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Background: 14-3-3epsilon regulates a wide range of biological processes, including cell cycle control, proliferation, and apoptosis, and plays a significant role in neurogenesis and the formation of malignant tumours. However, the exact function and regulatory mechanism of 14-3-3epsilon in carcinogenesis have not been elucidated.Methods: The expression of 14-3-3epsilon was assessed by RT-PCR and western blotting. The invasiveness and viability of Hep-2 cells were determined by the transwell migration assay and MTT assay, respectively. Cell cycle and apoptosis of Hep-2 cells were detected by flow cytometry.Results: The mRNA and protein expression of 14-3-3epsilon in larynx squamous cell carcinoma (LSCC) tissues were significantly lower than those in clear surgical margin tissues. Statistical analysis showed that the 14-3-3epsilon protein level in metastatic lymph nodes was lower than that in paired tumour tissues. In addition, the protein level of 14-3-3epsilon in stage III or IV tumours was significantly lower than that in stage I or II tumours. Compared with control Hep-2 cells, the percentages of viable cells in the 14-3-3epsilon-GFP and negative control GFP groups were 36.68 +/- 14.09% and 71.68 +/- 12.10%, respectively. The proportions of S phase were 22.47 +/- 3.36%, 28.17 +/- 3.97% and 46.15 +/- 6.82%, and the apoptotic sub-G1 populations were 1.23 +/- 1.02%, 2.92 +/- 1.59% and 13.72 +/- 3.89% in the control, negative control GFP and 14-3-3epsilon-GFP groups, respectively. The percentages of the apoptotic cells were 0.84 +/- 0.25%, 1.08 +/- 0.24% and 2.93 +/- 0.13% in the control, negative control GFP and 14-3-3epsilon-GFP groups, respectively. The numbers of cells that penetrated the filter membrane in the control, negative control GFP and 14-3-3epsilon-GFP groups were 20.65 +/- 1.94, 17.63 +/- 1.04 and 9.1 +/- 0.24, respectively, indicating significant differences among the different groups.Conclusions: Decreased expression of 14-3-3epsilon in LSCC tissues contributes to the initiation and progression of LSCC. 14-3-3epsilon can promote apoptosis and inhibit the invasiveness of LSCC. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to analyze the effect of optical coherence tomography angiography (OCTA) on diabetic macular edema (DME) staging and assess the efficacy of laser photocoagulation., Methods: Eighty-six patients (141 eyes) with suspected DME who visited our hospital from August 2019 to March 2022 were selected and underwent fundus angiography and OCTA. The two examination methods were compared in terms of their efficacy in macular edema staging. Subsequently, the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of OCTA in diagnosing DME were assessed using fundus angiography as the gold standard. In patients with clinically significant macular edema (CSME) treated with laser photocoagulation, the central concave non-perfused zone (FAZ), vascular density (VD), central macular retinal thickness (CRT), whole retinal blood flow density (FD-300), superficial capillary plexus (SCP), and deep capillary plexus (DCP) were measured using the OCTA 3 mm × 3 mm mode before treatment, at 3 months after treatment, and at 6 months after treatment. SCP, deep capillary plexus (DCP), blood flow density (VD), best corrected visual acuity (BCVA), and central retinal thickness (CRT) were recorded before treatment, 3 months after treatment, and 6 months after treatment. The correlation between BCVA and pre-treatment OCTA parameters at 6 months after treatment was analyzed using Pearson's correlation., Results: Fundus angiography was performed in 86 patients (141 eyes) with suspected DME. Of the 141 eyes, 44 had no leakage, 52 had diffuse edema, 40 had focal macular edema, and 5 had eyes ischemia. A total of 97 eyes showed CSME on fundus angiography. Using fundus angiography as the gold standard, OCTA exhibited a sensitivity of 97.94 %, a specificity of 63.64 %, and an accuracy of 87.23 % in diagnosing CSME. The Kappa value between OCTA and fundus angiography was 0.674. The receiver operating characteristic curve revealed that the area under the curve (AUC) of OCTA in diagnosing CSME was 0.808 (95 % confidence interval: 0.717-0.899). The BCVA was higher, while the CRT was lower in CSME patients at 3 and 6 months after treatment (P<0.05). No significant difference was observed in the OCTA parameters in CSME patients at 3 months after treatment compared with that before treatment (P>0.05). Similarly, no significant difference was found in the FD300 of CSME patients at 6 months after treatment compared with that before treatment (P>0.05). However, the FAZ area, DCP-VD (overall, central concave, and paracentral concave), and SCP-VD (overall, central concave, and paracentral concave) were higher in CSME patients at 6 months after treatment compared with that before treatment (P<0.05). Pearson's correlation showed that BCVA was positively correlated with pre-treatment FAZ area, DCP-VD, and SCP-VD (r>0, P<0.05), and negatively associated with CRT (r<0, P<0.05)., Conclusion: OCTA exhibited high sensitivity and specificity in diagnosis and staging DME. It adeptly captures the microvascular and visual changes in the central macular recess before and after laser photocoagulation therapy, which can quantitatively guide the follow-up treatment of DME., Competing Interests: Declaration of competing interest The authors declared no conflict of interest existing in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: The abnormality of chromosomal karyotype is one factor causing poor prognosis of lymphoma. In the analysis of abnormal karyotype of lymphoma patients, three smallest overlap regions were found, in which MYCT1 was located. MYCT1 is the first tumor suppressor gene cloned by our research team, but its studies relating to the occurrence and development of lymphoma have not been reported., Methods: R banding analyses were employed to screen the abnormality of chromosomal karyotype in clinical specimen and MYCT1 over-expression cell lines. FISH was to monitor MYCT1 copy number aberration. RT-PCR and Western blot were to detect the mRNA and protein levels of the MYCT1 and RUNX1 genes, respectively. The MYCT1 and RUNX1 protein levels in clinical specimen were evaluated by immunohistochemical DAB staining. The interaction between MYCT1 and MAX proteins was identified via Co-IP and IF. The binding of MAX on the promoter of the RUNX1 gene was detected by ChIP and Dual-luciferase reporter assay, respectively. Flow cytometry and CCK-8 assay were to explore the effects of MYCT1 and RUNX1 on the cell cycle and proliferation, respectively., Results: MYCT1 was located in one of three smallest overlap regions of diffuse large B-cell lymphoma, it altered chromosomal instability of diffuse large B-cell lymphoma cells. MYCT1 negatively correlated with RUNX1 in lymphoma tissues of the patients. MAX directly promoted the RUNX1 gene transcription by binding to its promoter region. MYCT1 may represses RUNX1 transcription by binding MAX in diffuse large B-cell lymphoma cells. MYCT1 binding to MAX probably suppressed RUNX1 transcription, leading to the inhibition of proliferation and cell cycle of the diffuse large B-cell lymphoma cells., Conclusion: This study finds that there is a MYCT1-MAX-RUNX1 signaling pathway in diffuse large B-cell lymphoma. And the study provides clues and basis for the in-depth studies of MYCT1 in the diagnosis, treatment and prognosis of lymphoma., (© 2023. The Author(s).)

Tubulointerstitial abnormalities contribute to the progression of diabetic kidney disease (DKD). However, the underlying mechanism of the pathobiology of tubulointerstitial disease is largely unknown. Here, we showed that MYCT1 expression was downregulated in in vitro and in vivo DKD models. Adeno-associated virus (AAV)-Myct1 significantly attenuated renal dysfunction and tubulointerstitial fibrosis in diabetic db/db mice and downregulated Sp1 transcription and TGF-β1/SMAD3 pathway activation. In human proximal tubular epithelial cells, high glucose-induced high expression of SP1 and TGF-β1/SMAD3 pathway activation as well as overaccumulation of extracellular matrix (ECM) were abrogated by MYCT1 overexpression. Mechanistically, the binding of VDR to the MYCT1 promoter was predicted and confirmed using dual-luciferase reporter and ChIP analysis. VDR transcriptionally upregulates MYCT1. Our data reveal MYCT1 as a new and potential therapeutic target in treating DKD., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Background: Although breastfeeding is one of the top priorities for public health in China, the rate of breastfeeding is still low and a large number of women do not initiate breastfeeding due to various reasons. Hence, this study aimed to understand the decision-making of non-initiation and reveal the underlying reasons in order to protect, support, and promote breastfeeding., Methods: In-depth, exploratory interviews were carried out with 13 new mothers who did not initiate breastfeeding. The collected data were analyzed by inductive content analysis., Results: Although mothers generally understood the protective effects of breastfeeding, they believed that formula milk is a good alternative to human milk and even better in some aspects. Five core themes related to non-initiation decision-making emerged: (1) expected breastfeeding stress; (2) maladjustment to the maternal role; (3) concerns about physiological issues; (4) the lack of knowledge about the risks of artificial feeding; (5) belief that it is better not to initiate than to interrupt., Conclusions: The environment for mothers to breastfeed in China is not supportive enough, and the medical staff and families should be held responsible for the non-initiation of breastfeeding. More professionals are needed to support mothers to solve their problems and breastfeeding education should be further expanded., (© 2022. The Author(s).)

MYCT1 has been shown to function as a tumor suppressor in various tumors, but its role in metabolism has never been reported. Here, we showed that global inactivation of Myct1 in mice led to progressive accumulation of glycogen in the liver, which was accompanied by aberrant changes in intermediates of the glycogen metabolic pathway. Mechanistically, MYCT1 appeared to promote translation efficiency of PGM1, UGP2 and GSK3A in hepatic cells in a RACK1-dependent manner. Consequently, upregulation of the three enzymes enhanced the glycogen shunt. Our data reveal a critical role of MYCT1 as a switch for the glycogen shunt in tumor cells., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Objective: We aimed to identify the role of DOCK6 in oral squamous cell cancer (OSCC) in this study., Design: DOCK6 expression in OSCC was analyzed using TCGA and GEO datasets and was verified by quantitative real-time PCR, Western blotting, and immunohistochemistry. Statistical analyses were performed to evaluate the relationships between DOCK6 expression and the clinicopathological characteristics of OSCC patients. Wound healing and Transwell assays were performed to assess OSCC cell migration and invasion, respectively. STRING and GO analyses and gene set enrichment analysis were used to identify DOCK6-interacting proteins, their functions and their potential pathways., Results: DOCK6 was significantly upregulated at both the mRNA and protein levels in OSCC tissues (all P < 0.05). DOCK6 levels were positively correlated with age (P < 0.05), lymph node metastasis status (P < 0.001), clinical stage (P < 0.001), differentiation (P < 0.05), and poor clinical outcome (P < 0.05) in OSCC patients. Furthermore, univariate and multivariate analyses revealed that high DOCK6 expression (P < 0.01) and clinical stage III-IV (P < 0.05) might serve as independent prognostic factors for OSCC patients. Functionally, DOCK6 silencing significantly suppressed OSCC cell migration and invasion (all P < 0.05). Ten proteins that interact with DOCK6, more than ten functions related to cancer, and more than six pathways related to DOCK6 in OSCC were identified via bioinformatic methods., Conclusion: DOCK6 is upregulated in OSCC, is associated with a poor prognosis in OSCC patients and increases OSCC cells migration and invasion. These findings suggest that DOCK6 may be a potential therapeutic target with prognostic implication in patients with OSCC., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: The high morbidity, high mortality and low survival rate of cardiac arrest (CA) cause a heavy global burden. We aimed to analyze the changes in scientific output related to CA over the past two decades., Methods: We analyzed the scientific output related to CA from 2000 to 2020 via the Web of Science. The data were analyzed using CiteSpace software., Results: In total, 28,312 articles relating to CA were identified in the Web of Science. The volume of scientific research output in the field of global CA research was mainly distributed in the Americas, Europe and Asia, covering a wide range. Of the 28,312 articles, the research content of the highly cited literature mainly focused on CA, mild hypothermia treatment, and prognosis of CA patients., Conclusion: Various scientific methods were applied to reveal scientific productivity, collaboration, and research hotspots in the CA research field. Cardiopulmonary resuscitation (CPR), extracorporeal membrane oxygenation (ECMO), survival and target temperature management are research hotspots. Future research on CA will continue to focus on its treatment and prognosis to improve the survival rate of CA patients., Competing Interests: Conflicts of interest: The authors declare that they have no conflicts of interest., (Copyright: © World Journal of Emergency Medicine.)

MYCT1, a target of c-Myc, inhibits laryngeal cancer cell migration, but the underlying mechanism remains unclear. In the study, we detected differentially expressed genes (DEGs) from laryngeal cancer cells transfected by MYCT1 using RNA-seq (GSE123275). DEGs from head and neck squamous cell carcinoma (HNSCC) were first screened by comparison of transcription data from the Gene Expression Omnibus (GSE6631) and the Cancer Genome Atlas (TCGA) datasets using weighted gene co-expression network analysis (WGCNA). GO and KEGG pathway analysis explained the functions of the DEGs. The DEGs overlapped between GSE6631and TCGA datasets were then compared with ours to find the key DEGs downstream of MYCT1 related to the adhesion and migration of laryngeal cancer cells. qRT-PCR and Western blot were applied to validate gene expression at mRNA and protein levels, respectively. Finally, the cell adhesion, migration, and wound healing assays were to check cell adhesion and migration abilities, respectively. As results, 39 overlapping genes were enriched in the GSE6631 and TCGA datasets, and most of them revealed adhesion function. Thirteen of 39 genes including COL6 members COL6A1, COL6A2, and COL6A3 were overlapped in GSE6631, TCGA, and GSE123275 datasets. Similar to our RNA-seq results, we confirmed that COL6 is a target of MYCT1 in laryngeal cancer cells. We also found that MYCT1 inhibited the adhesion and migration of laryngeal cancer cells via COL6. These indicate that COL6 is a potential target of MYCT1 and participates the adhesion and migration of laryngeal cancer cells, which provides an important clue for further study on how MYCT1 regulating COL6 in laryngeal cancer progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Ding, Sun, Li and Fu.)

MYCT1 has an inhibitory effect on the migration of laryngeal cancer cells, although the underlying molecular mechanism remains unknown. In this study, we aimed to explore the mechanism of MYCT1 in the epithelial-mesenchymal transition (EMT) and migration of laryngeal cancer cells. We found that MYCT1 significantly decreased the expression of miR-629-3p but increased the expression of ESRP2 in laryngeal cancer cells. The expression of miR-629-3p and ESRP2 in laryngeal cancer tissues showed significantly positive and negative correlations with patient metastasis, respectively. miR-629-3p was confirmed to repress the expression of ESRP2 by targeting its 3'UTR. SP1 was verified to be a direct transcription factor for miR-629-3p and a downstream target of MYCT1. Moreover, MYCT1 inhibited the EMT and migration of laryngeal cancer cells through the SP1/miR-629-3p/ESRP2 pathway. Taken together, our results establish a novel MYCT1 signaling pathway in the EMT and migration of laryngeal cancer cells, thus providing important insights for further studying the pathway in the diagnosis and treatment of laryngeal cancer., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

MYCT1 is an important gene known to regulate cell viability and apoptosis of laryngeal cancer cells. However, the underlying molecular mechanism remains unclear. Here, we show that MAX enhances the expression of miR-181a by directly binding to its promoter, whereas miR-181a targets NPM1 and suppresses its expression in laryngeal cancer cells. MYCT1 and miR-181a decrease cell viability and colony formation through enhanced apoptosis, whereas NPM1 displays opposite effects in laryngeal cancer cells. Their opposing functions are further supported by the findings (a) that miR-181a is down-regulated, while NPM1 is up-regulated in laryngeal cancer, and (b) that either inhibition of miR-181a or overexpression of NPM1 can revert the pro-apoptotic effects of MYCT1 on laryngeal cancer cells through extracellular and intracellular apoptotic pathways. Our data suggest that MYCT1 may synergistically interact with MAX as a co-transcription factor or a component of MAX transcriptional complex, to transcriptionally regulate the expression of miR-181a, which, in turn, decreases NPM1 expression at post-transcriptional levels, leading to enhanced apoptosis in laryngeal cancer cells. These factors may serve as potential targets for early diagnosis and treatment of laryngeal cancer., (© 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

It is currently believed that the TBX1 gene is one of the core genes of congenital heart disease (CHD). However, there are few studies on the abnormal regulation of TBX1 gene expression. The purpose of this work was to investigate the role of miR-144 and TBX1 in cardiac development by studying the regulatory relationship and mechanism of miR-144 on TBX1/JAK2/STAT1 in cardiomyocytes. Cell proliferation was detected by MTT and clone formation assay and cell cycle and apoptosis by flow cytometry. The levels of miR-144 and TBX1 in H9c2 cells were assessed by qRT-PCR. Dual luciferase reporter assay was used to validate the direct targeting of TBX1 with miR-144. The protein expression levels of TBX1 and its downstream proteins were measured by Western blot analysis. miR-144 inhibited H9c2 cell proliferation by arresting cells in G1 phase. Furthermore, miR-144 induced H9c2 cell apoptosis and activated the JAK2/STAT1 signaling pathway. Bioinformatic predictions and luciferase reporter assay showed that miR-144 directly targets TBX1. Co-overexpression of miR-144 and TBX1 upregulated cell proliferation by accelerating G1 to S phase transition and downregulated cell apoptosis through inhibiting the JAK2/STAT1 signaling pathway. miR-144 acts as a proliferation inhibitor in cardiomyocytes via the TBX1/JAK2/STAT1 axis and is therefore a potential novel therapeutic target for CHD treatment., (© 2020 S. Karger AG, Basel.)

Purpose: Systemic lupus erythematosus (SLE) is a complex autoimmune disease with marked disparities in prevalence and disease severity among different ethnic groups. The purpose of this study is to characterize a Latin American cohort and identify genetic risk factors for developing SLE and its end-organ manifestations in this Latin Hispanic cohort., Methods: A total of 201 SLE cases and 205 non-diseased controls were recruited in the Dominican Republic (DR). Cases were defined according to the 1997 revised American College of Rheumatology criteria for the classification of SLE. Genomic DNA was prepared from whole blood and applied to genotyping analyses for 42 single nucleotide polymorphisms (SNPs) that have been implicated in autoimmune diseases, including SLE, in other ethnic populations. Data were analyzed by Fisher's Exact Probability Test., Results: In this cohort, SNP rs9271366 (tag SNP for HLA-DRB1*15:01) confers the highest risk for SLE among the 13 MHC gene alleles that display association with SLE (p = 8.748E-10; OR = 3.5). Among the 26 non-MHC gene alleles analyzed, SNP rs2476601 in PTPN22 gene confers the highest risk for SLE (p = 0.0001; OR = 5.6). ITGAM, TNFSF4, TNIP1, STAT4, CARD11, BLK, and TNXB gene alleles were confirmed as SLE-susceptible alleles in the DR cohort. However, IRF5 and TNFAIP3 gene alleles, established risk factors for SLE in populations of European and Asian ancestry, are not significantly associated with SLE in this cohort. We also defined a novel HLA-DRA haplotype that confers an increased risk for lupus nephritis (LN) and alleles in HLA-DRA2 and TNFSF4 genes as genetic risk factors for developing neuropsychiatric (NP) SLE., Conclusion: Our data suggest that the Latin American population shares some common genetic risk factors for SLE as other populations, but also has distinct risk gene alleles that contribute to SLE susceptibility and development of LN and NPSLE. This is the first study focusing on genetic risk factors for SLE in the DR, a Latin American population that has never been characterized before., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

YY1 is a key transcription factor and plays different roles in various cancers. However, role and mechanism of YY1 in laryngeal cancer are still unknown. YY1 and MYCT1 mRNA and protein levels were detected by Real-time RT-PCR and Western Blot methods, respectively. Binding of YY1 to MYCT1 promoter was predicted and confirmed by bioinformatics and chromatin immunoprecipitation assays, respectively. MYCT1 promoter activity was assessed by dual luciferase assay system. Laryngeal cancer cell proliferation, migration, and apoptosis were evaluated by cell viability, colony formation, cell scratch assay, transwell assay, and flow cytometry methods, respectively. YY1 and MYCT1 were upregulated and downregulated at transcriptional level in laryngeal cancer, respectively, which showed a negative correlation between YY1 and MYCT1 expression in laryngeal cancer. Significantly higher expression of YY1 and lower expression of MYCT1 were found in laryngeal cancer tissues of patients with lymphatic metastasis than those without metastasis.YY1 directly bound to MYCT1 promoter region and inhibited its promoter activity. YY1 silence had similar biological functions as MYCT1 overexpression in repressiveness of proliferation and migration, and promotion of apoptosis in laryngeal cancer cells. However, the effects of YY1 silence were recovered by MYCT1 knockdown. YY1 promotes proliferation and migration with suppression of apoptosis via directly inhibiting MYCT1 in laryngeal cancer cells, suggesting that YY1 is a useful target as a potential oncogene in laryngeal cancer development and progression., (© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

miR-27a regulates cell differentiation in a variety of diseases. However, whether and how miR-27a participates in laryngeal cancer cell differentiation remains unknown. Therefore, we explored role and molecular mechanism of miR-27a in laryngeal cancer differentiation in the study. We found that miR-27a expression was inversely correlated with laryngeal cancer differentiation degree based on the clinical pathological diagnosis of each patient. miR-27 asignificantly rescued differentiation and inhibited β-catenin, LEF1, OCT4 and SOX2 in Wnt/β-catenin pathway in all-trans-retinoic acid (ATRA)-induced laryngeal cancer cells. Bindings of RARα to miR-27a and miR-27a to GSK-3β were confirmed by ChIP and Luciferase reporter assays, respectively. In conclusion, miR-27a is a negative regulator in laryngeal cancer differentiation. RARα-mediated miR-27a transcriptional inactivation releases the inhibition of miR-27a on GSK-3β leading to laryngeal cancer differentiation through GSK-3β-involved Wnt/β-catenin pathway, suggesting that miR-27a is a usefully therapeutic target at least in ATRA-induced laryngeal cancer differentiation.

Purpose: LncRNA HOTAIR plays an important role in many cancer. Several studies have shown that some HOTAIR SNPs might be associated with tumor risk in case-control studies, but the results are inconsistent and inconclusive. Therefore, it is necessary to better evaluate association between the HOTAIR SNPs and the risk of cancer., Results: rs920778, rs7958904 and rs874945 but not rs4759314 and rs1899663 loci were significantly related to cancer risk, among of which rs920778 and rs874945 increased and rs7958904 decreased cancer risk, respectively. Moreover, rs920778 is significantly susceptible in both Asian population and digestive cancer risks., Materials and Methods: Data were collected from PubMed, Embase and Web of Science. A total of 11 case-control studies were selected for the quantitative analysis. Software Stata (Version 12) was used to calculate Odds ratios (ORs) and 95% confidence intervals (CIs) to evaluate the strength of the associations. Subgroup analysis, sensitivity analysis, and publication bias were also performed. Five HOTAIR SNPs were finally enrolled in the study., Conclusions: HOTAIR SNP rs920778, rs7958904 and rs874945 are susceptible to cancer risk. SNP rs920778 is also a useful risk factor in evaluation of Asian population and digestive cancer. In addition, the cancer risk SNP rs874945 is first reported in the meta-analysis.

DNA methylation plays critical roles in regulation of microRNA expression and function. miR-23a-27a-24-2 cluster has various functions and aberrant expression of the cluster is a common event in many cancers. However, whether DNA methylation influences the cluster expression and function is not reported. Here we found a CG-rich region spanning two SP1 sites in the cluster promoter region. The SP1 sites in the cluster were demethylated and methylated in Hep2 cells and HEK293 cells, respectively. Meanwhile, the cluster was significantly upregulated and downregulated in Hep2 cells and HEK293 cells, respectively. The SP1 sites were remethylated and the cluster was significantly downregulated in Hep2 cells into which methyl donor, S-adenosyl-L-methionine, was introduced. Moreover, S-adenosyl-L-methionine significantly increased Hep2 cell viability and repressed Hep2 cell early apoptosis. We also found that construct with two SP1 sites had highest luciferase activity and SP1 specifically bound the gene cluster promoter in vitro. We conclude that demethylated SP1 sites in miR-23a-27a-24-2 cluster upregulate the cluster expression, leading to proliferation promotion and early apoptosis inhibition in laryngeal cancer cells.

In this article, the mechanism of inheritance behind inherited hearing loss and genetic susceptibility in noise-induced hearing loss are reviewed. Conventional treatments for sensorineural hearing loss (SNHL), i.e. hearing aid and cochlear implant, are effective for some cases, but not without limitations. For example, they provide little benefit for patients of profound SNHL or neural hearing loss, especially when the hearing loss is in poor dynamic range and with low frequency resolution. We emphasize the most recent evidence-based treatment in this field, which includes gene therapy and allotransplantation of stem cells. Their promising results have shown that they might be options of treatment for profound SNHL and neural hearing loss. Although some treatments are still at the experimental stage, it is helpful to be aware of the novel therapies and endeavour to explore the feasibility of their clinical application.

MicroRNA-23a (miR-23a) is a potential biomarker for laryngeal cancer. Apoptotic protease activating factor 1 ( APAF-1 ) was recently demonstrated to be a target of miR-23a. However, whether miR-23a exerts its effects via APAF-1 in laryngeal cancer, remains unknown. In the present study, miR-23a expression was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). APAF-1 mRNA and protein expression levels were assayed by RT-qPCR and western blotting, respectively. Binding of miR-23a to APAF-1 was monitored by a luciferase reporter assay. Gain-of-function and loss-of-function studies were performed in order to investigate the roles of miR-23a and APAF-1 in Hep2 cell proliferation and apoptosis. miR-23a and APAF-1 were found to be significantly upregulated and downregulated, respectively, in laryngeal cancer tissues, and there was a significant negative correlation between APAF-1 and miR-23a expression. The results of the luciferase reporter assay demonstrated that miR-23a bound directly to the APAF-1 mRNA 3'-untranslated region. Ectopic expression of miR-23a and knockdown of APAF-1 significantly promoted cell proliferation and colony formation, and inhibited early apoptosis in Hep2 cells. In conclusion, miR-23a acts as an oncogenic regulator in laryngeal carcinoma by directly targeting APAF-1 , and may be a useful biomarker in the diagnosis and treatment of laryngeal carcinoma.

Background: miR-23a, which participates in invasion of pancreatic ductal adenocarcinoma cells into the mesothelial barrier, is a critical regulator in many cancers. It, however, is still unknown whether miR-23a regulates pancreatic cell proliferation and apoptosis or not., Aims: We sought to investigate the role of miR-23a in regulation of pancreatic cell proliferation and apoptosis., Methods: miRNA, mRNA, and protein expressions were determined by qRT-PCR and Western blot, respectively. Dual-luciferase reporter assay was used in detection for binding ability of miR-23a to APAF1. Ectopic miR-23a and APAF 1 were introduced to pancreatic cells, and their roles in proliferation and apoptosis were detected by MTT, colony formation, and apoptosis assays, respectively., Results: Up-regulation of miR-23a and down-regulation of APAF 1 were found in pancreatic ductal cancer, respectively. miR-23a significantly inhibited the luciferase activity by targeting APAF 1 3'UTR. Ectopic miR-23a significantly suppressed the APAF 1 gene expression in pancreatic cancer cells. Similar to siAPAF1, miR-23a significantly promoted pancreatic cancer cell proliferation and repressed apoptosis. Furthermore, miR-23a inhibitor and exogenous APAF 1 could recover the effects., Conclusions: It is suggested that miR-23a, acting as an oncogenic regulator by directly targeting APAF 1 in pancreatic cancer, is a useful potential biomarker in diagnosis and treatment of pancreatic cancer.

Background: MicroRNA-23a (miR-23a) has been demonstrated to play an important role in the development of several types of cancer, but its role in tumorigenesis of laryngeal carcinoma is still unclear. The aim of this study was to investigate the expression patterns and clinical implications of miR-23a in laryngeal cancer., Methods: Quantitative RT-PCR was performed to evaluate the expression level of miR-23a in 52 pairs of laryngeal cancer. Analysis between miR-23a expression and clinical features of laryngeal carcinomas was performed by appropriate statistical methods. Role of miR-23a in laryngeal cancer cell migration and invasion was detected via transwell and matrigel assays, respectively., Results: miR-23a was significantly up-regulated in laryngeal cancer tissues compared to normal adjacent laryngeal tissues (P < 0.01). Tumors with high miR-23a expression had significantly greater extent of lymph node metastasis (P < 0.01), worse clinical stage (P < 0.05) and shorter overall five-year survival (P < 0.01) than those with low miR-23a expression. Both univariate and multivariate Cox hazard regression analysis results showed that clinical stage and miR-23a expression were significantly correlated with patient five-year survival (P < 0.01). miR-23a overexpression also significantly promoted laryngeal cancer cell migration and invasion in vitro., Conclusions: miR-23a, an independent prognostic factor for laryngeal cancer, participates in the onset and progression of laryngeal cancer., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2021488014982305.

Background: miRNA-27a has been confirmed as an important regulator in carcinogenesis and other pathological processes. Whether and how it plays a role in the laryngeal carcinoma is unknown., Methods: Mature miRNA-27a expression in laryngeal cancer was detected by qRT-PCR. Gain-of-function studies using mature miR-27a were performed to investigate cell proliferation and apoptosis in the Hep2 cells. In silico database analysis and luciferase reporter assay were applied to predict and validate the direct target, respectively. Loss-of-function assays were performed to investigate the functional significance of the miR-27a target gene. qRT-PCR and Western blot were used to evaluate mRNA and protein levels of the target, respectively., Results: miR-27a was significantly up-regulated in the laryngeal tumor tissues compared to the adjacent non-tumor tissues. In silico database analysis result revealed that PLK2 is a potential target of miR-27a. luciferase reporter assay result showed the direct inhibition of miR-27a on PLK2-3'UTR. In the cases with miR-27a up-regulation, PLK2 protein expression level was significantly lower in cancer tissues than that in the adjacent non-tumor tissues, which showed a negative correlation with miR-27a expression level. Both miR-27a and knockdown of PLK2 caused the increase of the cell viability and colony formation and inhibition of the late apoptosis in the Hep2 cell lines. Moreover, miR-27a but not PLK2 also repressed the early apoptosis in the Hep2 cells. Additionally, no alteration of the Hep2 cell cycle induced by miR-27a was detected., Conclusions: miR-27a acts as an oncogene in laryngeal squamous cell carcinoma through down-regulation of PLK2 and may provide a novel clue into the potential mechanism of LSCC oncogenesis or serve as a useful biomarker in diagnosis and therapy in laryngeal cancer.

TRAF2 promotes cancer cell survival, proliferation and metastasis through the NF-κB pathway by directly interacting with various TNF recepors. However, the molecular mechanism of TRAF2 dysregulation in breast cancer remains to be elucidated. In the present study, miR-502-5p was predicted as a potential regulator of TRAF2. miR-502-5p was significantly downregulated in breast cancer tissues when compared to the level in paired normal breast tissues. The breast cancer cell lines including MCF-7 and MDA-MB-231 expressed a lower level of miR-502-5p when compared to the level in the non-malignant breast epithelial cell line MCF-10A. In vitro, miR-502-5p enhanced early apoptosis and inhibited proliferation of breast cancer cells. Luciferase reporter assay results showed that miR-502-5p could bind to the 3'-untranslated region of the TRAF2 gene, thus, exerting an inhibitory effect on TRAF2. Furthermore, silencing of TRAF2 exhibited effects similar to those of exogenous miR‑502-5p, while overexpression of TRAF2 partially abrogated miR-502-5p-mediated suppression in breast cancer cells. In conclusion, miR-502-5p may act as a tumor-suppressor gene by targeting oncogenic TRAF2 in breast cancer and, therefore, may be a potential diagnostic and anticancer therapeutic marker for breast cancer.

DNA hypomethylation is correlated with the overexpression of the S100A4 gene in several types of cancers including laryngeal cancer, but the molecular mechanism is unknown. We speculated that the methylation status of the promoter affects its binding to the corresponding transcription factors. In the present study, luciferase reporter assay results indicated that the sequences -485 - +73 and -486 - -530 of the S100A4 promoter may harbor the positive and negative cis-acting elements, respectively; and moreover, the luciferase activity promoted by the sequence -485 - +73 increased and the S100A4 gene was significantly upregulated in 5-Aza-induced HEp2 cells. This implies that the methylation status of the sequence is important in regulating the expression of S100A4. Four transcription factor binding motifs including c-Myb, C/EBpα, Ap2 and Msx-1 in the region were predicted by P-Match software. c-Myb and C/EBpα but not Ap2 and Msx-1 were confirmed by EMSA and ChIP as transcription factors of S100A4. The decreased luciferase activity in methylation-free HEp2 cells transfected by the mutant c-Myb motif related to the methylated cytosine suggests that the hypomethylation of the c-Myb motif upregulates the S100A4 expression in laryngeal cancer.

Background: MYCT1, previously named MTLC, is a novel candidate tumor suppressor gene. MYCT1 was cloned from laryngeal squamous cell cancer (LSCC) and has been found to be down-regulated in LSCC; however, the regulatory details have not been fully elucidated., Methods: Here, we sought to investigate the methylation status of the CpG islands of MYCT1 and mRNA levels by bisulfite-specific PCR (BSP) based on sequencing restriction enzyme digestion, reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR). The function of specific sites in the proximal promoter of MYCT1 in LSCC was measured by transient transfection, luciferase assays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP)., Results: The results suggested hypermethylation of 12 CpG sites of the promoter in both laryngeal cancer tissues and the laryngeal cancer line Hep-2 cell. The hypermethylation of the site CGCG (-695 to -692), which has been identified as the c-Myc binding site, was identified in laryngeal cancer tissues (59/73) compared to paired mucosa (13/73); in addition, statistical analysis revealed that the methylation status of this site significantly correlated with cancer cell differentiation(p < 0.01). The mRNA level of MYCT1 increased in Hep-2 cells treated with 5-aza-C (p < 0.01). The luciferase activity from mutant transfectants pGL3-MYCT1m (-852/+12, mut-695-C > A, mut-693-C > G) was significantly reduced compared with the wild type pGL3-MYCT1 (-852/+12), while the luciferase activity from wild transfectants pGL3-MYCT1 (-852/+12) rose after 5-aza treatment in Hep-2 cells. Finally, EMSA and ChIP confirmed that the methylation of the CGCG (-695 to -692) site prevented c-Myc from binding of the site and demethylation treatment of the 5' flanking region of MYCT1 by 5-aza induced the increased occupation of the core promoter by c-Myc (p < 0.01)., Conclusion: In summary, this study concluded that hypermethylation contributed to the transcriptional down-regulation of MYCT1 and could inhibit cancer cell differentiation in LSCC. DNA methylation of the CGCG site (-695 to -692) of MYCT1 altered the promoter activity by interfering with its binding to c-Myc in LSCC. Epigenetic therapy of reactivating MYCT1 by 5-aza should be further evaluated in clinical trails of LSCC.

Chromodomain helicase DNA-binding protein 5 (CHD5) has been found to be a candidate tumor suppressor gene (TSG) in malignant neural tumors. In mice heterozygous for chd5 deficiency, the first tumor observed was pathological squamous cell carcinoma. More than 95% of primary laryngeal cancer is squamous cell carcinoma. Thus, we explored the expression of CHD5 in 65 patients with laryngeal squamous cell carcinoma (LSCC) using real-time PCR, immunohistochemistry and Western blotting. DNA methylation was detected using bisulfate-specific sequencing. The potential function of CHD5 was determined using MTT, apoptosis and transwell migration assays in CHD5-transfected Hep-2 cells. Our results revealed that the mRNA and protein expression levels of CHD5 in LSCC tissues were significantly lower than those in clear surgical margin tissues (p<0.05), and there is a significant correlation between the mRNA and protein expression levels of CHD5 (p<0.01). In addition, there were significant differences in CHD5 mRNA and protein levels with respect to the patient's clinical stage (p<0.05). Aberrant methylation of the CHD5 promoter was frequently found in the Hep-2 cell line and LSCC tumor tissues, especially tumor tissues from advanced TNM (p<0.05) or older patients (p<0.05). Finally, ectopic expression of CHD5 in laryngeal cancer cells led to significant inhibition of growth and invasiveness. Our data suggest that CHD5 is a tumor suppressor gene that is epigenetically downregulated in LSCC., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: MYCT1, a putative target of c-Myc, is a novel candidate tumor suppressor gene cloned from laryngeal squamous cell carcinoma (LSCC). Its transcriptional regulation and biological effects on LSCC have not been clarified., Methodology/principal Findings: Using RACE assay, we cloned a 1106 bp transcript named Myc target 1 transcript variant 1 (MYCT1-TV) and confirmed its transcriptional start site was located at 140 bp upstream of the ATG start codon of MYCT1-TV. Luciferase, electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed c-Myc could regulate the promoter activity of MYCT1-TV by specifically binding to the E-box elements within -886 to -655 bp region. These results were further verified by site-directed mutagenesis and RNA interference (RNAi) assays. MYCT1-TV and MYCT1 expressed lower in LSCC than those in paired adjacent normal laryngeal tissues, and overexpression of MYCT1-TV and MYCT1 could inhibit cell proliferation and invasion and promote apoptosis in LSCC cells., Conclusions/significance: Our data indicate that MYCT1-TV, a novel MYCT1 transcript, is regulated by c-Myc and down-regulation of MYCT1-TV/MYCT1 could contribute to LSCC development and function.

Fhl1 (Four and a Half LIM domain 1) regulates muscle growth and development. In addition, skeletal myoblast growth is significantly affected by gender differences, implicating estrogen in the regulation of muscle development. We sought to determine if estrogen influences Fhl1 gene expression levels in rat L6GNR4 myoblastocytes that express the estrogen receptor beta (ERbeta), while luciferase assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay were employed to confirm the interaction between ERbeta and Fhl1. Treatment of L6GNR4 cells with physiological levels of 17beta-estradiol (E2) results in markedly decreased endogenous Fhl1 expression. Tamoxifen, an ER antagonist, partially reverses E2-mediated Fhl1 down-regulation in L6GNR4 cells. Furthermore, luciferase assay and EMSA identified a novel promoter region of Fhl1 that directly interacts with ERbeta. ChIP of the ERbeta-Fhl1 promoter complex from L6GNR4 cells confirmed that endogenous ERbeta interacts with this region. These data indicate that E2 down-regulates Fhl1 expression through its binding to the ERbeta. This is the first report of a small molecule that can affect Fhl1 expression. E2 may therefore be useful in developing new strategies for regulating Fhl1 expression and understanding the influence of estrogen on muscle growth and development.

The present study aimed to identify genes related to 5AZA-CdR in laryngeal squamous cell carcinoma (LSCC) and to investigate the role of S100A4 in the development and aggression of LSCC. Differentially expressed proteins were identified in Hep-2 cells treated with 5AZA-CdR by two-dimensional gel electrophoresis combined with MALDI-TOF-MS. mRNA, protein levels and DNA methylation status of S100A4 were assessed by RT-PCR, Western blotting and methylation-specific PCR, respectively. The invasiveness of Hep-2 cells transfected by siRNA S100A4 was determined by transwell migration assay. Protein profiles from Hep-2 cells treated with 5AZA-CdR were obtained, and several differentially expressed proteins such as S100 calcium-binding protein A4 (S100A4) were identified. Results of RT-PCR and Western blotting revealed that both mRNA and protein levels of S100A4 were significantly higher in the metastatic lymph nodes than those in paired adjacent normal laryngeal (PANL) or tumor tissues. The DNA methylation status displayed significant differences between the LSCC and the PANL tissues. The expression level of S100A4 decreased in Hep-2 cells undergoing RNA interference of S100A4. The number of cells which crossed the basement membrane filter was significantly lower in the RNAi S100A4 group when compared with the number in the control group. The abnormal expression of S100A4 identified in Hep-2 cells treated with an inhibitor of DNA methyltransferase appeared to result from the aberrant DNA methylation status of S100A4. The abnormal expression of S100A4 altered the invasiveness of LSCC.

Idiopathic congenital clubfoot (CCF) is a type of congenital foot malformation, and previous studies using the extended transmission disequilibrium testing (ETDT) analysis have confirmed the HOXD13 gene as the susceptible gene of CCF. This study aimed to verify whether Hoxd13 directly regulates skeletal muscle LIM protein 1 (Fhl1) expression during limb development in rat embryo, which will serve as a basis for comprehending etiological research of idiopathic CCF. Immunofluorescence staining was utilized to detect Hoxd13 and Fhl1 expression in 12.5d rat embryo, while luciferase assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay were used to confirm the interaction between the two genes. Both Hoxd13 and Fhl1 were expressed in the interdigital tissues of E12.5 rat embryo. Luciferase assay and EMSA identified a novel promoter region of Fhl1 that directly interacts with Hoxd13. ChIP of the Hoxd13-Fhl1 promoter complex from the developing limb confirmed that endogenous Hoxd13 interacts with this region. Thus, Hoxd13 directly regulates Fhl1 expression in rat embryo. These findings suggest that HOXD13 may regulate the expression of FHL1 in the development of idiopathic CCF., (Copyright 2008 S. Karger AG, Basel.)

To investigate the relationship of HOXD13 and FHL1 in idiopathic congenital talipes equinovarus(ICTEV), 84 samples from patients with ICTEV were used in the study. Mutation in the coding region of HOXD13 was detected by denaturing gradinent electrophoresis. The mRNA and protein levels of HOXD13 and FHL1 were evaluated by RT-PCR and immunohistochemistry, respectively. The binding site of FHL1 to HOXD13 predicted by PMATCH software was validated by EMSA( Electrophoretic mobility shift assay,EMSA).No mutation was found in the coding region of HOXD13 in 84 samples from patients with ICTEV. Both HOXD13(33.3%) and FHL1(46.6%) were down-regulated in ICTEV muscle tissue. The result of EMSA showed that the special binding band appeared when HOXD13 existed. The results shows that HOXD13 gene mutation was not involved in outbreak in idiopathic congenital talipes equinovarus, but changes of HOXD13 and FHL1 gene expression related to the development of talipes equinovarus malformation. HOXD13 might play an role in ICTEV through regulating FHL1 expression.

Background: The role of epigenetics in gene expression regulation and development significantly enhances our understanding of carcinogenesis. All the tumor related genes may be the target of epigenetical or genetic regulation. We selected some epigenetically regulated genes for cDNA array analysis and observed variability in the expression of the DICER1 gene in distinct stages of gastric cancer. The aim of this study was to assess the correlation between the expression of DICER1, an epigenetically regulated gene, and gastric cancer., Methods: To detect the expression of 506 tumor-associated genes, including DICER1, in the matched cancerous mucosa, pre-malignant lesion (adjacent mucosa), non-cancerous gastric mucosa and distant lymphocyte metastatic lesion in 3 cases of gastric cancers using cDNA array. DICER1 mRNA expression and DICER1 protein expression were further analyzed by Real-time PCR and Western blot in 32 cases of progressive gastric cancer. DICER1 protein expression was also detected in 33 early and 30 progressive gastric cancers by the immunohistochemistry (IHC) method., Results: In 3 cases of gastric cancer cDNA array showed dramatically decreased expression of DICER1 in pre-malignant lesion, cancerous mucosa and distant lymphocyte metastatic lesions compared with matched noncancerous gastric mucosa, pre-malignant lesion and cancerous mucosa. Real-time PCR results showed that the expression level of DICER1 mRNA in gastric cancer was significantly down-regulated compared to normal gastric tissue (P < 0.05). The IHC assay also showed that the expression of DICER1 was significantly decreased in progressive gastric cancer. Among the 63 cases of gastric cancers, 13/33 early (39.4%) and 19/30 (63.3%) progressive cancers showed negative expression of DICER1 (50.8%). The difference in expression of DICER1 between early and progressive gastric cancers was significant (P < 0.01). The result of Western blotting showed that DICER1 protein was down-regulated significantly in advanced gastric cancer (P < 0.05)., Conclusions: DICER1 expression is decreased during the progression of gastric cancer, especially in progressive gastric cancers, which indicating DICER1 may play an important role in the development of cancer and the epigenetical regulation involved.

Objective: To explore mechanism of S100A8 in the oncogenesis and development of laryngeal cancer., Methods: Proteins interacting with S100A8 were isolated from laryngeal cancer cell lines Hep-2 by immunoprecipitation assay with anti-S100A8 antibody. The target bands were cut out and identified by maxtrix assisted laser desorption/ionization time of flight (MALDI-TOF). The peptide mass fingerprinting data of the proteins identified were analyzed based on the Mascot database. The NF-kappa B binding sites of the proteins were predicted by P-Match software. The binding ability of one of the proteins to S100A8 was confirmed by co-immunoprecipitation and immunocytochemistry methods., Results: Four proteins interacting with S100A8 were obtained, which were hypothetical protein LOC80154, MHC class I HLA-B, similar to T-box 1 isoform C and sarcolemmal associated protein 1. The four genes were predicted to have NF-kappa B binding sites. MHC class I HLA-B, which is one of targets in NF-kappa B pathway, was first confirmed to have the binding ability to S100A8., Conclusion: The novel partners of S100A8 identified in the study might be involved in NF-kappa B pathway. The binding ability of MHC class I HLA-B to S100A8 implies that S100A8 might function as a new member with other proteins including HLA-B in NF-kappa B pathway. These findings provide a new clue to further study on the molecular mechanism of S100A8 in the genesis of laryngeal carcinomas.

To investigate the influence of B-RAF-specific RNA interference on the proliferation and apoptosis of gastric cancer BGC823 cell line. B-RAF-siRNA and scramble-siRNA were synthesized and transfected into BGC823 cells by TransMessenger. The expression of B-RAF gene and Bcl-2 gene in BGC823 cells was detected by RT-PCR and the level of apoptosis was evaluated in transfected cells by flow cytometry. Results showed that TransMessenger could effectively transfect B-RAF-siRNA and scramble-siRNA into BGC823 cells. B-RAF-siRNA significantly inhibited the expression of B-RAF gene and Bcl-2 gene in BGC823 cells by more than 90% to 100%. B-RAF-siRNA inhibited BGC823 cell prolifera-tion and induced apoptosis (P < 0.01). In conclusion, B-RAF-siRNA can effectively inhibit the expression of B-RAF gene and Bcl-2 gene, induce cell apoptosis and inhibit the proliferation of gastric cancer BGC823 cells.

Objective: To search for characteristic chromosome changes in primary laryngeal squamous cell carcinoma (LSCC) and Hep-2 cell line and to realize the relationship between the cytogenetic abnormality and the pathogenetic mechanism in LSCC., Methods: The fresh resulted samples of LSCC were analyzed with an improved primary cell culture for chromosome preparation and G-banding technique. Hep-2 cell line was analyzed by high resolution banding technique. Molecular cytogenetics analysis was made by chromosome 6 painting probe., Results: Four primary LSCC succeeded in primary cell culture and obtained metaphases, one was tetraploid, the other three were triploid. The chromosome mode of Hep-2 cell line was from 68 to 75 and fifteen marker chromosomes were found. The most structural abnormalities of chromosome in primary LSCC and HEP-2 cell line were unbalance translocation, terminal deletion and isochromosome. The complicate aberration in chromosome 6 was common in LSCC and Hep-2., Conclusion: 6q-, I(5p), 17p-, 5q- are considered as characteristic chomosome changs in LSCC. Fluorescence in situ hybridization (FISH) may enhance the ability of detecting complicated chromosome rearrangements and marker chromosomes, which could provide more value data to verify the chromosome characteristic aberration in LSCC.