Rab8a serves as a valuable biomarker of esophageal squamous cell carcinoma

Rab8a serves as a valuable biomarker of esophageal squamous cell carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rab8a serves as a valuable biomarker of esophageal squamous cell carcinoma Rong Liu, Zhanting Kang, Ting Gan, Yangruonan Wang, Zhiyuan Huangfu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3839052/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 Background Esophageal carcinoma (ESCA) is a digestive tract malignancy with high morbidity in China, among which esophageal squamous cell carcinoma (ESCC) accounts for 90% of the confirmed cases. Rab8a is a member of the Ras small GTPase superfamily, and it has been shown to play an important role in endometrial cancer (EC) and hepatocellular carcinoma (HCC). However, the function of Rab8a in ESCC are currently unclear. This study aimed to investigate Rab8a as a biomarker for the diagnosis of ESCC. Methods The study first used TIMER2.0, GEPIA and UALCAN to analyze the expression of Rab8a in a variety of clinically common malignancies including ESCC, followed by real-time PCR (quantitative real-time PCR, qPCR), Western blot, immunohistochemical (IHC) ，and a series of in vitro biological experiments Results Rab8a is highly expressed in the esophageal cancer cells and tissues.overexpression of Rab8a can promote the proliferation and migration of ESCC ,while knockdown its expression can inhibit the proliferation and migration of ESCC. Correlation analysis revealed the positive correlation between the expression of Rab8a and NDUFA1, CYC1 in public GEO databases. Therefore, Rab8a may promote ESCC progression by activating mitochondrial respiration. Conclusions This study demonstrated that Rab8a is upregulated in ESCC and may promote ESCC cell proliferation and migration by activating mitochondrial respiration. This study provides a rationale for clinical diagnosis and screening of new therapeutic targets for ESCC. esophageal squamous cell carcinoma Rab8a proliferation migration biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Esophageal carcinoma (ESCA) is one common malignant tumor of digestive system with the seventh incidence and sixth mortality worldwide[ 1 ]. And the main histologic subtype is esophageal squamous cell carcinoma (ESCC) in China, which accounting for about 90% of all diagnosed cases[ 2 ]. Although there has been improvement of therapeutic approaches, the outcome of ESCC patients remains poor because of the late diagnosis[ 3 ]. Therefore, it is of great significance to find a valuable biomarker for ESCC diagnosis. Rab8a protein is a member of RAS superfamily which participates in membrane traffic processes [ 4 – 7 ]. Previous study showed that Rab8a was up-regulated in endometrial cancer and might be a new biomarker [ 8 ]. In addition, it was reported that the enhanced expression of Rab8a could improve the immunotherapy efficacy of hepatocellular carcinoma, which suggested that it might be a novel therapeutic target [ 9 ]. However, the expression and biological functions remain unknown in other human cancers including ESCC. In the current study, we first analyzed the expression of Rab8a in many cancer types and their normal tissues by conducting pan-cancer analysis from some public databases. And according to the result of the analysis, we then investigated the expression and biological function of Rab8a in ESCC progression and at last explored the potential molecular mechanism of Rab8a in ESCC. Materials and methods Rab8a expression and survival analysis in different cancers The normalized transcripts per million (nTPM) levels of Rab8a in different types of tumors were identified based on the data of TIMER, Gene Expression Profiling Interactive Analysis (GEPIA) and UALCAN online resources. The relationship of Rab8a mRNA expression and the pathological features were available from UALCAN online databases. Also, we analyzed the correlation between Rab8a expression and overall survival (OS) outcomes of the patients with different cancers using the web database of Kaplan-Meier plotter. Tissue specimens, Cell lines and transfection 62 cases of formalin-fixed paraffin-embedded and 12 cases of fresh ESCC samples and their matched adjacent normal tissues were collected from the Department of pathology, Third Affiliated Hospital of Xinxiang Medical University (Xinxiang City, Henan Province, China) from June 2016 to July 2022. The fresh tissues were frozen in liquid nitrogen before extracting the total RNA. None of the patients underwent chemotherapy, radiotherapy or immunotherapy before surgery. Prior approval for the study had been obtained from Xinxiang Medical University Institutional Board (Xinxiang, China). ESCC cell lines of ECA109 and ECA9706 were obtained from the Cell Bank of the Chinese Academy (Shanghai, China). All ESCC cells were cultured in RPMI-1640 with 10% FBS at 37°C in a humidified atmosphere with 5% CO2. The overexpression and knockdown plasmids of Rab8a were the products of Public Protein/Plasmid Library (PPL). Lentivirus was produced by HEK293FT cells using the calcium phosphate method. The transfected cells were selected in medium containing puromycin. Immunohistochemistry (IHC) The formalin-fixed paraffin-embedded sample tissues were cut into 4-µm sections and baked at 60°C for 2 h. IHC was conducted by the SP kits (ZSGB-BIO, Beijing, China). The sections were incubated overnight at 4°C with the rabbit polyclonal antibody of Rab8a (1:100, Proteintech, USA). PBS was used as the negative control. The sections were stained with 3,3-diaminobenzidine (DAB) and sealed with neutral balsam. Finally, we observed the sections by two pathologists used immunoreactive score (IRS) which was mainly based on the proportion and intensity of the stained tumor cells[ 10 ]. Real-time quantitative PCR (qPCR) TRIzol reagent (Invitrogen, USA) was used to extract the total RNA from human tissues and cultured cells. qPCR was carried out according to the manufacturer’s protocol with SYBR Green I. The data were normalized to the the housekeeping gene of GAPDH, and calculated as 2 –ΔΔCT . The follows were primer sequences: GAPDH (F: ACA GTC AGC CGC ATC TTC TT, R: GAC AAG CTT CCC GTT CTC AG), Rab8a (F: ACG CCT TCA ACT CCA CTT; R: ACC AGC ATG ATG CCC ATT). Western blot The protein was extracted from the corresponding cells with the sample buffer lysate solution. Then the lysates were subjected to SDS-PAGE and transferred to PVDF membranes. 5% non-fat dry milk were used to block the membranes and incubated overnight at 4°C with the primary antibodies of polyclonal rabbit anti-Rab8a and mouse anti-GAPDH (Proteintech, USA). The appropriate secondary antibodies were used to incubate the membranes at room temperature for 1 h and then exposed for autoradiography. CCK-8 assay Cells with stable over- or knockdown expression of Rab8a were seeded in 96-well plates at the density of 1×10 3 cells per well and cultured for 24 h. Then, the culture medium was discarded and 10 µL of CCK-8 reagent and 100 µL of medium were added to every well. After 2 h incubation, the absorbance at 450 nm was detected. The experiment was conducted repeatedly for three times. Colony formation assay Cells with stable over- or knockdown expression of Rab8a were plated on 6-well plates (200 cells/well) and cultured for 2 weeks. The colonies were stained with Hematoxylin for 30 min after fixation with 4% paraformaldehyde for 10 minutes. The number of colonies, defined as > 50 cells/colony, were counted. Three independent experiments were performed. Transwell migration assay Boyden chambers were used to conducted Transwell migration assays. Cells (1×10 5 ) in culture medium without FBS were seeded to the upper chamber, and culture medium containing 20% FBS as a chemoattractant was added to the lower chamber. After incubation for 48 h, the chamber was fixed in 4% paraformaldehyde and stained with Hematoxylin. Cells on the upper sides of the filters were removed with cotton swabs. Cells that migrated to the lower sides were stained with Hematoxylin. The migratory cells on the lower surfaces of the filters were counted. Three independent experiments were performed. Wound-healing assay Cells were seeded to 6-well plates and incubated under permissive conditions until the cells reached 90% confluence. After serum starvation for 24 h, wounds were generated in the confluent cells using a pipette tip. Wound healing within the scrape line was then observed and photographed at indicated time points. Each experiment was repeated at least three times. Gene set enrichment analysis Gene set enrichment analysis (GSEA) is a knowledge-based approach for interpreting genome-wide expression differences between two biological states. We performed GSEA to explore the potential biological functions affected by Rab8a expression in ESCC. Statistical analysis All statistical analyses were performed using SPSS20.0 for Windows. The data are expressed as mean ± standard deviations (s.d.) from at least three independent experiments. The two-tailed paired Student's t-test was conducted for the analysis of two groups. Mann-Whitney U test was conducted to analyze the relationship between Rab8a expression and the clinicopathologic characteristics of ESCC. p < 0.05 was considered as statistically different. * represented p < 0.05; **represented p < 0.01. Results Rab8a expression in pan-cancer We analyzed the expression of Rab8a by applying the TIMER2.0 and found that it was significantly increased in multiple tumor types compared with those in normal tissues, including ESCA, bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cholangio carcinoma (CHOL), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carcinoma (LIHC), stomach adenocarcinoma (STAD) and uterine corpus endometrial carcinoma (UCEC) (Fig. 1 A). The results of GEPIA online database analysis demonstrated that the expression levels were obviously increased in ESCA, BRCA, STAD, glioblastoma (GBM), acute myeloid leukemia (LAML), lower grade glioma (LGG), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD) and testicular germ cell tumors (TGCT) than those in their normal tissues (Fig. 1 B). Also, the expression of Rab8a was analyzed across 24 types of human cancer samples compared with their normal controls in UALCAN online database and revealed that it was increased in the following cancers: ESCA, CHOL, BLCA, HNSC, KIRC, LIHC, STAD, UCEC, BRCA and cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC)(Fig. 1 C). Summarized the above results, it was found that the expression of Rab8a was significantly up-regulated in the common solid cancers of BRCA, STAD and ESCA. Expression and prognostic significance of Rab8a in BRCA, STAD and ESCA Then we explored the Rab8a expression with different histological subtypes and the OS significance in BRCA, STAD and ESCA. It was revealed that Rab8a expression was significantly increased in all of the histological subtypes of BRCA, STAD and ESCA (Fig. 2 A,C,E). And the survival analysis revealed that Rab8a expression had no prognostic value in BRCA and STAD (Fig. 2 B,D). But in ESCA, Rab8a was the valuable prognostic marker. It was found that the high expression of Rab8a revealed poor prognosis of ESCC patients but good prognosis of esophageal adenocarcinoma (EAC) (Fig. 2 F,G). So, the above analytical results implied that Rab8a might be an important diagnostic and prognostic marker of ESCC. Rab8a expression and its correlation with the clinicopathological characteristics in human ESCC tissues We then detected Rab8a mRNA expression in 12 cases of ESCC and their paired adjacent normal tissues by qPCR. It was found Rab8a mRNA was obviously increased (T/N > 0.5) in 9 cases (Fig. 3 A,B). The IHC results showed that the rate of high expression of Rab8a protein was obviously increased in ESCC (77.42%) than that in their paired adjacent normal tissues (8.06%) (Fig. 3 C,D). Further analysis of the correlation of Rab8a expression with ESCC clinicopathological characteristics demonstrated that the expression of Rab8a was significantly associated with the size and infiltration depth of ESCC (Table 1 ). Therefore, the higher expression of Rab8a implied greater ability of proliferation and invasion. Table 1 Clinicopathologic Characteristics of Rab8a Expression in ESCC Patients Characteristics STX2 Expression χ 2 Value p Value Low High Age < 60 9 19 2.671 0.102 ≥ 60 5 29 Gender Male 8 32 0.429 0.512 Female 6 16 Differentiation Well-Moderate 11 34 0.326 0.568 Poor 3 14 Tumor Size < 2 cm 7 8 6.566 0.010 ≥ 2 cm 7 40 T classification T1 + T2 9 15 4.986 0.026 T3 + T4 5 33 N classification N0 4 16 0.112 0.737 N1 10 32 Overexpression of Rab8a increased the proliferation and migration of ESCC To elucidate the biological roles of Rab8a in ESCC progression, we established the ESCC cells of ECA109-Rab8a and ECA9706-Rab8a with stable Rab8a overexpression (Fig. 4 A,B). Then, we conducted a series of in vitro biological experiments with these cells. The CCK-8 and colony formation assays showed that the overexpression of Rab8a obviously promoted the abilities of ESCC proliferation (Fig. 4 C-F). Transwell migration and wound-healing assays revealed that the migrated abilities of these ESCC cells were significantly increased compared with their vector cells (Fig. 4 G-J). Repressed expression of Rab8a decreased the proliferation and migration of ESCC To further verify the biological roles of Rab8a in ESCC progression, we also established the ESCC cells of ECA109-shRab8a and ECA9706-shRab8a with stable knockdown expression of Rab8a (Fig. 5 A,B). Then we conducted in vitro biological experiments with these cells. The CCK-8 and colony formation assays demonstrated that the knockdown expression of Rab8a could inhibit the proliferation of ESCC (Fig. 4 C-F). Transwell migration and wound-healing assays confirmed that the migrated abilities of these ESCC cells were markedly repressed compared with their control cells (Fig. 4 G-J). So, the above data verified that the increased expression of Rab8a could facilitate ESCC progression. Rab8a promoted ESCC progression by activating mitochondrial mitochondrial respiration To investigate the molecular mechanism of Rab8a in promoting ESCC progression, we conducted the GSEA using the publicly available portal of LinkedOmics. The results showed that the mitochondrial respiration pathway-related genes were significantly enriched, including the mitochondrial gene expression, mitochondrial respiratory chain complex assembly and NADH dehydrogenase complex assembly pathways (Fig. 6 A-D)༎NADH ubiquinone oxidoreductase subunit A1 (NDUFA1) and Cytochrome C1 (CYC1) had been identified as key genes of these pathways. So, we analyzed the correlation between the expression of Rab8a and NDUFA1, CYC1 in GSE21293, GSE33426 and GSE38129 and found the expression of Rab8a was positively correlated with NDUFA1 and CYC1 (Fig. 5 E-G). These data implied that Rab8a promoted ESCC progression by activating mitochondrial mitochondrial respiration. Rab8a-associated therapeutic drugs The results of gene-drug interaction network showed that Rab8a expression could be decreased by many popular clinical anticancer drugs (Fig. 7 A). The development of acquired resistance to therapy remains the current stumbling blocks in our fighting against cancer. Therefore, Rab8a might be a potential therapeutic target in combating ESCC. Discussion There are notable differences in the morbidity of ESCA among countries and more than half of the cases occur in China[ 11 ]. The carcinogenesis of ESCA is a complex process involving numerous gene alterations. And the two major histologic types of ESCA are ESCC and ECA. And they have different geographic patterns, causes and prognosis[ 12 ]. Patients with cancers have poor prognosis mostly because of the late-stage at diagnosis[ 13 , 14 ]. So it is very important to find the valuable diagnostic marker for combating cancers. Rab8 is a member of Ras superfamily and key regulator of intracellular membrane trafficking [ 6 , 15 , 16 ]. Previous studies demonstrated that Rab8 played important roles in the cell migration, polarization and intracellular signal transduction[ 17 , 18 ]. Rab8a is one major subtype of Rab8 and is reported to play important roles in endometrial cancer and hepatocellular carcinoma and might be a potential tumor marker[ 19 ]. In this study, we first analyzed the expression of Rab8a by employing multiple public cancer genomics programs and found that Rab8a was obviously increased in some common solid cancers including ESCA. As more than 70% of ESCA cases occurred in China, and the main histology type was ESCC, we mainly explored the expression and significance of Rab8a in ESCC [ 2 , 20 ]. The analytical results based on the online web resource of UALCAN and Kaplan-Meier plotter showed that Rab8a was significantly up-regulated in ESCC and the high expression of Rab8a indicated poor prognosis of ESCC patients which implied that Rab8a may serve as a diagnostic and prognostic marker of ESCC. Therefore, we next detected the mRNA and protein expression of Rab8a in human ESCC tissues by qPCR and IHC and found that the expression Rab8a mRNA and protein was obviously increased in ESCC compared with their paired adjacent normal tissues. And the further analysis of the relationship of Rab8a protein expression and the clinicopathological characteristics showed that the high expression of Rab8a implied larger size and deeper invasion of ESCC. So, Rab8a could be a valuable marker for the diagnosis and prognosis of ESCC. In addition, we constructed the ESCC cells with stable over or knockdown expression of Rab8a and conducted the CCK-8, colony formation, transwell migration and wound-healing assays. The results identified Rab8a as a key promoter of ESCC proliferation and migration. To further explore the molecular mechanisms of Rab8a in promoting ESCC progression, we conducted the analysis of GSEA and found that Rab8a the mitochondrial respiration pathway-related genes were enriched significantly. It became increasingly clear that the increased metabolic activity of mitochondria could promote the immortalization of tumor cells[ 21 ]. And the dysregulation of cellular metabolism is a hallmark of cancer, which is mainly to meet the bioenergetic demands of the high proliferation rates of cancer cells[ 22 ]. As we know, the normal bioenergetics of mitochondrial respiration is a vital process that produces ATP and provides energy to support the growth of cancers[ 23 , 24 ]. NDUFA1 and CYC1 are important genes of mitochondrial electron transport chain which had been identified to play important roles in the mitochondrial respiratory chain by transferring electrons[ 25 , 26 ]. Next, the further correlation analysis revealed the positive correlation between the expression of Rab8a and NDUFA1, CYC1 in public GEO databases. So, the data implied that Rab8a promoted ESCC progression by activating mitochondrial mitochondrial respiration. As the excessive mitochondrial respiration could increase tumor cells oxygen consumption, which triggers hypoxia and irregular blood vessels formation and eventually results in the resistance to therapy [ 27 ]. To explore the role of Rab8a in cancer therapy, we conducted the analysis of gene-drug interaction network constructed via CTD and Cytoscape and found that Rab8a could be decreased by many popular clinical anticancer drugs. That is to say, the acquired resistance to these drugs might be overcome by the interference expression of Rab8a. Therefore, Rab8a might be a potential therapeutic target for ESCC therapy. Conclusions In conclusion, this study confirmed that Rab8a was up-regulated in ESCC and served as a key promoter in ESCC progression by activating mitochondrial respiration. Our current study uncovered a novel tumor-promoting function of Rab8a in ESCC and offered a potential diagnostic and therapeutic biomarker. Abbreviations ESCA,esophageal carcinoma ESCC,esophageal squamous cell carcinoma TIMER,Tumor Immune Estimation Resource TCGA,The Cancer Genome Atlas GEPIA,Gene Expression Profiling Interactive Analysis UALCAN,The University of Alabama at Birmingham Cancer data analysis Portal GEO, Gene Expression Omnibus IHC,Immunohistochemistry NDUFA1,NADH:Ubiquinone Oxidoreductase Subunit A1 CYC1,Cytochrome C1 Ntpm,The normalized transcripts per million OS,overall survival DAB,diaminobenzidine IRS,immunoreactive score PVDF, polyvinylidene difluoride SDS-PAGE,sodium dodecyl sulfate polyacrylamide gel electropheresis GSEA,Gene set enrichment analysis BRCA,breast invasive carcinoma BLCA,bladder urothelial carcinoma CHOL,cholangio carcinoma HNSC,head and neck squamous cell carcinoma KIRC,kidney renal clear cell carcinoma LIHC,liver hepatocellular carcinoma STAD,stomach adenocarcinoma UCEC,uterine corpus endometrial carcinoma GBM,glioblastoma LAML,acute myeloid leukemia LGG,lower grade glioma OV,ovarian serous cystadenocarcinoma TGCT,testicular germ cell tumors PAAD,pancreatic adenocarcinoma CESC,cervical squamous cell carcinoma and endocervical adenocarcinoma EAC,esophageal adenocarcinoma Declarations Ethics approval and consent to participate All participants provided written informed consent to participate and the tissue acquisition protocol was approved by the Ethic Institutional Board of Xinxiang Medical University. Consent for publication The relevant patients were informed and agreed to publication. Availability of data and materials The datasets used during the present study are available from the corresponding authors on reasonable request. Competing interests The authors declare that they have no competing interests. Authors' contributions YX -W and JC: conseptualization, methodology, validation, formal analysis, investigation, writing-original draft;RL: investigation,formal analysis, investigation, writing-original draft; ZT-K: methodology, investigation; TG,YRN-W,ZY-HF: investigation.All authors read and approved the final manuscript. Acknowledgements Not applicable. References Sung H, Ferlay J, Siegel RL , et al . Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries . CA Cancer J Clin 2021, 71 (3):209-249. Chen W, Zheng R, Baade PD , et al . Cancer statistics in China, 2015 . CA Cancer J Clin 2016, 66 (2):115-132. 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Bonnay F, Veloso A, Steinmann V , et al . Oxidative Metabolism Drives Immortalization of Neural Stem Cells during Tumorigenesis . Cell 2020, 182 (6):1490-1507.e1419. Rabe P, Liebing AD, Krumbholz P , et al . Succinate receptor 1 inhibits mitochondrial respiration in cancer cells addicted to glutamine . Cancer Lett 2022, 526 :91-102. Huang D, Jing G, Zhu S. Regulation of Mitochondrial Respiration by Hydrogen Sulfide . Antioxidants (Basel, Switzerland) 2023, 12 (8). Magrì A, Lipari CLR, Risiglione P , et al . ERK1/2-dependent TSPO overactivation associates with the loss of mitophagy and mitochondrial respiration in ALS . Cell Death Dis 2023, 14 (2):122. Au HC, Seo BB, Matsuno-Yagi A , et al . The NDUFA1 gene product (MWFE protein) is essential for activity of complex I in mammalian mitochondria . Proc Natl Acad Sci U S A 1999, 96 (8):4354-4359. Gaignard P, Menezes M, Schiff M , et al . Mutations in CYC1, encoding cytochrome c1 subunit of respiratory chain complex III, cause insulin-responsive hyperglycemia . Am J Hum Genet 2013, 93 (2):384-389. Akter Z, Salamat N, Ali MY , et al . The promise of targeting heme and mitochondrial respiration in normalizing tumo Additional Declarations No competing interests reported. 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-3839052","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265807435,"identity":"435b9c8a-a78c-4fe5-9784-6d529f551a8e","order_by":0,"name":"Rong Liu","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Liu","suffix":""},{"id":265807436,"identity":"1de74918-472e-4038-aee6-f3bb2ae94fe0","order_by":1,"name":"Zhanting Kang","email":"","orcid":"","institution":"Xinxiang Medical 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University","correspondingAuthor":true,"prefix":"","firstName":"Yongxia","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-01-06 06:44:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3839052/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3839052/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49386885,"identity":"66a89059-74db-46c7-97c1-80878c5e9a19","added_by":"auto","created_at":"2024-01-09 20:02:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1104225,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRab8a expression in pan-cancer. \u003c/strong\u003e(A)Expression levels of Rab8a in TCGA cancers were analyzed by TIMER2.0 database.(B) Expression levels of Rab8a were analyzed by GEPIA.(C) Expression levels of Rab8a in TCGA cancers were analyzed by UALCAN.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3839052/v1/b225772ef4e73f49359753a3.jpg"},{"id":49386243,"identity":"fbcb3441-4061-49e9-9f32-4dcab142763e","added_by":"auto","created_at":"2024-01-09 19:54:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":621698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression and prognostic significance of Rab8a in BRCA, STAD and ESCA.\u003c/strong\u003e(A) Rab8aexpression in all of the histological subtypes of BRCA, STADand ESCA.(B) KM survival curves showed that Rab8a expression was highly associated with clinical outcomes in BRCA.(C) Rab8a expression in all of the histological subtypes of STAD.(D) KM survival curves showed that Rab8a expression was highly associated with clinical outcomes in STAD.(E) Rab8a expression in all of the histological subtypes of ESCA.(F,G) KM survival curves showed that Rab8a expression was highly associated with clinical outcomes in EAC and ESCC.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3839052/v1/37b8f619d4ec3800fe34b824.jpg"},{"id":49386247,"identity":"1ada93f9-d9fa-4b01-8a28-add0eb249a76","added_by":"auto","created_at":"2024-01-09 19:54:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":369539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRab8a expression and its correlation with the clinicopathological characteristics in\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ehuman ESCC tissues.\u003c/strong\u003e(A,B) qPCR of Rab8a in 12 cases of fresh human ESCC and normal tissues (2\u003csup\u003e–ΔΔCt\u003c/sup\u003e, T/N, dotted line represents two-fold difference.(C,D) IHC of Rab8a in ESCC and normal tissues. ** p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3839052/v1/01ded078b907bd2661f0961b.jpg"},{"id":49386884,"identity":"c06943f1-3ffd-43d4-b11a-5fc73624a66b","added_by":"auto","created_at":"2024-01-09 20:02:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":960775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of Rab8a increased the proliferation and migration of ESCC.\u003c/strong\u003e (A, B) The confirmation of Rab8a overexpressed ESCC cells by qPCR and Western blot analysis.(C-F)The proliferative ability of the indicated cells detected by CCK-8 assays and colony formation assays.Histograms represent the average number of the colonies from three independent experiments. Error bars represent mean±s.d.(G-J)The migration ability of the indicated cells detected by Transwell migration and wound-healing assays.** p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3839052/v1/d9d5cbc75a75a846906b2f1a.jpg"},{"id":49386244,"identity":"566f9667-ccaf-485d-9a50-fccb5e0a9d00","added_by":"auto","created_at":"2024-01-09 19:54:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":928286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepressed expression of Rab8a decreased the proliferation and migration of ESCC .\u003c/strong\u003e(A, B) The construction and confirmation of stable knockdown ECA109 and ECA9706 cells of Rab8a by qPCR and Western blot analysis. (C-F)The proliferative ability of the indicated cells detected by CCK-8 assays and colony formation assays.Histograms represent the average number of the colonies from three independent experiments. Error bars represent mean±s.d. (G-J)The migration ability of the indicated cells detected by Transwell migration and wound-healing assays.** p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3839052/v1/873334a87a9ab6bf1a173c96.jpg"},{"id":49386249,"identity":"ecbbfc3a-0c21-4d28-9290-b65ff5a4334e","added_by":"auto","created_at":"2024-01-09 19:54:12","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":825659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRab8a promoted ESCC progression by activating mitochondrial mitochondrial respiration.\u003c/strong\u003e (A-C) GO and GSEA analysis results in Linkedomics.(E-G) The expression correlation between Rab8a and NDUFA1,CYC1.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3839052/v1/6caba9fb5e7683f6854332d0.jpg"},{"id":49386246,"identity":"39ed3ce0-eee1-413e-8b65-939f8f2002b3","added_by":"auto","created_at":"2024-01-09 19:54:11","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":240358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRab8a-associated therapeutic drugs.\u003c/strong\u003e blue arrows: Chemotherapeutic drugs relevant to lower GNB2 expression, and count of arrows: specific interaction supported numbers of studies in literature.\u003c/p\u003e","description":"","filename":"Fig7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3839052/v1/628b29e903bcb83766fa1961.jpeg"},{"id":50804769,"identity":"c17ad38f-5258-441f-a25b-aac4276ae3f7","added_by":"auto","created_at":"2024-02-07 15:02:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2133735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3839052/v1/18919f2c-3843-46b5-89c6-dc9671532bf0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rab8a serves as a valuable biomarker of esophageal squamous cell carcinoma","fulltext":[{"header":"Background","content":"\u003cp\u003eEsophageal carcinoma (ESCA) is one common malignant tumor of digestive system with the seventh incidence and sixth mortality worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. And the main histologic subtype is esophageal squamous cell carcinoma (ESCC) in China, which accounting for about 90% of all diagnosed cases[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although there has been improvement of therapeutic approaches, the outcome of ESCC patients remains poor because of the late diagnosis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, it is of great significance to find a valuable biomarker for ESCC diagnosis.\u003c/p\u003e \u003cp\u003eRab8a protein is a member of RAS superfamily which participates in membrane traffic processes [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previous study showed that Rab8a was up-regulated in endometrial cancer and might be a new biomarker [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition, it was reported that the enhanced expression of Rab8a could improve the immunotherapy efficacy of hepatocellular carcinoma, which suggested that it might be a novel therapeutic target [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the expression and biological functions remain unknown in other human cancers including ESCC.\u003c/p\u003e \u003cp\u003eIn the current study, we first analyzed the expression of Rab8a in many cancer types and their normal tissues by conducting pan-cancer analysis from some public databases. And according to the result of the analysis, we then investigated the expression and biological function of Rab8a in ESCC progression and at last explored the potential molecular mechanism of Rab8a in ESCC.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRab8a expression and survival analysis in different cancers\u003c/h2\u003e \u003cp\u003eThe normalized transcripts per million (nTPM) levels of Rab8a in different types of tumors were identified based on the data of TIMER, Gene Expression Profiling Interactive Analysis (GEPIA) and UALCAN online resources. The relationship of Rab8a mRNA expression and the pathological features were available from UALCAN online databases. Also, we analyzed the correlation between Rab8a expression and overall survival (OS) outcomes of the patients with different cancers using the web database of Kaplan-Meier plotter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTissue specimens, Cell lines and transfection\u003c/h2\u003e \u003cp\u003e62 cases of formalin-fixed paraffin-embedded and 12 cases of fresh ESCC samples and their matched adjacent normal tissues were collected from the Department of pathology, Third Affiliated Hospital of Xinxiang Medical University (Xinxiang City, Henan Province, China) from June 2016 to July 2022. The fresh tissues were frozen in liquid nitrogen before extracting the total RNA. None of the patients underwent chemotherapy, radiotherapy or immunotherapy before surgery. Prior approval for the study had been obtained from Xinxiang Medical University Institutional Board (Xinxiang, China).\u003c/p\u003e \u003cp\u003eESCC cell lines of ECA109 and ECA9706 were obtained from the Cell Bank of the Chinese Academy (Shanghai, China). All ESCC cells were cultured in RPMI-1640 with 10% FBS at 37\u0026deg;C in a humidified atmosphere with 5% CO2.\u003c/p\u003e \u003cp\u003eThe overexpression and knockdown plasmids of Rab8a were the products of Public Protein/Plasmid Library (PPL). Lentivirus was produced by HEK293FT cells using the calcium phosphate method. The transfected cells were selected in medium containing puromycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eThe formalin-fixed paraffin-embedded sample tissues were cut into 4-\u0026micro;m sections and baked at 60\u0026deg;C for 2 h. IHC was conducted by the SP kits (ZSGB-BIO, Beijing, China). The sections were incubated overnight at 4\u0026deg;C with the rabbit polyclonal antibody of Rab8a (1:100, Proteintech, USA). PBS was used as the negative control. The sections were stained with 3,3-diaminobenzidine (DAB) and sealed with neutral balsam. Finally, we observed the sections by two pathologists used immunoreactive score (IRS) which was mainly based on the proportion and intensity of the stained tumor cells[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eReal-time quantitative PCR (qPCR)\u003c/h2\u003e \u003cp\u003eTRIzol reagent (Invitrogen, USA) was used to extract the total RNA from human tissues and cultured cells. qPCR was carried out according to the manufacturer\u0026rsquo;s protocol with SYBR Green I. The data were normalized to the the housekeeping gene of GAPDH, and calculated as 2\u003csup\u003e\u0026ndash;ΔΔCT\u003c/sup\u003e. The follows were primer sequences: GAPDH (F: ACA GTC AGC CGC ATC TTC TT, R: GAC AAG CTT CCC GTT CTC AG), Rab8a (F: ACG CCT TCA ACT CCA CTT; R: ACC AGC ATG ATG CCC ATT).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eThe protein was extracted from the corresponding cells with the sample buffer lysate solution. Then the lysates were subjected to SDS-PAGE and transferred to PVDF membranes. 5% non-fat dry milk were used to block the membranes and incubated overnight at 4\u0026deg;C with the primary antibodies of polyclonal rabbit anti-Rab8a and mouse anti-GAPDH (Proteintech, USA). The appropriate secondary antibodies were used to incubate the membranes at room temperature for 1 h and then exposed for autoradiography.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCCK-8 assay\u003c/h2\u003e \u003cp\u003eCells with stable over- or knockdown expression of Rab8a were seeded in 96-well plates at the density of 1\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well and cultured for 24 h. Then, the culture medium was discarded and 10 \u0026micro;L of CCK-8 reagent and 100 \u0026micro;L of medium were added to every well. After 2 h incubation, the absorbance at 450 nm was detected. The experiment was conducted repeatedly for three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay\u003c/h2\u003e \u003cp\u003eCells with stable over- or knockdown expression of Rab8a were plated on 6-well plates (200 cells/well) and cultured for 2 weeks. The colonies were stained with Hematoxylin for 30 min after fixation with 4% paraformaldehyde for 10 minutes. The number of colonies, defined as \u0026gt;\u0026thinsp;50 cells/colony, were counted. Three independent experiments were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTranswell migration assay\u003c/h2\u003e \u003cp\u003eBoyden chambers were used to conducted Transwell migration assays. Cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e) in culture medium without FBS were seeded to the upper chamber, and culture medium containing 20% FBS as a chemoattractant was added to the lower chamber. After incubation for 48 h, the chamber was fixed in 4% paraformaldehyde and stained with Hematoxylin. Cells on the upper sides of the filters were removed with cotton swabs. Cells that migrated to the lower sides were stained with Hematoxylin. The migratory cells on the lower surfaces of the filters were counted. Three independent experiments were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWound-healing assay\u003c/h2\u003e \u003cp\u003eCells were seeded to 6-well plates and incubated under permissive conditions until the cells reached 90% confluence. After serum starvation for 24 h, wounds were generated in the confluent cells using a pipette tip. Wound healing within the scrape line was then observed and photographed at indicated time points. Each experiment was repeated at least three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGene set enrichment analysis\u003c/h2\u003e \u003cp\u003eGene set enrichment analysis (GSEA) is a knowledge-based approach for interpreting genome-wide expression differences between two biological states. We performed GSEA to explore the potential biological functions affected by Rab8a expression in ESCC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using SPSS20.0 for Windows. The data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (s.d.) from at least three independent experiments. The two-tailed paired Student's t-test was conducted for the analysis of two groups. Mann-Whitney U test was conducted to analyze the relationship between Rab8a expression and the clinicopathologic characteristics of ESCC. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically different. * represented \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **represented \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRab8a expression in pan-cancer\u003c/h2\u003e \u003cp\u003eWe analyzed the expression of Rab8a by applying the TIMER2.0 and found that it was significantly increased in multiple tumor types compared with those in normal tissues, including ESCA, bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cholangio carcinoma (CHOL), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carcinoma (LIHC), stomach adenocarcinoma (STAD) and uterine corpus endometrial carcinoma (UCEC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results of GEPIA online database analysis demonstrated that the expression levels were obviously increased in ESCA, BRCA, STAD, glioblastoma (GBM), acute myeloid leukemia (LAML), lower grade glioma (LGG), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD) and testicular germ cell tumors (TGCT) than those in their normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Also, the expression of Rab8a was analyzed across 24 types of human cancer samples compared with their normal controls in UALCAN online database and revealed that it was increased in the following cancers: ESCA, CHOL, BLCA, HNSC, KIRC, LIHC, STAD, UCEC, BRCA and cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC)(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Summarized the above results, it was found that the expression of Rab8a was significantly up-regulated in the common solid cancers of BRCA, STAD and ESCA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eExpression and prognostic significance of Rab8a in BRCA, STAD and ESCA\u003c/h2\u003e \u003cp\u003eThen we explored the Rab8a expression with different histological subtypes and the OS significance in BRCA, STAD and ESCA. It was revealed that Rab8a expression was significantly increased in all of the histological subtypes of BRCA, STAD and ESCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,C,E). And the survival analysis revealed that Rab8a expression had no prognostic value in BRCA and STAD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB,D). But in ESCA, Rab8a was the valuable prognostic marker. It was found that the high expression of Rab8a revealed poor prognosis of ESCC patients but good prognosis of esophageal adenocarcinoma (EAC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF,G). So, the above analytical results implied that Rab8a might be an important diagnostic and prognostic marker of ESCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRab8a expression and its correlation with the clinicopathological characteristics in human ESCC tissues\u003c/h2\u003e \u003cp\u003eWe then detected Rab8a mRNA expression in 12 cases of ESCC and their paired adjacent normal tissues by qPCR. It was found Rab8a mRNA was obviously increased (T/N\u0026thinsp;\u0026gt;\u0026thinsp;0.5) in 9 cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B). The IHC results showed that the rate of high expression of Rab8a protein was obviously increased in ESCC (77.42%) than that in their paired adjacent normal tissues (8.06%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC,D). Further analysis of the correlation of Rab8a expression with ESCC clinicopathological characteristics demonstrated that the expression of Rab8a was significantly associated with the size and infiltration depth of ESCC (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, the higher expression of Rab8a implied greater ability of proliferation and invasion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinicopathologic Characteristics of Rab8a Expression in ESCC Patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSTX2 Expression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003eValue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;60\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e2.671\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.102\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026ge;\u0026thinsp;60\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e29\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e32\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.429\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.512\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDifferentiation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWell-Moderate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e34\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.326\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.568\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePoor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor Size\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;2 cm\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e6.566\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.010\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026ge;\u0026thinsp;2 cm\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e40\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT classification\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT1\u0026thinsp;+\u0026thinsp;T2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e4.986\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.026\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT3\u0026thinsp;+\u0026thinsp;T4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e33\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eN classification\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eN0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.112\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e0.737\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eN1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e32\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOverexpression of Rab8a increased the proliferation and migration of ESCC\u003c/h2\u003e \u003cp\u003eTo elucidate the biological roles of Rab8a in ESCC progression, we established the ESCC cells of ECA109-Rab8a and ECA9706-Rab8a with stable Rab8a overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B). Then, we conducted a series of in vitro biological experiments with these cells. The CCK-8 and colony formation assays showed that the overexpression of Rab8a obviously promoted the abilities of ESCC proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-F). Transwell migration and wound-healing assays revealed that the migrated abilities of these ESCC cells were significantly increased compared with their vector cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRepressed expression of Rab8a decreased the proliferation and migration of ESCC\u003c/h2\u003e \u003cp\u003eTo further verify the biological roles of Rab8a in ESCC progression, we also established the ESCC cells of ECA109-shRab8a and ECA9706-shRab8a with stable knockdown expression of Rab8a (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,B). Then we conducted in vitro biological experiments with these cells. The CCK-8 and colony formation assays demonstrated that the knockdown expression of Rab8a could inhibit the proliferation of ESCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-F). Transwell migration and wound-healing assays confirmed that the migrated abilities of these ESCC cells were markedly repressed compared with their control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-J). So, the above data verified that the increased expression of Rab8a could facilitate ESCC progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eRab8a promoted ESCC progression by activating mitochondrial mitochondrial respiration\u003c/h2\u003e \u003cp\u003eTo investigate the molecular mechanism of Rab8a in promoting ESCC progression, we conducted the GSEA using the publicly available portal of LinkedOmics. The results showed that the mitochondrial respiration pathway-related genes were significantly enriched, including the mitochondrial gene expression, mitochondrial respiratory chain complex assembly and NADH dehydrogenase complex assembly pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D)༎NADH ubiquinone oxidoreductase subunit A1 (NDUFA1) and Cytochrome C1 (CYC1) had been identified as key genes of these pathways. So, we analyzed the correlation between the expression of Rab8a and NDUFA1, CYC1 in GSE21293, GSE33426 and GSE38129 and found the expression of Rab8a was positively correlated with NDUFA1 and CYC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-G). These data implied that Rab8a promoted ESCC progression by activating mitochondrial mitochondrial respiration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eRab8a-associated therapeutic drugs\u003c/h2\u003e \u003cp\u003eThe results of gene-drug interaction network showed that Rab8a expression could be decreased by many popular clinical anticancer drugs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The development of acquired resistance to therapy remains the current stumbling blocks in our fighting against cancer. Therefore, Rab8a might be a potential therapeutic target in combating ESCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere are notable differences in the morbidity of ESCA among countries and more than half of the cases occur in China[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The carcinogenesis of ESCA is a complex process involving numerous gene alterations. And the two major histologic types of ESCA are ESCC and ECA. And they have different geographic patterns, causes and prognosis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Patients with cancers have poor prognosis mostly because of the late-stage at diagnosis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. So it is very important to find the valuable diagnostic marker for combating cancers.\u003c/p\u003e \u003cp\u003eRab8 is a member of Ras superfamily and key regulator of intracellular membrane trafficking [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Previous studies demonstrated that Rab8 played important roles in the cell migration, polarization and intracellular signal transduction[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Rab8a is one major subtype of Rab8 and is reported to play important roles in endometrial cancer and hepatocellular carcinoma and might be a potential tumor marker[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we first analyzed the expression of Rab8a by employing multiple public cancer genomics programs and found that Rab8a was obviously increased in some common solid cancers including ESCA. As more than 70% of ESCA cases occurred in China, and the main histology type was ESCC, we mainly explored the expression and significance of Rab8a in ESCC [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The analytical results based on the online web resource of UALCAN and Kaplan-Meier plotter showed that Rab8a was significantly up-regulated in ESCC and the high expression of Rab8a indicated poor prognosis of ESCC patients which implied that Rab8a may serve as a diagnostic and prognostic marker of ESCC. Therefore, we next detected the mRNA and protein expression of Rab8a in human ESCC tissues by qPCR and IHC and found that the expression Rab8a mRNA and protein was obviously increased in ESCC compared with their paired adjacent normal tissues. And the further analysis of the relationship of Rab8a protein expression and the clinicopathological characteristics showed that the high expression of Rab8a implied larger size and deeper invasion of ESCC. So, Rab8a could be a valuable marker for the diagnosis and prognosis of ESCC. In addition, we constructed the ESCC cells with stable over or knockdown expression of Rab8a and conducted the CCK-8, colony formation, transwell migration and wound-healing assays. The results identified Rab8a as a key promoter of ESCC proliferation and migration.\u003c/p\u003e \u003cp\u003eTo further explore the molecular mechanisms of Rab8a in promoting ESCC progression, we conducted the analysis of GSEA and found that Rab8a the mitochondrial respiration pathway-related genes were enriched significantly. It became increasingly clear that the increased metabolic activity of mitochondria could promote the immortalization of tumor cells[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. And the dysregulation of cellular metabolism is a hallmark of cancer, which is mainly to meet the bioenergetic demands of the high proliferation rates of cancer cells[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As we know, the normal bioenergetics of mitochondrial respiration is a vital process that produces ATP and provides energy to support the growth of cancers[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. NDUFA1 and CYC1 are important genes of mitochondrial electron transport chain which had been identified to play important roles in the mitochondrial respiratory chain by transferring electrons[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Next, the further correlation analysis revealed the positive correlation between the expression of Rab8a and NDUFA1, CYC1 in public GEO databases. So, the data implied that Rab8a promoted ESCC progression by activating mitochondrial mitochondrial respiration.\u003c/p\u003e \u003cp\u003eAs the excessive mitochondrial respiration could increase tumor cells oxygen consumption, which triggers hypoxia and irregular blood vessels formation and eventually results in the resistance to therapy [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To explore the role of Rab8a in cancer therapy, we conducted the analysis of gene-drug interaction network constructed via CTD and Cytoscape and found that Rab8a could be decreased by many popular clinical anticancer drugs. That is to say, the acquired resistance to these drugs might be overcome by the interference expression of Rab8a. Therefore, Rab8a might be a potential therapeutic target for ESCC therapy.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study confirmed that Rab8a was up-regulated in ESCC and served as a key promoter in ESCC progression by activating mitochondrial respiration. Our current study uncovered a novel tumor-promoting function of Rab8a in ESCC and offered a potential diagnostic and therapeutic biomarker.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eESCA,esophageal carcinoma\u003c/p\u003e\n\u003cp\u003eESCC,esophageal squamous cell carcinoma\u003c/p\u003e\n\u003cp\u003eTIMER,Tumor\u0026nbsp;Immune Estimation Resource\u003c/p\u003e\n\u003cp\u003eTCGA,The Cancer Genome Atlas\u003c/p\u003e\n\u003cp\u003eGEPIA,Gene Expression Profiling Interactive Analysis\u003c/p\u003e\n\u003cp\u003eUALCAN,The\u0026nbsp;University of\u0026nbsp;Alabama at Birmingham\u0026nbsp;Cancer data analysis Portal\u003c/p\u003e\n\u003cp\u003eGEO, Gene Expression Omnibus\u003c/p\u003e\n\u003cp\u003eIHC,Immunohistochemistry\u003c/p\u003e\n\u003cp\u003eNDUFA1,NADH:Ubiquinone Oxidoreductase Subunit A1\u003c/p\u003e\n\u003cp\u003eCYC1,Cytochrome C1\u003c/p\u003e\n\u003cp\u003eNtpm,The normalized transcripts per million\u003c/p\u003e\n\u003cp\u003eOS,overall survival\u003c/p\u003e\n\u003cp\u003eDAB,diaminobenzidine\u003c/p\u003e\n\u003cp\u003eIRS,immunoreactive score\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePVDF, polyvinylidene difluoride\u003c/p\u003e\n\u003cp\u003eSDS-PAGE,sodium dodecyl sulfate polyacrylamide gel electropheresis\u003c/p\u003e\n\u003cp\u003eGSEA,Gene set enrichment analysis\u003c/p\u003e\n\u003cp\u003eBRCA,breast invasive carcinoma\u003c/p\u003e\n\u003cp\u003eBLCA,bladder urothelial carcinoma\u003c/p\u003e\n\u003cp\u003eCHOL,cholangio carcinoma\u003c/p\u003e\n\u003cp\u003eHNSC,head and neck squamous cell carcinoma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKIRC,kidney renal clear cell carcinoma\u003c/p\u003e\n\u003cp\u003eLIHC,liver hepatocellular carcinoma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSTAD,stomach adenocarcinoma\u003c/p\u003e\n\u003cp\u003eUCEC,uterine corpus endometrial carcinoma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGBM,glioblastoma\u003c/p\u003e\n\u003cp\u003eLAML,acute myeloid leukemia\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLGG,lower grade glioma\u003c/p\u003e\n\u003cp\u003eOV,ovarian serous cystadenocarcinoma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTGCT,testicular germ cell tumors\u003c/p\u003e\n\u003cp\u003ePAAD,pancreatic adenocarcinoma\u003c/p\u003e\n\u003cp\u003eCESC,cervical squamous cell carcinoma and endocervical adenocarcinoma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEAC,esophageal adenocarcinoma\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants provided written informed consent to participate and the tissue acquisition protocol was approved by the Ethic Institutional Board of Xinxiang Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe relevant patients were informed and agreed to publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used during the present study are available from the corresponding authors on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYX -W and JC: conseptualization, methodology, validation, formal analysis, investigation, writing-original draft;RL: investigation,formal analysis, investigation, writing-original draft; ZT-K: methodology, investigation; TG,YRN-W,ZY-HF: investigation.All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot 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\u003cstrong\u003e96\u003c/strong\u003e(8):4354-4359.\u003c/li\u003e\n\u003cli\u003eGaignard P, Menezes M, Schiff M\u003cem\u003e, et al\u003c/em\u003e.\u003cstrong\u003eMutations in CYC1, encoding cytochrome c1 subunit of respiratory chain complex III, cause insulin-responsive hyperglycemia\u003c/strong\u003e. \u003cem\u003eAm J Hum Genet \u003c/em\u003e2013, \u003cstrong\u003e93\u003c/strong\u003e(2):384-389.\u003c/li\u003e\n\u003cli\u003eAkter Z, Salamat N, Ali MY\u003cem\u003e, et al\u003c/em\u003e.\u003cstrong\u003eThe promise of targeting heme and mitochondrial respiration in normalizing tumo\u003c/strong\u003e\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":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":"esophageal squamous cell carcinoma, Rab8a, proliferation, migration, biomarker","lastPublishedDoi":"10.21203/rs.3.rs-3839052/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3839052/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\n\u003cp\u003eEsophageal carcinoma (ESCA) is a digestive tract malignancy with high morbidity in China, among which esophageal squamous cell carcinoma (ESCC) \u0026nbsp;accounts for 90% of the confirmed cases. Rab8a is a member of the Ras small GTPase superfamily, and it has been shown to play an important role in endometrial cancer (EC) and hepatocellular carcinoma (HCC). However, the function of Rab8a in ESCC are currently unclear. This study aimed to investigate Rab8a as a biomarker for the diagnosis of ESCC.\u003c/p\u003e\n\u003cp\u003eMethods\u003c/p\u003e\n\u003cp\u003eThe study first used TIMER2.0, GEPIA and UALCAN to analyze the expression of Rab8a in a variety of clinically common malignancies including ESCC, followed by real-time PCR (quantitative real-time PCR, qPCR), Western blot, immunohistochemical (IHC) ，and a series of in vitro biological experiments\u003c/p\u003e\n\u003cp\u003eResults\u003c/p\u003e\n\u003cp\u003eRab8a is highly expressed in the esophageal cancer cells and tissues.overexpression of Rab8a can promote the proliferation and migration of ESCC ,while knockdown its expression can inhibit the proliferation and migration of ESCC. Correlation analysis revealed the positive correlation between the expression of Rab8a and NDUFA1, CYC1 in public GEO databases. Therefore, Rab8a may promote ESCC progression by activating mitochondrial respiration.\u003c/p\u003e\n\u003cp\u003eConclusions\u003c/p\u003e\n\u003cp\u003eThis study demonstrated that Rab8a is upregulated in ESCC and may promote ESCC cell proliferation and migration by activating mitochondrial respiration. This study provides a rationale for clinical diagnosis and screening of new therapeutic targets for ESCC.\u003c/p\u003e","manuscriptTitle":"Rab8a serves as a valuable biomarker of esophageal squamous cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-09 19:54:07","doi":"10.21203/rs.3.rs-3839052/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":"74e88c50-51d1-47c6-82bc-21a9d055b270","owner":[],"postedDate":"January 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-14T07:50:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-09 19:54:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3839052","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3839052","identity":"rs-3839052","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}