Transcriptomic Time-Course Sequencing: Insights into the Cell Wall Macromolecule-Mediated Fruit Dehiscence during Ripening in Camellia oleifera.

Camellia oleifera (C. oleifera), one of the world's four major edible woody oil crops, has been widely planted in southern China's subtropical region for the extremely high nutritional and health benefits of its seed oil. Timing and synchronization of fruit dehiscence are critical factors influencing the oil output and quality, as well as the efficiency and cost of harvesting C. oleifera, yet they extremely lack attention. To gain an understanding of the molecular basis underlying the dehiscence of C. oleifera fruit, we sampled pericarp-replum tissues containing dehiscence zones from fruits at different developmental stages and performed time-series transcriptomic sequencing and analysis for the first time. Statistical and GO enrichment analysis of differentially expressed genes revealed that drastic transcriptional changes occurred over the last short sampling interval (4 days, 18th-22nd October), which directed functional classifications link to cell wall and cell wall macromolecule activity. WGCNA further showed that factors controlling cell wall modification, including endo-1,3;1,4-beta-D-glucanase, WAT1-like protein 37, LRR receptor-like serine/threonine-protein kinase, and cellulose synthase A catalytic subunit, were identified as core members of the co-expression network of the last stage highly related modules. Furthermore, in these modules, we also noted genes that were annotated as coding for polygalacturonase and pectinesterase, two pectinases that were expected to be major players in cell separation during dehiscence. qRT-PCR further confirmed the expression profiles of these cell wall modification relating factors, which possessed a special high transcriptional abundance at the final stage. These results suggested the cell wall associated cell separation, one of the essential processes downstream of fruit dehiscence, happened in dehiscing fruit of C. oleifera during ripening. Hydrolases acting on cell wall components are good candidates for signal mediating dehiscence of C. oleifera fruit. In conclusion, our analysis provided insights into the cell wall macromolecule-mediated fruit dehiscence during ripening in C. oleifera.

Effects of traditional Chinese herbal feed supplement on growth performance, immunity, antioxidant levels, and intestinal health in chickens: a study on Ningdu yellow chickens.

Traditional Chinese herbs have been widely researched as a green, safe, and effective feed additive for poultry. The purpose of this study was to investigate the effects of traditional Chinese prescription (TCP) based on various herbs in a specific ratio on the growth performance, carcass traits, immunity, antioxidant level, and intestinal health of Ningdu yellow chickens. A total of 420 female Ningdu yellow chickens were randomly divided into 5 groups, with 6 replicates of 14 each. The chickens were fed with a basal diet supplemented with 0 (CON), 0.2, 0.4, 0.6, or 0.8% TCP from d 43 to 105. Body weight, feed intake, and serum biochemical indicators were recorded at d 70 and 105, intestinal morphology and microflora of the carcass were determined at d 105. Compared to the control group, chickens fed with TCP, particularly at the level of 0.6%, showed improved average daily gain and breast muscle percentage, as well as a lower feed-to-gain ratio with statistical significance (P < 0.05). Between 43 and 70 d of age, chickens fed with TCP exhibited higher levels of serum glutathione peroxidase activity, total antioxidant capacity, and superoxide dismutase, particularly in the group fed with the 0.6% level of TCP (P < 0.05). Between 43 and 105 d of age, feeding chickens with 0.4 and 0.6% TCP resulted in a decrease in serum IL-2 concentration, and increase in the IL-4 content (P < 0.05). Chickens fed with 0.4, 0.6, and 0.8% TCP had significantly higher jejunum villous height (P < 0.05), TCP supplementation also led to a marked increase in the relative abundance of Bacteroidota compared to the control group (P < 0.05). Collectively, the study suggests that TCP supplementation can enhance immune and antioxidant functions, improve jejunum morphology, and positively impact cecum microflora in chickens. Based on these results, a level of 0.6% TCP could be considered an optimum level as a feed supplement for Ningdu yellow chickens aged 43 to 105 d.

Genome-wide characterization and gene expression analyses of ALDH gene family in response to drought stress in moso bamboo (Phyllostachys edulis).

Aldehyde dehydrogenase (ALDH) superfamily, comprising enzymes dependent on NAD+ or NADP+, plays an important role in controlling plant growth and development, as well as in responsing to phytohormone and environmental stress. These enzymes possess the ability to prevent toxic effects of aldehydes by converting them into their corresponding carboxylic acids. However, the potential function of ALDH genes in moso bamboo (Phyllostachys edulis) remains largely unknown. In this study, the ALDH gene superfamily in moso bamboo was analyzed through genome-wide screening, the evolutionary relationship of expansion genes was conducted. Tissue-specific expression patterns of ALDH genes were observed in 26 different tissues. Plant hormone and environmental stress responsive cis-elements were identified in the promoter of ALDH genes, which were supported by public databases data on the expression patterns under various abiotic stresses and hormone treatments. ALDH activity was increased in moso bamboo seedlings exposed to drought, compared to control condition. Furthermore, PeALDH2B2 was found to physically interact with PeGPB1 in response to drought. Overall, the study provides a comprehensive analysis of the ALDH family in moso bamboo and contributes to our understanding of the function of ALDH genes in growth, development, and adaptation to drought stresses.

Tumor budding and tumor-infiltrating lymphocytes can predict prognosis in pT1b esophageal squamous cell carcinoma.

BACKGROUND: Tumor budding (TB) and tumor-infiltrating lymphocyte (TIL) are significant predictive indicators of lymph node metastasis (LNM) and unfavorable prognosis in various tumors. Currently, there is no gold standard for TB and TIL evaluation in esophageal squamous cell carcinoma (ESCC). This study aimed to identify the standard of TB and TIL evaluations and build a predictive model for prognosis among patients with pT1b ESCC. METHODS: We retrospectively analyzed the prognostic values of TB and TIL in 150 pT1b ESCC cases. Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) of anti-pan cytokeratin (AE1/AE3) were used to analyze the threshold of TB, and intratumoral TIL and peritumoral TIL (pTIL) were evaluated using the receiver operating characteristic curves (ROC). RESULTS: We found that TB in a three-tiered grading system (low-TB: 0-4; middle-TB: 5-15; high-TB: ≥16) displayed an excellent prognosis prediction for LNM and survival based on IHC staining using a 20× objective lens. Low pTIL level (≤20%) was a significant indicator of LNM and unfavorable prognosis (p < 0.05). Moreover, lower tumor location and lymphovascular invasion (LVI) were correlated with an unfavorable prognosis (p < 0.05). A nomogram developed based on TB, pTIL, LVI, and tumor location showed good discrimination, as shown by the area under the ROC and calibration curves. CONCLUSION: We therefore recommend identifying TB using a 20× objective lens under IHC staining and TIL adjacent to the tumor. Additionally, a nomogram was built for facilitating individualized prediction of survival for patients with pT1b ESCC.

Genome-Wide Detection of SPX Family and Profiling of CoSPX-MFS3 in Regulating Low-Phosphate Stress in Tea-Oil Camellia.

Camellia oleifera a member of the family Theaceae, is a phosphorus (P) tolerator native to southern China. The SPX gene family critically regulates plant growth and development and maintains phosphate (Pi) homeostasis. However, the involvement of SPX genes in Pi signaling in Tea-Oil Camellia remains unknown. In this work, 20 SPX genes were identified and categorized into four subgroups. Conserved domains, motifs, gene structure, chromosomal location and gene duplication events were also investigated in the SPX gene family. Defense and stress responsiveness cis-elements were identified in the SPX gene promoters, which participated in low-Pi stress responses. Based on transcriptome data and qRT-PCR results, nine CoSPX genes had similar expression patterns and eight genes (except CoPHO1H3) were up-regulated at 30 days after exposure to low-Pi stress. CoSPX-MFS3 was selected as a key candidate gene by WGCNA analysis. CoSPX-MFS3 was a tonoplast protein. Overexpression of CoSPX-MFS3 in Arabidopsis promoted the accumulation of total P content and decreased the anthocyanin content. Overexpression of CoSPX-MFS3 could enhance low-Pi tolerance by increased biomass and organic acid contents in transgenic Arabidopsis lines. Furthermore, the expression patterns of seven phosphate starvation genes were higher in transgenic Arabidopsis than those in the wild type. These results highlight novel physiological roles of the SPX family genes in C. oleifera under low-Pi stress, and lays the foundation for a deeper knowledge of the response mechanism of C. oleifera to low-Pi stress.

Prognostic significance of lymphovascular invasion in patients with pT1b esophageal squamous cell carcinoma.

BACKGROUND: Lymphovascular invasion (LVI) is a crucial predictor of lymph node metastasis (LNM). However, few studies have investigated the LVI positivity rate and its clinical significance in pT1b esophageal squamous cell carcinoma (ESCC) using immunohistochemistry and elastin staining. METHODS: We collected data from158 patients with pT1b ESCC who had undergone radical esophagectomy. All paraffin blocks of invasive carcinoma from each patient were subjected to HE staining, elastin staining + CK (AE1/AE3) immunohistochemistry (E&IHC), and CD31/D2-40 + CK (AE1/AE3) double immunohistochemistry (D-IHC). The LVI was classified into types, i.e., vascular invasion (VI) and lymphatic vessel invasion (LI), and its location, quantity, and clinical significance were explored. RESULTS: The positivity rates of VI by E&IHC (E-VI), VI by CD31D-IHC (CD31-VI), and LI by D2-40 D-IHC (D2-40-LI) were significantly higher than those obtained by HE staining (P < 0.001, respectively). CD31-VI and E-VI were independent adverse prognostic factors for recurrence-free survival (RFS), and they were significantly associated with poor distant metastasis-free survival and overall survival in pT1b ESCC. Intratumoral LVI was also crucial in pT1b ESCC, and L2 (the count of D2-40-LI was 5 or more) was the strongest predictor for LNM and RFS in pT1b ESCC. CONCLUSION: E&IHC and D-IHC can dramatically improve the detection rate of LVI in pT1b ESCC, and the classification and grading of LVI can help to improve the prediction of LNM and prognosis.

Integrated molecular characterization of esophageal basaloid squamous cell carcinoma: a subtype with distinct RNA expression pattern and immune characteristics, but no specific genetic mutations.

Esophageal basaloid squamous cell carcinoma (bSCC) is a subtype of squamous cell carcinoma (SCC) with a different behavior and poor prognosis. Exploring bSCC's molecular characteristics and treatment strategies are of great clinical significance. We performed multi-omics analysis of paired bSCC and common SCC (cSCC) using whole exome sequencing and a NanoString nCounter gene expression panel. Immunohistochemistry was used for verification of candidate biomarkers. Different treatment response was analyzed on both patients receiving neoadjuvant treatment and late-stage patients. The common genetically-clonal origin of bSCC and cSCC was confirmed. No significant differences between their genetic alterations or mutation spectra were observed. Mutation signature 15 (associated with defective DNA damage repair) was less prominent, and tumor mutational burden (TMB) was lower in bSCC. bSCC with an RNA expression pattern resembling cSCC had a better survival than other bSCCs. Moreover, bSCC showed significant upregulation of expression of genes associated with angiogenesis response, basement membranes, and epithelial-mesenchymal transition, and downregulation of KRT14 (squamous differentiation) and CCL21 (associated with immune response). Immunohistochemistry for SFRP1 was shown to be highly sensitive and specific for bSCC diagnosis (p < 0.001). In addition, bSCC receiving neoadjuvant immuno-chemotherapy had a worse pathological response than bSCC receiving neoadjuvant chemotherapy (but without statistical significance), even in bSCC positive for PD-L1. Our results demonstrated the molecular characteristics of esophageal bSCC as a subtype with a distinct RNA expression pattern and immune characteristics, but no specific genetic mutations. We provided a useful biomarker, SFRP1, for diagnosis. After outcome analysis for six bSCCs with neoadjuvant immunotherapy treatment and four late-stage bSCCs with immunotherapy, we found that immunotherapy may not be an effective treatment option for most bSCCs. This may also provide a clue for the same subtypes of lung and head and neck cancer. Our study highlighted the heterogeneity among bSCC patients, and might explain the conflicting results of bSCC outcomes in existing studies. © 2022 The Pathological Society of Great Britain and Ireland.

The efficacy of additional surgical resection after endoscopic resection in pT1b esophageal squamous cell carcinoma: A multi-institutional retrospective study in China.

BACKGROUND: pT1b esophageal squamous cell carcinoma (ESCC) patients treated by endoscopic resection (ER) required additional treatment with surgical resection (SR) or chemoradiotherapy (CRT) according to 2020 Japan Gastroenterological Endoscopy Society (JGES) guideline. Given the evidences for this recommendation were largely based on small-size studies, our study collected 166 cases of ER-treated pT1b patients in order to investigate the efficacy of additional SR as compared to ER-alone treatment. METHODS: A multi-institutional retrospective study in China was conducted. The pT1b ESCC treated by ER + SR (n = 42) and ER-alone (n = 124) from 2007 to 2018 were recruited. Meanwhile, patients with positive lymphovascular invasion (LVI(+)) and/or with positive vertical margin (VM(+)) were put into high-risk group, and those with both VM(-) and LVI(-) were selected into low-risk group. The clinicopathological parameters, lymph node metastasis (LNM), and survival between ER + SR and ER-alone groups were analyzed. RESULTS: In high-risk group, concurrent LNM revealed in surgically resected specimens accounted for 52.6% cases in ER + SR group. After surgical removal, the incidence of post-resection LNM dropped down to 5.6%. However, in low-risk group, patients with ER + SR treatment did not exhibit any concurrent LNM in surgically resected specimens, and the incidence of their overall LNM was similar to that in ER-alone group (0% vs. 2.8%, p = 1.000). More importantly, these cases demonstrated significantly shorter overall survival (OS) than that in ER-alone group (81.8% and 100.0%, respectively, at 3 years; log-Rank: P = 0.010). CONCLUSIONS: For ER-treated pT1b patients in high-risk group, additional SR is strongly recommended. However, for those in low-risk group, additional SR does not generate much benefit for clearance of LNM, but brings harm to shorten their OS. Therefore, additional SR is not recommended for ER-treated pT1b patient in low-risk group.

Overexpression of dihydroflavonol 4-reductase (CoDFR) boosts flavonoid production involved in the anthracnose resistance.

The outbreak of anthracnose caused by Colletotrichum spp. represents a devastating epidemic that severely affects oil tea (Camellia oleifera) production in China. However, the unknown resistance mechanism to anthracnose in C. oleifera has impeded the progress of breeding disease-resistant varieties. In this study, we investigated the physiological responses of resistant and susceptible lines during C. gloeosporioides infection. Our results showed that the accumulation of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in both disease-resistant and susceptible lines increased by C. gloeosporioides infection. Also, disease-resistant lines exhibited lower MDA, but higher POD, SOD, and CAT activities compared to susceptible lines. The accumulation of flavonoids in both resistant and susceptible C. oleifera leaves increased following C. gloeosporioides infection, and the increase was greater in resistant lines. Further, we identified and functionally characterized the dihydroflavonol 4-reductase (CoDFR) from the resistant C. oleifera line. We showed that the full-length coding sequence (CDS) of CoDFR is 1044 bp encoding 347 amino acids. The overexpression of CoDFR in tobacco altered the expression of flavonoid biosynthetic genes, resulting in an increased flavonoid content in leaves. CoDFR transgenic tobacco plants exhibited increased anthracnose resistance. Furthermore, the transgenic plants had higher salicylic acid content. These findings offer potential insights into the pivotal role of CoDFR involved in flavonoid-mediated defense mechanisms during anthracnose invasion in resistant C. oleifera.

A liquid biopsy signature predicts lymph node metastases in T1 oesophageal squamous cell carcinoma: implications for precision treatment strategy.

BACKGROUND: The treatment strategies for T1 oesophageal squamous cell carcinoma (ESCC) patients with or without lymph node metastasis (LNM) are different. Given the advantages of the minimally invasive, sensitive and real-time detection, liquid biopsy has become an important cancer diagnostic and prognostic tool. METHODS: MiRNA array and small-RNA sequencing were performed. Then, 222 formalin-fixed and paraffin-embedded tumour samples and 229 pretreatment serum samples from T1 ESCC patients were used to verify and evaluate the results. RESULTS: We demonstrated that serum miR-20b-5p could predict LNM in T1 ESCC patients. The AUC for serum miR-20b-5p was higher (0.827) than those for lymphovascular invasion (LVI) (0.751, P = 0.2128), invasion depth (0.662, P = 0.0027) and tumour differentiation grade (0.634, P = 0.0019). A nomogram for predicting LNM with three independent significant predictors (miR-20b-5p, LVI and invasion depth) was constructed with a concordance index of 0.931. Serum miR-20b-5p was also significantly correlated with disease-free survival (P < 0.001). An algorithm of improved T1 ESCC treatment strategy after biopsy and/or after endoscopic resection based on serum miR-20b-5p level was constructed. CONCLUSIONS: This study suggests that serum miR-20b-5p is a potential biomarker for predicting LNM and can be helpful for precise clinical decision-making strategies and improve treatment outcomes for T1 ESCC patients.

Integration of small RNA, degradome, and transcriptome sequencing data illustrates the mechanism of low phosphorus adaptation in Camellia oleifera.

Phosphorus (P) is an indispensable macronutrient for plant growth and development, and it is involved in various cellular biological activities in plants. Camellia oleifera is a unique high-quality woody oil plant that grows in the hills and mountains of southern China. However, the available P content is deficient in southern woodland soil. Until now, few studies focused on the regulatory functions of microRNAs (miRNAs) and their target genes under low inorganic phosphate (Pi) stress. In this study, we integrated small RNA, degradome, and transcriptome sequencing data to investigate the mechanism of low Pi adaptation in C. oleifera. We identified 40,689 unigenes and 386 miRNAs by the deep sequencing technology and divided the miRNAs into four different groups. We found 32 miRNAs which were differentially expressed under low Pi treatment. A total of 414 target genes of 108 miRNAs were verified by degradome sequencing. Gene ontology (GO) functional analysis of target genes found that they were related to the signal response to the stimulus and transporter activity, indicating that they may respond to low Pi stress. The integrated analysis revealed that 31 miRNA-target pairs had negatively correlated expression patterns. A co-expression regulatory network was established based on the profiles of differentially expressed genes. In total, three hub genes (ARF22, WRKY53, and SCL6), which were the targets of differentially expressed miRNAs, were discovered. Our results showed that integrated analyses of the small RNA, degradome, and transcriptome sequencing data provided a valuable basis for investigating low Pi in C. oleifera and offer new perspectives on the mechanism of low Pi tolerance in woody oil plants.

High lymphocyte­to­monocyte ratio is associated with low α­fetoprotein expression in patients with hepatitis B virus­associated hepatocellular carcinoma.

The association of the peripheral lymphocyte­to­monocyte ratio (LMR) with α­fetoprotein (AFP) status in patients with AFP­positive and AFP­negative hepatocellular carcinoma (HCC) has not been investigated in detail. The aim of the present study was to examine the association between the LMR and AFP status in these patients. The samples were obtained from patients with a hepatitis B virus (HBV) infection, who were negative for non­HBV hepatitis viruses and who did not suffer from autoimmune hepatitis. These patients were retrospectively reviewed and the differences of test indicators in the AFP­negative and AFP­positive groups were assessed. Flow cytometry was used to detect the expression levels of CD4, CD8 and programmed cell death protein 1 (PD­1), and ELISAs were used to analyze the expression levels of interleukin (IL)­10 and transforming growth factor (TGF)­ß1. In addition, luciferase reporter assays were used to assess binding of the IL­10 promoter to the glucocorticoid receptor (GR) gene. Receiver operating characteristic curve and Spearman correlation analyses demonstrated that the AFP­negative HCC group exhibited a higher LMR, lower D­dimer and lower fibrin degradation products compared with the AFP­positive HCC group. The cut­off value of the LMR was 2.01 for AFP detection, with a sensitivity of 68.6% and a specificity of 75%. The high LMR noted in the AFP­negative HCC group was accompanied by a lower proportion of CD4+ T lymphocytes and CD8­PD­1 expression compared with the corresponding levels of these parameters in the AFP­positive HCC group. Furthermore, the high levels of IL­10 and low levels of TGF­ß1 were expressed in the AFP­positive HCC group. The data indicated that the IL­10­592 promoter exhibited a potent induction of luciferase activity in 293T cells cotransfected with a GR­overexpressing vector compared with the control cells. However, the relative luciferase activity was not altered following a mutation or polymorphism in the IL­10 gene. These results suggested that a high LMR was indicative of low AFP expression in HBV­associated HCC patients.

Identification of second primary tumors from lung metastases in patients with esophageal squamous cell carcinoma using whole-exome sequencing.

Esophageal squamous cell carcinoma (ESCC) patients with a synchronous or metachronous lung tumor can be diagnosed with lung metastasis (LM) or a second primary tumor (SPT), but the accurate discrimination between LM and SPT remains a clinical dilemma. This study aimed to investigate the feasibility of using the whole-exome sequencing (WES) technique to distinguish SPT from LM. Methods: We performed WES on 40 tumors from 14 patients, including 12 patients with double squamous cell carcinomas (SCCs) of the esophagus and lung (lymph node metastases were sequenced as internal controls) diagnosed as LM according to pathological information and 2 patients with paired primary ESCC and non-lung metastases examined as external controls. Results: Shared genomic profiles between esophageal (T) and lung (D) tumors were observed in 7 patients, suggesting their clonal relatedness, thus indicating that the lung tumors of these patients should be LM. However, distinct genomic profiles between T and D tumors were observed in the other 5 patients, suggesting the possibility of SPTs that were likely formed through independent multifocal oncogenesis. Conclusions: Our data demonstrate the limitations and insufficiency of clinicopathological criteria and that WES could be useful in understanding the clonal relationships of multiple SCCs.

Liver metastatic basaloid squamous cell carcinoma with negative expression of pancytokeratin: a case report and literature review.

BACKGROUND: Basaloid squamous cell carcinoma (BSCC) is a rare subtype of squamous cell carcinoma with a high rate of distant metastasis. BSCC occurs most commonly in the esophagus, lungs, and head and neck. However, BSCC occurring in an atypical site without a known primary tumor and/or with the presence of atypical immunohistochemical features can result in delayed diagnosis or misdiagnosis. CASE PRESENTATION: Here, we report a case of a 67-year-old man with liver metastatic BSCC with negative pancytokeratin (AE1/AE3) expression. He presented with a chief complaint of epigastric discomfort. Imaging examination revealed a subcapsular mass in the right anterior lobe of the liver. Then, the patient underwent an irregular right hepatectomy. Grossly, the mass was gray, with a size of 7 × 7 × 4 cm. Microscopically, the mass comprised epithelioid tumor cells with both solid and pseudoadenoid structures, accompanied by necrosis. Immunohistochemical staining showed that the tumor cells were negative for AE1/AE3, CK18, CK7, CK19, Hepatocyte Paraffin-1, Glypican-3, Arginase-1, CD56, Chromogranin A, Synaptophysin, Vimentin, and Carcinoembryonic antigen. The Ki-67 index was 80%.The mass was diagnosed as a malignant tumor but could not be classified further. One month after surgery, the patient's reexamination revealed esophageal tumor, and biopsy revealed BSCC. The slides of the liver tumor were reviewed, and the morphology was similar to that of the esophageal tumor. Moreover, supplementary immunohistochemical staining of liver tumor indicated p63 and p40 were strongly positive, that confirmed the liver tumor was metastatic BSCC. Previous studies have reported that 3.7% of esophageal BSCCs did not express AE1/AE3. CONCLUSION: When a malignant tumor comprises epithelioid cells with solid and/or pseudoadenoid structures, but not adenocarcinoma or neuroendocrine carcinoma, even if the tumor cells are negative or weakly positive for AE1/AE3, we should consider BSCC. For a definite diagnosis, immunohistochemical staining for squamous cell carcinoma markers, including p63 and p40, and examination of common primary sites of BSCC should be performed.

ISG15 promotes esophageal squamous cell carcinoma tumorigenesis via c-MET/Fyn/ß-catenin signaling pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors in China with a poor prognosis. Most ESCC patients were diagnosed at advanced stages, losing the opportunity for surgical excision. Hence, it remains a pressing work to identify biomarkers for early detection, prognosis prediction and targeting therapies in ESCC. Interferon-stimulated gene 15 (ISG15) encodes a 15-kDa protein, and is involved in the post-translational modification (PTMs) of multiple proteins. However, the molecular functions of ISG15 in ESCC remain unclear. In this work, we found that ISG15 was aberrantly expressed in ESCC tissues and cell lines. Enhanced protein level of ISG15 promoted cellular malignant phenotypes including proliferation, migration, invasion and tumor formation in vivo. Consistently, reduction of ISG15 attenuated the cellular malignant phenotype in ESCC cell lines. Furthermore, gene-expression profiles suggested that the differentially expressed ISG15 affected the expression of a panel of genes enriched in the cell adherens junction, such as c-MET. Notably, as a secreted protein, the concentration of ISG15 was elevated in ESCC plasma than healthy individuals, acting as a potential diagnostic marker. Taken together, our results suggested a tumor promotion role of ISG15 in ESCC via c-MET/Fyn/ß-catenin pathway.