Role of miR-182 in neoplastic progression of colorectal cancer

Numerous studies have demonstrated that aberrant expressions of specific microRNAs (miRNAs) are involved in many cancer types including colorectal cancer (CRC). In particular, in a previous study we integrated miRNA and target gene expression data obtained from chip array by comparing normal colon tissue, primary tumor and liver metastasis, and we focused our attention on post-transcriptional regulatory networks with differentially expressed miRNAs and their supported relations with target genes. We demonstrated that miR-182 was one of the most up-regulated miRNAs in primary CRC compared to normal colon mucosa. Starting from these premises, the project was focused on the following tasks: 1) Identification of miRNA biomarkers for CRC monitoring and screening in colon cancer patients; 2) Analysis of the functional effects of miR-182 inhibition in CRC cell lines characterized by a different in vivo tumorigenic behavior. Regarding the first task in my first year of PhD we published a paper (68, Appendix 1) to confirm the involvement of miR-182 in CRC development and progression. In particular, a total of 240 histopathological and 51 plasma samples were included in this study. We observed a significant overexpression of miR-182 in CRC primary tumor compared to normal colon mucosa, which is also maintained in CRC liver metastases. Then, we also demonstrated that plasma miR-182 levels are significantly higher in CRC patients than in healthy controls. Moreover, miR-182 plasma levels were significantly reduced in post-operative samples after radical hepatic metastasectomy, compared to pre-operative samples. These results indicated that the evaluation of circulating miR-182 levels could be a promising approach to improve the repertoire of non-invasive blood based biomarkers for CRC monitoring and screening. To strengthen these evidences, we carried out a prospective study in stage I-II (N0 M0) colon cancer. We preselected four strongly up-regulated miRNAs involved in the same post-transcriptional sub-network (miR-18a, miR-21, miR-182 and miR-183) and the most down-regulated miRNA (miR-139) and we confirmed that all the selected miRNAs are significantly modulated in colon cancer compared to normal colon mucosa. Moreover, we observed that miR-182, miR-183 and miR-139 were not modulated in inflammatory tissue compared to colon mucosa; by contrast miR-18a and miR-21 are significantly up-regulated also in the inflammation-related process. To investigate whether the selected miRNAs could be useful to predict tumor relapse the patients were subdivided in Recurrent and Non Recurrent groups within 55 months. We calculated 10 ratios between the expression values of all possible miRNA pairs, applying the miRNA ratio approach both in the tumor tissue and in the adjacent normal mucosa. None of the miRNA ratios resulted predictive when evaluated in the colon cancer tissue, instead three miRNA ratios evaluated in the tumor-adjacent mucosa were found to be significant predictors of relapse by 55 months from resection: miR-21/miR-183, miR-18a/miR-182 and miR-18a/miR-183. Manuscript submitted. Regarding the second task, to gain insights in the functional role played by miR-182 in the tumorigenesis we investigated the effects of miR-182 inhibition. To this end, we used two CRC cell lines as in vitro models: MICOL-14h-tert (an in vivo non-tumorigenic cell line derived from a lymph node metastasis of rectal cancer,) and its in vivo tumorigenic variant MICOL-14tum (or TC22). We carried out transfection experiments for the transient inhibition of miR-182 and we observed a significant increase of cell apoptosis in both cell lines after the treatment. We confirmed the results with cleaved PARP and Caspase-3 proteins detection by Western Blot. Therefore, we evaluated the effect of miR-182 inhibition on in vivo tumor growth. To this end, we injected subcutaneously the TC22 cells treated with the anti-miR-182 in NOD/SCID mice and, after a week, we performed also an in vivo intra-tumor injection of miR-182 inhibitor to maintain the silencing. Interestingly, the inhibition of miR-182 significantly reduced the size of the tumor, and the obtained mass exhibited a pattern of features as less aggressive tumors compared to controls. Manuscript in preparation. In conclusions: - miR-182 expression levels can be followed in tissues and plasma of CRC patients. In particular, circulating miR-182 evaluation could be a promising approach to enhance the repertoire for blood based biomarkers in non-invasive CRC monitoring and screening; - the panel of selected miRNAs were significantly regulated also in the early phases of the CRC tumor process extending to stage I-II the results obtained in our previous work in stage IV CRC; - not a single miRNA, but rather a coordinated alteration of four miRNAs may be useful to predict recurrence after resection in early CRC when evaluate in the normal mucosa adjacent to tumor; - in CRC cell lines the expression level of miR-182 is higher in in vivo tumorigenic variant, suggesting a role of this miRNA in tumor aggressiveness. MiR-182 seems to be involved in increasing the survival of cancer cells and enhance the tumor growth

A coordinate deregulation of microRNAs expressed in mucosa adjacent to tumor predicts relapse after resection in localized colon cancer

Up to 20% of colorectal cancer (CRC) node-negative patients develop loco-regional or distant recurrences within 5 years from surgery. No predictive biomarker able to identify the node-negative subjects at high risk of relapse after curative treatment is presently available.Forty-eight localized (i.e. stage I-II) colon cancer patients who underwent radical tumor resection were considered. The expression of five miRNAs, involved in CRC progression, was investigated by qRT-PCR in both tumor tissue and matched normal colon mucosa.Interestingly, we found that the coordinate deregulation of four miRNAs (i.e. miR-18a, miR-21, miR-182 and miR-183), evaluated in the normal mucosa adjacent to tumor, is predictive of relapse within 55 months from curative surgery.Our results, if confirmed in independent studies, may help to identify high-risk patients who could benefit most from adjuvant therapy. Moreover, this work highlights the importance of extending the search for tissue biomarkers also to the tumor-adjacent mucosa

Profiling Insulin Like Factor 3 (INSL3) Signaling in Human Osteoblasts

Abstract BACKGROUND: Young men with mutations in the gene for the INSL3 receptor (Relaxin family peptide 2, RXFP2) are at risk of reduced bone mass and osteoporosis. Consistent with the human phenotype, bone analyses of Rxfp2(-/-) mice showed decreased bone volume, alterations of the trabecular bone, reduced mineralizing surface, bone formation, and osteoclast surface. The aim of this study was to elucidate the INSL3/RXFP2 signaling pathways and targets in human osteoblasts. METHODOLOGY/PRINCIPAL FINDINGS: Alkaline phosphatase (ALP) production, protein phosphorylation, intracellular calcium, gene expression, and mineralization studies have been performed. INSL3 induced a significant increase in ALP production, and Western blot and ELISA analyses of multiple intracellular signaling pathway molecules and their phosphorylation status revealed that the MAPK was the major pathway influenced by INSL3, whereas it does not modify intracellular calcium concentration. Quantitative Real Time PCR and Western blotting showed that INSL3 regulates the expression of different osteoblast markers. Alizarin red-S staining confirmed that INSL3-stimulated osteoblasts are fully differentiated and able to mineralize the extracellular matrix. CONCLUSIONS/SIGNIFICANCE: Together with previous findings, this study demonstrates that the INSL3/RXFP2 system is involved in bone metabolism by acting on the MAPK cascade and stimulating transcription of important genes of osteoblast maturation/differentiation and osteoclastogenesis

From Heterogeneous Sensor Networks to Integrated Software Services: Design and Implementation of a Semantic Architecture for the Internet of Things at ARCES@UNIBO

The Internet of Things (IoTs) is growing fast both in terms of number of devices connected and of complexity of deployments and applications. Several research studies an- alyzing the economical impact of the IoT worldwide identify the interoperability as one of the main boosting factor for its growth, thanks to the possibility to unlock novel commercial opportunities derived from the integration of heterogeneous systems which are currently not interconnected. However, at present, interoperability constitutes a relevant practical issue on any IoT deployments that is composed of sensor platforms mapped on different wireless technologies, network protocols or data formats. The paper addresses such issue, and investigates how to achieve effective data interoperability and data reuse on complex IoT deployments, where multiple users/applications need to consume sensor data produced by heterogeneous sensor networks. We propose a generic three-tier IoT architecture, which decouples the sensor data producers from the sensor data consumers, thanks to the intermediation of a semantic broker which is in charge of translating the sensor data into a shared ontology, and of providing publish-subscribe facilities to the producers/consumers. Then, we describe the real-world implementation of such architecture devised at the Advanced Research Center on Electronic System (ARCES) of the University of Bologna. The actual system collects the data produced by three different sensor networks, integrates them through a SPARQL Event Processing Architecture (SEPA), and supports two front- end applications for the data access, i.e. a web dashboard and an Amazon Alexa voice service

Role of miR-182 in neoplastic progression of colorectal cancer

Numerous studies have demonstrated that aberrant expressions of specific microRNAs (miRNAs) are involved in many cancer types including colorectal cancer (CRC). In particular, in a previous study we integrated miRNA and target gene expression data obtained from chip array by comparing normal colon tissue, primary tumor and liver metastasis, and we focused our attention on post-transcriptional regulatory networks with differentially expressed miRNAs and their supported relations with target genes. We demonstrated that miR-182 was one of the most up-regulated miRNAs in primary CRC compared to normal colon mucosa. Starting from these premises, the project was focused on the following tasks: 1) Identification of miRNA biomarkers for CRC monitoring and screening in colon cancer patients; 2) Analysis of the functional effects of miR-182 inhibition in CRC cell lines characterized by a different in vivo tumorigenic behavior. Regarding the first task in my first year of PhD we published a paper (68, Appendix 1) to confirm the involvement of miR-182 in CRC development and progression. In particular, a total of 240 histopathological and 51 plasma samples were included in this study. We observed a significant overexpression of miR-182 in CRC primary tumor compared to normal colon mucosa, which is also maintained in CRC liver metastases. Then, we also demonstrated that plasma miR-182 levels are significantly higher in CRC patients than in healthy controls. Moreover, miR-182 plasma levels were significantly reduced in post-operative samples after radical hepatic metastasectomy, compared to pre-operative samples. These results indicated that the evaluation of circulating miR-182 levels could be a promising approach to improve the repertoire of non-invasive blood based biomarkers for CRC monitoring and screening. To strengthen these evidences, we carried out a prospective study in stage I-II (N0 M0) colon cancer. We preselected four strongly up-regulated miRNAs involved in the same post-transcriptional sub-network (miR-18a, miR-21, miR-182 and miR-183) and the most down-regulated miRNA (miR-139) and we confirmed that all the selected miRNAs are significantly modulated in colon cancer compared to normal colon mucosa. Moreover, we observed that miR-182, miR-183 and miR-139 were not modulated in inflammatory tissue compared to colon mucosa; by contrast miR-18a and miR-21 are significantly up-regulated also in the inflammation-related process. To investigate whether the selected miRNAs could be useful to predict tumor relapse the patients were subdivided in Recurrent and Non Recurrent groups within 55 months. We calculated 10 ratios between the expression values of all possible miRNA pairs, applying the miRNA ratio approach both in the tumor tissue and in the adjacent normal mucosa. None of the miRNA ratios resulted predictive when evaluated in the colon cancer tissue, instead three miRNA ratios evaluated in the tumor-adjacent mucosa were found to be significant predictors of relapse by 55 months from resection: miR-21/miR-183, miR-18a/miR-182 and miR-18a/miR-183. Manuscript submitted. Regarding the second task, to gain insights in the functional role played by miR-182 in the tumorigenesis we investigated the effects of miR-182 inhibition. To this end, we used two CRC cell lines as in vitro models: MICOL-14h-tert (an in vivo non-tumorigenic cell line derived from a lymph node metastasis of rectal cancer,) and its in vivo tumorigenic variant MICOL-14tum (or TC22). We carried out transfection experiments for the transient inhibition of miR-182 and we observed a significant increase of cell apoptosis in both cell lines after the treatment. We confirmed the results with cleaved PARP and Caspase-3 proteins detection by Western Blot. Therefore, we evaluated the effect of miR-182 inhibition on in vivo tumor growth. To this end, we injected subcutaneously the TC22 cells treated with the anti-miR-182 in NOD/SCID mice and, after a week, we performed also an in vivo intra-tumor injection of miR-182 inhibitor to maintain the silencing. Interestingly, the inhibition of miR-182 significantly reduced the size of the tumor, and the obtained mass exhibited a pattern of features as less aggressive tumors compared to controls. Manuscript in preparation. In conclusions: - miR-182 expression levels can be followed in tissues and plasma of CRC patients. In particular, circulating miR-182 evaluation could be a promising approach to enhance the repertoire for blood based biomarkers in non-invasive CRC monitoring and screening; - the panel of selected miRNAs were significantly regulated also in the early phases of the CRC tumor process extending to stage I-II the results obtained in our previous work in stage IV CRC; - not a single miRNA, but rather a coordinated alteration of four miRNAs may be useful to predict recurrence after resection in early CRC when evaluate in the normal mucosa adjacent to tumor; - in CRC cell lines the expression level of miR-182 is higher in in vivo tumorigenic variant, suggesting a role of this miRNA in tumor aggressiveness. MiR-182 seems to be involved in increasing the survival of cancer cells and enhance the tumor growth.Numerosi studi hanno dimostrato che l’alterata espressione di specifici microRNA (miRNAs) è coinvolta in molti tipi di tumore, incluso il cancro del colon-retto (CRC). In particolare, in un precedente studio abbiamo integrato dati di espressione di miRNA e geni ottenuti da chip array, confrontando tessuto colico normale, tumore primario e metastasi epatiche, e abbiamo concentrato la nostra attenzione sulle reti regolatorie post-trascrizionali con miRNA differenzialmente espressi e le loro relazioni supportate con i geni bersaglio. Abbiamo dimostrato che il miR-182 risultava essere uno dei più up-regolati nel CRC primario rispetto alla mucosa normale di colon. Partendo da questi persupposti, gli obiettivi del progetto sono stati i seguenti: 1) Identificazione di miRNAs come possibili biomarcatori per il monitoraggio e lo screening nei pazienti con tumore del colon; 2) Analisi degli effetti funzionali indotti dall'inibizione del miR-182 in linee cellulari di tumore del colon caratterizzate da un diverso comportamento tumorigenico in vivo. Per quanto riguarda il primo obiettivo, abbiamo pubblicato un lavoro (68, appendice 1) in cui si conferma il coinvolgimento del miR-182 nello sviluppo e nella progressione del CRC. In particolare, in questo studio sono stati inclusi complessivamente 240 campioni istopatologici e 51 plasmatici. Abbiamo osservato una significativa sovra-espressione di miR-182 nel tumore primario rispetto alla mucosa normale di colon, la quale si mantiene anche nelle metastasi epatiche. Abbiamo anche dimostrato che i livelli plasmatici di miR-182 sono significativamente più elevati nei pazienti con CRC rispetto ai controlli sani. Inoltre, i livelli di miR-182 nel plasma si riducono significativamente dopo la metastasectomia radicale epatica. Questi risultati suggeriscono che la valutazione dei livelli di miR-182 circolante potrebbero essere un approccio promettente per ampliare la disponibilità di biomarcatori non invasivi per il monitoraggio e lo screening di questi pazienti. Per rafforzare queste evidenze, abbiamo effettuato uno studio prospettico su una casistica indipendente di CRC allo stadio I-II (N0 M0) e abbiamo pre-selezionato alcuni miRNA fortemente regolati: il miR-139 e 4 coinvolti nella stessa rete post-trascrizionale (miR-18a, miR-21, miR-182 e miR-183). Questi 5 miRNA sono significativamente modulati nel tessuto tumorale rispetto alla mucosa normale di colon. Inoltre, abbiamo osservato che miR-182, miR-183 e miR-139 non risultano modulati nel tessuto infiammatorio rispetto alla mucosa normale; per contro, il miR-18a e il miR-21 sono significativamente regolati anche nel processo legato all'infiammazione. Per valutare il possibile ruolo predittivo di questi miRNA i pazienti sono stati suddivisi in gruppi ricorrenti e non ricorrenti entro 55 mesi. Abbiamo quindi calcolato 10 ratio tra i valori di espressione di tutte le possibili coppie di miRNA, sia nel tessuto tumorale che nella mucosa normale adiacente. Nessuno dei miRNA ratio è risultato predittivo quando valutato nel tessuto tumorale; tre miRNA ratio valutati nella mucosa adiacente al tumore risultano, invece, predittori significativi della ricaduta a 55 mesi dalla resezione: miR-21/miR-183, miR-18a/miR-182 e miR-18a/miR-183. Manuscript submitted. Per quanto riguarda il secondo obiettivo, per approfondire il ruolo funzionale svolto dal miR-182 nella tumorigenesi abbiamo valutato gli effetti indotti dalla sua inibizione. A questo scopo, abbiamo utilizzato due linee di CRC come modello in vitro: MICOL-14h-tert (una linea cellulare non tumorigenica in vivo derivata da metastasi linfonodali del cancro rettale), e la sua variante tumorigenica in vivo MICOL-14tum ( o TC22). Abbiamo effettuato esperimenti di trasfezione per l'inibizione transitoria del miR-182 e abbiamo osservato un significativo aumento dell'apoptosi in entrambe le linee cellulari dopo il trattamento. Abbiamo confermato i risultati mediante l’identificazione di PARP e Caspasi-3 in Western Blot. Inoltre abbiamo valutato l'effetto dell'inibizione di miR-182 sulla crescita tumorale in vivo. A tal fine abbiamo eseguito inoculi sottocute di cellule TC22 trattate con anti-miR-182 in topi NOD/SCID, e dopo una settimana abbiamo eseguito un'ulteriore iniezione intra-tumorale in vivo di anti-miR-182 per mantenere il silenziamento. È interessante notare che l'inibizione del miR-182 riduce significativamente la dimensione del tumore, il quale presenta caratteri di minore aggressività rispetto ai controlli. Manuscript in preparation. In conclusione: - i livelli di espressione del miR-182 possono essere rilevati nel tessuto e nel plasma dei pazienti affetti da CRC. In particolare, la valutazione del miR-182 circolante potrebbe essere un approccio promettente per migliorare il repertorio di biomarcatori non invasivi per il monitoraggio e lo screening; - il gruppo di miRNA selezionati è risultato significativamente regolato anche nelle fasi iniziali del processo tumorale, estendendo alla fase I-II i risultati già ottenuti nei CRC fase IV; - non un singolo miRNA, ma piuttosto un'alterazione coordinata di quattro miRNA può essere utile per prevedere la ricorrenza dopo la resezione nel CRC precoce quando valutata nella mucosa normale adiacente al tumore; - nelle linee cellulari di CRC il livello di espressione del miR-182 è più elevato nella variante tumorigenica in vivo, suggerendo un suo ruolo nell’aggressività tumorale. In particolare sembra essere coinvolto nel sostenere la sopravvivenza delle cellule tumorali e la crescita del tumore stesso

How the human spermatozoa sense the oocyte: a new role of SDF1-CXCR4 signalling.

For fertilization to occur in mammals, ejaculated spermatozoa must reach the egg which, following ovulation has moved from the ovary into the Fallopian tube. Two active mechanisms of spermatozoa guidance have been shown in mammals: thermotaxis and chemotaxis. The identity of most of human spermatozoa chemoattractants is unknown, and herein we tested if SDF1 (chemokine stromal cell-derived factor-1) and its pathway is involved in spermatozoa chemotaxis. We found that SDF1 is expressed in the oocytes, endometrium and follicular fluid, as well as its specific receptor CXCR4 (chemokine CXC motif receptor 4) is expressed in the head of spermatozoa. By SDF1 gradient experiments, we stated that SDF1 is able to induce hyperactivation in spermatozoa leading to accumulation, to give rise to an increase in intracellular calcium concentration, and to preserve the mitochondrial status and not to induce acrosome reaction. Our findings suggest these phenomena could reflect spermatozoa chemotaxis, and that SDF1 action could represent an important event leading to egg fertilization, even if further studies regarding the link between spermatozoa accumulation and chemotaxis are mandatory. These data suggest that the SDF-1/CXCR4 signalling could be used to manipulate the human fertilization, to improve both the outcome of physiological or assisted reproduction, and to develop new contraceptive methods, by development of SDF1 or CXCR4 antagonist

Western blotting for Col6A1, osteonectin, and M-CSF of human osteoblasts stimulated by INSL3.

<p>A dose-dependent increase in protein level is evident, confirming quantitative RT-PCR results.</p