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Concerns are raised over the risk to digestive system's tumors from the N-nitrosamines (NAs) exposure in drinking water. Albeit considerable studies are conducted to explore the underlying mechanism responsible for NAs-induced esophageal squamous cell carcinoma (ESCC), the exact molecular mechanisms remain largely unknown, especially at the epigenetic regulation level. In this study, it is revealed that the urinary concentration of N-Nitrosodiethylamine is higher in high incidence area of ESCC, and the lncRNA-UCA1(UCA1) is significantly decreased in ESCC tissues. In vitro and in vivo experiments further show that UCA1 is involved in the malignant transformation of Het-1A cells and precancerous lesions of the rat esophagus induced by N-nitrosomethylbenzylamine (NMBzA). Functional gain and loss experiments verify UCA1 can affect the proliferation, migration, and invasion of ESCC cells in vitro and in vivo. Mechanically, through binding to heterogeneous nuclear ribonucleoprotein F (hnRNP F) protein, UCA1 regulates alternative splicing of fibroblast growth factor receptor 2 (FGFR2), which promotes the FGFR2IIIb isoform switching to FGFR2 IIIc isoform, and the latter activates epithelial-mesenchymal transition via PI3K-AKT signaling pathways impacting tumorigenesis. Therefore, NAs-mediated downregulation of UCA1 promotes ESCC progression through targeting hnRNP F/FGFR2/PI3k-AKT axis, which provides a new chemical carcinogenic target and establishes a previously unknown mechanism for NAs-induced ESCC.

The long intergenic noncoding RNA, regulator of reprogramming (linc-ROR) has been reported to participate in tumorigenesis, while its functions and fundamental mechanisms in esophageal squamous cell carcinoma (ESCC) remain unclear. In this study, gain-of-function assays showed that linc-ROR upregulation enhanced cell viability, promoted cell proliferation, and inhibited apoptosis. Mechanistically, the regulatory network of linc-ROR/miR-204-5p/MDM2 was established with bioinformatics analysis and online databases, then validated via dual-luciferase reporter assays, RNA immunoprecipitation assays in ESCC cells. Linc-ROR positively regulates the expression of MDM2 as a molecular sponge of miR-204-5p. Moreover, results of western blot and coimmunoprecipitation indicated that linc-ROR overexpression enhanced the ubiquitination level of p53, and its downstream apoptosis-related genes have showed higher bcl-2 expression, lower bax, and cleaved caspase-3 expressions, while miR-204-5p could counteract with this effect. Finally, small interfering RNAs tailored to linc-ROR were established to further evaluate its effects on ESCC comprehensively. In summary, this study revealed that linc-ROR modulated cell apoptosis and regulated p53 ubiquitination via targeting miR-204-5p/MDM2 axis, which provides a novel therapeutic insight into treatments for ESCC.

Esophageal cancer is one of the most common cancers worldwide. It is critical to find early diagnostic biomarkers for esophageal cancer. MicroRNAs (miRNAs) play important regulatory roles in occurrence and development of esophageal cancer, which has the diagnostic and prognostic values. The aim of the study was to evaluate the potential diagnostic value of the novel miRNA. The novel miRNA was predicted using miRDeep2 software and validated by quantitative reverse transcription PCR (qRT-PCR) and the TA-cloning sequencing of the PCR products. The expression of the novel miRNA in esophageal cancer tissues and adjacent tissues was also analyzed by qRT-PCR. The EdU staining and transwell method were used to detect the capacity of cell proliferation, migration, and invasion. Besides, the target gene CTNNA2 of novel-miR-4885 was verified via qRT-PCR, western blot, luciferase reporter assay, and RNA immunoprecipitation. We identified the novel-miR-4885, the expression level was confirmed by qRT-PCR in the esophageal cancer cells. The result of TA-cloning sequencing was consistent with the prediction and the pre-miRNA had a standard hairpin stem-loop structure. In addition, the expression of novel-miR-4885 was upregulated in esophageal cancer tissue compared with that in adjacent tissue (p 0.05). Further, the assays showed that overexpression novel-miR-4885 could improve the cell proliferation, migration, and invasion with an average fold change of 1.19, 1.59, and 2.34, respectively. Novel-miR-4885 can bind to 3' untranslated region of CTNNA2 to reduce cell adhesion and promote epithelial-mesenchymal transition in esophageal cancer cell. The expression of N-cadherin, β-catenin, Vimentin, and α-smooth muscle actin was upregulated, while that of E-cadherin and ZO-1 proteins was downregulated through western blot. Novel miRNA present in esophageal cancer cells was validated, supplementing the miRNA database. Meantime, the possible functional mechanisms were explored, and the results showed that the novel miRNA may serve as potential biomarker for the diagnosis of esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) has a high degree of malignancy, and there is currently no effective treatment. Circular RNA is a novel endogenous noncoding RNA with a stable loop structure. Several theories have been proposed regarding its biogenesis and usefulness as a biomarker in various cancers. Here we explore the predictive value and potential functions of a candidate circRNA (hsa_circ_0071106) in ESCC.The altered expression of hsa_circ_0071106 was validated in clinical tissue samples from 64 patients with ESCC. The correlation between the clinical characteristics of these patients and the expression of hsa_circ_0071106 was analyzed by the chi-square test. The receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were used to evaluate the diagnostic value of hsa_circ_0071106. The human esophageal carcinoma cell lines ECA109 and ECA9706 and the normal human esophageal epithelial cell line (HET-1A) were applied for functional analysis of hsa_circ_0071106. qRT-PCR, sanger sequencing, agarose gel electrophoresis, RNase R digestion assay, Actinomycin D inhibition assay, nucleoplasm separation assay, and FISH assays were utilized to verify the circular characteristics and subcellular localization of hsa_circ_0071106. SiRNA and circRNA interference lentivirus was constructed to inhibit the expression of hsa_circ_0071106 in the ECA109 and ECA9706 cell lines. We used the EdU assay, Transwell assay, and flow cytometry to detect changes in cell proliferation, invasion, migration, and apoptosis.We identified that hsa_circ_0071106 showed significantly decreased expression levels in ESCC cells (P 0.001) and tissue samples (P 0.001). The correlation analysis of clinicopathological features and gene expression revealed that low expression of hsa_circ_0071106 was related to poor differentiation grade (P 0.05). ROC curves indicated that hsa_circ_0071106 might have a diagnostic value for ESCC (AUC = 0.826, 95% CI: 0.748-0.904) and a predictive value for a high degree of histological differentiation (AUC = 0.730, 95% CI: 0.607-0.854). Hsa_circ_0071106 has a stable circular structure and was primarily localized in the cytoplasm. A transient knockdown of hsa_circ_0071106 by siRNA promotes proliferation, migration, and invasion and inhibits apoptosis of ESCC cells. A steady knockdown of hsa_circ_0071106 by a lentivirus knockout vector significantly promoted cell migration and invasion in ECA109 and ECA9706 cell lines.Hsa_circ_0071106 was a downregulated circRNA related to ESCC and promoted ESCC progression by regulating cell migration and invasion. These findings suggest that hsa_circ_0071106 could be a promising diagnostic biomarker and potential therapeutic target and might predict ESCC's histological differentiation degree.

Cancer stem cells (CSCs) are closely related to tumor resistance and tumor recurrence in esophageal squamous cell carcinoma (ESCC). The lack of specific biomarkers to identify and isolate CSCs has led to the slow progression of research on CSCs in ESCC. Here, we established a method to identify and isolate CSCs in ESCC using fluorescence-activated cell sorting with combined surface biomarkers including CD71, CD271, and CD338. CD71&minus, /CD271+/CD338+ subpopulation cells possessed more stem cell properties in proliferation, self-renewal, differentiation, metastasis, drug resistance, and tumorigenesis. We further explored possible roles that microRNAs played in stem cells. Using microarrays, we identified that has-miR-21-3p was highly expressed in positive sorted cells, and further functional and Luciferase reporter assays verified that has-miR-21-3p promoted proliferation and anti-apoptosis by regulating TRAF4. We further analyzed the relationship between hsa-miR-21-3p and ESCC in 137 patients with ESCC. Statistical analysis showed that up-regulation of hsa-miR-21-3p was associated with a high risk of ESCC. Collectively, we identified surface biomarkers of stem cells in esophageal squamous cell carcinoma, and discovered thathsa-miR-21-3p may be involved in stemness maintenance by regulating TRAF4.

MicroRNA (miRNA) clusters are expressed universally across different types of organisms, and an accumulating number of studies have demonstrated that miRNA clusters function more efficiently compared with single miRNAs during the development of certain cancer types. miRNA clusters may have increased stability and reliability over individual miRNAs as diagnostic or therapeutic biomarkers. In the present study, the expression levels of mature miRNAs within the miR-144/451 cluster were examined using stem‑loop reverse transcription‑quantitative polymerase chain reaction in 102 patients pathologically diagnosed with esophageal carcinoma. Bioinformatics tools were used to identify a possible miRNA‑mediated network of the miR‑144/451 cluster. The expression levels of hsa‑miR‑451a, hsa‑miR‑144‑3p and hsa‑miR‑144‑5p in tumor tissues were significantly lower compared with those in adjacent non‑tumor tissues (P

Muhe Shang, Xianghu Wang, Ying Zhang, Zhikui Gao, Tian Wang, Ran Liu Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu, China Background and objective: In an attempt to discover a new biomarker for early diagnosis and prognosis of esophageal squamous cell carcinoma (ESCC), the regulation mechanism of large intergenic non-coding RNA–regulator of reprogramming (lincRNA-ROR) as a microRNA (miRNA) sponge was studied.Patients and methods: ROR expression in 91 pairs of ESCC tissue samples and matched adjacent tissues was quantified with real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The ROR–miRNA–mRNA regulatory network was built with 161 esophageal cancer (EC) tissues and 11 adjacent tumor tissues from The Cancer Genome Atlas (TCGA) database. A total of 96 cases of ESCC from TCGA database were collected for analysis on survival rates. The regulatory relationship between ROR, miR-145 and FSCN1 was verified in ESCC cells via qRT-PCR, dual luciferase reporter (DLR) assay, RNA immunoprecipitation (RIP) and Western blotting. The transwell method was used to detect cell migration and invasion.Results: ROR expression in ESCC tumor tissues was significantly higher than in the adjacent tissues, p

Additional file 3: Table S3. Relative expression of proteins.

Esophageal squamous-cell carcinoma (ESCC) is one of the most common tumors worldwide. Recent studies suggested that long noncoding RNAs (lncRNAs) might play a key role in regulating cellular processes and cancer progression. One of the lncRNAs, urothelial carcinoma associated 1 (UCA1), is known to be dysregulated in several cancers, including bladder carcinoma, colorectal, melanoma, breast, gastric, and ESCC. However, contributions of UCA1 to ESCC remain largely undiscovered. In order to understand the role and mechanisms underlying UCA1 in ESCC, the association of UCA1 expression with risk of esophageal cancer development was determined in 106 esophageal cancer tissues of ESCC patients and adjacent normal tissues using real-time reverse-transcription polymerase chain reaction (PCR). The relative expression of UCA1 was significantly reduced in cancer versus adjacent normal tissues suggesting an enhanced risk of esophageal cancer. To investigate the biological functions of UCA1 in ESCC, it was of interest to examine whether overexpression of UCA1 might influence cell proliferation, apoptosis, cell cycle distribution, migration, and invasion in vitro using EC109 cells. Our results demonstrated that UCA1 decreased cell proliferation, migration, invasion, and cell cycle progression of EC109 cells. Further, mRNA microarray analysis of overexpressed UCA1 in EC109 cells revealed that abnormal expression of UCA1 also inhibited the Wnt signaling pathway. Gene levels of DKK1 were elevated while C-myc fell significantly in overexpressed UCA1 EC109 cells. Interestingly, Western blot demonstrated no significant differences in relative expression of CTNNB1 (β-catenin) but marked reduction in β-catenin (active form) levels in both total and nuclear proteins. These results suggest that UCA1 may inhibit ESCC growth by regulating the Wnt signaling pathway. In conclusion, UCA1 may be a novel biomarker involved in ESCC development that may provide a potential therapeutic target for ESCC.

Purpose This study was aimed to conduct a proteomics profiling analysis on plasma obtained from ESCC patients with the goal of identifying appropriate plasma protein biomarkers in the progression of ESCC. Experimental design Plasma from 28 ESCC patients and 28 Healthy controls (HC) were analyzed by iTRAQ combined with 2D-LC-MS/MS. ProteinPilot software was used to identify the differentially expressed plasma proteins in ESCC compared to HC. Western blot was performed to verify the expression of selected proteins in 37 independent ESCC patients and 37 HC. Transwell and MTT assays were used to detect the biological function of ECM1 protein in vitro. Results Nineteen (four up regulated and fifteen down regulated) proteins were identified as differentially expressed between ESCC and HC (P

The study uses Fo’ model of PAM 2100 to measure the Chlorophyll a Fluorescence Kinetics of O’-P’-F-C-S multi-stage change in plants leaves under steady state of photosynthesis which is induced through alternation of far-red light, darkness, and actinic light. We call it “Post-Steady-State Chlorophyll a Fluorescence Kinetics” (PSF). The results have demonstrated that the above process has gradually evolved into the steady-state with the light induction process moving on. It is closely related with the redox state of the photosynthetic electron chain and the activation of the carbon assimilation. At the same time, this method has provided an effective means of research into the electronic transfer of photosynthesis and the dynamic change process of the carbon assimilation when the stomata of the CAM plants closes in light.

In this paper, arbuscular mycorrhizal fungi (AMF) Glomus versiforme (GV), Glomus mosseae (GM), Glomus intraradices (GI) and their mixtures were applied to inoculate plastic greenhouse Cucumis sativus seedlings to investigate the effects of AMF on C. sativus growth, development, yield, and quality. The results showed that all test AMF could form mycorrhiza with cucumber roots, and the infected rate reached 41.74% - 55.69%. Compared with the control, treatments GV, GM, GM + GV, GM + GV + GI and GV + GI enhanced the healthy seedling index by 58.14% - 123.6%, and increased early yield by 21.71% - 37.87% and total yield by 19.72% - 34.41%. AMF also improved fruit quality. The Vc content of cucumber fruit increased by 22.84% and 21.95% in treatments GM + GV and GV + GI, soluble sugar content increased by 13.29%, 8.25%, and 10.20% in treatments GV, GI and GV + GI, and amino acid content increased by 47.66% and 23.19% in treatments GV and GM + GI, respectively, while soluble protein content increased by 17.67% - 34.29% in all AMF treatments. The results suggested that AMF inoculation could significantly promote the seedling growth of cucumber and improve its fruit quality, and the effects differed with different AMF and their combinations.