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Chromatin regulators constitute a fundamental means of transcription regulation, which have been implicated in neurodevelopment and neurodevelopment disorders (NDDs). Supt16, one of candidate genes for NDDs, encodes the large subunit of facilitates chromatin transcription. However, the underlying mechanisms remain poorly understood. Here, Supt16+/− mice was generated, modeling the neurodevelopment disorder. Abnormal cognitive and social behavior was observed in the Supt16 +/− mice. Simultaneously, the number of neurocytes in the cerebral cortex and hippocampus is decreased, which might be resulted from the impairment of mouse neural stem cells (mNSCs) in the SVZ. Supt16 haploinsufficiency affects the proliferation and apoptosis of mNSCs. As the RNA-seq and chromatic immunoprecipitation sequencing assays showed, Supt16 haploinsufficiency disrupts the stemness of mNSCs by inhibiting MAPK signal pathway. Thus, this study demonstrates a critical role of Supt16 gene in the proliferation and apoptosis of mNSCs and provides a novel insight in the pathogenesis of NDDs.

The maintenance of neural stem cells (NSCs) plays a critical role in neurodevelopment and has been implicated in neurodevelopmental disorders (NDDs). However, the underlying mechanisms linking defective human neural stem cell self-renewal to NDDs remain undetermined. Our previous study found that Supt16 haploinsufficiency causes cognitive and social behavior deficits by disrupting the stemness maintenance of NSCs in mice. However, its effects and underlying mechanisms have not been elucidated in human neural stem cells (hNSCs). Here, we generated Supt16+/− induced pluripotent stem cells (iPSCs) and induced them into hNSCs. The results revealed that Supt16 heterozygous hNSCs exhibit impaired proliferation, cell cycle arrest, and increased apoptosis. As the RNA-seq analysis showed, Supt16 haploinsufficiency inhibited the PI3K/AKT/mTOR pathway, leading to rising autophagy, and further resulted in the dysregulated expression of multiple proteins related to cell proliferation and apoptotic process. Furthermore, the suppression of Supt16 heterozygous hNSC self-renewal caused by autophagy activation could be rescued by MHY1485 treatment or reproduced in rapamycin-treated hNSCs. Thus, our results showed that Supt16 was essential for hNSC self-renewal and its haploinsufficiency led to cell cycle arrest, impaired cell proliferation, and increased apoptosis of hNSCs by regulating the PI3K/AKT/mTOR/autophagy pathway. These provided a new insight to understand the causality between the Supt16 heterozygous NSCs and NDDs in humans.

Lactate provides signals that modulate neuronal functions, including excitability, plasticity and memory consolidation. This process is mediated by its specific receptor, G-protein-coupled receptor 81 (GPR81), which activates phosphatidylinositol 3 kinase /protein kinase B/cyclic adenosine monophosphate response element binding protein (PI3K/Akt/CREB) signaling pathway. However, it is recognized that cerebral lactate content is reduced at the early stage of Alzheimer’s disease (AD). Besides, continiously production of amyloid beta (Aβ) induces synaptic dysfunction. Here, this study focuses on the decrease of cerebral lactate content, and its relation with Aβ production at early stage of AD. Firstly, it identifies a decrease of lactate level, GPR81 and phosphorylation of PI3K/Akt/CREB, a reduction of deoxyribonucleic acid methyltransferase 1 (DNMT1), an increase of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and Aβ in the cortex and hippocampus of early AD model mice. Then, through correlation analysis, it is found that reduction of cerebral lactate level is positively correlative with the decreased phosphorylation of PI3K/Akt/CREB. Especially, phosphorylation of CREB is positively correlative with DNMT1 expression, and negatively correlated with BACE1 and Aβ level. These results signify that reduction of cerebral lactate content contributes to suppression of PI3K/Akt/CREB signaling pathway, leading to DNMT1 downregulation. As a result, BACE1 and Aβ are increased in the cortex and hippocampus of early AD model mice. Hence, this study proposes a novel insight that lactate reduction contributes to Aβ production at early period of AD. The underlying mechanism is that reduced lactate can hardly activate GPR81-PI3K/Akt/CREB, which results in DNMT1 reduction, and then increasing BACE1 level and Aβ production (Graphic Abstract).

Objective: Intellectual disability (ID) is one of the most common developmental disabilities. To identify the genetic etiology of IDs in Chongqing, we conducted a multistage study in Chinese Han patients.Methods: We collected the clinical and etiological data of 1665 ID patients, including 1,604 from the disabled children evaluation center and 61 from the pediatric rehabilitation unit. Routine genetic screening results were obtained, including karyotype and candidate gene analysis. Then 105 idiopathic cases with syndromic and severe ID/developmental delay (DD) were selected and tested by chromosomal microarray (CMA) and whole exome sequencing (WES) sequentially. The pathogenicity of the CNVs and SNVs were evaluated according to ACMG guidelines.Results: Molecular diagnosis was made by routine genetic screening in 216 patients, including 196 chromosomal syndromes. Among the 105 idiopathic patients, 49 patients with pathogenic/likely pathogenic CNVs and 21 patients with VUS were identified by CMA. Twenty-six pathogenic CNVs underlying well-known syndromic cases, such as Williams-Beuren syndrome, were confirmed by multiplex ligation-dependent probe amplification (MLPA). Nine novel mutations were identified by WES in thirty-fix CNV-negative ID cases.Conclusions: The study illustrated the genetic aberrations distribution of a large ID cohort in Chongqing. Compared with conventional or single methods, a tiered high-throughput diagnostic strategy was developed to greatly improve the diagnostic yields and extend the variation spectrum for idiopathic syndromic ID cases.

Scope Choline is an essential nutrient and a primary dietary source of methyl groups that are vital for brain development. Low choline (LC) in the maternal diet during pregnancy alters neurogenesis in the fetal brain and leads to low cognitive performance. However, the key signaling pathways that are sensitive to maternal choline supply during neural progenitor cell (NPC) development and the epigenetic mechanisms by which choline availability regulates gene expression are unclear. Methods and results Timed-pregnant Nestin-CFPnuc transgenic mice are fed either a control diet or LC diet during E11-17. Gene expression changes in sorted E17 NPCs are identified by RNA sequencing. A maternal LC diet significantly increases Tlr4 transcription, causing premature neuronal differentiation and enhanced ethanol-induced NLRP3 inflammasome activation. No changes in DNA methylation at the Tlr4 gene promoter region are detected; however, a 70% decrease in H3K27me3 is observed in the LC-treated NPCs. Inhibition of EZH2 decreases H3K27me3 levels and increases Tlr4 expression. Conversely, the application of catalytically inactive Cas9 with EZH2 to increase H3K27me3 at the Tlr4 promoter causes reduced Tlr4 expression. Conclusion These data reveal an epigenetic mechanism for the effect of maternal choline availability on brain development, suggesting a likely intervention for neurodevelopmental diseases.

Background SOX2 overlapping transcript (SOX2OT) has been reported to be an important lncRNA in various cancers. SOX2 is embedded in an intron of the SOX2OT gene. But the role of SOX2OT in esophageal squamous cell carcinoma (ESCC) and the association between SOX2OT and SOX2 remain unclear. Methods Quantitative PCR (qPCR) was used to detect the expression of SOX2OT and SOX2 in ESCC tissues and cells. The isoforms of SOX2OT were identified by PCR and confirmed by sequencing. CCK-8 and Edu assays were performed to investigate the effects of SOX2OT on cell growth. The relationship between SOX2OT and SOX2 was explored by luciferase reporter assay. Results Both SOX2OT and SOX2 were upregulated in ESCC tissues and cells. SOX2OT expression was positively associated with SOX2 expression in ESCC tissues. NR_004053 was one of the major SOX2OT transcripts aberrantly expressed in ESCC tissues and cells. Overexpression of SOX2OT (NR_004053) promoted ESCC cell growth, antagonized the effect of DDP and increased cell proliferation ratio. Ectopic expression of SOX2 could increase the luciferase activity of SOX2OT-pGL3/Basic and SOX2OT expression, while overexpression of SOX2OT (NR_004053) had no effect on SOX2 expression. Conclusion Our study demonstrates that the major isoform of SOX2OT in ESCC, SOX2OT (NR_004053) contributes to cell growth. SOX2 promotes SOX2OT expression at transcriptional level. Electronic supplementary material The online version of this article (10.1186/s12935-018-0570-7) contains supplementary material, which is available to authorized users.

Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal cancer. Long noncoding RNAs (lncRNAs) are thought to play a critical role in cancer development. Recently, lncRNA CASC9 was shown to be dysregulated in many cancer types, but the mechanisms whereby this occurs remain largely unknown. In this study, we found that CASC9 was significantly upregulated in ESCC tissues, with further analysis revealing that elevated CASC9 expression was associated with ESCC prognosis and metastasis. Furthermore, we found that CASC9 knockdown significantly repressed ESCC migration and invasion in vitro and metastasis in nude mice in vivo. A microarray analysis and mechanical experiments indicated that CASC9 preferentially affected gene expression linked to ECM-integrin interactions, including LAMC2, an upstream inducer of the integrin pathway. We demonstrated that LAMC2 was consistently upregulated in ESCC and promoted ESCC metastasis. LAMC2 overexpression partially compromised the decrease of cell migration and invasion capacity in CASC9 knockdowns. In addition, we found that both CASC9 and LAMC2 depletion reduced the phosphorylation of FAK, PI3K, and Akt, which are downstream effectors of the integrin pathway. Moreover, the reduction in phosphorylation caused by CASC9 depletion was rescued by LAMC2 overexpression, further confirming that CASC9 exerts a pro-metastatic role through LAMC2. Mechanistically, RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assay indicated that CASC9 could bind with the transcriptional coactivator CREB-binding protein (CBP) in the nucleus. Chromatin immunoprecipitation (ChIP) assay additionally illustrated that CASC9 increased the enrichment of CBP and H3K27 acetylation in the LAMC2 promoter, thereby upregulating LAMC2 expression. In conclusion, we demonstrate that CASC9 upregulates LAMC2 expression by binding with CBP and modifying histone acetylation. Our research reveals the prognostic and pro-metastatic roles for CASC9 in ESCC, suggesting that CASC9 could serve as a biomarker for prognosis and a target for metastasis treatment.

An increasing number of long noncoding RNAs (lncRNAs) have been discovered, and dysregulation of lncRNAs plays critical roles in tumorigenesis and tumor progression. In this study, we identified a novel lncRNA LINC01980, located in both the cytoplasm and nucleus, which was significantly upregulated in esophageal squamous cell carcinoma (ESCC) tissues through microarray profiling. Further analysis revealed that LINC01980 overexpression was positively correlated with deeper invasion of cancer, positive lymph node metastasis, and advanced TNM stage. Additionally, high LINC01980 expression in ESCC tissues was associated with poor prognosis. In vitro and in vivo experiments demonstrated that LINC01980 promoted ESCC growth. EdU incorporation assay implied that LINC01980 accelerated ESCC proliferation. Flow cytometry analysis showed that knockdown of LINC01980 induced cell cycle arrest and increased apoptosis. Microarray analysis indicated that LINC01980 upregulated the expression of growth arrest and DNA damage inducible 45 alpha (GADD45A). Further experiments demonstrated that GADD45A promoted ESCC cell growth, indicating that GADD45A may be a downstream target of LINC01980. In conclusion, this study identified LINC01980 as a novel potential oncogene in ESCC, which can be a promising biomarker for prognosis and therapeutic targeting in ESCC.

Trichostatin A (TSA) has been demonstrated to exhibit various anticancer effects that influence cell cycle arrest, cell proliferation and apoptosis of cancer cells. A potential association between TSA and endoplasmic reticulum (ER) function has been suggested but its anticancer mechanism involving the induction of ER stress is unknown. p53 has previously been demonstrated to regulate ER function in response to stress but its role involving TSA and ER stress in cancer cells is poorly understood. The current study identified that TSA induced ER stress in wild type (WT) HCT116 human colon cancer cells. Following TSA treatment, the ER stress markers GRP78 and GRP94 significantly increased without hyperacetylation of their promoter regions. The inositol-requiring enzyme 1 α (IRE1α)/X-box binding protein 1 (XBP1) pathway was implicated due to an association of phosphorylated IRE1α and spliced XBP1 with ER stress. However, luciferase reporter assay indicated that splicing events were attenuated in HCT116 TP53(−/-) cells. Furthermore, cell viability and apoptosis were revealed to depend on p53 during TSA treatment. Cell viability increased and the apoptosis rate decreased in HCT116 TP53(−/-) cells compared with WT HCT116 cells undergoing TSA treatment. In conclusion, the current study revealed that TSA may induce ER stress via a p53-dependent mechanism in colon cancer cells. This provides information that may assist the development of treatments that exploit the anticancer function of TSA.

Additional file 2: Figure S1. The association between mRNA expression and clinical characteristics of ESCC. A and B qPCR analysis of SOX2OT and SOX2 expression in 46 ESCC tissues. Neither SOX2OT expression nor SOX2 expression was observed to be associated with ESCC tumor size, lymphatic metastasis and TNM stage. C and D Online analysis of SOX2OT and SOX2 expression in 184 ESCC tissues using TCGA data. ( http://www.linkedomics.org/login.php ) Neither SOX2OT expression nor SOX2 expression was correlated with pathologic stage, N stage, M stage and prognosis. Figure S2. SOX2OT isoforms confirmed by sequencing. A SOX2OT isoforms in ESCC tissue and cells. The first column was No.342 ESCC tissue. From the above to the bottom, there were NR_075092, NR_075093, NR_004053, NR_075089 and NR_075090. NR_075093 and NR_075090 were also found in No.342 ESCC adjacent normal tissue (the second column). NR_075092, NR_075093 and NR_004053 were detected both in KYSE150 and KYSE450 cells, while NR_075089 was only detected in KYSE450. SOX2OT_new was uniquely found in ESCC cells rather than tissues. Its partial structure was demonstrated in B. Figure S3. SOX2 expression after overexpressing SOX2OT. A qPCR was used to detect SOX2 mRNA and SOX2OT expression. Overexpression of SOX2OT had no effect on SOX2 mRNA expression in KYSE150 and KYSE450 cells. ***P

MiR-196a could play important roles in carcinogenesis by targeting many protein coding genes. However, little is known about whether miR-196a can target any long non-coding RNAs (lncRNAs). In the present study, we screen lncRNAs which are regulated by miRNA-196a in human esophageal squamous cell carcinoma (ESCC). We found that miR-196a could suppress the expression of lncRNA growth arrest-specific 5(GAS5). GAS5 is frequently down-regulated in 86 paired human ESCC tissues. Importantly, there was lower GAS5 expression in the late stage of ESCC patients. The reduced expression of GAS5 in ESCC may not be related to DNA methylation but related to the high expression of miR-196a. In vitro and in vivo studies indicated that GAS5 could inhibit the growth of ESCC cells. Using Chromatin Isolation by RNA Purification-qPCR, we found that miR-196a could bind to GAS5. The Luciferase Reporter Assay indicated that miR-196a could bind to the seventh exon of GAS5. Additionally, both GAS5 and miR-196a could bind to Ago2 which is a key component of the RNA-induced silencing complex (RISC). Together, these results suggest that GAS5 functions as a tumor suppressor gene in ESCC and is regulated by miR-196a involved in RISC.

Esophageal cancer is one of the leading causes of cancer-related mortality because of poor prognosis. Long noncoding RNAs (lncRNAs) have been gradually demonstrated to play critical roles in cancer development. We identified a novel long noncoding RNA named linc00460 by microarray analysis using esophageal squamous cell carcinoma (ESCC) clinical samples, which has not been studied before. Our research indicated that linc00460 was overexpressed in the majority of tumor tissues and ESCC cell lines. Linc00460 expression was positively correlated with ESCC TNM stage, lymph node metastasis, and predicted poor prognosis. In vitro experiments showed that linc00460 depletion suppressed ESCC cell growth through regulating cell proliferation and cell cycle; in additional, linc00460 depletion accelerated ESCC cell apoptosis. We further revealed that linc00460 overexpression was manipulated by transcriptional co-activator CBP/P300 through histone acetylation. Given the high expression and important biological functions of linc00460, we suggest that linc00460 works as an oncogene and might be a valuable prognostic biomarker for ESCC diagnosis and treatment.

Background Abnormal expression of numerous long non-coding RNAs (lncRNAs) has been reported in esophageal squamous cell carcinoma (ESCC) recently, but the great majority of their roles and mechanisms remain largely unclear. We aim to identify the critical ESCC-associated lncRNAs and elucidate the functions and mechanisms in detail. Methods Microarrays were used to analyze the differentially expressed lncRNAs in ESCC tissues. qRT-PCR was used to verify the result of microarrays. The effects of the most up-regulated lncRNA, cancer susceptibility candidate 9(CASC9), on cell growth, proliferation and cell cycle were investigated by in vivo and in vitro assays. Microarrays and recovery tests were used to discover the regulatory targets of CASC9. RNA FISH and subcellular fractionation assays were used to detect the subcellular location of CASC9. Finally, the mechanism of CASC9 regulating PDCD4 was explored by RIP, RNA-protein pull down and ChIP assays. Results ESCC tissue microarrays showed that CASC9 was the most up-regulated lncRNA. qRT-PCR analysis indicated that CASC9 expression was positively associated with tumor size and TNM stage, and predicted poor overall survival of ESCC patients. Knockdown of CASC9 inhibited ESCC cell growth in vitro and tumorigenesis in nude mice. Furthermore interfering CASC9 decreased cell proliferation and blocked cell cycle G1/S transition. CASC9-associated microarrays indicated that PDCD4 might be the target of CASC9. Consistent with this, PDCD4 expression was negatively associated with CASC9 expression in ESCC tissues and predicted good prognosis. Manipulating CASC9 expression in ESCC cells altered both PDCD4 mRNA and protein levels and cell cycle arrest caused by CASC9 knockdown could be rescued by suppressing PDCD4 expression. CASC9 located both in the nucleus and cytoplasm. Mechanistically, enhancer of zeste homolog2 (EZH2) could bind to both CASC9 and PDCD4 promoter region. Interfering CASC9 reduced the enrichment of EZH2 and H3K27me3 in the PDCD4 promoter region. Conclusions Our study firstly demonstrates that lncRNA CASC9 functions as an oncogene by negatively regulating PDCD4 expression through recruiting EZH2 and subsequently altering H3K27me3 level. Our study implicates lncRNA CASC9 as a valuable biomarker for ESCC diagnosis and prognosis. Electronic supplementary material The online version of this article (10.1186/s12943-017-0715-7) contains supplementary material, which is available to authorized users.

Dysregulation of c-Myc is one of the most common abnormalities in human malignancies, including esophageal cancer, one of the world's most lethal cancers. MicroRNA-26 family, including miR-26a and miR-26b, is transcriptionally suppressed by c-MYC. Our previous microarray data indicated a decreased-expression of miR-26 family in esophageal squamous cell carcinoma (ESCC). However, its roles in c-MYC pathway regulation and esophageal cancer tumorigenesis have yet not been elucidated. In this study, we expanded the detection of miR-26 expression in ESCC patients and found that the great majority of ESCC tissues showed an > 50% reduction, even in the early-staged tumor. Furthermore, ectopic expression of miR-26a or miR-26b induced ESCC cell growth inhibition and G1 phase arrest. MYC binding protein (MYCBP) was identified as a direct target of miR-26. MiR-26 could dramatically decrease MYCBP mRNA and protein levels, as well as the expression of luciferase carrying MYCBP 3′-untranslated region. Moreover, knock-down of MYCBP mimicked the effect of miR-26. More importantly, miR-26 overexpression could downregulate a series of c-MYC target genes as MYCBP silence did. Taken together, these results indicate that miR-26 family can suppress esophageal cancer cell proliferation by inhibition of MYCBP, subsequently downregulate c-MYC pathway. Besides, we also found that reduction of miR-26 expression in ESCC was not due to DNA methylation. Hence, our study reveals a novel feedback loop for c-MYC pathway and implicates miR-26 as a potential target for prevention and treatment of esophageal cancer.

Correlation between CASC9 expression and clinicopathological characteristics of ESCC patients. Table S2. General clinical characteristics of ESCC patients used for microarrays. Table S3. All primers and siRNA sequences used in this study (DOCX 17 kb)

The STR identification of KYSE150 and KYSE450. Fig. S2. HE staining of ESCC and adjacent tissues. Fig. S3. Cell apoptosis was determined by flow cytometry analysis. Fig. S4. Correlation analysis of CASC9 intensities and candidate genes intensities provided by the ESCC tissue profiles. Fig. S5. qRT-PCR was used to detect the mRNA expression of target genes after interfering and overexpressing CASC9. Fig. S6. Correlation between transcripts identified in Fig.1 and CASC9 or PDCD4. Fig. S7. ChIP-seq data from Encode indicate that the promoter region of PDCD4 is enriched in the H3K27me3 and EZH2 binding sites. Fig. S8. ChIP assay showed that EHZ2 could bind to the region of PDCD promoter. Fig. S9. PDCD4 mRNA expression after interfering EZH2 (DOCX 14348 kb)

Scope Alzheimer's disease (AD) is a detrimental neurodegenerative disease and has no known effective treatment. The essential nutrient choline potentially plays an important role in cognition. Perinatal choline supplementation (CS) is critical for memory performance. Findings have shown that postnatal choline-containing compounds enhance memory functions in populations with memory impairments. However, whether CS can be targeted to decelerate the progression of AD remains unknown. Methods and results APP/PS1 mice and their wild-type littermates are fed either a control or CS diet from 2 to 11 months of age. As compared to WT mice, APP/PS1 mice on the control diet are characterized by the reduction in the number of cholinergic neurons in the basal forebrain, reduced cholinergic fiber staining intensity in the amygdala, and reduced hippocampal and cerebral cortical levels of choline and acetylcholine. CS partially prevents these changes and ameliorates cognitive deficits and anxiety. Furthermore, amyloid-β deposition and microgliosis are decreased in the APP/PS1 mice fed a CS diet. These effects may have been due to inhibition of NLRP3 inflammasome activation and restoration of synapse membrane formation. Conclusion These findings reveal a beneficial effect of CS on AD progression during adulthood and provide a likely therapeutic intervention for AD patients.

Pachyonychia congenita (PC), a rare autosomal dominant disorder characterized by hypertrophic nail dystrophy, is classified into two main clinical subtypes: PC-1 and PC-2. PC-1 is associated with mutations in the KRT6A or KRT16 genes, whereas PC-2 is linked to KRT6B or KRT17 mutations. Blood samples were collected from three generations of a new Chinese PC-1 family, including three PC patients and five unaffected family members. A novel missense mutation p.Leu128Pro (c.383T>C) was identified in a highly conserved helix motif in domain 1A of K16. The disease haplotype carried the mutation and cosegregated with the affection status. PolyPhen2 and SIFTS analysis rated the substitution as probably damaging; Swiss-Model analysis indicated that the structure of the mutant protein contained an unnormal α-helix. Overexpression of mutant protein in cultured cells led to abnormal cell morphology. Conclusion: The wider spectrum of KRT16 mutations suggests that changes in codons 125, 127, and 132 are most commonly responsible for PC-1 and that proline substitution mutations at codons 127 or 128 may produce more severe disease. This study extends the KRT16 mutation spectrum and adds new information on the clinical and genetic diversity of PC.

Recent studies indicated that the aberrant gene expression of peroxiredoxin-6 (prdx6) was found in various kinds of cancers. Because of its biochemical function and gene expression pattern in cancer cells, the association between genetic polymorphism of Prdx6 and cancer onset is interesting. In this report, we have developed and implemented a serial experiment in molecular biology laboratory course to teach single nucleotide polymorphism (SNP) to undergraduate students majoring in molecular biology or genetics. The flanking sequence of rs4382766 was located in Prdx6 gene, which contained a restriction site of SspI, and was used as a target in this lab course. The students could mimic real research by integrating different techniques, such as database retrieving, genomic DNA isolation, PCR, and restriction enzyme assay. This serial experiment of PCR-RFLP helps students set up intact idea of molecular biology and understand the relation among individual experiments. Students were found to be more enthusiastic during the laboratory classes than those in the former curriculum. © 2013 by The International Union of Biochemistry and Molecular Biology, 41(4):262–266, 2013

Background: Rab37 encodes a Rab GTPase which regulates the vesicular transport of exocytosis. But the different findings in two types of cancers made its roles in oncology more confused. In this study, a clinical research on genetic polymorphisms was performed to evaluate the association between Rab37 and esophageal squamous cell carcinoma (ESCC).Methods: The mRNA expression was tested by reverse transcription-polymerase chain reaction (RT-PCR) in four ESCC cell lines. A case-control study including 212 ESCC patients and 213 cancer-free controls was genotyped by PCR-restriction fragment length polymorphism. The Hardy-Weinberg equilibrium test, association analysis and haplotype analysis were performed with SPSS and SHEsis software respectively.Results: Rab37 mRNA could be specifically detected in two ESCC cell lines, EC109 and EC9706, but not in KYS150 and KYS450. The allele, genotype and haplotype frequencies of rs9904078G>A, rs2034310T>C and rs5018106T>C, located in Rab37, did not significantly differ between the patients and the controls. No association between the polymorphisms and the TNM stages of patients was found.Conclusions: Rab37 mRNA was specifically expressed in some ESCC cell lines but its genetic polymorphisms were not associated with ESCC.

Background: Rab37 encodes a Rab GTPase which regulates the vesicular transport of exocytosis. But the different findings in two types of cancers made its roles in oncology more confused. In this study, a clinical research on genetic polymorphisms was performed to evaluate the association between Rab37 and esophageal squamous cell carcinoma (ESCC). Methods: The mRNA expression was tested by reverse transcription-polymerase chain reaction (RT-PCR) in four ESCC cell lines. A case-control study including 212 ESCC patients and 213 cancer-free controls was genotyped by PCR-restriction fragment length polymorphism. The Hardy-Weinberg equilibrium test, association analysis and haplotype analysis were performed with SPSS and SHEsis software respectively. Results: Rab37 mRNA could be specifically detected in two ESCC cell lines, EC109 and EC9706, but not in KYS150 and KYS450. The allele, genotype and haplotype frequencies of rs9904078G>A, rs2034310T>C and rs5018106T>C, located in Rab37, did not significantly differ between the patients and the controls. No association between the polymorphisms and the TNM stages of patients was found. Conclusions: Rab37 mRNA was specifically expressed in some ESCC cell lines but its genetic polymorphisms were not associated with ESCC.

MicroRNAs (miRNAs) are small, non-protein-coding RNAs that function as tumour suppressors or oncogenes. A single nucleotide change in the sequence of pre-miRNA can affect miRNA expression, so single-nucleotide polymorphisms (SNPs) in pre-miRNA may be biomarkers for biomedical applications. In this study, we performed a genetic association study between the SNP (rs11614913) in pre-miRNA-196a and esophageal squamous cell carcinoma (ESCC) susceptibility in a case-control study. We found that the homozygote CC of this SNP increased the risk of ESCC compared with the homozygote TT and the risk was more evident among smokers than non-smokers. Therefore, this functional SNP may be a biomarker for ESCC outcome.

MicroRNAs are a new class of non-proteincoding, small RNAs that function as tumor suppressors or oncogenes. They participate in diverse biological pathways and function as gene regulators. A G>C polymorphism (rs2910164), which is located in the sequence of miR-146a precursor, results in a change from G:U to C:U in its stem region. However, it remains largely unknown whether this single nucleotide polymorphism (SNP) may alter esophageal squamous cell carcinoma (ESCC) susceptibility. In the current study, we evaluated association between rs2910164 and ESCC susceptibility in a case-control study of 444 sporadic ESCC patients and 468 matched cancer-free controls in a Chinese Han population. Compared with rs2910164 variant genotype CC, genotype GG was associated with increased risk of ESCC (Odds Ratio, 2.39, 95% Confidence Interval, 1.36-4.20). In the smokers, the risk of rs2910164 GG genotype was more notable (Odds Ratio, 3.17, 95% Confidence Interval, 1.71-4.46). In the stratification analyses, we also found there was a strong correlation between rs2910164 C/G variant and the clinical TNM stage (P < 0.01). These findings suggest that this functional SNP in pre-miR-146a could contribute to ESCC susceptibility and clinical outcome.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers in the world. Epidemiological survey studies have verified that the development of ESCC relates to a complex interactive process between multiple genetic susceptibilities and environmental exposure. Serpins are a broadly distributed family of protease inhibitors and have been recognized as tumor suppressors in multiple cancer types. While previous studies have reported that Serpin polymorphisms are associated with tumorigenesis, the genetic and functional single nucleotide polymorphisms (SNP) in these genes appear to be complex and remain to be elucidated. In this study, a total of 500 ESCC cases and 500 matched controls in a Southwest China population were evaluated for six SNPs in the exons of three Serpin genes (SerpinB5, SerpinB2, and SerpinE1). Among the six SNPs, the C allele of rs2289519 and rs2289520 in SerpinB5 showed decreased risk of ESCC and the variants might interact with smoking status. Haplotype analysis showed that the T-G haplotype (corresponding to rs2289519-rs2289520) increased the risk of ESCC, while the C-C haplotype decreased the risk. We also found that SerpinB5 gene mRNA expression was significantly downregulated in ESCC cell lines and patient specimen while there is no change in protein structure with different haplotypes. Our results demonstrated that the expression of SerpinB5 was downregulated in ESCC, and the positive SNPs might be associated with a risk of ESCC development.

MicroRNAs comprise a family of small non-coding RNA molecules that have emerged as key post-transcriptional regulators of gene expression. Aberrant miRNA expression has been linked to various human tumors. This study was aimed to identify novel miRNAs involved in the carcinogenesis of esophageal squamous cell carcinoma (ESCC) and their potential functions. We performed miRNA microarray and found that miR-330-3p was highly expressed in ESCC tumor tissues. qRT-PCR further confirmed the result in other 35 pairs of ESCC tumor tissues and ESCC cell lines. Ectopic expression of miR-330-3p significantly promoted ESCC cell proliferation, survival, migration, invasion in vitro and stimulated tumor formation in nude mice. Knockdown of miR-330-3p leaded to the opposite effects. The luciferase assay confirmed that miR-330-3p directly interacted with the PDCD4 mRNA 3’ un-translated region (UTR). Moreover, expression of PDCD4 was inversely associated with miR-330-3p in ESCC tissues. Silencing of PDCD4 significantly promoted cell growth, cell migration, invasion and inhibited cisplatin-induced apoptosis in ESCC cells. This study suggested that miR-330-3p might play an oncogenic role in the development of ESCC partially via suppression of PDCD4 expression.

Epithelial-mesenchymal transition (EMT) is a developmental program that is associated with esophageal squamous cell carcinoma (ESCC) progression and metastasis. Recently, C/EBPβ has been reported to be an EMT inducer in cancer. However, the detailed molecular mechanisms remain unclear. Here, we report for the first time, that the truncated CCAAT-enhancer-binding protein β (C/EBPβ) LIP isoform is abnormally overexpressed and correlated with cancer metastasis in clinical specimens of human ESCC. Furthermore, we demonstrate that C/EBPβ LIP mediates epithelial growth factor (EGF)-induced EMT and increases migration and invasion of esophageal cancer cells in a manner that is dependent on miR-203 inactivation. Finally, we identified miR-203 as a direct target of C/EBPβ LIP. Disruption of C/EBPβ LIP attenuated the EGF-mediated decrease in miR-203, whereas overexpression of C/EBPβ LIP alone markedly suppressed miR-203. In addition, we demonstrated that C/EBPβ LIP inhibited miR-203 transcription by directly interacting with a conserved distal regulatory element upstream of the miR-203 locus, and in doing so, orchestrated chromatin remodeling. In conclusion, our results have revealed a new regulatory mechanism that involves C/EBPβ-LIP-mediated downregulation of miR-203, which plays a key role in EMT and metastasis.

Epithelial-mesenchymal transition (EMT) is a developmental program that is associated with esophageal squamous cell carcinoma (ESCC) progression and metastasis. Recently, C/EBPβ has been reported to be an EMT inducer in cancer. However, the detailed molecular mechanisms remain unclear. Here, we report for the first time, that the truncated CCAAT-enhancer-binding protein β (C/EBPβ) LIP isoform is abnormally overexpressed and correlated with cancer metastasis in clinical specimens of human ESCC. Furthermore, we demonstrate that C/EBPβ LIP mediates epithelial growth factor (EGF)-induced EMT and increases migration and invasion of esophageal cancer cells in a manner that is dependent on miR-203 inactivation. Finally, we identified miR-203 as a direct target of C/EBPβ LIP. Disruption of C/EBPβ LIP attenuated the EGF-mediated decrease in miR-203, whereas overexpression of C/EBPβ LIP alone markedly suppressed miR-203. In addition, we demonstrated that C/EBPβ LIP inhibited miR-203 transcription by directly interacting with a conserved distal regulatory element upstream of the miR-203 locus, and in doing so, orchestrated chromatin remodeling. In conclusion, our results have revealed a new regulatory mechanism that involves C/EBPβ-LIP-mediated downregulation of miR-203, which plays a key role in EMT and metastasis.

The serine/threonine kinase 11 (STK11) gene is the main causal gene in Peutz–Jeghers syndrome (PJS). Abnormal STK11 may increase cancer risk of PJS patients via affecting its target proteins such as P53, AMPK, and PTEN. In this study, we investigated the molecular basis of six Chinese PJS patients. Blood samples were collected from four Chinese PJS families and two sporadic patients. The entire coding region of the STK11 gene was amplified by polymerase chain reaction and analyzed by direct sequencing. Functions of mutants were assessed by PolyPhen-2, Swiss-Model software, and luciferase reporter assay. Novel mutations (c.842_843insC, c.804_805insG, and c.922T>G) and recurrent mutations (c.526G>A, c.180C>G, and c.1062C>G) were identified. Missense mutation c.922T>G and c.526G>A were predicted as probably damaging by PolyPhen-2, while c.1062C>G was benign. Mutation c.108C>G was a nonsense mutation. The 284Ter mutants of c.842_843insC and c.804_805insG significantly diminished the capacity of P53 activity in 293FT cells. Our results support that STK11 gene mutations underlie Chinese patients with PJS. Mutation involving partial kinase domain disrupts normal function of STK11. Our results also enlarge the spectrum of STK11 variants in PJS patients.

Recent studies indicated that the aberrant gene expression of peroxiredoxin-6 (prdx6) was found in various kinds of cancers. Because of its biochemical function and gene expression pattern in cancer cells, the association between genetic polymorphism of Prdx6 and cancer onset is interesting. In this report, we have developed and implemented a serial experiment in molecular biology laboratory course to teach single nucleotide polymorphism (SNP) to undergraduate students majoring in molecular biology or genetics. The flanking sequence of rs4382766 was located in Prdx6 gene, which contained a restriction site of SspI, and was used as a target in this lab course. The students could mimic real research by integrating different techniques, such as database retrieving, genomic DNA isolation, PCR, and restriction enzyme assay. This serial experiment of PCR-RFLP helps students set up intact idea of molecular biology and understand the relation among individual experiments. Students were found to be more enthusiastic during the laboratory classes than those in the former curriculum.

Polymorphisms in 3' untranslated region (UTR) of cancer-related genes might affect regulation by microRNA (miRNA) and contribute to carcinogenesis. In this study, we screened several single nucleotide polymorphisms (SNPs) in 3'UTR of cancer-related genes and investigated their effects on the risk of esophageal squamous cell carcinoma (ESCC). First, we used SNaPshot assay to genotype seven 3'UTR SNPs in 537 ESCC cases and 608 normal controls in a Chinese Han population and found that SNP rs6573 in 3'UTR of RAS-related proteins (RAP1A) was significantly associated with ESCC risk [P = 0.02, odds ratio (OR) = 0.43; 95% confidence interval (CI): 0.21-0.91] and pathologic stage (P = 0.03, OR = 1.89; 95% CI: 1.06-3.36). A putative binding site for miRNA-196a (miR-196a) exists in the 3'UTR of RAP1A, and the genetic variant, rs6573 A→C, is present in this binding region. We confirmed that miR-196a regulated the expression of RAP1A by luciferase reporter assay and that the regulation was affected by the RAP1A genotype. SNP rs6573 A to C change interfere in the interaction of miR-196a binding to RAP1A 3'UTR, resulting in higher constitutive expression of RAP1A. Moreover, we observed that RAP1A was overexpressed in the majority of ESCC tissues and correlated with RAP1A genotype and lymph node metastasis. In vitro study indicated RAP1A might function as a promoter for esophageal cancer cell migration and invasion through matrix metalloproteinase 2. Our study highlights RAP1A and SNP rs6573 functioning as potential personal diagnostic and prognosis markers for ESCC.

Previous studies on esophageal squamous cell carcinoma (ESCC) indicated that it contains much dysregulation of microRNAs (miRNAs). DNA hypermethylation in the miRNA 5′ regulatory region is a mechanism that can account for the downregulation of miRNA in tumors (Esteller, N Engl J Med 2008;358:1148–59). Among those dysregulated miRNAs, miR-203, miR-34b/c, miR-424 and miR-129-2 are embedded in CpG islands, as is the promoter of miR-34a. We investigated their methylation status in ESCC by bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP). The methylation frequency of miR-203 and miR-424 is the same in carcinoma and in the corresponding non-tumor tissues. The methylation ratio of miR-34a, miR-34b/c and miR-129-2 is 66.7% (36/54), 40.7% (22/54) and 96.3% (52/54), respectively in ESCC, which are significantly higher than that in the corresponding non-tumor tissues(p < 0.01). Quantitative RT-PCR analysis in clinical samples suggested that CpG island methylation is significantly correlated with their low expression in ESCC, 5-aza-2′-deoxycytidine (DAC) treatment partly recovered their expression in EC9706 cell line. We conclude that CpG island methylation of miR-34a, miR-34b/c and miR-129-2 are frequent events and important mechanism for their low expression in ESCC. DNA methylation changes have been reported to occur early in carcinogenesis and are potentially good early indicators of carcinoma (Laird, Nat Rev Cancer 2003;3:253–66). The high methylation ratio of miR-129-2 indicated its potential as a methylation biomarker in early diagnosis of ESCC.

Decoy receptor 3 (DcR3) is a soluble receptor without transmembrane and intracellular sequences in its peptide. It can bind to and inactivate the apoptosis-inducing ligand FasL, LIGHT, and TL1A. The aims of this study are to genotype the two polymorphisms in the promotor of DcR3 in esophageal squamous cell cancer patients and controls and analyze the association between individual genetic variation and susceptibility to esophageal squamous cell carcinoma (ESCC). A total of 444 Chinese ESCC patients and 468 matched cancer-free controls were evaluated for the two single nucleotide polymorphisms (SNPs) in DCR3. Polymorphisms were genotyped using the SNPShot technique. The expression of DCR3 in cancer tissues was detected by reverse transcription-polymerase chain reaction. There were significant differences in the allele's distribution of the two SNPs among ESCC cases and controls (P < 0.01). Compared with the 147TT genotype, the 147CC genotype was associated with significant elevated risk of ESCC (odds ratio, 1.89; 95% confidence interval, 1.30-2.96). The risk of 147CC genotype was more notable (odds ratio, 2.19; 95% confidence interval, 1.32-3.64) in the smokers than in the nonsmokers (odds ratio, 1.25; 95% confidence interval, 0.59-2.69). In the stratification analyses, we also found there was a strong correlation between 147 CC and the clinical TNM stage (P < 0.01). Furthermore, significant difference in DCR3 expression in ESCC tissues was found between subgroups with different 147C/T variant. Our finding suggested that DcR3 promoter polymorphism 147C/T might influence the susceptibility to ESCC in the Chinese Han population, maybe by influencing DcR3 expression.