// Gianluigi Mazzoccoli 1 , Tommaso Colangelo 2, * , Anna Panza 3, * , Rosa Rubino 1, * , Cristiana Tiberio 1 , Orazio Palumbo 4 , Massimo Carella 4 , Domenico Trombetta 5 , Annamaria Gentile 3 , Francesca Tavano 3 , Maria Rosa Valvano 3 , Clelia Tiziana Storlazzi 6 , Gemma Macchia 6 , Angelo De Cata 1 , Giovanni Bisceglia 7 , Daniele Capocefalo 8, * , Vittorio Colantuoni 2, # , Lina Sabatino 2, # , Ada Piepoli 9, # , Tommaso Mazza 8 1 Division of Internal Medicine and Chronobiology Unit, IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy 2 Department of Sciences and Technologies, University of Sannio, Benevento, Italy 3 Division of Gastroenterology and Research Laboratory, IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy 4 Medical Genetics Service, IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy 5 Oncology-Research Laboratory, IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy 6 Department of Biology, University of Bari, Bari, Italy 7 Department of Surgical Sciences, IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy 8 Bioinformatics Unit, IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy 9 Division of Epidemiology and Health Statistics, IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy * These junior authors contributed equally to this work # These senior authors contributed equally to this work Correspondence to: Gianluigi Mazzoccoli, email: g.mazzoccoli@operapadrepio.it Tommaso Mazza, email: t.mazza@css-mendel.it Keywords: miRNA-mRNA, clock genes, circadian, colorectal cancer Received: April 29, 2016 Accepted: May 29, 2016 Published: June 14, 2016 ABSTRACT Altered functioning of the biological clock is involved in cancer onset and progression. MicroRNAs (miRNAs) interact with the clock genes modulating the function of genetically encoded molecular clockworks. Collaborative interactions may take place within the coding-noncoding RNA regulatory networks. We aimed to evaluate the cross-talk among miRNAs and clock genes in colorectal cancer (CRC). We performed an integrative analysis of miRNA-miRNA and miRNA-mRNA interactions on high-throughput molecular profiling of matched human CRC tissue and non-tumor mucosa, pinpointing core clock genes and their targeting miRNAs. Data obtained in silico were validated in CRC patients and human colon cancer cell lines. In silico we found severe alterations of clock gene–related coding-noncoding RNA regulatory networks in tumor tissues, which were later corroborated by the analysis of human CRC specimens and experiments performed in vitro . In conclusion, specific miRNAs target and regulate the transcription/translation of clock genes and clock gene-related miRNA-miRNA as well as mRNA-miRNA interactions are altered in colorectal cancer. Exploration of the interplay between specific miRNAs and genes, which are critically involved in the functioning of the biological clock, provides a better understanding of the importance of the miRNA-clock genes axis and its derangement in colorectal cancer.
Circadian disruption and deranged molecular clockworks are involved in carcinogenesis. The cryptochrome genes (CRY1 and CRY2) encode circadian proteins important for the functioning of biological oscillators. Their expression in human colorectal cancer (CRC) and in colon cancer cell lines has not been evaluated so far. We investigated CRY1 and CRY2 expression in fifty CRCs and in the CaCo2, HCT116, HT29, SW480 cell lines. CRY1 (p = 0.01) and CRY2 (p < 0.0001) expression was significantly changed in tumour tissue, as confirmed in a large independent CRC dataset. In addition, lower CRY1 mRNA levels were observed in patients in the age range of 62-74 years (p = 0.018), in female patients (p = 0.003) and in cancers located at the transverse colon (p = 0.008). Lower CRY2 levels were also associated with cancer location at the transverse colon (p = 0.007). CRC patients displaying CRY1 (p = 0.042) and CRY2 (p = 0.043) expression levels over the median were hallmarked by a poorer survival rate. Survey of selected colon cancer cell lines evidenced variable levels of cryptochrome genes expression and time-dependent changes in their mRNA levels. Moreover, they showed reduced apoptosis, increased proliferation and different response to 5-fluorouracil and oxaliplatin upon CRY1 and CRY2 ectopic expression. The relationship with p53 status came out as an additional layer of regulation: higher CRY1 and CRY2 protein levels coincided with a wild type p53 as in HCT116 cells and this condition only marginally affected the apoptotic and cell proliferation characteristics of the cells upon CRY ectopic expression. Conversely, lower CRY and CRY2 levels as in HT29 and SW480 cells coincided with a mutated p53 and a more robust apoptosis and proliferation upon CRY transfection. Besides, an heterogeneous pattern of ARNTL, WEE and c-MYC expression hallmarked the chosen colon cancer cell lines and likely influenced their phenotypic changes. Cryptochrome gene expression is altered in CRC, particularly in elderly subjects, female patients and cancers located at the transverse colon, affecting overall survival. Altered CRY1 and CRY2 expression patterns and the interplay with the genetic landscape in colon cancer cells may underlie phenotypic divergence that could influence disease behavior as well as CRC patients survival and response to chemotherapy.
The clock gene machinery controls cellular metabolism, proliferation, and key functions, such as DNA damage recognition and repair. Dysfunction of the circadian clock is involved in tumorigenesis, and altered expression of some clock genes has been found in cancer patients. The aim of this study was to evaluate the expression levels of core clock genes in colorectal cancer (CRC). Quantitative real-time polymerase chain reaction (qPCR) was used to examine ARNTL1, CLOCK, PER1, PER2, PER3, CRY1, CRY2, Timeless (TIM), TIPIN, and CSNK1? expression levels in the tumor tissue and matched apparently healthy mucosa of CRC patients. In the tumor tissue of CRC patients, compared to their matched healthy mucosa, expression levels of ARNTL1 (p=.002), PER1 (p=.002), PER2 (p=.011), PER3 (p=.003), and CRY2 (p=.012) were lower, whereas the expression level of TIM (p=.044) was higher. No significant difference was observed in the expression levels of CLOCK (p=.778), CRY1 (p=.600), CSNK1 (p=.903), and TIPIN (p=.136). As to the clinical and pathological features, a significant association was found between low CRY1 expression levels in tumor mucosa and age (p=.026), and female sex (p=.005), whereas high CRY1 expression levels in tumor mucosa were associated with cancer location in the distal colon (p?=?.015). Moreover, high TIM mRNA levels in the tumor mucosa were prevalent whenever proximal lymph nodes were involved (p= .013) and associated with TNM stages III-IV (p=.005) and microsatellite instability (p=.015). Significantly poorer survival rates were evidenced for CRC patients with lower expression in the tumor tissue of PER1 (p=.010), PER3 (p= .010), and CSNKIE (p=.024). In conclusion, abnormal expression levels of core clock genes in CRC tissue may be related to the process of tumorigenesis and exert an influence on host/tumor interactions.
Idiopathic achalasia is a disease that is characterized by the absence of peristalsis and incomplete relaxation of the lower esophageal sphincter, which is accompanied by dysphagia, regurgitation, chest pain and weight loss. The role of inflammatory infiltrates in the pathogenesis of achalasia remains controversial, although the infiltrating cell profile in the tissue has been previously characterized histologically and immunohistochemically. The present study aimed to evaluate the serum levels of 27 protein biomarkers to determine their association with achalasia and the clinical disease characteristics. The cytokine, chemokine and growth factor serum profiles of 68 patients with achalasia and 39 healthy individuals were explored using the 27‑Bio‑Plex Pro Human Cytokine assay. Reductions in the levels of inflammatory mediators IL‑1β, IL‑2, IL‑5, IL‑6, IL‑8, IL‑10, IL‑12p70, IL‑13, IL‑15, IL‑17, fibroblast growth factor, granulocyte colony‑stimulating factor, granulocyte‑macrophage colony‑stimulating factor, interferon‑γ, monocyte chemoattractant protein‑1, macrophage inflammatory protein‑1 (MIP‑1)α and MIP‑1β, regulated upon activation normal T cell expressed and presumably secreted, TNF‑α and VEGF were detected in the serum samples of patients with achalasia compared with those in the control group (P<0.05). However, significant associations between the expression in the levels of inflammatory factors and clinical characteristics of the patients were not found (P>0.05). These results suggest that achalasia is a disease that has a local but not a systemic inflammatory pattern. Further studies are required to improve the current understanding of the mechanism underlying this disease.
Abstract Background The malignant transformation of precancerous colorectal lesions involves progressive alterations at both the molecular and morphologic levels, the latter consisting of increases in size and in the degree of cellular atypia. Analyzing preinvasive tumors of different sizes can therefore shed light on the sequence of these alterations. Methods We used a molecular pathway-based approach to analyze transcriptomic profiles of 59 colorectal tumors representing early and late preinvasive stages and the invasive stage of tumorigenesis. Random set analysis was used to identify biological pathways enriched for genes differentially regulated in tumors (compared with 59 samples of normal mucosa). Results Of the 880 canonical pathways we investigated, 112 displayed significant tumor-related upregulation or downregulation at one or more stages of tumorigenesis. This allowed us to distinguish between pathways whose dysregulation is probably necessary throughout tumorigenesis and those whose involvement specifically drives progression from one stage to the next. We were also able to pinpoint specific changes within each gene set that seem to play key roles at each transition. The early preinvasive stage was characterized by cell-cycle checkpoint activation triggered by DNA replication stress and dramatic downregulation of basic transmembrane signaling processes that maintain epithelial/stromal homeostasis in the normal mucosa. In late preinvasive lesions, there was also downregulation of signal transduction pathways (e.g., those mediated by G proteins and nuclear hormone receptors) involved in cell differentiation and upregulation of pathways governing nuclear envelope dynamics and the G2>M transition in the cell cycle. The main features of the invasive stage were activation of the G1>S transition in the cell cycle, upregulated expression of tumor-promoting microenvironmental factors, and profound dysregulation of metabolic pathways (e.g., increased aerobic glycolysis, downregulation of pathways that metabolize drugs and xenobiotics). Conclusions Our analysis revealed specific pathways whose dysregulation might play a role in each transition of the transformation process. This is the first study in which such an approach has been used to gain further insights into colorectal tumorigenesis. Therefore, these data provide a launchpad for further exploration of the molecular characterization of colorectal tumorigenesis using systems biology approaches.
Celiac disease (CD) is an autoimmune disease with the destruction of small intestinal villi, which occurs in genetically predisposed individuals. At the present moment, a gluten-free diet (GFD) is the only way to restore the functionality of gut mucosa. However, there is an open debate on the effects of long-term supplementation through a GFD, because some authors report an unbalance in microbial taxa composition.For microbiome analysis, fecal specimens were collected from 46 CD individuals in GFD for at least 2 years and 30 specimens from the healthy controls (HC). Data were analyzed using an ensemble of software packages: QIIME2, Coda-lasso, Clr-lasso, Selbal, PICRUSt2, ALDEx2, dissimilarity-overlap analysis, and dysbiosis detection tests.The adherence to GFD restored the alpha biodiversity of the gut microbiota in celiac people but microbial composition at beta diversity resulted as different to HC. The microbial composition of the CD subjects was decreased in a number of taxa, namely Bifidobacterium longum and several belonging to Lachnospiraceae family, whereas Bacteroides genus was found to be more abundant. Predicted metabolic pathways among the CD bacterial communities revealed an important role in tetrapyrrole biosynthesis.CD patients in GFD had a non-dysbiotic microbial composition for the crude alpha diversity metrics. We found significant differences in beta diversity, in certain taxon, and pathways between subjects with inactive CD in GFD and controls. Collectively, our data may suggest the development of new GFD products by modulating the gut microbiota through diet, supplements of vitamins, and the addition of specific prebiotics.
Abstract Background Achalasia is a rare idiopathic disease with a complex etio‐pathogenesis still unknown. This study aimed to identify microRNA (miRNA)‐mRNA regulatory networks underlying achalasia. Methods The investigation was performed in tissue specimens from 11 patients and five controls using the microarray technology followed by an integrated bioinformatics analysis. Key Results One hundred and six miRNAs were significantly up‐regulated and 64 were down‐regulated in achalasia patients. The expression of the most 10 differential expressed miRNAs (miR‐122‐5p, miR‐133a‐3p, miR‐504‐5p, miR‐187‐3p, miR‐133b, miR‐200c‐3p, miR‐375, miR‐200b‐5p, miR‐200b‐3p, and miR203a) was confirmed by droplet digital PCR in an independent cohort. The interactions between the significant miRNAs and their targets uncovered 14 miRNA‐mRNA interacting pairs with experimentally predicted genes (ie, FN1 , ROCK2 , DPYSL2 ), and 35 pairs with not experimentally target genes (ie, SULF1 , MRVI1 , PRKG1 ); all genes were involved in immune cell trafficking , skeletal and muscular system development , nervous system development macro‐processes. Conclusion & Inferences The mRNA–miRNA regulatory networks described in this study provide new insights in the genetic background of the disease, suggesting further investigations in novel pathogenic mechanisms.
This study was conducted to evaluate the association of the leucine-rich repeat (LRR) gene family with colorectal cancer (CRC). The expression of members of the LRR gene family were analyzed in 17 CRC specimens and in 59 healthy colorectal tissues by using Human Exon1.0ST microarray, and in 25 CRC specimens and 32 healthy colorectal tissues by U133Plus2.0 microarray. An association was found for 25 genes belonging to the plant-specific (PS) class of LRR genes ( P = 0.05 for Exon1.0 ST and P = 0.04 for U133Plus2.0). In both data-sets, in CRC, we found down-regulation of SHOC2 ( P < 0.00003) and LRRC28 ( P < 0.01) and up-regulation of LRSAM1 ( P < 0.000001), while up-regulation of MFHAS1 ( P = 0.0005) and down-regulation of WDFY3 ( P = 0.026) were found only in the Exon1.0 ST data-set. The PS LLR gene class encodes proteins that activate immune cells and might play a key role in programmed cell death and autophagy. SHOC2 and LRRC28 genes involved in RAS-mediated signaling, which hinders nutrient deprivation-induced autophagy, might be a possible link between the negative control of autophagy and tumorigenesis.
