The cellular mechanisms governing the change from pulmonary nodules to early-stage lung adenocarcinoma (LUAD) at single-cell resolution are not yet fully elucidated. In the present study, we employed single-nucleus RNA sequencing (snRNA-seq) on 44 pulmonary nodule and paired normal specimens to investigate this process. Within the epithelial compartment, alveolar type 2 (AT2) cells were categorized into high- and low-malignancy subclusters. Our differential expression analysis identified a significant upregulation of the transcription factor SMAD family member 3 (SMAD3) in the high-malignancy group, which was shown to enhance LUAD proliferation and migration. Mechanistically, intercellular adhesion molecule 1 (ICAM1) was established as a downstream target of SMAD3, playing a pivotal role in mediating intercellular crosstalk between malignant AT2 cells and lymphocytes. Specifically, the interaction between ICAM1 and immunosuppressive CD4+ T cells and immune-eliminating CD8+ T cells was found to dampen T cell-mediated antitumor response. In conclusion, these results reveal that the SMAD3-ICAM1 axis drives the malignant change from pulmonary nodules to early-stage LUAD, underscoring the potential of these molecules as biomarkers to inform diagnosis and surgical decision-making.

Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer. Glutamine plays a critical role in the progression of LUAD. However, the function of pyrroline-5-carboxylate reductase 1 (PYCR1) and its regulatory role in glutamine metabolism remain unclear. Transcriptomic and clinical data for LUAD were obtained from The Cancer Genome Atlas (TCGA) and validated using Gene Expression Omnibus (GEO) datasets (GSE19188, GSE13213). Glutamine metabolism-related genes were analyzed for differential expression and prognostic significance. Functional enrichment was performed via gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses. Single-cell RNA-seq data (GSE117570) were processed using Seurat, and cell-cell communication was inferred with CellChat. In vitro, lentiviral overexpression, Western blotting, EdU, CCK-8, and glutamine uptake assays were conducted. An orthotopic xenograft model was established in nude mice to assess tumor growth in vivo. Six glutamine-metabolism-related genes were found significantly overexpressed in LUAD tissues and associated with poor overall survival. Single-cell sequencing revealed predominant PYCR1 expression in malignant cells. Functional assays demonstrated that PYCR1 overexpression enhanced glutamine uptake, proliferation, and inhibited apoptosis in LUAD cells, effects mediated via suppression of the P53 pathway. PYCR1 promoted tumor growth in a xenograft model and was found to transcriptionally upregulate 5-oxoprolinase (OPLAH), which augmented its oncogenic effects. Our findings identify the PYCR1/OPLAH axis as a key driver of LUAD progression via p53 signaling, revealing a promising therapeutic target.

Long noncoding RNA (lncRNA) cancer susceptibility 9 (CASC9) has been found to be overexpressed and functions as an oncogene in many cancer types. We investigated the molecular mechanism underlying CASC9 overexpression in esophageal squamous cell carcinoma (ESCC). Transcripts containing exons 2 and 6 and exons 4 and 6 showed the highest CASC9 expression levels in ESCC, no transcripts were detected in the normal esophageal epithelial Het1A cell line. The Long Interspersed Nuclear Element-1 (LINE1 or L1) element in the genome was found to participate in the evolution of lncRNA CASC9, the antisense promoter (ASP) of L1 provides the cis-regulatory elements necessary for CASC9 activation, and the antisense chain of L1 participates in the formation of exons of CASC9. The activation of the antisense promoter was due to the aberrant hypomethylation of L1 elements. An active enhancer element was identified in the downstream region of CASC9 gene by ChIP-seq and ChIP-qPCR. The interaction between ASP and the enhancer elements was confirmed by chromosome conformation capture (3C). Thus, our results suggest that the L1 ASP activation due to aberrant hypomethylation and downstream enhancer interaction plays a key role in the overexpression of lncRNA CASC9 in ESCC.

This paper investigated the mechanism of Huoxue Dingxuan Capsules(HXDX) on autophagy in vascular endothelial cells based on the "crosstalk" of Bcl-2 and mTOR protein. bEnd.3 cells were divided into a blank control group, a model group, and an HXDX group. CO-IP experiments were conducted, and then Western blot(WB) was used to detect the binding of Bcl-2 and mTOR. Co-localization of Bcl-2 with mTOR protein was observed by laser confocal microscopy after staining. The model of Bcl-2 siRNA in bEnd.3 cells was constructed, and the bEnd.3 cells were divided into five groups, including blank control group, oxygen-glucose deprivation(OGD) group, transfected Bcl-2 siRNA group, negative control(NC) group, and HXDX group. The model of mTOR siRNA in bEnd.3 cells was constructed, and the bEnd.3 cells were divided into five groups, including blank control group, OGD group, transfected mTOR siRNA group, NC group, and HXDX group. The expression of autophagy-related proteins was detected by WB. The results of CO-IP experiments showed that Bcl-2 and mTOR proteins could be co-localized and expressed in bEnd.3 cells, and the expression of Bcl-2 and mTOR proteins increased after the intervention of the HXDX-containing serum. After screening and transfection with Bcl-2-mus-384, autophagy of bEnd.3 cells was induced. The expression of Bcl-2 in the Bcl-2 siRNA group was significantly decreased compared with the blank control group and model group. The expression of mTOR protein was significantly lower than that of the OGD group, and the expression of human microtubule-associated protein light chain 3Ⅱ/Ⅰ was significantly higher than that of the model group. After the intervention of the HXDX-containing serum, the expression of Bcl-2 and mTOR was increased in the HXDX group compared with that in the Bcl-2 siRNA group, and the expression of LC3Ⅱ/LC3Ⅰ and Beclin1 was decreased in the HXDX group compared with that in the Bcl-2 siRNA group. After transfecting mTOR-mus-7061 and inducing autophagy of bEnd.3 cells, in the mTOR siRNA group, mTOR protein expression was decreased compared with the blank control and model groups, and LC3Ⅱ/LC3Ⅰ and Beclin1 protein expression was increased compared with the model group. After the intervention of the HXDX-containing serum, the expression of Bcl-2 and mTOR was increased in the HXDX group compared with that in the mTOR siRNA group, and the expression of LC3Ⅱ/LC3Ⅰ and Beclin1 proteins was decreased compared with the mTOR siRNA group. This study explored the "crosstalk" of Bcl-2 and mTOR during autophagy in bEnd.3 cells at the cellular level and illustrated the modulating effect of the HXDX-containing serum, which provided a basis for the treatment of cervical spondylosis of vertebral artery type.

Background: Protein extracts from chicken egg whites were prepared by utilizing different solvents and we studied the differences in proliferation. Methods: The egg white extract was prepared by using lysate, phosphate-buffered saline (PBS), saline and pure water. The in vitro mixture experiment was carried out to observe the effect of different egg white preparations on the proliferation of cells. The samples were divided into the following groups: the control group with media, the original lysate group, the new lysate group, the PBS group, the saline group and the pure water group. Result: The study found that at final concentrations of 10%, 20%, 30%, 40% and 50%, the differences among the six groups were statistically significant (P less than 0.01, F greater than 100). The results of pairwise comparison showed that the proliferative effect of chicken egg albumin extract prepared by PBS was significantly higher than that of the medium at final concentrations of 20%, 30% and 50% (P less than 0.05). When the fetal bovine serum concentration was only 8%, 7% and 5%, the cell proliferation effect was better than that of the control group with 10% fetal bovine serum, indicating that the chicken egg white extract promoted cell proliferation. This result indicated that the best chicken egg albumin extract was obtained through PBS addition. The quantity of expensive fetal bovine serum could be considerably reduced by supplementing the media with chicken egg albumin extract. Among the solvents tested, PBS was the best solvent for preparing chicken egg albumin extract.

BackgroundInsulin-like growth factor binding protein-1 (IGFBP-1) is considered a decline in polycystic ovary syndrome (PCOS), but it remains controversial that whether such reduction is attributed to obesity.AimsThis systematic review aims to explore whether IGFBP-1 is reduced in PCOS, and whether such reduction is associated with obesity.ResultsOur pooled study included 12 studies with a total of 450 participants. IGFBP-1 levels in PCOS were significantly lower than that in non-PCOS (SMD (95%CI)=-0.49(-0.89, -0.09), P=0.02). No significant difference in IGFBP-1 levels between patients with or without PCOS classified by BMI. Whilst, stratification by PCOS status revealed a significant decrease in IGFBP-1 in overweight (SMD (95%CI)=-0.92(-1.46, -0.37), P=0.001). When comparing fasting insulin in the same way, PCOS patients had significantly elevated fasting insulin level but not statistically declined IGFBP-1 after classified by BMI.ConclusionThis meta-analysis provides evidence that the decrease of IGFBP-1 in PCOS was more strongly influenced by comorbid obesity than by PCOS itself. Additionally, contrast to previous findings that insulin significantly suppresses IGFBP-1, our results suggested that the suppression of PCOS-related hyperinsulinemia on IGFBP-1 seemed diminished. Overall, our work may provide a novel perspective on the mechanism between insulin and IGFBP-1 underlying PCOS development.

Abstract Lactate arouses an increasing concern in recent studies of Alzheimer’s disease (AD). At late stage of AD, amyloid beta (Aβ) directly promotes lactate level, continuously activating microglial inflammation and aggravating neuronal damage. However, whether lactate fluctuation can effect on Aβ hasn’t been recognized. Especially, it is observed a reduction of lactate in the hippocampus and cortex of early AD mouse model. Hence, this study discusses the relation between decreased lactate and Aβ production at early stage of AD, and its possibly mechanism. As results showed, a specific receptor of lactate, G-protein-coupled receptor 81 (GPR81) and its downstream of phosphatidylinositol 3 kinase /protein kinase B (PI3K/Akt)-cyclic-adenosine-monophosphate response element-binding protein (CREB) is suppressed in the cortex and hippocampus of early AD mouse model. Moreover, deoxyribonucleic acid methyltransferase 1 (DNMT1) is reduced in the presence of increased beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and Aβ. Previously, it is observed that PI3K/Akt-CREB inhibition leads to DNMT1 downregulation. As an indispensable methyltransferase, DNMT1 reduction can directly inhibit the methylation of specific loci within BACE1 gene promoter, leading to increased BACE1 and Aβ production. 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).

Chronic alcoholism often causes liver injuries characterized by hepatic steatosis, inflammation as well as oxidative stress and finally leads to advanced cirrhosis and liver cancer. Fas-activated serine/threonine kinase (FASTK) and its homologs are gradually known as multifunctional proteins involved in various biological processes; however, the role of FASTK and its family members in alcoholic liver disease (ALD) is still unexplored. Here we found that, among FASTK family members, the expression of FASTK was specifically induced both in livers of mice received chronic ethanol ingestion and in ethanol-stimulated hepatocytes. Animal studies showed that genetic deletion of FASTK attenuated chronic ethanol ingestion-induced liver damage, steatosis, and inflammation. Moreover, FASTK deficiency was associated with improved oxidative/anti-oxidative system homeostasis and reduced reactive oxygen species (ROS) generation in livers upon chronic ethanol stimulation. Importantly, FASTK ablation preserved hepatic sirtuin-1 (SIRT1) expression/activity upon chronic ethanol ingestion and SIRT1 silencing via adenovirus-mediated small interfering RNA transfer diminished FASTK deletion-elicited beneficial effects on alcohol-associated hepatic steatosis, inflammation, and oxidative stress. Mechanistically, ethanol increased the phosphorylation of human antigen R (HuR, a RNA binding protein that stabilizes SIRT1 mRNA) and triggered the dissociation of HuR-SIRT1 mRNA complex, in turn promoting SIRT1 mRNA decay. Genetic deletion of FASTK diminished ethanol-induced HuR phosphorylation and HuR-SIRT1 mRNA complex dissociation, thereby enhancing SIRT1 mRNA stability. Collectively, these findings for the first time highlight a critical role of FASTK in the pathogenesis of ALD and implicate HuR-SIRT1 mRNA complex involves in this process.

Breast cancer is one of the most common malignant cancers among women. Cancer cells and adjacent cells determine the development of the disease. Tumor associated macrophages (TAMs) are involved in the regulation of different stages of cancer progression. LncRNAs play an important role in tumor growth and metastasis. However, the function of lncRNA in macrophage and tumor cell interaction is poorly described. Here we reported that lncRNA SNHG1 functioned as a modulator of M2 macrophage polarization and regulated tumor growth and angiogenesis. We indicated that knockdown of SNHG1 inhibited M2 macrophage polarization by suppression of STAT6 phosphorylation. SNHG1 silencing significantly alleviated migration of MCF-7 cells and tube formation of Human Umbilical Vein Endothelial Cells (HUVEC). Furthermore, we found that implantation of cell mixture of MCF-7 cells and macrophages promoted tumor growth and angiogenesis. However, knockdown of SNHG1 in macrophages reversed that effect. Collectively, we demonstrated the important role of lncRNA SNHG1 in macrophages and breast cancer cells interaction. We highlight the essential effect of lncRNA in tumor progression and provide a new method for the prevention and treatment of breast tumor metastasis.

Chronic alcoholism disrupts mitochondrial function and often results in alcoholic cardiomyopathy (ACM). Fas-activated serine/threonine kinase (FASTK) is newly recognized as a key post-transcriptional regulator of mitochondrial gene expression. However, the modulatory role of FASTK in cardiovascular pathophysiology remains totally unknown. In experimental ACM models, cardiac FASTK expression markedly declined. Ethanol directly suppressed FASTK expression at post-transcriptional level through NADPH oxidase-derived reactive oxygen species (ROS). Ethanol destabilized FASTK mRNA 3′-untranslated region (3′-UTR) and accelerated its decay, which was blocked by the clearance of ROS. Regnase-1 (Reg1), a ribonuclease regulating mRNA stability, was induced by ROS in ethanol-stimulated cardiomyocytes. Reg1 directly bound to FASTK mRNA 3′-UTR and promoted its degradation, whereas silencing of Reg1 reversed ethanol-induced FASTK downregulation. Compared to wild type control, alcohol-related myocardial morphological (hypertrophy, fibrosis and cardiomyocyte apoptosis) and functional (reduced ejection fraction and compromised cardiomyocyte contraction) anomalies were worsened in FASTK deficient mice. Mechanistically, FASTK ablation repressed NADH dehydrogenase subunit 6 (MTND6, a mitochondrial gene encoding a subunit of complex I) mRNA production and reduced complex I-supported respiration. Importantly, cardiomyocyte-specific upregulation of FASTK through intra-cardiac AAV9-cTNT injection mitigated myocardial mitochondrial dysfunction and restrained ACM progression. In vitro study showed that overexpression of FASTK ameliorated ethanol-induced MTND6 mRNA downregulation, complex I inactivation, and cardiomyocyte death, whereas these beneficial effects were counteracted by rotenone, a complex I inhibitor. Collectively, ROS-accelerated FASTK mRNA degradation via Reg1 underlies chronic ethanol ingestion-associated mitochondrial dysfunction and cardiomyopathy. Restoration of FASTK expression through genetic approaches might be a promising therapeutic strategy for ACM.

Mitochondrial dynamics, determining mitochondrial morphology, quality and abundance, have recently been implicated in myocardial ischemia and reperfusion (MI/R) injury. The roles of κ-opioid receptor activation in cardioprotection have been confirmed in our previous studies, while the underlying mechanism associated with mitochondrial dynamics remains unclear. This study aims to investigate the effect of κ-opioid receptor activation on the pathogenesis of MI/R and its underlying mechanisms. MI/R mouse model and hypoxia-reoxygenation cardiomyocyte model were established in this study. Mitochondrial dynamics were analyzed with transmission electron microscopy in vivo and confocal microscopy in vitro. STAT3 phosphorylation and OPA1 expression were detected by Western blotting. We show here that κ-opioid receptor activation with its selective receptor agonist U50,488H promoted mitochondrial fusion and enhanced myocardial resistance to MI/R injury, while these protective effects were blockaded by nor-BNI, a selective κ-opioid receptor antagonist. In addition, κ-opioid receptor activation increased STAT3 phosphorylation and OPA1 expression, which were blockaded by nor-BNI. Furthermore, inhibition of STAT3 phosphorylation by stattic, a specific STAT3 inhibitor, repressed the effects of κ-opioid receptor activation on promoting OPA1 expression and mitochondrial fusion, as well as inhibiting cell apoptosis and oxidative stress both in vivo and in vitro during MI/R injury. Overall, our data for the first time provide evidence that κ-opioid receptor activation promotes mitochondrial fusion and enhances myocardial resistance to MI/R injury via STAT3-OPA1 pathway. Targeting the pathway regulated by κ-opioid receptor activation may be a potential therapeutic strategy for MI/R injury.

Background: Thyroid cancer (TC) is the most common type of endocrine malignancy and its incidence is increasing over years. Conventional surgery, radiotherapy and chemotherapy are difficult to improve the significant effects of it due to aggression and metastasis of poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC), and these are regarded as the most malignant types of TC. Glucose-regulated protein (GRP78) is the key molecule of tumor growth, apoptosis and metastasis. However, the underlying mechanisms of GRP78 in TC still require discussion. This study aimed to explore the role of GRP78 and its potential mechanism in TC. Results: GRP78 expression was increased in TC tissues when compared with adjacent normal tissues. Besides, down-regulation of GRP78 significantly inhibited the metastatic and proliferative ability of ATC cells in in vitro studies. In addition, tunicamycin-induced ER stress up-regulated the expression of GRP78, PERK and XBP1 as well as reversed the metastatic ability of GRP78 in ATC cells. Bioinformatics and statistical analysis of gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for RNA-sequencing data with regard to si-GRP78 and si-control showed that GRP78 might regulate the ability of metastasis through extracellular matrix (ECM) remodeling in ATC cells, as well as the expression of ECM components such as COL1A1 and MMP13, which were highly relevant to ATC cells. The analysis of GEPIA database confirmed that high genomic amplification of MMP13 and COL1A1 in TC tissues showed correlation with TNM stage. Further western blotting analysis showed that MMP13 might be the target of GRP78 in ATC cells and ER stress could activate the expression of MMP13 that is suppressed by GRP78 depletion. Conclusions: GRP78 acts as an important regulator of metastasis under ER stress. In addition, the function of GRP78 might be mediated by ECM remodeling in ATC cells, implicating it as a therapeutic target in TC.

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.

Background/Aims: Autophagy is essential for maintaining cellular homeostasis and the survival of terminally differentiated cells as neurons. In this study, we aim to investigate whether mitofusin 2, a mitochondrial fusion protein, mediates autophagy in cerebral ischemia/reperfusion (I/R) injury. Methods: Primary cultured neurons were treated with oxygen-glucose deprivation/reperfusion to mimic cerebral I/R injury in vitro. Autophagosomes were visualized upon TEM. Autophagy-markers were then detected to monitor autophagy by western-blot and real-time PCR, and the autophagic flux was tracked with a mRFP-GFP-LC3 construct by fluorescence as well as autophagy inhibitors and agonists. The up- and downregulation of Mfn2 were through transfecting a lentivirusexpression vector respectively. And neuronal injury was detected by cell counting kit and TUNEL assay. Results: Results showed I/R increased autophagosome formation and inhibited autolysosome degradation. Furthermore, use of autophagy related agents demonstrated that I/R injury was caused by insufficient autophagy and aggravated by impaired autophagic degradation. The results also indicated that mitofusin 2 could ameliorate I/R injury through increasing autophagosome formation and promoting the fusion of autophagosomes and lysosomes. In contrast, downregulation of mitofusin 2 aggravated the I/R injury by inhibiting autophagosome formation and the fusion of autophagosomes and lysosomes. Additionly, mitofusin 2 overexpression did not lead to autolysosome accumulation induced by I/R. Conclusions: In summary, this study explicitly demonstrated that mitofusin 2 could ameliorate I/R injury mainly through promoting autophagy, which represented a potential novel strategy for neuroprotection against cerebral I/R damage.

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.

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.

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.

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 miR26 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 miR26 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 miR26 expression in ESCC was not due to DNA methylation. Hence, our study reveals a novel feedback loop for cMYC pathway and implicates miR-26 as a potential target for prevention and treatment of esophageal cancer.

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.

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.