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The transcription factor forkhead box D3 (FOXD3) is an important member of the FOX family, which can maintain the pluripotent properties of cell clusters, neural crest, and trophoblastic progenitor cells in vivo. It has been shown that FOXD3 could affect proliferation, migration, and angiogenesis of various tumors and its deletion and overexpression in organisms will undoubtedly have important influence on the change of cell fate and the occurrence of tumors. However, the underlying functions and molecular mechanisms of FOXD3 in esophageal squamous cell carcinoma (ESCC) have not been fully clarified. According to the present study, the expression levels and functional roles of FOXD3 were investigated, and its prognostic value and molecular mechanisms in tumorigenesis and progression of ESCC were clarified. The expression level of FOXD3 was significantly downregulated in ESCC tissues and cell lines, and correlated with gender, family history of upper gastrointestinal cancer, TNM stage, depth of invasion, lymph node metastasis, and ESCC patients' survival. Moreover, FOXD3 inhibited cells migration and invasion as well as participated in TGF-β1 induced epithelial-mesenchymal transition process. Furthermore, a positive correlation between FOXD3 and SMAD family member 7 (SMAD7) was explored in ESCC. FOXD3 could directly bind to promoter regions of SMAD7 gene, leading to transcriptional promotion of SMAD7 in human esophageal cancer cells. Taken together, FOXD3 may play a tumor suppressor role in ESCC and may be applied as a new therapeutic target and prognostic marker for ESCC.

Increasing evidence demonstrates that long non-coding RNAs (lncRNA) play a vital role in the progression of tumors, containing esophageal squamous cell carcinoma (ESCC). LINC00239 was reported as an oncogene in diverse kinds of cancers, whereas its specific role is still unclear in ESCC. In this study, we detected the expression and functional role of LINC00239 in ESCC specimens and cells, and investigated the molecular mechanisms of it. LINC00239 was highly expressed in ESCC tissues and cells, and was related to poor prognosis of patients with ESCC. The proliferation, metastasis, and invasion ability as well as epithelial–mesenchymal transition (EMT) process were all enhanced in LINC00239-overexpressed ESCC cells. LINC00239 was upregulated in TGF-β1–treated ESCC cells. Furthermore, LINC00239 was found to bind directly to the transcription factor c-Myc promoter–binding protein-1 (MBP-1). MBP-1 was detected to inhibit the transcription of c-Myc in ESCC. Moreover, LINC00239 could activate c-Myc transcription through influencing MBP-1–binding ability to c-Myc promoter. These data suggest that LINC00239 may act as an oncogene to promote the transcription of c-Myc by competitively combining with MBP-1 in ESCC, and may serve as a potential target for antitumor therapy in ESCC. Implications: LINC00239 may function as an oncogenic lncRNA in ESCC through the LINC00239/MBP-1/c-Myc axis to activate EMT process.

Countless evidence suggests that long noncoding RNAs (lncRNAs) are involved in human malignant cancers, including esophageal squamous cell carcinoma (ESCC), although their exact function remains unclear. In the present study, we aimed to investigate the roles and molecular mechanisms of the lncRNA LOC440173 in ESCC progression. Microarray analysis and quantitative real-time polymerase chain reaction were conducted to measure the expression levels of LOC440173 and miR-30d-5p. The biological function of this lncRNA was investigated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, clone formation, and transwell assays, as well as flow cytometry and Western blot analysis. The function of LOC440173 was validated in vivo using tumor xenografts. The regulatory network of LOC440173/miR-30d-5p/HDAC9 was established using bioinformatic analysis and verified with dual-luciferase reporter assays, RNA immunoprecipitation assay, and rescue experiments. The expression level of LOC440173 was significantly increased in ESCC tissues and esophageal carcinoma cells. High LOC440173 expression was correlated with histological grade, tumor invasion depth, lymph node metastasis, and TNM stage. Overexpression of LOC440173 promoted esophageal cancer cell proliferation, migration, and invasion, as well as the epithelial-mesenchymal transition (EMT) process in vitro, and facilitated tumor growth in vivo. MicroRNA-30d-5p (miR-30d-5p) was downregulated in ESCC tissues and acted as a direct binding target of LOC440173 during the regulation of HDAC9 expression in esophageal carcinoma cells. In conclusion, LOC440173 exerts a promotive role in ESCC tumorigenesis by targeting the miR-30d-5p/HDAC9 axis and regulating the EMT process. LOC440173 might be a new therapeutic target for the treatment of ESCC.

The gut microbiota play an important role in the growth and intestinal health of broilers. The present study was to investigate the gut microbiota, short-chain fatty acids, and intestinal morphology of broilers at different ages. A total of 320 one-day-old male broilers were raised in 8 replicates and fed the same corn–soybean diets for 42 D. The duodenal, jejunal, and ileal segments and their and cecal microbiota were collected on day 1, 7, 14, 21, and 42, respectively. The villous height (VH), crypt depth (CD), and their ratio of VH:CD in the duodenum, jejunum, and ileum all increased (P

This study was conducted to investigate the effect of dietary supplementation with pyrroloquinoline quinone (PQQ) in the form of PQQ disodium (PQQ▪Na2) on the growth performance, carcass traits, meat quality and antioxidative ability of broilers. A total of 720 one-d-old Arbor Acres male broilers were randomly allocated to 1 of 6 treatments with 8 replicates of 15 birds per replicate in a completely randomized design. Birds were fed a PQQ▪Na2-unsupplemented corn-soybean meal basal diet (control) or the basal diet supplemented with 0.1, 0.2, 0.3, 0.4 or 0.5 mg PQQ▪Na2 kg−1 for 42 d. Compared with the control chicks, the chicks fed the diets supplemented with PQQ▪Na2 had lower (P

Persistent exposure to ambient fine particulate matter (PM2.5) can exacerbate allergic diseases in humans. Mast cells play an important role in allergic inflammation in peripheral tissues, such as skin, mucosa, and lung. Engagement of the high-affinity Fc receptor leads to mast cell degranulation, releasing a variety of highly active mediators including histamine, leukotrienes, and inflammatory cytokines. How PM2.5 exposure affects mast cell activation and function remains largely unknown. To characterize the effect of PM2.5 on mast cells, we used bone marrow-derived mast cells (BMMCs) to examine whether PM2.5 affected FceRI-mediated signaling, cytokine production, and degranulation. Exposure to high doses of PM2.5 caused pronounced apoptosis and death of BMMCs. In contrast, exposure to low doses of PM2.5 enhanced mast cell degranulation and FceRI-mediated cytokine production. Further analysis showed that PM2.5 treatment increased Syk activation and subsequently phosphorylation of its substrates including LAT, PLC-γ1, and SLP-76. Moreover, PM2.5 treatment led to activation of the PI3K and MAPK pathways. Intriguingly, water-soluble fraction of PM2.5 were found responsible for the enhancement of FceRI-mediated signaling, mast cell degranulation, and cytokine production. Our data suggest that PM2.5, mainly water-soluble fraction of PM2.5, could affect mast cell activation through enhancing FceRI-mediated signaling.

Frequent deletions of tumor-suppressor genes at chromosome 3p21.3 have been detected in esophageal squamous cell carcinoma (ESCC). As a candidate tumor suppressor gene, semaphorin 3B (SEMA3B) is located at 3p21.3 and is frequently inactivated in several tumors. However, the role and inactivation mechanisms of SEMA3B and its antisense long non-coding RNA (lncRNA) SEMA3B-AS1 in the carcinogenesis of ESCC have not been fully elucidated. The present study was conducted to investigate the role, epigenetic inactivation mechanisms, and prognostic value of SEMA3B and SEMA3B-AS1 in ESCC tumorigenesis and prognosis. Frequent downregulation of SEMA3B and SEMA3B-AS1 was detected in esophageal cancer cells and ESCC tissues, and the expression level of SEMA3B and SEMA3B-AS1 in ESCC tissues was correlated with TNM stage and lymph node metastasis. SEMA3B and SEMA3B-AS1 shared the same CpG island in the promoter region and the expression of both genes might be regulated by the promoter methylation status. Furthermore, transcription factor Sp1 activated SEMA3B or SEMA3B-AS1 transcription and the promoter hypermethylation of SEMA3B and SEMA3B-AS1 influenced Sp1 binding ability. Moreover, over-expression of SEMA3B and SEMA3B-AS1 suppressed the viability and invasion of esophageal cancer cells in vitro. SEMA3B-AS1 influenced the protein expression of SEMA3B. SEMA3B or SEMA3B-AS1 expression and promoter methylation status were correlated with ESCC patients' survival. Thus, these findings suggest that SEMA3B and SEMA3B-AS1 may act as tumor suppressors and may serve as potential targets for antitumor therapy.

Chicken ovaries are known to develop asymmetrically and only the left ovary fully develops. Although both have been greatly investigated, a gap in scientific reports is still felt between 2-mo-old and sexual maturity. In this study, we aimed at investigating the changes in components that occur during growth to analyze the morphohistological correlation between the left ovary and the follicle development at different age stages in Gallus domesticus. The ovaries were harvested from 60 chickens aged 1 and 3-wk-old, 1, 2, 3, and 4-mo-old (n = 10 per age group), then fixed in AAF solution. Hematoxylin-and Eosin protocol was used to stain the tissue for microscopic observations. Results revealed that the left ovary exhibited an ovarian tissue, a site of follicular growth that displayed various shapes from smooth to greatly indented as the follicles differentiated. Atretic follicles at various regression stages were noticed frequently as the chicks grew in age from 3-wk-old onward along with their differentiation. Rete ovarii, remnants from the male homologs were observed throughout the whole study showing epoophoron, connecting rete, and gland-like structures that tend to diminish with age. The feature of the left ovary is closely related to the follicular developmental stage, and the bigger and differentiated the follicles are, the more indented and irregular its epithelium appears. Atresia is a normal physiological process that we observed throughout the whole study. Also that, rete ovarii do not spontaneously arise in the ovary but it develops and grows in juvenile chicken as well as in adult ones.

Steroid hormone levels are associated with estrous behavior, which affects timely mating and reproductive efficiency in pigs. 17&beta, hydroxysteroid dehydrogenase type 14 (HSD17B14) modulates steroid synthesis and metabolism. To identify the functional single nucleotide polymorphisms (SNPs) in the porcine HSD17B14 gene, ear tissues from Large White and Mi gilts were collected to extract genomic DNA. Variable lengths of truncated promoter of HSD17B14 gene were used to determine the promoter activity by a dual luciferase reporter system. The vector HSD17B14Phe or HSD17B14Val was transfected into porcine granulosa cells (GCs). The core promoter region was identified between -72bp and -218bp. Six of seven SNPs had significant differences of allele frequency between Large White and Mi gilts. The plasmids with the wild genotype AA of rs329427898 maintained a smaller fraction of promoter activity compared with the plasmids with the mutant genotype GG, while the plasmids with wild the genotype TT of rs319864566 had a greater promoter activity than the plasmids with the mutant genotype CC. A missense mutation (Phe73Val) caused changes in the structural dynamics and function of the HSD17B14 protein. The highly expressed HSD17B14Val degraded less estradiol into estrone, while the relatively lowly expressed HSD17B14Phe degraded more estradiol into estrone, suggesting the protein activity of HSD17B14Phe was greater than that of HSD17B14Val. Moreover, the HSD17B14Phe group has a greater apoptosis rate of porcine GCs. The HSD17B14 gene could been used as a candidate molecular marker for estrus behavior in pigs.

Zinc (Zn) has been shown to attenuate the adverse effects of heat stress on broilers, but the mechanisms involving this process remain unclear. We aimed to investigate possible protective mechanisms of Zn on primary cultured hepatocytes of broiler embryos subjected to heat stress. Three experiments were conducted. In Exp. 1, hepatocytes were treated with 0, 50, 100, 200, or 400 μmol/L added Zn as inorganic Zn sulfate (iZn) for 12, 24 or 48 h. In Exp. 2, cells were exposed to 40 °C (a normal temperature [NT]) and 44 °C (a high temperature [HT]) for 1, 2, 4, 6, or 8 h. In Exp. 3, cells were preincubated with 0 or 50 μmol/L Zn as iZn or organic Zn lysine chelate (oZn) for 8 h under NT, and then incubated with the same Zn treatments under NT or HT for 4 or 6 h. The biomarkers of antioxidative status and heat stress in cells were measured. The results in Exp. 1 indicated that 50 μmol/L Zn and 12 h incubation were the optimal conditions for increasing antioxidant ability of hepatocytes. In Exp. 2, the 4 or 6 h incubation under HT was effective in inducing heat shock responses of hepatocytes. In Exp. 3, HT elevated (P

LINC01503 has been reported to act as a candidate oncogenic lncRNA in several types of human cancer. However, the functions and underlying mechanisms of LINC01503 in gastric cardia adenocarcinoma (GCA) remain unclear. To investigate the roles and underlying mechanisms of LINC01503 in GCA progression. Gene expressions were detected by quantitative real-time PCR (qRT-PCR). Gain-of-function assays were performed to evaluate the function of LINC01503 in gastric cancer cells. Bioinformatics analysis, luciferase reporter assay, and RIP assay were performed to identify associations among LINC01503, miR-133a-5p, and VIM. The expression level of LINC01503 was significantly elevated in GCA tissues and cell lines. High expression of LINC01503 was correlated with lymph node metastasis, TNM stage, and poor prognosis of GCA patients. Knockdown of LINC01503 significantly reduced proliferation, migration, and invasion ability in GC cells. LINC01503 might function as a competing endogenous RNA (ceRNA) via sponging miR-133a-5p to upregulate the expression of VIM. Furthermore, overexpression of LINC01503 promoted the progression of epithelial mesenchymal transition (EMT) in vitro. LINC01503 serves as an oncogenic lncRNA to promote GCA progression via affecting LINC01503/miR-133a-5p/VIM axis and EMT process. LINC01503 not only has a critical role in GCA progression but also provide a novel potential biomarker in predicting prognosis for GCA patients.

The effects of rice bran and corn on ensiling characteristics of potato vines were investigated. Ruminal fermentation of potato vine silage was measured using a rumen simulation technique (Rusitec)...

Lianhua Zhu,1 Luofu Wang,2 Yu Liu,1 Dan Xu,1 Kejing Fang,1 Yanli Guo1 1Department of Ultrasound, Southwest Hospital, Third Military Medical University (Army Medical University), Shapingba District, Chongqing, China; 2Department of Urology, Daping Hospital, Third Military Medical University (Army Medical University), Yuzhong District, Chongqing, China Purpose: Targeted nanobubbles can penetrate the tumor vasculature and achieve ultrasound molecular imaging (USMI) of tumor parenchymal cells. However, most targeted nanobubbles only achieve USMI of tumor parenchymal cells from one organ, and their distribution, loading ability, and binding ability in tumors are not clear. Therefore, targeted nanobubbles loaded with carbonic anhydrase IX (CAIX) aptamer were fabricated for USMI of various tumors, and the morphological basis of USMI with targeted nanobubbles was investigated.Materials and methods: The specificity of CAIX aptamer at the cellular level was measured by immunofluorescence and flow cytometry. Targeted nanobubbles loaded with CAIX aptamer were prepared by a maleimide-thiol coupling reaction, and their binding ability to CAIX-positive tumor cells was analyzed in vitro. USMI of targeted and non-targeted nanobubbles was performed in tumor-bearing nude mice. The distribution, loading ability, and binding ability of targeted nanobubbles in xenograft tumor tissues were demonstrated by immunofluorescence.Results: CAIX aptamer could specifically bind to CAIX-positive 786-O and Hela cells, rather than CAIX-negative BxPC-3 cells. Targeted nanobubbles loaded with CAIX aptamer had the advantages of small size, uniform distribution, regular shape, and high safety, and they could specifically accumulate around 786-O and Hela cells, while not binding to BxPC-3 cells in vitro. Targeted nanobubbles had significantly higher peak intensity and larger area under the curve than non-targeted nanobubbles in 786-O and Hela xenograft tumor tissues, while there was no significant difference in the imaging effects of targeted and non-targeted nanobubbles in BxPC-3 xenograft tumor tissues. Immunofluorescence demonstrated targeted nanobubbles could still load CAIX aptamer after penetrating the tumor vasculature and specifically binding to CAIX-positive tumor cells in xenograft tumor tissues.Conclusion: Targeted nanobubbles loaded with CAIX aptamer have a good imaging effect in USMI of tumor parenchymal cells, and can improve the accuracy of early diagnosis of malignant tumors from various organs. Keywords: targeted ultrasound contrast agents, molecular imaging, malignant tumors, aptamers, morphological basis

BACKGROUND Although downregulation of caveolin-1 (Cav-1), which is a key constituent of membrane caveolae and a regulator of cellular processes, is associated with colorectal cancer (CRC), its involvement in the disease progression is largely unknown. This study aimed to explore the role of Cav-1 in CRC and the associated mechanisms. MATERIAL AND METHODS Fresh tissues from patients with CRC and human CRC SW480 cells were used to evaluate Cav-1 and Ki-67 expression using immunohistochemistry and Western blotting. The MTS and Transwell assays were performed to determine the effects of Cav-1 overexpression via pcDNA3.1/Cav-1 plasmid on cell proliferation and metastasis. The effect of Cav-1 on the epidermal growth factor receptor (EGFR) activation was evaluated by Western blotting. The correlation of Cav-1 expression with clinicopathological factors was statistically analyzed. RESULTS Overexpression of Cav-1 significantly reduced proliferation, migration, and invasion of SW480 cancer cells in vitro. The EGF-induced phosphorylation of EGFR and activations of the RAF-MEK-ERK and PI3K-AKT pathways were adversely regulated by Cav-1 overexpression in vitro. In 76 cases of CRC patients with EGFR expression, a negative correlation was observed between the level of Cav-1 and tumor-node-metastasis stage, lymph node metastasis, and distant metastasis (All p

Cinnamon essential oil-mesoporous silica nanoparticles (CEO-MSNPs)/potato starch films have better antifungal activity against molds commonly found in post-harvest white mushrooms. The antifungal effects of CEO-MSNPs against Mucor sp. FJ09 species and Mucor circinelloides CNRMA 03.0371 were investigated. The antifungal mechanisms of CEO-MSNPs against molds were elucidated. Results showed that after adding CEO-MSNPs at the minimum inhibitory concentration level, there were obvious changes in membrane permeability, reactive oxygen species (ROS) content, cell component leakage, cell component leakage, soluble sugar, and malondialdehyde (MDA) content. The scanning electron microscope assay showed that CEO-MSNPs effectively destroyed the mycelial morphology of Mucor sp. FJ09 species and Mucor circinelloides CNRMA 03.0371, and interfered with their normal growth. Notably, CEO-MSNPs could be in the up-regulation of nox1 and nox2 genes of Mucor sp. FJ09 species and Mucor circinelloides CNRMA 03.0371 to generate a large number of reactive oxygen species, thereby inhibiting spore germination and growth. The results of this study could provide a reference for screening key targets of drug resistance of molds in post-harvest white mushrooms.

In the present study, the effect of manganese (Mn) on antioxidant status and the expression of the manganese superoxide dismutase (MnSOD) gene in cultured primary myocardial cells collected from the chick embryos was investigated. The hypothesis that Mn supplementation would enhance the expression of MnSOD in cultured primary myocardial cells of chick embryos was tested. Eggs collected from Mn-depleted Arbor Acres laying breeder hens were incubated for 10 days and then myocardial cells were isolated and cultivated for 8 days. The embryonic myocardial cells on day 6 were treated with Mn in the cell culture medium at different time points when the proportion of cells showing spontaneous contraction was over 95% after the 3-day primary culture. A completely randomized design involving a 3 Mn levels (0, 0.5 and 1.0 mmol L−1)×3 incubation time points (12, 24 and 48 h) factorial arrangement of treatments (n=6) was used in the current experiment. The results showed that MnSOD activity and mRNA expression level were induced by Mn and increased with incubation time, which supported the hypothesis that Mn would enhance the expression of the MnSOD gene, and thus might protect myocardial cells from oxidative stress during the chick embryonic development.

Maternally expressed gene 3 (MEG3), a long non-coding RNA (lncRNA), has tumor-suppressor properties and its expression is lost in several human tumors. However, its biological role in esophageal squamous cell carcinoma (ESCC) tumorigenesis is poorly defined. The present study determined the role and methylation status of MEG3 in esophageal cancer cells and ESCC clinical specimens, and further observed the competing endogenous RNA (ceRNA) activity of MEG3 in the pathogenesis and development of ESCC. Significant downregulation of MEG3 was detected in esophageal cancer cells and ESCC tissues and the expression level of MEG3 was significantly increased in cancer cells after treated with the DNA methyltransferase inhibitor 5-Aza-dC. Upregulation of MEG3 led to the inhibition of proliferation and invasiveness of the cancer cells. The aberrant promoter hypermethylation of MEG3 indicates silencing of its expression. Furthermore, MEG3 acts as a ceRNA to regulate the expression of E-cadherin and FOXO1 by binding hsa-miR-9. Upregulation of miR-9 was detected in esophageal cancer cell lines and ESCC tissues, and miR-9 promoted esophageal cancer cell proliferation and invasion. Finally, downregulation and hypermethylation of MEG3 was associated with ESCC patients' survival. Implications: MEG3 functions as a tumor-suppressive lncRNA and aberrant promoter hypermethylation is critical for MEG3 gene silencing in ESCC. In addition, MEG3 acts as a ceRNA to regulate expression of E-cadherin and FOXO1 by competitively binding miR-9 and may be used as a potential biomarker in predicting ESCC patients' progression and prognosis. Mol Cancer Res; 15(7); 800–10. ©2017 AACR.

Traditional imaging examinations have difficulty in identifying benign and malignant changes in renal masses. This difficulty may be solved by ultrasound molecular imaging based on targeted nanobubbles, which could specifically enhance the ultrasound imaging of renal cell carcinomas (RCC) so as to discriminate benign and malignant renal masses. In this study, we aimed to prepare anti-G250 nanobody-functionalized targeted nanobubbles (anti-G250 NTNs) by coupling anti-G250 nanobodies to lipid nanobubbles and to verify their target specificity and binding ability to RCC cells that express G250 antigen and their capacity to enhance ultrasound imaging of RCC xenografts. Anti-G250 nanobodies were coupled to the lipid nanobubbles using the biotin-streptavidin bridge method. The average particle diameter of the prepared anti-G250 NTNs was 446 nm. Immunofluorescence confirmed that anti-G250 nanobodies were uniformly distributed on the surfaces of nanobubbles. In vitro experiments showed that the anti-G250 NTNs specifically bound to G250-positive 786-O cells and HeLa cells with affinities of 88.13% ± 4.37% and 71.8% ± 5.7%, respectively, and that they did not bind to G250-negative ACHN cells. The anti-G250 NTNs could significantly enhance the ultrasound imaging of xenograft tumors arising from 786-O cells and HeLa cells compared with blank nanobubbles, while the enhancement was not significant for xenograft tumors arising from ACHN cells. Immunofluorescence of tumor tissue slices confirmed that the anti-G250 NTNs could enter the tissue space through tumor blood vessels and bind to tumor cells specifically. In conclusion, anti-G250 nanobody-functionalized targeted nanobubbles could specifically bind to G250-positive RCC cells and enhance the ultrasound imaging of G250-positive RCC xenografts. This study has high-potential clinical application value for the diagnosis and differential diagnosis of renal tumors.

Understanding the role of Long non-coding RNAs (lncRNAs) in tumorigenesis in diverse human malignancies would helpful for targeted therapies, containing esophageal squamous cell carcinoma (ESCC). However, the specific role and molecular mechanisms of LINC01980 in ESCC remain unclarified. In this study, we investigated the expression level, function role, and molecular mechanisms of LINC01980 in esophageal cancer cells and ESCC tissues. The high expression of LINC01980 was detected in ESCC tissues and cells, and predicted poor prognosis. LINC01980 promoted the cell proliferation, migration, invasion ability and epithelial-mesenchymal transition (EMT) progress in ESCC cells. In addition, a negative correlation between LINC01980 and miR-190a-5p or miR-190a-5p and MYO5A was observed in ESCC. We found that miR-190a-5p could directly bind with the mRNA of LINC01980 and MYO5A, and it was detected low expression in ESCC. We further demonstrated that the downregulation of MYO5A caused by overexpressing miR-190a-5p was released via upregulation of LINC01980. Functionally, LINC01980 acted as a competitively endogenous RNA (ceRNA) to impact the expression of MYO5A by sponging miR-190a-5p in ESCC. Therefore, these findings suggest that LINC01980 may act as an oncogenic lncRNA in ESCC and LINC01980/miR-190a-5p/MYO5A pathway contributes to the development of ESCC.

Natural antisense lncRNAs can interfere with their corresponding sense transcript to elicit concordant or discordant regulation. LncRNA ZNF667-AS1 and its sense gene ZNF667 were found to be downregulated in esophageal squamous cell carcinoma (ESCC) tissues by RNA sequencing; however, the exact roles of both genes in ESCC occurrence and development have not been clarified. This study was to investigate the expression patterns, epigenetic inactivation mechanisms, function, and prognostic significance of ZNF667-AS1 and ZNF667 in ESCC tumorigenesis. Frequent downregulation of ZNF667-AS1 and ZNF667 was detected in esophageal cancer cells and ESCC tissues. The expression levels of ZNF667-AS1 and ZNF667 were significantly reversed by treatment with 5-Aza-dC and TSA in esophageal cancer cell lines. The CpG sites hypermethylation within proximal promoter influenced the binding ability of transcription factor E2F1 to the binding sites and then affected the transcription and expression of ZNF667-AS1 and ZNF667. Overexpression of ZNF667-AS1 and ZNF667 suppressed the viability, migration, and invasion of esophageal cancer cells in vitro. Overexpression of ZNF667-AS1 increased mRNA and protein expression level of ZNF667. ZNF667-AS1 interacts with and recruits TET1 to its target gene ZNF667 and E-cadherin to hydrolyze 5′-mc to 5′-hmc and further activates their expression, meanwhile, ZNF667-AS1 also interacts with UTX to decrease histone H3K27 tri-methylation to activate ZNF667 and E-cadherin expression. Furthermore, ZNF667-AS1 or ZNF667 expression and promoter methylation status were correlated with ESCC patients’ survival. Thus, these findings suggest that ZNF667-AS1 and ZNF667 may act as tumor suppressors and may serve as potential targets for antitumor therapy.

Long non‐coding RNAs (lncRNAs) have been well demonstrated to emerge as crucial regulators in cancer progression, and they can function as regulatory network based on their interactions. Although the biological functions of FAM83H‐AS1 have been confirmed in various tumour progressions, the underlying molecular mechanisms of FAM83H‐AS1 in oesophageal squamous cell carcinoma (ESCC) remained poorly understood. To address this, we treated human oesophageal cancer cell line Eca109 cells with TGF‐β and found FAM83H‐AS1 was notably overexpressed. In the present study, FAM83H‐AS1 was observed to be significantly up‐regulated in ESCC tissues and was associated with TNM stage, pathological differentiation and lymph node metastasis. FAM83H‐AS1 reinforced oesophageal cancer cell proliferation, migration and invasion, and participated in epithelial‐to‐mesenchymal transition (EMT) process at mRNA and protein levels. In addition, a concordant regulation between FAM83H‐AS1 and its sense strand FAM83H was detected at the transcriptional and translational levels. Furthermore, FAM83H‐AS1 could act as competing endogenous RNA to affect the expression of Girdin by sponging miR‐10a‐5p verified by RIP and luciferase reporter assays. Consequently, the study provided a unique perspective of FAM83H‐AS1 in ESCC progression, which may be considered as potential biomarker and therapeutic target for ESCC therapy.

Trypsin is an important proteolytic enzyme in the digestive system and its activity is a major indicator for evaluating diseases such as chronic pancreatitis. Here, we present a novel label-free method to detect trypsin kinetics using a nanopore technique. A mutant α-hemolysin (M113R)7 protein nanopore equipped with a polyamine decorated β-cyclodextrin (am7β-CD) was employed as a sensing platform for the real-time monitoring of the process of trypsin enzymatic cleavage of a substrate Nα-benzoyl-l-arginine ethyl ester (BAEE) at the single molecule level. Significantly, this sensor can exclusively respond to the current modulation caused by the product and prevent interference from the substrate, thus improving detection sensitivity, and it provides a new scheme to detect enzyme activity for cleaving small molecules.

Esophageal squamous cell carcinoma (ESCC) may be caused by a combination of environmental factors and genetic variants. The present study was to evaluate the association between haplotype-tagging SNPs (htSNPs) of lincRNA-NR_024015 and the risk of ESCC. We selected htSNPs across the whole 1469 bp lincRNA-NR_024015 locus and 2 kb upstream as well as 2 kb downstream regions of the gene and conducted a case-control study in 581 ESCC cases and 677 healthy controls to test the effects of functional lincRNA-NR_024015 htSNPs on ESCC susceptibility. Of the seven potential functional htSNPs, rs8506 AA genotype was found to be associated with increased risk of ESCC. Further stratification analysis showed that the risk effect was more pronounced in male patients and patients with TNM stage III and IV. LincRNA-NR_024015 was predominantly expressed in cytoplasm of esophageal cancer cells. The expression level of lincRNA-NR_024015 in ESCC tumor tissues was significantly higher than that in corresponding normal tissues and rs8506 genotype has a genotype-specific effect on lincRNA-NR_024015 expression. Furthermore, rs8506 G to A variant might influence lincRNA-NR_024015 expression and function by disrupting the binding of hsa-miR-526b to the site. High expression level of lincRNA-NR_024015 and rs8506 A allele were associated with poor ESCC patients' survival. These findings indicate that functional polymorphism rs8506 G>A in lincRNA-NR_024015 exon may be a genetic modifier for the development of ESCC and lincRNA-NR_024015 may be a useful marker for the prediction of the biological behavior of ESCC. © 2016 Wiley Periodicals, Inc.

Triterpenic acids possess rich biological activity. Due to slight differences in structure and polarity, the simultaneous determination of isomeric triterpenic acids is challenging. In the present work, a simple and effective approach to chromatographic separation of such compounds based on conventional C18 stationary phase with gradient elution was developed, which allowed the simultaneous separation of eleven analytes including euscaphic, arjunic, tormentic, arjunolic, asiatic, pomolic, maslinic, corosolic, oleanolic, ursolic and 2-Epi tormentic acid (internal standard). This approach with mass spectrometric detection and ultrasonic extraction was fast, sensitive and accurate for analyzing isomeric triterpenic acids in O. fragrans fruits with a toal duration of the analytical cycle (including pretreatment) within one hour. The LODs lie in ranges of 0.8–12 ng/mL (30 ng/mL for asiatic and corosolic acid). The developed method was validated and successfully applied in ten batches of O. fragrans fruits, which could reflect the detail content difference of triterpenic acid components.

Background Accumulation of storage compounds during seed development plays an important role in the life cycle of oilseed plants; these compounds provide carbon and energy resources to support the establishment of seedlings. Results In this study, we show that BnCIPK9 has a broad expression pattern in Brassica napus L. tissues and that wounding stress strongly induces its expression. The overexpression of BnCIPK9 during seed development reduced oil synthesis in transgenic B. napus compared to that observed in wild-type (WT) plants. Functional analysis revealed that seed oil content (OC) of complementation lines was similar to that of WT plants, whereas OC in Arabidopsis thaliana (L.) Heynh. Atcipk9 knockout mutants (cipk9) was higher than that of WT plants. Seedling of cipk9 mutants failed to establish roots on a sugar-free medium, but root establishment could be rescued by supplementation of sucrose or glucose. The phenotype of complementation transgenic lines was similar to that of WT plants when grown on sugar-free medium. Mutants, cipk9, cbl2, and cbl3 presented similar phenotypes, suggesting that CIPK9, CBL2, and CBL3 might work together and play similar roles in root establishment under sugar-free condition. Conclusion This study showed that BnCIPK9 and AtCIPK9 encode a protein kinase that is involved in sugar-related response and plays important roles in the regulation of energy reserves. Our results suggest that AtCIPK9 negatively regulates lipid accumulation and has a significant effect on early seedling establishment in A. thaliana. The functional characterization of CIPK9 provides insights into the regulation of OC, and might be used for improving OC in B. napus. We believe that our study makes a significant contribution to the literature because it provides information on how CIPKs coordinate stress regulation and energy signaling. Electronic supplementary material The online version of this article (10.1186/s13068-018-1122-z) contains supplementary material, which is available to authorized users.

HPV type was evaluated in a select group of Chinese women that were positive with hybrid capture, and correlations were performed between the pathology found, the type of virus and a semiquantitation from the hybrid capture results. Totally 394 referred high-risk-HPV-positive women evaluated by Hybrid Capture 2 (HC-2) assay were enrolled. Before colposcopy, cervical specimens were collected from all participants and suspended into PreservCytcollection medium (Hologic Inc., Marlborough, MA), and tested with the APTIMA HPV16 18/45 mRNA assay. Colposcopy and diagnostic biopsies were done on all participants. Viral load was assessed by HC2 assay. Totally 55 women were diagnosed as CIN 3 plus cancer (≥CIN3), and the prevalence of HPV16/18/45 was 65.5 % (95 % confidence interval [CI], 52.9-78.0 %) among these ≥CIN3 women. Compared with the group with positive HC2 but negative HPV16/18/45, the odds ratio (OR) to identify ≥CIN3 was 6.3 (95 % CI, 3.2-12.3) for HPV16 and 3.2 (95 % CI, 1.4-7.2) for HPV18/45. When using ≥CIN3 as an endpoint, the sensitivity and specificity was 65.5 % (95 % CI, 52.9-78.0 %) and 72.0 % (95 % CI, 67.2-76.8 %). In the case of HPV16/18/45 negative, no high HPV load had a statistically significant increased risk for the prevalence of ≥CIN3. HPV16, 18 and 45 infection is a major cause for ≥CIN3 in Chinese women. Women with positive HPV16/18/45 should be referred to colposcopy immediately. HPV load was not suitable for the further triaged of the HPV16/18/45 negative cases.

The RAS-association domain family (RASSF) consists of 10 members, and several members act as tumor suppressor genes and epigenetically inactivated in different tumor types. The present study investigated the role and methylation status of RASSF2, RASSF3, RASSF4, and RASSF6 in the pathogenesis and prognosis of GCA. Quantitative real-time RT-PCR, Western blot, and immunohistochemistry (IHC) methods were used respectively to detect the expression of RASSF2, RASSF3, RASSF4, and RASSF6 in 135 GCA cases and BS-MSP method was used to clarify the methylation status of these four genes. Decreased mRNA and protein expression of RASSF2, RASSF3, RASSF4, and RASSF6 were detected in GCA tumor tissues. Aberrant CpG island methylation of RASSF2, RASSF4, and RASSF6 were detected in GCA tumor tissues and were inversely correlated with the expression levels of these genes. Both of RASSF2 and RASSF6 expression and methylation were associated with TNM stage, depth of invasion, LN metastasis, distant metastasis or recurrence, and UGIC family history. GCA patients with simultaneous negative protein expression of RASSF2 and RASSF6 or with simultaneous methylation of both genes demonstrated poor patient survival. These results suggest that down-regulation of RASSF2, RASSF3, RASSF4, and RASSF6 is a tumor-specific phenomenon and the inactivation of RASSF2 and RASSF6 may be associated with tumor progression. Inactivation of RASSF2, RASSF4, and RASSF6 through CpG island methylation may play important roles in GCA carcinogenesis. A combination of RASSF2 and RASSF6 expression or hypermethylation may serve as useful prognostic biomarker for GCA. © 2015 Wiley Periodicals, Inc.

As a result of alternative splicing and differential promoter usage, RASSF5 exists in at least three isoforms (RASSF5A-RASSF5C), which may play different roles in tumorigenesis. The present study was to detect the role of RASSF5A, B and C in esophageal squamous cell carcinoma (ESCC) and clarify the critical CpG sites of RASSF5A, in order to clarify more information on the role of RASSF5 with regard to the pathogenesis of ESCC. Frequent silencing of RASSF5A but not RASSF5B and RASSF5C were found in esophageal cancer cell lines and the silencing of RASSF5A may be reversed by 5-Aza-dC or TSA treatment. The aberrant CpG island 1 methylation of RASSF5A induces silencing of its expression in TE13 cell line. Decreased mRNA and protein expression of RASSF5A was observed in ESCC tumor tissues and was associated with RASSF5A CpG island 1 methylation status. Unlike RASSF5A, expression variation of RASSF5B and RASSF5C was not found in ESCC tissues. Aberrant promoter methylation of RASSF5C was also not found in ESCC. RASSF5A methylation and protein expression were independently associated with ESCC patients' survival. These data indicated that the inactivation of RASSF5A through CpG island 1 methylation may play an important role in ESCC carcinogenesis, RASSF5A may be a functional tumor suppressor and may serve as a prognostic biomarker for ESCC.

As a long non-coding RNA, C5orf66-AS1 is located at 5q31.1. Downregulation and aberrant hypermethylation of C5orf66-AS1 have been detected in a limited several tumors. However, the biological role and distribution of methylated CpG sites of C5orf66-AS1 in gastric cardia adenocarcinoma (GCA) development and prognosis are poorly clarified. The present study was to investigate the expression status and function of C5orf66-AS1 in GCA, and to detect the distribution of methylated CpG sites within the three CpG islands of the promoter and gene body of C5orf66-AS1, further to clarify its prognostic value in GCA patients. C5orf66-AS1 was significantly downregulated in GCA tissues and cell lines, and the expression level was associated with TNM stage, pathological differentiation, lymph node metastasis, and distant metastasis or recurrence. The expression level of C5orf66-AS1 was significantly increased in cancer cells after treated with 5-Aza-dC. Further methylation analysis demonstrated that the aberrant hypermethylation of the regions around the transcription start site of C5orf66-AS1 was more tumor specific and was associated with its expression. Moreover, Sp1 may upregulate C5orf66-AS1 expression and CpG sites hypermethylation within the binding sites may abrogate Sp1 binding. In addition, C5orf66-AS1 inhibited gastric cancer cell proliferation and invasion, and the dysregulation and hypermethylation of the regions around the transcription start site of C5orf66-AS1 were associated with poorer GCA patients' survival. These findings suggest that aberrant hypermethylation-mediated downregulation of C5orf66-AS1 may play important roles in GCA tumorigenesis and C5orf66-AS1 may serve as a potential prognostic marker in predicting GCA patients' survival.

Background Ultrasound molecular imaging is a novel diagnostic approach for tumors, whose key link is the construction of targeted ultrasound contrast agents. However, available targeted ultrasound contrast agents for molecular imaging of tumors are only achieving imaging in blood pool or one type tumor. No targeted ultrasound contrast agents have realized targeted ultrasound molecular imaging of tumor parenchymal cells in a variety of solid tumors so far. Carbonic anhydrase IX (CAIX) is highly expressed on cell membranes of various malignant solid tumors, so it’s a good target for ultrasound molecular imaging. Here, targeted nanobubbles carrying CAIX polypeptides for targeted binding to a variety of malignant tumors were constructed, and targeted binding ability and ultrasound imaging effect in different types of tumors were evaluated. Results The mean diameter of lipid targeted nanobubbles was (503.7 ± 78.47) nm, and the polypeptides evenly distributed on the surfaces of targeted nanobubbles, which possessed the advantages of homogenous particle size, high stability, and good safety. Targeted nanobubbles could gather around CAIX-positive cells (786-O and Hela cells), while they cannot gather around CAIX-negative cells (BxPC-3 cells) in vitro, and the affinity of targeted nanobubbles to CAIX-positive cells were significantly higher than that to CAIX-negative cells (P

As an important long noncoding RNA, Hox transcript antisense intergenic RNA (HOTAIR) is involved in the development and progression of various carcinomas. However, the role and genetic alterations of HOTAIR in gastric cardia adenocarcinoma (GCA) occurrence and progression have not been elucidated. We performed a case-control study in a population of north China to evaluate the possible association between haplotype-tagging SNPs (htSNPs) of the whole HOTAIR sequence and the risk of GCA as well as functional effect of the susceptibility single nucleotide polymorphism (SNP) rs12826786 on gene expression. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to examine the genotype of htSNPs in 515 GCA patients and 654 control subjects, and the quantitative real-time reverse transcription PCR (RT-PCR) method was used to examine the expression of HOTAIR in 102 GCA patients. A family history of upper gastrointestinal cancer (UGIC) significantly increased the risk of developing GCA. Among three htSNPs of the HOTAIR gene (rs12826786 C>T, rs4759314 A>G, and rs10783618 C>T), only the T allele of rs12826786 was found to increase the risk of developing GCA and was associated with smoking habit and tumor-node-metastasis (TNM) stage. In addition, higher expression levels of HOTAIR were found in tumor tissues and rs12826786 SNP has a genotype-specific effect on HOTAIR expression. A high HOTAIR expression level was associated with poor GCA patients’ survival. These results indicate that functional genotype alteration of rs12826786 SNP may influence the expression of HOTAIR, and HOTAIR may be a useful marker to predict the biological behavior of tumors and potentially a therapeutic target in GCA treatment.

Due to alternative splicing and differential promoter usage, RASSF5 exists in at least three isoforms, RASSF5A, RASSF5B, and RASSF5C. Expression and epigenetic inactivation of different transcripts of RASSF5 in gastric cardia adenocarcinoma (GCA) progression have not been evaluated. Quantitative real-time RT-PCR and immunohistochemistry (IHC) methods were used respectively to detect the role of RASSF5A, RASSF5B, and RASSF5C in 132 GCA cases and BS-MSP method was used to clarify the critical CpG sites of RASSF5A. Expression of RASSF5A and RASSF5C transcripts were easily detectable in all normal gastric cardia epithelial tissues; however, expression of RASSF5B was rare detected in normal gastric cardia epithelial tissues and tumor tissues. Both RASSF5A and RASSF5C expression were frequently downregulated in GCA tumor tissues and RASSF5A was more commonly down-regulated compared to RASSF5C. Abnormal reduction of RASSF5A was more commonly observed in advanced stage and poor differentiated tumors. The methylation frequency of CpG island 1 region of RASSF5A in GCA tumor tissues was significantly higher than that in corresponding normal tissues and was inversely correlated with RASSF5A expression. Aberrant promoter methylation of RASSF5C was not found in GCA. RASSF5A methylation and protein expression were independently associated with GCA patients' survival. These results indicate that down-regulation of RASSF5A and RASSF5C expression is a tumor-specific phenomenon and RASSF5A may be a more common target for inactivation in GCA. Inactivation of RASSF5A through CpG island 1 methylation may play an important role in GCA carcinogenesis and may serve as a prognostic biomarker for GCA.

BACKGROUND F-box protein 32 (FBXO32) (also known as atrogin-1), a member of the F-box protein family, has recently been identified as a transforming growth factor beta (TGF-β)/Smad target gene involved in regulating cell survival, and it may be transcriptionally silenced by epigenetic mechanisms in some kinds of carcinomas, yet its role in esophageal squamous cell carcinoma (ESCC) has not been defined. METHODS The role of FBXO32 in ESCC and the correlation of FBXO32 methylation with a series of pathologic parameters were studied in a large cohort of patients with ESCC. RESULTS Decreased messenger RNA (mRNA) expression and protein expression of FBXO32 were observed in esophageal cancer cell lines, and the silencing of FBXO32 could be reversed by treatment with 5-aza-2′-deoxycytidine or trichostatin A in the TE13 cell line. In addition, aberrant methylation of FBXO32 and histone deacetylation was capable of suppressing FBXO32 mRNA and protein expression in TE13 cells. Decreased mRNA and protein expression of FBXO32 was observed in ESCC tumor tissues and was associated with FBXO32 promoter methylation status. A positive correlation between FBXO32 and phosphorylated SMAD family members 2 and 3 expression and Smad4 protein expression also was observed in clinical specimens. FBXO32 methylation status and protein expression were independently associated with survival in patients with ESCC. CONCLUSIONS FBXO32 may be a functional tumor suppressor. Its inactivation through promoter methylation could play an important role in ESCC carcinogenesis, and reactivation of the FBXO32 gene may have therapeutic potential and might be used as a prognostic marker for patients with ESCC. Cancer 2014;120:2412–2423. © 2014 American Cancer Society.

The Ras-Association Domain Family 1 (RASSF1) gene, which is located on the small arm of chromosome 3, contains two CpG islands and generates seven transcripts (RASSF1A-RASSF1G) by differential promoter usage and alternative splicing. As the main transcript, RASSF1A, B and C may play different roles in tumorigenesis. The present study was to detect the role of RASSF1A, B and C in esophageal squamous cell carcinoma (ESCC) and clarify the critical CpG sites of RASSF1A, in order to clarify more information on the role of RASSF1 with regard to the pathogenesis of ESCC. Frequent silencing of RASSF1A but not RASSF1B and RASSF1C were found in esophageal cancer cell lines and the silencing of RASSF1A may be reversed by 5-Aza-dC treatment. The aberrant promoter and exon 1 especially exon 1 methylation of RASSF1A induces silencing of its expression in TE13 cell line. Decreased mRNA and protein expression of RASSF1A was observed in ESCC tumor tissues and was associated with RASSF1A promoter and exon 1 methylation status. Unlike RASSF1A, methylation and expression variation of RASSF1B was not found in ESCC tissues. However, RASSF1C is highly expressed in ESCC tissues. RASSF1A methylation and protein expression were independently associated with ESCC patients' survival. These data indicated that the inactivation of RASSF1A through promoter and exon 1 methylation may play an important role in ESCC carcinogenesis and reactivation of RASSF1A gene may has therapeutic potential and may be used as a prognostic marker for ESCC patients.

Gao, S.; Yin, F.; Chen, J., and Guo, Y., 2019. The Mechanism of Inter-Organizational Collaboration Network on Innovation Performance: Evidences from East Coastal Enterprises in China. In: ...

Rationale: Transplantation of stem cells into damaged hearts has had modest success as a treatment for ischemic heart disease. One of the limitations is the poor stem cell survival in the diseased microenvironment. Prolyl hydroxylase domain protein 2 (PHD2) is a cellular oxygen sensor that regulates 2 key transcription factors involved in cell survival and inflammation: hypoxia-inducible factor and nuclear factor-κB. Objective: We studied whether and how PHD2 silencing in human adipose-derived stem cells (ADSCs) enhances their cardioprotective effects after transplantation into infarcted hearts. Methods and Results: ADSCs were transduced with lentiviral short hairpin RNA against prolyl hydroxylase domain protein 2 (shPHD2) to silence PHD2. ADSCs, with or without shPHD2, were transplanted after myocardial infarction in mice. ADSCs reduced cardiomyocyte apoptosis, fibrosis, and infarct size and improved cardiac function. shPHD2-ADSCs exerted significantly more protection. PHD2 silencing induced greater ADSC survival, which was abolished by short hairpin RNA against hypoxia-inducible factor-1α. Conditioned medium from shPHD2-ADSCs decreased cardiomyocyte apoptosis. Insulin-like growth factor-1 (IGF-1) levels were significantly higher in the conditioned medium of shPHD2-ADSCs versus ADSCs, and depletion of IGF-1 attenuated the cardioprotective effects of shPHD2-ADSC–conditioned medium. Nuclear factor-κB activation was induced by shPHD2 to induce IGF-1 secretion via binding to IGF-1 gene promoter. Conclusions: PHD2 silencing promotes ADSCs survival in infarcted hearts and enhances their paracrine function to protect cardiomyocytes. The prosurvival effect of shPHD2 on ADSCs is hypoxia-inducible factor-1α dependent, and the enhanced paracrine function of shPHD2-ADSCs is associated with nuclear factor-κB–mediated IGF-1 upregulation. PHD2 silencing in stem cells may be a novel strategy for enhancing the effectiveness of stem cell therapy after myocardial infarction.

The growth arrest DNA damage-inducible gene (GADD45) family, which is composed of GADD45A, GADD45B, and GADD45G, may play similar but not identical roles in tumorigenesis. Genetic changes associated with or responsible for their dysregulation are in general uncommon. This study was to detect the role of GADD45 gene family in gastric cardia adenocarcinoma (GCA) and the relationship of GADD45A and GADD45G methylation to a series of pathological parameters in a large GCA sample, in order to elucidate more information on the role of GADD45 gene family with regard to the pathogenesis of GCA. Decreased mRNA and protein expression of GADD45A and GADD45G but not GADD45B were found in 138 GCA tumor tissues. The methylation frequency of 5' 4 CpG region located in distal promoter of GADD45A and proximal promoter of GADD45G in GCA tumor tissues was significantly higher than that in corresponding normal tissues. The expression levels of GADD45A and GADD45G were inversely correlated with methylation levels. GADD45B expression was not correlated with GCA patients survival, while GADD45A and GADD45G methylation status and protein expression were independently associated with GCA patients' survival. These results suggest that GADD45A and GADD45G gene may act as functional tumor suppressor but being frequently inactivated epigenetically in patients with GCA. Silencing of GADD45A and GADD45G, negative regulator of cell growth, is most likely responsible for conferring a selective growth advantage during GCA evolution and outgrowth.

ObjectiveIn the present study, we investigated the relationship between the single-nucleotide polymorphism (SNP) of caspase-3 rs1049216 (C > T), a miRNA target site, and the risk and progression of cervical cancer.Materials and MethodsUsing polymerase chain reaction–restriction fragment length polymorphism, we evaluated the genotype and distribution of caspase-3 rs1049216 in 515 patients with cervical squamous cell cancer and 415 controls. In additional experiments, we transfected luciferase reporter plasmids carrying T or C allele and/or miRNA mimics into the human cervical cell lines (HeLa and C-33A) to analyze its roles in the regulation of caspase-3 expression. By immunohistochemistry, the protein level of caspase-3 expression was examined in tumor tissues from 515 patients with cervical squamous cell cancer.ResultsWe found that the TT genotype of caspase-3 rs1049216 conferred a significantly decreased risk of cervical cancer (adjusted odds ratio, 0.35; 95% confidence interval, 0.154–0.581) and may be associated with the progression of this cancer. Although the expression of caspase-3 in the TT genotype was higher than that in CC/CT genotype in peripheral blood mononuclear cells and tumor tissues. Additional luciferase analysis showed that the rs1049216 variant T allele was associated with significantly higher luciferase activity, compared with the C allele in the transfected cells, and when cotransfected with miRNAs, miRNA-181a could downregulate the luciferase activity in the cells that transfected the construct containing C allele, compared with T allele, which had not happened in the presence of other miRNAs selected.ConclusionsThese data indicate that through upregulating the expression of caspase-3, the TT genotype of caspase-3 rs1049216 can be associated with not only the risk of cervical cancer but also the progression of this cancer.

Background As seed oil content (OC) is a key measure of rapeseed quality, better understanding the genetic basis of OC would greatly facilitate the breeding of high-oil cultivars. Here, we investigated the components of genetic effects and genotype × environment interactions (GE) that govern OC using a full diallel set of nine parents, which represented a wide range of the Chinese rapeseed cultivars and pure lines with various OCs. Results Our results from an embryo-cytoplasm-maternal (GoCGm) model for diploid seeds showed that OC was primarily determined by genetic effects (VG) and GE (VGE), which together accounted for 86.19% of the phenotypic variance (VP). GE (VGE) alone accounted for 51.68% of the total genetic variance, indicating the importance of GE interaction for OC. Furthermore, maternal variance explained 75.03% of the total genetic variance, embryo and cytoplasmic effects accounted for 21.02% and 3.95%, respectively. We also found that the OC of F1 seeds was mainly determined by maternal effect and slightly affected by xenia. Thus, the OC of rapeseed was simultaneously affected by various genetic components, including maternal, embryo, cytoplasm, xenia and GE effects. In addition, general combining ability (GCA), specific combining ability (SCA), and maternal variance had significant influence on OC. The lines H2 and H1 were good general combiners, suggesting that they would be the best parental candidates for OC improvement. Crosses H3 × M2 and H1 × M3 exhibited significant SCA, suggesting their potentials in hybrid development. Conclusions Our study thoroughly investigated and reliably quantified various genetic factors associated with OC of rapeseed by using a full diallel and backcross and reciprocal backcross. This findings lay a foundation for future genetic studies of OC and provide guidance for breeding of high-oil rapeseed cultivars.

The effects of different substrate stiffness were investigated on epithelial-mesenchymal transition (EMT) of cervical cancer cell lines and the role of miR-106b and its target protein DAB2 therein. Cervical cancer cell lines HeLa and SiHa were cultured on artificial substrates with different stiffness prepared using different ratios of acrylamide and bis-acrylamide. Changes of microRNA profiles were detected using microRNA chip analysis, and the expression levels of EMT-related markers E-cadherin and vimentin were detected using western blotting and real-time PCR. In addition, the effects of miR-106b overexpression as well as miR-106b and DAB2 knockdown on expression of E-cadherin and vimentin were also examined using western blotting and real-time PCR. The results showed that i) cervical cancer cell lines SiHa and HeLa cultured on substrate with stiffness of 20 kPa had the strongest EMT ability, showed the highest levels of vimentin and lowest levels of E-cadherin, compared with cells cultured on substrate with stiffness of 1 kPa; ii) miR-106b knockdown reversed the effects of substrate stiffness on EMT of cervical cancer cells, while miR-106 overexpression and DAB2 knockdown induced EMT of cervical cancer cells cultured on substrate with stiffness of 20 kPa. Overall, the results indicated that substrate stiffness could regulate EMT of cervical cancer cell lines HeLa and SiHa at least partially through miR-106b and its downstream target DAB2.

An experiment was conducted to estimate the optimal dietary zinc (Zn) level of brown-egg laying hens fed a corn-soybean meal diet from 20 to 40 weeks of age. A total of 120 20-week-old Beijing Red commercial laying hens were randomly allotted by bodyweight to one of five treatments with six replicates of four birds each in a completely randomized design, and fed a Zn-unsupplemented corn-soybean meal basal diet containing 27.95 mg Zn/kg by analysis and the basal diets supplemented with 30, 60, 90, or 120 mg Zn/kg as Zn sulfate (reagent grade ZnSO4·7H2O) for a duration of 20 weeks. Laying performance, egg quality, tissue Zn concentrations, and activities of serum alkaline phosphatase (AKP), and liver copper-Zn superoxide dismutase (CuZnSOD) were measured. Regression analyses were performed to estimate an optimal dietary Zn level whenever a significant quadratic response (P

Background and Aims Caveolin-1 (CAV1) is a multifunctional scaffolding protein and plays an important role in tumorigenesis. However, the epigenetic changes of CAV1 in gastric cardia adenocarcinoma (GCA) have not been investigated so far. The purpose of this study was to clarify the contribution of critical CpG sites in CAV1 to progression/prognosis of GCA and to further elucidate the effect of critical CpG sites on the ectopic expression of β-catenin in GCA. Methods Methylation-specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS) methods were, respectively, applied to examine the methylation status of CAV1 . RT-PCR and immunohistochemistry methods were used to determine the mRNA and protein expression of CAV1 and β-catenin. Results Decreased mRNA and protein expression of CAV1 were observed in GCA tumor tissues and were associated with hypermethylation of CpG island shore and transcription start site (TSS) regions in CAV1 . Hypermethylation of the other two regions within CpG islands in CAV1 was observed both in tumor and corresponding adjacent tissues but was not related to the transcriptional inhibition of CAV1 . The methylation status of CpG island shore region in CAV1 was associated with the ectopic expression of β-catenin and was independently associated with survival in GCA patients. Conclusions Hypermethylation of CpG island shore and TSS regions is cancer specific and is closely associated with reduced expression of CAV1 . The CpG island shore methylation of CAV1 may play an important role in progression of GCA and may serve as a prognostic methylation biomarker for GCA patients.

WWOX is a tumor suppressor gene that maps to the common fragile site FRA16D and is involved in carcinogenesis and cancer progression in many different carcinomas. Reduced WWOX expression is associated with more aggressive phenotypes and poor patient outcomes in several cancers. The present study was conducted in order to elucidate more precisely the genetic and epigenetic alterations of WWOX that play a role in gastric cardia adenocarcinoma (GCA) carcinogenesis in a population from Northern China. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), PCR-simple sequence length polymorphism (PCR-SSLP), and methylation specific PCR (MSP) methods were used to characterize polymorphisms in the rs3764340, rs2548861, and rs1079635 loci, the level of loss of heterozygosity (LOH), and methylation status of WWOX, respectively. Protein and mRNA expression of WWOX in GCA tissues was quantified by immunohistochemistry and reverse transcription-PCR (RT-PCR). Family history of upper gastrointestinal cancer (UGIC) significantly increased the risk of developing GCA. The CG+GG genotype of rs3764340 and GT or TT genotype of rs2548861 significantly elevated the risk of developing GCA. LOH at the WWOX locus was observed in 45.6% tumors. The promoter and exon 1 methylation frequency of WWOX in GCA tissues was significantly higher than in corresponding normal tissues and was associated with UGIC family history. Protein and mRNA expression of WWOX was reduced in GCA tumor tissues and was associated with LOH and methylation of the gene. These results indicate that WWOX may play an important role in GCA especially in individuals with UGIC family history. Environ. Mol. Mutagen., 2013. © 2012 Wiley Periodicals, Inc.

The loss of transforming growth factor-β (TGF-β) response due to the dysregulation of TGF-β receptor type I (TGFBR1), type II (TGFBR2) and Smad4 is well known for its contribution to oncogenesis, although the role of the genes of TGF-β/Smad signalling pathway in gastric cardia adenocarcinoma (GCA) is poorly understood. In the present study, the methylation status and expression of TGF-β receptor type I (TGFBR1), type II (TGFBR2), and Smad4 was investigated in GCA and dysplasia. MSP approach was used to detect the methylation status of TGFBR1, TGFBR2, and Smad4. Immunohistochemistry and quantitative RT-PCR methods were used respectively to examine the protein and mRNA expression of them in tissues. The methylation frequency of TGFBR1 and TGFBR2 in the tissues of high grade dysplasia and GCA was significantly higher than that in corresponding normal tissues (p < 0.01) and was significantly associated with mRNA and protein expression of the two genes (p < 0.05). The methylation frequency of Smad4 in the 30 ~ 171 sites was higher than that in the −248 ~ 26 sites and was associated with the loss of Smad4 expression. The decreased expression of TGFBR1, TGFBR2 and Smad4 was correlated with increased expression of TGF-β1 in GCA. In all, these data suggest that methylation of TGFBR1, TGFBR2 and Smad4 may exist in the gastric cardia dysplasia stages and plays an important role in these genes silencing and subsequently affect the TGF-β/Smad signaling pathway.

Transforming growth factor-β (TGF-β) is a pleiotropic growth factor with multiple functions through its type I (TGFBR1) and type II (TGFBR2) receptors. A reduction or loss of expression of TGFBRs enables cancer cells to escape the growth inhibitory effect of TGF-β and to gain a growth advantage.The promoter methylation status and expression of TGFBR1, TGFBR2 and Smad4 gene were investigated in esophageal squamous cell carcinoma (ESCC).Methylation-specific polymerase chain reaction approach was used to detect the methylation status. Immunohistochemistry and reverse transcription-polymerase chain reaction method were used to examine the protein and messenger RNA expression, respectively.Both the ESCC and the high-grade dysplastic tissues showed hypermethylation of TGFBR1 and TGFBR2, and the hyper-methylation of TGFBR1 and TGFBR2 in ESCC tissues was significantly associated with decreased messenger RNA and protein expression (P0.05). When stratified for tumor lymph node metastasis stages, TGFBR1 and TGFBR2 gene methylation was more frequent in stage III and stage IV tumor tissues than that in stage I and stage II tumor tissues (P0.05). Simultaneous methylation of the 2 receptors was progressively increased along with the increasing of tumor lymph node metastasis stage and decreasing of histological differentiation. Smad4 hypermethylation was only detected in 7 ESCC tumor tissues and associated with the loss of Smad4 expression. The decreased protein expression of TGFBR1, TGFBR2 and Smad4 was correlated with increased expression of TGF-β1 in ESCC.These data suggest that promoter methylation of TGFBR1 and TGFBR2 may exist in the early stage of ESCC and play important roles in TGFBR1 and TGFBR2 gene silencing.

The WW domain-containing oxidoreductase (WWOX) gene, located on chromosome 16q23.3–24.1 in the region recognized as the common fragile site FRA16D is considered to be a tumor suppressor gene involved in various carcinomas. The present study was to investigate the alterations of WWOX expression and its correlation with polymorphism, the level of WWOX loss of heterozygosity (LOH), and methylation status in esophageal squamous cell carcinoma (ESCC). Immunohistochemistry and RT-PCR methods were used, respectively, to examine the protein and mRNA expression of WWOX in ESCC tissues. PCR-RFLP, PCR-SSLP, and MSP approach were used, respectively, to detect polymorphisms of rs3764340, rs2548861, and rs1079635 site, the level of LOH, and WWOX methylation status. Family history of upper gastrointestinal cancer (UGIC) significantly increased the risk of developing ESCC. Protein and mRNA expression of WWOX was reduced in ESCC tumor tissues and was associated with LOH and hypermethylation of the gene. The G allele of rs3764340 significantly elevated the risk of developing ESCC and was associated with TNM stage. LOH at the WWOX loci was observed in 41.4% tumors. The hypermethylation of promoter and exon1 of WWOX was found to be occurred in dysplastic tissues and the methylation frequency of WWOX in ESCC tumor tissues was significantly higher than that in corresponding normal tissues and was associated with UGIC family history. In all, these results indicate that the WWOX gene may play an important role in the development of ESCC especially in individuals with UGIC family history. © 2011 Wiley Periodicals, Inc.

Both transforming growth factor-β receptor I (TGFBR1) and receptor II (TGFBR2) are serine/threonine kinases and play important roles in TGF-β/Smads signal pathway. The case-control study was performed to evaluate the possible association of Int7G24A and *6A polymorphisms of TGFBR1 and G-875A polymorphism of TGFBR2 with susceptibility to gastric cardia adenocarcinoma (GCA) in a population of North China. Polymerase-chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and PCR methods were used respectively to detect the genotype of Int7G24A, *6A and G-875A in 468 GCA and 584 healthy controls. Immunohistochemistry method was used to determine the protein expression of TGFBR1 and TGFBR2. Family history of upper gastrointestinal cancer (UGIC) significantly increased the risk of developing GCA. There were no differences in the genotype distribution of TGFBR1 *6A polymorphism among cases and controls. However, A allele of Int7G24A significantly elevated the risk of developing GCA (adjusted OR = 1.34, 95% CI 1.03-1.87) and A allele of G-875A significantly decreased the risk of developing GCA (adjusted OR = 0.73, 95% CI 0.49-0.92). When stratified for TNM stage, A allele of Int7G24A and G-875A allele carriers had a 1.41-fold (95% CI 1.05-1.98) increased and a 0.70-fold (95% CI 0.47-0.92) decreased risk of stage III and IV gastric cardia adenocarcinoma. The protein expression of TGFBR1 and TGFBR2 in GCA was not correlated with genotypes of them. In conclusions, TGFBR1 Int7G24A and TGFBR2 G-875A polymorphisms may play important roles in the risk of GCA in North China.

The canonical Wnt signalling pathway plays a key role during embryogenesis and pathogenesis of various types of tumors. Recently, several studies have shown that the promoter hypermethylation of Wnt-antagonist genes, including sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wif-1 and Dkk-3, have been certified to contribute to the tumorigenesis of several cancers. The aim of this study was to investigate the promoter methylation of Wnt-antagonist genes in gastric cardia adenocarcinoma (GCA) and corresponding adjacent non-cancerous tissues, and to establish the possible relationship between DNA methylation status and the pathogenesis of GCA. MSP, RT-PCR methods were applied respectively to examine the CpG methylation of the Wnt-antagonist genes and its mRNA expression in tumors and corresponding non-cancerous tissues, and immunohistochemistry method was used to determine protein expression of β-catenin(the key factor of the Wnt signalling pathway) and cyclin D1(the target gene of this pathway). The frequency of promoter methylation of sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wif-1 and Dkk-3 genes in GCA tumor tissues were 78.7%(74/94), 76.6%(72/94), 70.2%(66/94), 77.1%(73/94), 61.7%(58/94) and 21.3%(20/94), respectively, which were significantly higher than those in adjacent non-cancerous tissues. Furthermore, the frequencies of silenced mRNA expression of these six genes in GCA tumor tissues were significantly higher than those in adjacent non-cancerous tissues. Methylation levels of these six genes were all correlated with loss of mRNA expression. The ectopic expression of β-catenin and cyclin D1 was significantly more frequent in GCA tumor tissues than that in adjacent non-cancerous tissues and correlated with each Wnt-antagonist genes hypermethylation status. Epigenetic silencing of Wnt-antagonist genes expression by promoter hypermethylation may play an important role in gastric cardia adenocarcinoma.

Thrombospondin-1(TSP1) is an inhibitor of angiogenesis and its promoter hypermethylation has been found resulting in gene silencing in some primary human carcinomas. This study was to investigate the promoter methylation of TSP1 and its correlation with TGF-beta1 level and T cell immunity in gastric cardia adenocarcinoma (GCA).Methylation specific polymerase chain reaction (MSP) approach and immunohistochemistry method were used to examine the methylation status of the 5' CpG island and expression of TSP1 protein, respectively. The level of TGF-beta1 was measured by ELISA and T cell immunity of GCA by flow cytometry analysis.TSP1 methylation frequency was significantly higher in tumor specimens than in corresponding normal tissues (35.4% vs. 3.1%, P0.001) and significantly higher in Stages III and IV tumor tissues than in Stages I and II tumor tissues (P0.05). TSP1 protein expression was significantly lower in the tumor tissues than in corresponding normal tissues (P0.05) and statistically correlated with its methylation status (P0.01). The total level of TGF-beta1 was significantly higher in the GCA patients than in healthy controls(P0.05) and significantly higher in Stages III and IV GCA patients than in Stages I and II GCA patients (P0.05). The level of active TGF-beta1 was significantly higher in the GCA patients with hypermethylation of TSP1 than in the GCA patients without methylation of TSP1(P0.05), but there was no statistical difference(P0.05). The function of T cell immunity was significantly different between the GCA patients with hypermethylation of TSP1 and those without methylation of TSP1 (P0.05).Promoter hypermethylation of TSP1 may play an important role in the development of GCA and reflect the biological behaviours of GCA.