Hippo signaling pathway and YAP1 and correlated with prognosis and drug sensitivity in HCC patients. There is growing evidence that YAP1 is becoming an appealing target for cancer treatment and contributing to our chemotherapy resistance insights\u003csup\u003e[33]\u003c/sup\u003e. It is worth noting that we obtained similar results in this study by TMA, survival analysis, and cellular experiments. In this study, we first obtained immunohistochemical profiles of COPB2 and YAP1 in HCC tissues through database, and after preliminary analysis, we discovered that the cytoplasmic expression of COPB2 was positively associated with the cytosolic expression of YAP1. To verify this phenomenon, we retrospectively analyzed TMA specimens from HCC patients who underwent surgical treatment at two hospitals and divided them into a training group and a validation group. By immunohistochemical staining of the TMA, we obtained similar results. By chi-square test, we found that COPB2 expression was significantly associated with tumor size and TNM staging in HCC patients, and YAP1 expression was significantly correlated with the degree of tumor differentiation and TNM staging in HCC patients. This result suggests that there is a correlation between COPB2 and YAP1 expression and the tumor load and staging of the patients. It is well known that the prognosis of HCC patients is strongly influenced by tumor load and TNM staging. To further explore whether combining these two genes was associated with OS in HCC patients, we found that both high expression of COPB2 and YAP1 and TNM staging were independent prognostic factors for patients undergoing radical hepatic resection for HCC by univariate and multivariate analyses. Kaplan-Meier survival analysis showed a shorter OS for patients with high expression of both COPB2 and YAP1 than for those with low expression. To further investigate the prognostic value of combined COPB2 and YAP1 expression on the clinical outcome of HCC patients, we generated a nomogram, which in turn predicted 3- or 5-year survival rates for HCC patients. In recent years, artificial neural network technology has been widely used in the field of medical diagnosis and prediction. It is a mathematical model that simulates the human brain\u0026apos;s nervous system to synthesize complex information and has been shown to be superior to traditional discriminant analysis, and ANNs may be more accurate when multiple predictor variables involve multi-dimensional functions that interact with each other\u003csup\u003e[34][35]\u003c/sup\u003e. According to our model, YAP1 and COPB2 expression were second only to TNM staging and degree of differentiation in predicting overall survival in HCC patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurther data mining of HCC patients in TMA revealed that a total of 102 patients had undergone at least one TACE treatment after surgery for tumor recurrence or consolidation, of which DDP was the commonly used drug in TACE. We also performed univariate and multivariate analyses to understand whether double positive COPB2 and YAP1 expression is a prognostic guide for HCC patients undergoing postoperative TACE treatment. Surprisingly, we discovered that COPB2 combined with YAP1 expression was also an independent risk factor for this group of patients, suggesting that they play an essential role in assessing the prognosis of HCC patients. Moreover, Kaplan-Meier survival analysis also indicated that patients with high expression of both COPB2 and YAP1 had the worst prognosis. In colon cancer, activation of YAP has been shown to be associated with DDP resistance\u003csup\u003e[36]\u003c/sup\u003e. Therefore, we speculate that the high expression of COBP2 and YAP1 may be related to the chemical sensitivity of HCC patients to DDP.\u003c/p\u003e\n\u003cp\u003eThrough in vitro experiments, we observed that HCC cells were more sensitive to DDP after COPB2 knockdown. However, overexpression of YAP1 on top of this partially reversed this phenomenon. YAP1 can regulate the sensitivity of cancer cells to drugs through a variety of mechanisms, such as the HIPPO-YAP1-TEAD signaling pathway, stem cell markers and increased expression of ABC transporter proteins, but all of these mechanisms must rely on YAP1 entering the nucleus in order to achieve\u003csup\u003e[37][38][39]\u003c/sup\u003e. Consistent with our speculation, upregulation of YAP1 increased the short- and long-term viability of tumor cells under DDP treatment. Subsequent immunofluorescence experiments revealed a significant increase in YAP1 in the cytoplasm of HCC cells following the knockdown of COPB2. Activation of the Hippo pathway will promote phosphorylation of YAP1, which is degraded upon binding to the cytoplasmic 14-3-3 protein and is unable to enter the nucleus, losing transcriptional activity\u003csup\u003e[40]\u003c/sup\u003e. Furthermore, we discovered that the expression of pYAP1 and pLATS1 was increased after the knockdown of COPB2 by western blot.\u003c/p\u003e\n\u003cp\u003eThere are several limitations to this study that warrant further discussion. Firstly, all patients in our study were from Asia and the results obtained need to be validated in other populations and larger cohorts. Second, the detailed mechanism by which COPB2 regulates chemotherapy sensitivity through modulation of YAP1 needs to be further investigated.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we have identified increased COPB2/YAP1 expression as an independent risk factor in HCC patients and correlated with patient sensitivity to drugs. Inhibition of COPB2/YAP1 may be a promising new approach for the treatment of HCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe study was supported by Cancer research center Nantong.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eBiao Wu and Yumeng Wu contributed equally to this work and are co-first authors. BiaoWu made substantial contributions to conception and design of the work, in data analysis and drafting the manuscript. Yumeng Wu made substantial contributions to the interpretation of data. Xianlin Guo and Hongjian Chen conducted machine learning analysis. Yifei Liu, Wenjing Zhao and Yilang Wang took the patient\u0026apos;s clinical data. Xudong Chen and Suqing Zhang made substantial contributions guiding and revising the work critically for important intellectual content and has been involved in drafting the manuscript, answering the referees\u003cstrong\u003e\u0026apos;\u0026nbsp;\u003c/strong\u003ecomments and given final approval of the version to be published. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThis study was supported by scientific research project of Nantong Health Commission (JC22022084, JC22022011) and Scientific Research Project of Nantong Health Commission (MS2022058)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval statement\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThe study was approved by the institutional review boards of the two participating hospitals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThe raw data supporting the conclusions of this article will be made available by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung, H; Ferlay, J; Siegel, RL; et al.Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries.[J].CA Cancer J Clin. 20 21,71(3):209-249.\u003c/li\u003e\n\u003cli\u003eMerican, I; Guan, R; Amarapuka, D; et al.Chronic hepatitis B virus infection in Asian countries. 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Totty, NF; Irwin, MS; et al.Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis.[J].Mol Cell.2003,11(1):11-23.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\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":false,"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":"COPB2, YAP1, HCC, prognosis, DDP","lastPublishedDoi":"10.21203/rs.3.rs-4117273/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4117273/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Hippo pathway has been shown to be upregulated in many cancer patients. Yes-associated protein 1 (YAP1), an oncogene and core factor of the Hippo pathway, plays a key role in tumorigenesis and progression. Although YAP1 is a vital oncogene in HCC progression, its nuclear localization prevents its consideration as a potential therapeutic target. Recently, studies have reported that COPB2 also plays a critical role in HCC development, but its mechanism of action is unclear. Here, we found that COPB2 affects the drug sensitivity of HCC cells to DDP by regulating YAP1 nuclear translocation and stability. More importantly, COPB2 combined with YAP1 expression was related to overall postoperative survival in HCC patients and was an independent prognostic factor. The established nomogram and artificial neural network models also highlighted the prognostic value of these two genes for HCC patients. In summary, our findings suggest that COPB2/YAP1 affects the drug sensitivity of HCC cells to DDP and that targeting COPB2/YAP1 may be a promising strategy for the precision treatment of HCC.\u003c/p\u003e","manuscriptTitle":"COPB2 promotes hepatocellular carcinoma progression through regulation of YAP1 nuclear translocation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-26 18:10:22","doi":"10.21203/rs.3.rs-4117273/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":"8700f390-3468-4994-856e-dd2a8ef96f99","owner":[],"postedDate":"March 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-04T03:29:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-26 18:10:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4117273","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4117273","identity":"rs-4117273","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}