Cinzia Zucchini

Identification of circulating placental mRNA in maternal blood of pregnancies affected with fetal congenital heart diseases at the second trimester of pregnancy: implications for early molecular screening

To investigate whether a significantly aberrant expression of circulating placental mRNA genes related with cardiogenesis can be detected at the second trimester of pregnancy.

CD99 suppresses osteosarcoma cell migration through inhibition of ROCK2 activity

CD99, a transmembrane protein encoded by MIC2 gene is involved in multiple cellular events including cell adhesion and migration, apoptosis, cell differentiation and regulation of protein trafficking either in physiological or pathological conditions. In osteosarcoma, CD99 is expressed at low levels and functions as a tumour suppressor. The full-length protein (CD99wt) and the short-form harbouring a deletion in the intracytoplasmic domain (CD99sh) have been associated with distinct functional outcomes with respect to tumour malignancy. In this study, we especially evaluated modulation of cell–cell contacts, reorganisation of the actin cytoskeleton and modulation of signalling pathways by comparing osteosarcoma cells characterised by different metastasis capabilities and CD99 expression, to identify molecular mechanisms responsible for metastasis. Our data indicate that forced expression of CD99wt induces recruitment of N-cadherin and β-catenin to adherens junctions. In addition, transfection of CD99wt inhibits the expression of several molecules crucial to the remodelling of the actin cytoskeleton, such as ACTR2, ARPC1A, Rho-associated, coiled–coil containing protein kinase 2 (ROCK2) as well as ezrin, an ezrin/radixin/moesin family member that has been clearly associated with tumour progression and metastatic spread in osteosarcoma. Functional studies point to ROCK2 as a crucial intracellular mediator regulating osteosarcoma migration. By maintaining c-Src in an inactive conformation, CD99wt inhibits ROCK2 signalling and this leads to ezrin decrease at cell membrane while N-cadherin and β-catenin translocate to the plasma membrane and function as main molecular bridges for actin cytoskeleton. Taken together, we propose that the re-expression of CD99wt, which is generally present in osteoblasts but lost in osteosarcoma, through inhibition of c-Src and ROCK2 activity, manages to increase contact strength and reactivate stop-migration signals that counteract the otherwise dominant promigratory action of ezrin in osteosarcoma cells.

Performance of peripheral (serum and molecular) blood markers for diagnosis of endometriosis.

PurposeTo quantify the mRNA levels of MMP-3, MMP-9, VEGF and Survivin in peripheral blood and the serum levels of CA-125 and Ca19-9 in women with and without endometriosis and to investigate the performance of these markers to differentiate between deep and ovarian endometriosis.MethodsA case control study enrolled a series of 60 patients. Twenty controls have been matched with 20 cases of ovarian and 20 cases of deep endometriosis. Univariable and multivariable performance of serum CA125 and CA19-9, mRNA for Survivin, MMP9, MMP3 and VEGF genes have been evaluated by means of ROC curves and logistic regression, respectively.ResultsNo difference in markers’ concentration was detected between ovarian and deep endometriosis. In comparison with controls, serum CA125 and CA19 yielded the better sensitivity followed by mRNA for Survivin gene (81.5, 51.9 and 7.5% at 10% false positive rate, respectively). Multivariable estimated odds of endometriosis yielded a sensitivity of 87% at the same false positive rate.ConclusionsA combination of serum and molecular markers could allow a better diagnosis of endometriosis.

Matrix Metalloproteinase-3 mRNA: A Promising Peripheral Blood Marker for Diagnosis of Endometriosis

Background/Aims: Endometriosis is an invasive disease. Its diagnosis depends on laparoscopy, which is traumatic and associated with potential complications. The aim of this study was to develop a rapid, reliable, and less invasive diagnostic test for endometriosis. We hypothesized that genes related to cell invasion would be transcriptionally upregulated in endometriosis, and tested whether blood levels of their transcripts might be used as biomarkers of endometriosis.Methods:We used quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to quantify the mRNA levels of vascular endothelial growth factor A (VEGFA), matrix metalloproteinase-3 (MMP-3), and MMP-9 in peripheral blood from 20 patients with mild/intermediate endometriosis, 20 patients with severe endometriosis and 20 endometriosis-free subjects. Results:Our results indicate that circulating mRNA for MMP-3 is significantly higher in patients with endometriosis than in control patients, regardless of the degree of severity. Conversely, the level of circulating mRNA for VEGFA and MMP-9 did not distinguish patients from controls. Conclusion: MMP-3 mRNA is a promising peripheral blood marker that discriminates between patients with endometriosis and healthy subjects. Our results support the possibility of finding genes suitable for diagnostic qRT-PCR for endometriosis in peripheral blood and should be explored further.

Expression levels of insulin receptor substrate-1 modulate the osteoblastic differentiation of mesenchymal stem cells and osteosarcoma cells

Abstract The insulin-like growth factor-1 system, including its critical mediator insulin receptor substrate-1 (IRS-1), is involved in regulating osteosarcoma (OS) cell proliferation or differentiation. The aim of this study is to define the role of IRS-1 in OS cells by assessing the contribution of IRS-1 in the differentiation of human and murine OS cell lines and mouse mesenchymal stem cells (MSCs) and found that the basal level of IRS-1 is important for the initiation of differentiation. Both down-regulation and over-expression of IRS-1 inhibited osteoblastic differentiation. In vivo studies showed that OS cells over-expressing IRS-1 have increased metastatic potential and tumor growth. The proteasome inhibitor MG-132 led to an increase in IRS-1 protein level that inhibited osteoblastic differentiation, suggesting a role for proteasomal regulation in maintaining the appropriate expression level of IRS-1. Thus, precise regulation of IRS-1 expression level is critical for determining the differentiating capacity of MSCs and OS cells, and that derangement of IRS-1 levels can be a critical step in OS transformation.

Higher circulating mRNA levels of placental specific genes in a patient with placenta accreta

Giuliana Simonazzi1*, Antonio Farina1, Alessandra Curti1, Gianluigi Pilu1, Donatella Santini2, Cinzia Zucchini1, Akihiko Sekizawa3 and Nicola Rizzo1 1Department of Obstetrics and Gynecology, St. Orsola Malpighi Hospital, University of Bologna, Bologna, Italy 2Department of Pathology, St. Orsola Malpighi Hospital, University of Bologna, Bologna, Italy 3Department of Obstetrics and Gynecology, Showa University School of Medicine, Tokyo, Japan

Circulating mRNA for epidermal growth factor‐like domain 7 (EGFL7) in maternal blood and early intrauterine growth restriction. A preliminary analysis

To evaluate the alteration in epidermal growth factor‐like domain 7 (EGFL7) mRNA expression in maternal blood from pregnancies affected by early‐onset intrauterine growth restriction (IUGR) at 20–24u2009weeks.

Targeting ROCK2 rather than ROCK1 inhibits Ewing sarcoma malignancy

Understanding the molecular processes characterizing Ewing sarcoma (EWS) cell migration is crucial to highlight novel therapies for patients with disseminated disease. In this study we analyzed the role of ROCK kinases in the regulation of cell migration, growth and differentiation of EWS cells. Overexpression of ROCK promotes invasion and metastasis in many solid tumors. However, the effect of ROCK in EWS has not been extensively investigated. Expression of ROCK1 and ROCK2 was analyzed by western blotting in a representative panel of human EWS cell lines, in comparison with the parameters of in vitro malignancy. We investigated the effects of a ROCK2 specific inhibitor toward those of a pan-ROCK inhibitor on the growth, migration and differentiation of two EWS cell lines. ROCK2 but not ROCK1 expression was found to be associated with in vitro cell migration and anchorage-independent growth capabilities. Exposure of EWS cells to ROCK inhibitors significantly reduced migration and growth, while favoring morphology changes and neural differentiation. These effects were more striking when cells were specifically deprived of ROCK2 activity. Our findings lead to consider ROCK2, rather than ROCK1, as a possible molecular target for the treatment of EWS.

Fetal cardiac defects and third-trimester maternal serum placental growth factor.

Fetal echocardiography is considered the best screening tool for congenital heart defects (CHDs). However, the use of ultrasound is limited to a few specialized and experienced centers, in which it is offered to high-risk mothers1. Other strategies for screening have been proposed, including the use of biomarkers. In an observational study by Llurba et al., maternal placental growth factor (PlGF) was proposed as a first-trimester marker for the detection of conotruncal and valvular defects2. We performed a case–control study in the third trimester of pregnancy, involving 10 pregnant patients carrying a fetus affected by a CHD and 50 unaffected control pregnancies, matched for gestational age, at a 1:5 ratio. Maternal serum PlGF quantification was performed using the Alere PlGF Test, using Triage® MeterPro instrument (Alere Srl, Milan, Italy), a test based on a fluorescence immunoassay technique that allows measurement of PlGF over a range from 12 to 3000 pg/mL. The mean rank of PlGF concentration obtained from each case sample was significantly lower than that of the controls (2.00 vs 3.80; P = 0.003). Seven cases of valvular and conotruncal defects had very low rank values (Figure 1). Figure 2 reports the multiples of the median (MoM) stratified according to each set of data. As shown for each data set, one of the five controls had a PlGF-MoM value of 1, which represents the median (1 MoM) for that specific data set. The median MoM for CHD cases was 0.26, compared with the expected MoM of 1 for the 50 controls (P = 0.028). Speculation about levels of PlGF and other angiogenic factors such as vascular endothelial growth factor in the presence of CHD has been reported previously3. Intrinsically altered angiogenesis is thought not only to cause abnormal formation of the heart but also to result in abnormal placentation. We have reported previously that the placental tissue of fetuses affected with CHD shows dysregulated expression of several genes that regulate the extracellular matrix (ECM) of both endocardial cushions and valvular components4. We therefore speculate that such genes are somehow associated with the malformed features of CHD, and that extra-embryonic mesoderm (from which the mesenchymal axis of the villus arises) and intra-embryonic mesoderm (from which the heart, dermis and many other organs arise) have a similar origin. In support of this hypothesis, our previous data identified dysregulation in the expression pattern of selected genes implicated in the control of heart development and markers of functional abnormalities, such as contractile dysfunction and congestive heart failure, which may result from the presence of CHD. These results are 0 1 2 3 N um be r of c as es

Possible Gender-Related Modulation by the ROCK1 Gene in Colorectal Cancer Susceptibility

Aim: In view of accumulating evidence supporting a pivotal role of the Rho/ROCK pathway in cancer, we investigated Rho-kinase polymorphisms as potential susceptibility factors in colorectal cancer (CRC) in a representative sample of the Italian population. Methods: DNA obtained from the peripheral blood samples of 137 CRC patients and 141 healthy controls was genotyped for four ROCK1 (rs35996865; rs73963110; rs2127958; rs288980) and five ROCK2 (rs12692437; rs7563468; rs35768389; rs17463896; rs16857265) selected single nucleotide polymorphisms. Results: None of the allelic variants of the nine selected markers was associated with the occurrence of CRC or with the development of regional lymph node metastasis. By contrast, the ROCK1 rs35996865 G variant allele was significantly more frequent in male patients (p = 0.028) than in the control group. Conclusion: This finding is, at present, the first that points to a possible gender-related modulation by the ROCK1 gene in CRC susceptibility.