Recent studies underscore important roles of intestinal microbiota and the bacterial lipopolysaccharides (LPS) production in the pathogenesis of liver disease. However, how gut microbiota alters in response to the development of steatosis and subsequent progression to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) remains unclear. We aimed to study the gut microbial changes over liver disease progression using a streptozotocin-high fat diet (STZ-HFD) induced NASH-HCC C57BL/6J mouse model that is highly relevant to human liver disease. The fecal microbiota at various liver pathological stages was analyzed by 16S rDNA gene pyrosequencing. Both UniFrac analysis and partial least squares-discriminant analysis showed significant structural alterations in gut microbiota during the development of liver disease. Co-abundance network analysis highlighted relationships between genera. Spearman correlation analysis revealed that the bacterial species, Atopobium spp., Bacteroides spp., Bacteroides vulgatus, Bacteroides acidifaciens, Bacteroides uniformis, Clostridium cocleatum, Clostridium xylanolyticum and Desulfovibrio spp., markedly increased in model mice, were positively correlated with LPS levels and pathophysiological features. Taken together, the results showed that the gut microbiota was altered significantly in the progression of liver disease. The connection between the gut microbial ecology and the liver pathology may represent potential targets for the prevention and treatment of chronic liver disease and HCC.

As a naturally occurring inhibitor of mTOR, accumulated evidence has suggested that DEPTOR plays a pivotal role in suppressing the progression of human malignances. However, the function of DEPTOR in the development of esophageal squamous cell carcinoma (ESCC) is still unclear. Here we report that the expression of DEPTOR is significantly reduced in tumor tissues derived from human patients with ESCC, and the downregulation of DEPTOR predicts a poor prognosis of ESCC patients. In addition, we found that the expression of DEPTOR negatively regulates the tumorigenic activities of ESCC cell lines (KYSE150, KYSE510 and KYSE190). Furthermore, ectopic DEPTOR expression caused a significant suppression of the cellular proliferation, migration and invasion of KYSE150 cells, which has the lowest expression level of DEPTOR in the three cell lines. Meanwhile, CRISPR/Cas9 mediated knockout of DEPTOR in KYSE-510 cells significantly promoted cellular proliferation, migration and invasion. In addition, in vivo assays further revealed that tumor growth was significantly inhibited in xenografts with ectopic DEPTOR expression as compared to untreated KYSE150 cells, and was markedly enhanced in DEPTOR knockout KYSE-510 cells. Biochemical studies revealed that overexpression of DEPTOR led to the suppression of AKT/mTOR pathway as evidenced by reduced phosphorylation of AKT, mTOR and downstream SGK1, indicating DEPTOR might control the progression of ESCC through AKT/mTOR signaling pathway. Thus, these findings, for the first time, demonstrated that DEPTOR inhibits the tumorigenesis of ESCC cells and might serve as a potential therapeutic target or prognostic marker for human patients with ESCC.

Various studies have demonstrated the diagnostic value of microRNA (miRNA) for lung cancer, but miRNA signatures varied between different subtypes. Whether serum miRNAs could be used as biomarkers in lung squamous cell carcinoma (SCC) remains unknown. Using quantitative real-time polymerase chain reaction (qRT-PCR) based Exiqon panel, 38 differentially expressed miRNAs were identified from 3 male lung SCC pool samples and 1 normal control (NC) pool in the initial screening phase. After the training (24 SCC VS. 15 NCs), testing (44 SCC VS. 57 NCs) and external validation (34 SCC VS. 36 NCs VS. 10 pulmonary hamartoma) processes via qRT-PCR, we identified a three-miRNA panel ((miR-106a-5p, miR-20a-5p and miR-93-5p) to be a potential diagnostic marker for male lung SCC patients. The areas under the receiver operating characteristic (ROC) curve of the three-miRNA panel for the training, testing and validation phases were 0.969, 0.881 and 0.954 respectively. In addition, this signature could also differentiate lung SCC from pulmonary hamartoma (AUC=0.900). The 3 miRNAs were consistently up-regulated in lung SCC tissues (23 SCC VS. 23 NCs) and serum exosomes (17 SCC VS. 24 NCs). Moreover, expression of the 3 miRNAs was decreased in arterial serum (n = 3). In conclusion, we established a three-miRNA signature in the peripheral serum with considerable clinical value in the diagnosis of male lung SCC patients.

Breast cancer is the most common female cancer with considerable metastatic potential, explaining the need for new candidates that inhibit tumor metastasis. In our study, betulinic acid (BA), a kind of pentacyclic triterpenoid compound derived from birch trees, was evaluated for its anti-metastasis activity in vitro and in vivo. BA decreased the viability of three breast cancer cell lines and markedly impaired cell migration and invasion. In addition, BA could inhibit the activation of stat3 and FAK which resulted in a reduction of matrix metalloproteinases (MMPs), and increase of the MMPs inhibitor (TIMP-2) expression. Moreover, in our animal experiment, intraperitoneal administration of 10 mg/kg/day BA suppressed 4T1 tumor growth and blocked formation of pulmonary metastases without obvious side effects. Furthermore, histological and immunohistochemical analyses showed a decrease in MMP-9 positive cells, MMP-2 positive cells and Ki-67 positive cells and an increase in cleaved caspase-3 positive cells upon BA administration. Notably, BA reduced the number of myeloid-derived suppressor cells (MDSCs) in the lungs and tumors. Interestingly, in our caudal vein model, BA also obviously suppressed 4T1 tumor pulmonary metastases. These findings suggested that BA might be a potential agent for inhibiting the growth and metastasis of breast cancer.

The elevation of Nicotinamide N-methyltransferase (NNMT) has been reported in pancreatic cancer tissues and cell lines, but its clinical and prognostic implications remain controversial. This study aimed at investigating the expression of NNMT in pancreatic benign and malignant tissues and the prognostic value of NNMT in pancreatic cancer. The expression of NNMT in tissue specimens of 28 chronic pancreatitis patients and 178 pancreatic cancer patients were assayed with immunohistochemistry on tissue microarray. The NNMT expression levels of pancreatic patients were correlated with their clinicopathological characteristics. The influences of NNMT expression and patients' clinicopathological characteristics on overall survival (OS) were analyzed. The percentage of NNMT high expression (NNMTh) in pancreatic cancer (55.6%) was significantly higher than those in chronic pancreatitis (21.4%) and paracancerous tissues (14.8%) (p < 0.001). NNMTh tends to significantly correlate with unfavorable clinicopathological features such as age > 60 years old (p = 0.014), tumor diameter > 4 cm (p < 0.001), TNM stage III or IV (p < 0.001) and poor tumor differentiation (p = 0.004). The median OS of patients with NNMTh and NNMTl were 7.0 months (95% CI: 5.275-8.725) and 11.5 months (95% CI: 9.759-13.241) respectively (p = 0.005). On multivariate analysis, NNMTl (hazards ratio [HR]: 0.399; 95% CI: 0.284-0.560; p < 0.001), absence of neurological involvement (HR: 0.651; 95% CI: 0.421-0.947; p = 0.041), TNM stage I or II (HR: 0.506; 95% CI: 0.299-0.719; p = 0.015) and well tumor differentiation (HR: 0.592; 95% CI: 0.319-0.894; p = 0.044) were significant favorable prognostic factors of OS. In conclusion, NNMT is upregulated in pancreatic cancer, correlates with unfavorable clinicopathological features and may serve as an independent prognosticator of patients' survival.

Ascites syndrome (AS), also known as pulmonary artery hypertension, remains a challenging disease that severely affects both humans and broiler chickens. Pulmonary artery remodeling presents a key step in the development of AS. In this study, we obtained pulmonary artery tissues from broilers with and without AS to perform miRNA sequencing analysis, miRNA-mRNA association analysis and pathological examinations. 29 significantly differentially expressed miRNAs were found both in known and novel miRNAs with 18 up-regulated and 11 down-regulated miRNAs. Their predicted potential targets were involved in a wide range of functional clusters as indicated via GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses. The upregulation of miR-155, miR-23b-3p, miR-146b-5p and miR-146b-3p were found closely associated with the pathogenesis of pulmonary artery remodeling in AS progression. The association analysis for the miRNAs-mRNAs showed that these 29 significantly differentially expressed miRNAs regulate 162 differentially expressed target genes. Among them, 20 miRNAs correlated with 18 predicted target genes that appear to be involved in pulmonary artery remodeling, mainly in three broad physiological processes: the hypoxia sensing response (HIF1α, NHE1, STAT5 and STAT3), endothelial permeability dysfunction (CD44, TRAF2, CDK2AP1, LZTFL1, JAZF1, PEBP1, LRP1B, RPS14 and THBS2) and inflammation (MEOX2, STAT5, STAT3, IRF8, MAP3K8, IL-1BETA and TNFRSF1B). Pathological pulmonary artery remodeling in the AS broilers was consistently observed in the present study. Taken together, the current analysis further illuminates the molecular mechanism of pulmonary artery remodeling underlying AS progression.

The differential expression of microRNAs (miRNAs) in plasma of esophageal squamous cell carcinoma (ESCC) patients may serve as a diagnostic biomarker. A four-stage study was conducted to identify plasma miRNAs with potential in detecting ESCC. Exiqon panels (2 ESCC pools vs. 1 normal control (NC) pool) were applied in the screening phase to obtain miRNA profiles. The identified miRNAs were further evaluated through training (36 ESCC VS. 42 NCs) and testing stages (101 ESCC VS. 113 NCs) with qRT-PCR assays. A six-miRNA signature including up-regulated miR-106a, miR-18a, miR-20b, miR-486-5p, miR-584 and down-regulated miR-223-3p in ESCC was identified. The signature could accurately discriminate ESCC patients from NCs with areas under the receiver operating characteristic curve of 0.935, 0.959 and 0.966 for the training, testing and the additional validation stage (41 ESCC VS. 50 NCs), respectively. MiR-106a and miR-584 were significantly up-regulated in tumor tissues with qRT-PCR assays. And miR-584 was also up-regulated in ESCC tissues from TCGA database. In addition, exosomal miR-223-3p and miR-584 were consistently dysregulated with those in plasma and could also act as biomarkers in diagnosis of ESCC. In conclusion, we identified a six-miRNA signature in plasma which could act as a non-invasive biomarker in detection of ESCC.

Androgen receptor (AR) is a key transcription factor playing a critical role in prostate cancer (PCa) initiation and progression. However, the molecular mechanisms of AR action in prostate cancer are not very clear. CXCL13, known as B cell attracting chemokine1 (BCA-1), is a member of CXC chemokine family and relevant to cancer metastasis. This study shows that CXCL13 is an androgen-responsive gene and involved in AR-induced PCa cell migration and invasion. In clinical specimens, expression of CXCL13 in PCa tissues is markedly higher than that in adjacent normal tissues. In cultures, expression of CXCL13 is up-regulated by androgen-AR axis at both mRNA and protein levels. Furthermore, Chip-Seq assay identifies canonical androgen responsive elements (ARE) at CXCL13 enhancer and dual-luciferase reporter assays reveals that the ARE is highly responsive to androgen while mutations of the ARE abolish the reporter activity. Additional chromatin immunoprecipitation (ChIP) assays also identify that the ARE presents androgen responsiveness. In addition, CXCL13 promotes G2/M phase transition by increasing Cyclin B1 levels in PCa cells. Functional studies demonstrate that reducing endogenous CXCL13 expression in LNCaP cells largely weakens androgen-AR axis induced cell migration and invasion. Taken together, our study implicates for the first time that CXCL13 is an AR target gene and involved in AR-mediated cell migration and invasion in primary PCa.

FILIP1LΔC103 (COOH terminal truncation mutant 1-790 of Filamin A Interacting Protein 1-Like) has been identified to hold therapeutic potential for suppressing tumor growth. Cisplatin (DDP) is commonly used as a first-line drug in the treatment for ovarian cancer. The usage of polymeric nanoparticles to deliver functional genes intraperitoneally holds much promise as an effective therapy for ovarian cancer. In this study, a recombinant plasmid expressing FILIP1LΔC103 (FILIP1LΔC103-p) was constructed, and HPEI nanogels were prepared to deliver FILIP1LΔC103-p into SKOV3 cells. The expression of FILIP1LΔC103 in vitro and in vivo was determined using RT-PCR and Western Blotting. Moreover, in vivo treatment experiments were conducted on nude mice bearing SKOV3 ovarian cancer. The mice were treated with 5% glucose, HPEI+E-p, HPEI+FILIP1LΔC103-p, DDP or HPEI+FILIP1LΔC103-p plus DDP, respectively. Tumor weights were evaluated throughout the treatment duration. The cell proliferation and apoptosis were evaluated by Ki-67 immunochemical staining and TUNEL assay respectively, and the anti-angiogenic effect was assessed by CD31 immunochemical staining and alginate-encapsulated tumor cell assay. FILIP1LΔC103-p could be efficiently transfected into SKOV3 cells by HPEI nanogels. The combination of HPEI+FILIP1LΔC103-p with DDP exerted enhanced antitumor activity compared with HPEI+FILIP1LΔC103-p or DDP alone. Significant reduction of tumor cells proliferation, augmentation of tumor cells apoptosis and suppression of angiogenesis were observed in the combination group compared with controls. Our results demonstrated synergistic antineoplastic activity of combined FILIP1LΔC103 and low-dose DDP with no apparent toxicity, indicating a potential application of the combined approach in the treatment of ovarian cancer.

Differently expressed microRNAs (miRNAs) in the plasma of lung adenocarcinoma (LA) patients might serve as biomarkers for LA detection. MiRNA expression profiling was performed using Exiqon panels followed by the verification (30 LA VS. 10 healthy controls (HCs)) with quantitative reverse transcription polymerase chain reaction (qRT-PCR) in the screening phase. Identified miRNAs were confirmed through training (42 LA VS. 32 HCs) and testing stages (66 LA VS. 62 HCs) by using qRT-PCR based absolute quantification methods. A total of six up-regulated plasma miRNAs (miR-19b-3p, miR-21-5p, miR-221-3p, miR-409-3p, miR-425-5p and miR-584-5p) were identified. The six-miRNA panel could discriminate LA patients from HCs with areas under the receiver operating characteristic curve of 0.72, 0.74 and 0.84 for the training, testing and the external validation stage (33 LA VS. 30 HCs), respectively. All the miRNAs identified except miR-584-5p were significantly up-regulated in LA tissues. MiR-19-3p, miR-21-5p, miR-409-3p and miR-425-5p showed high expression in arterial plasma with borderline significance. Additionally, miR-19-3p, miR-21-5p and miR-221-3p were significantly up-regulated in exosomes extracted from LA peripheral plasma samples. In conclusion, we identified a six-miRNA panel in peripheral plasma which might give assistance to the detection of LA at least for Asian population to a certain extent.

Macular corneal dystrophy (MCD) is an autosomal recessive disorder mainly caused by gene mutations of carbohydrate sulfotransferase (CHST6) leading to bilateral visual impairment. Because the mechanism underlying this degeneration remains poorly understood, we investigated molecular alterations and pathways that may be involved in MCD in this issue. Different mutation sites were screened by direct sequencing of the coding region of CHST6. In addition, we described morphological changes in MCD keratocytes by light microscopy and electron microscopy and determined the relationship between the development of this disease and the occurrence of apoptosis through flow cytometry, cell counting kit-8, colony formation assay and other experiments. Western blotting and quantitative real-time polymerase chain reaction were used to determine if endoplasmic reticulum (ER) stress was activated. We found 10 kinds of mutations among these families with 3 novel mutations included. The percentage of apoptotic keratocytes increased in MCD patients; furthermore, the expression of apoptosis related protein B-cell lymphoma-2 (Bcl-2) was down-regulated while Bcl-2 associated X protein was upregulated. Finally, ER stress was activated with the upregulation of glucose-regulated protein 78 and CCAAT-enhancer-binding protein homologous protein. Our clinical and in vitro results suggest that the CHST6 mutation associated with MCD is associated with apoptosis, and ER stress is probably involved in this apoptosis pathway.

Some reports have evaluated the prognostic relevance of microRNAs (miRNAs) in patients with pancreatic cancer (PC). However, most studies focused on limited miRNAs with small number of patients. The aim of the study is to identify a panel of miRNA signature that could predict prognosis in PC with the data from The Cancer Genome Atlas (TCGA). A total of 167 PC patients with the corresponding clinical data were enrolled in our study. The miRNAs significantly associated with overall survival (OS) in PC patients were identified with Cox proportional regression model. A risk score formula was developed to evaluate the prognostic value of the miRNA signature in PC. Thirteen miRNAs were identified to be significantly related with OS in PC patients. Patients with high risk score suffered poor overall survival compared with patients who had low risk score. The multivariate Cox regression analyses showed that the miRNA signature could act as an independent prognostic indicator. In addition, the signature might serve as a predicator for treatment outcome. Our study identified a miRNA signature including 13 miRNAs which could serve as an independent marker in prognosis of PC.

Tissue differentiation-inducing non-protein coding RNA (TINCR) is required for normal epidermal differentiation. TINCR is also strongly overexpressed in human gastric cancer (GC) and contributes to carcinogenesis and tumor progression. However, the association between TINCR polymorphisms and the risk of any diseases, such as GC, remains unknown. In the present study, the tag single nucleotide polymorphisms rs8113645, rs2288947, rs8105637, and rs12610531 were analyzed in 602 patients with GC and 602 age- and sex-matched controls. Polymorphisms were genotyped using TaqMan technology. Carriers of variant rs8113645 and rs2288947 alleles indicated reduced risks of GC (p = 0.003 and 0.037, respectively). A allele genotypes of rs8113645 and G allele genotypes of rs2288947 (rs8113645 GA and AA; rs2288947 AG and GG) were also significantly associated with decreased GC risk (p < 0.05). Stratification analysis displayed that the correlations between GC risk and variant genotypes of both rs8113645 and rs2288947were more evident in younger individuals, men, nonsmokers, and individuals from rural areas. We also demonstrated that rs8113645 GA+AA genotype carriers had lower TINCR mRNA expression levels compared with common genotype in both normal and GC tissues (p < 0.05). These results suggest that long non-coding RNA TINCR polymorphisms may be implicated in GC development.

Dysregulated expression of specific microRNAs (miRNAs) in serum has been recognised as promising diagnostic biomarkers for colorectal cancer (CRC). In the initial screening phase, a total of 32 differentially expressed miRNAs were selected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) based Exiqon panel with 3 CRC pool samples and 1 normal control (NC) pool. Using qRT-PCR, selected serum miRNAs were further confirmed in training (30 CRC VS. 30 NCs) and testing stages (136 CRC VS. 90 NCs). We identified that serum levels of miR-19a-3p, miR-21-5p and miR-425-5p were significantly higher in patients with CRC than in NCs. The areas under the receiver operating characteristic (ROC) curve of the three-miRNA panel were 0.86, 0.74 and 0.87 for the training, testing and the external validation stages (30 CRC VS. 18 NCs), respectively. Significantly, elevated expression of the three miRNAs was also observed in CRC tissues (n = 24). Furthermore, the expression levels of the three miRNAs were significantly elevated in exosomes from CRC serum samples (n = 10). In conclusion, we identified a serum three-miRNA panel for the diagnosis of CRC.

In contrast to normal differentiated cells that depend on aerobicoxidation for energy production, cancer cells use aerobic glycolysis as the main source (Warburg's effect). The M2 splice isoform of pyruvate kinase (PKM2) is the key regulator for the aerobic glycolysis, high expression of PKM2 contributes to the aerobic glycolysis, promotes the growth of tumors. In the present study, we found that PKM2 was highly expressed in gastric cancer (GC) tissues and had a strongly inverse correlation with the expression of microRNA-let-7a (miR-let-7a). Furthermore, we found that the overexpression of miR-let-7a markedly suppressed the proliferation, migration, and invasion of GC cells by down-regulating the expression of PKM2. MicroRNAs (miRNAs) are important regulators play key roles in tumorigenesis and tumor progression. Although previous reports showed that let-7 family members act as tumor suppressors in many cancers. The specific regulatory mechanism of miR-let-7a to PKM2 in gastric cancer is still unclear. In this study, we revealed that miR-let-7a function as the antitumor and gene regulatory effects of PKM2 in GC cells.

Beneficial effects of the Chinese herbal medicine Qushi Huayu Decoction (QHD) were observed with non-alcoholic fatty liver disease (NAFLD) patients and animal models. The impact of QHD or its active components (geniposide and chlorogenic acid, GC) on NAFLD liver transcriptome and gut microbiota was examined with NAFLD rats. Increased expression for genes required for glutathione production and decreased expression for genes required for lipid synthesis was observed in NAFLD livers treated with QHD and GC. GC treatment decreased serum LPS, which could be explained by reduced mucosal damage in the colon of GC-treated rats. Further, our data suggest an increased abundance of Treg-inducing bacteria that stimulated the Treg activity in GC treated colon, which in turn down-regulated inflammatory signals, improved gut barrier function and consequently reduced hepatic exposure to microbial products. Our study suggests that QHD simultaneously enhanced the hepatic anti-oxidative mechanism, decreased hepatic lipid synthesis, and promoted the regulatory T cell inducing microbiota in the gut.

We investigated the differential expression of circular RNAs (circRNAs) in plasma samples from three coronary artery disease (CAD) patients to identify putative therapeutic targets. We identified 24 differentially expressed circRNAs (18 up-regulated and 6 down-regulated) and 7 differentially expressed mRNAs (6 up-regulated and 1 down-regulated) in CAD patients based on competing endogenous RNA (ceRNA) microarray analysis. MiR-221(p = 0.001), miR-155(p = 0.049), and miR-130a (p = 0.001) were downregulated in CAD patients based on qRT-PCR analysis of another independent population of 932 study subjects (648 CAD subjects and 284 controls). We constructed a hsa-miR-130a-3p-mediated circRNA-mRNA ceRNA network using the miRanda database. This included 9 circRNAs (hsa_circ_0089378, hsa_circ_0083357, hsa_circ_0082824, hsa_circ_0068942, hsa_circ_0057576, hsa_circ_0054537, hsa_circ_0051172, hsa_circ_0032970, and hsa_circ_0006323) and 1 mRNA (transient receptor potential cation channel subfamily M member 3 [TRPM3]). We have shown that 9 circRNAs promote TRPM3 expression by inhibiting hsa-miR-130a-3p in CAD patients.

Over-expression or amplification of ERBB2 is observed in multifarious carcinomas. However, the molecular mechanism of ERBB2 downregulation in ERBB2-positive cancers remains obscure. This experiment investigated the suppressive role of miR-3622b-5p in ERBB2-positive breast and gastric cancers. The luciferase activity of ERBB2 3'-untranslated region-based reporters constructed in HEK-293T, SK-BR-3 and MCF-10A cells suggested that ERBB2 was the target gene of miR-3622b-5p. Over-expressed miR-3622b-5p reduced the protein level of ERBB2, weakened the activation of mTORC1/S6, and induced the apoptosis of ERBB2-positive cancer cells. MiR-3622b-5p was significantly down-regulated in breast and gastric cancer tissues. This down-regulation in ERBB2-positive breast and gastric cancer tissues was more obvious than that in ERBB2-negative breast and gastric cancer tissues. MiR-3622b-5p turned ERBB2-positive cancer cells more vulnerable to the apoptosis induced by cisplatin and 5-fluorouracil. Taken together, miR-3622b-5p is involved in the proliferation and apoptosis of human ERBB2-positive cancer cells via targeting ERBB2/mTORC1 signaling pathway.