Molecular characterization of exosomes and their microRNA cargo in human follicular fluid: bioinformatic analysis reveals that exosomal microRNAs control pathways involved in follicular maturation.

OBJECTIVE: To characterize well-represented microRNAs in human follicular fluid (FF) and to ascertain whether they are cargo of FF exosomes and whether they are involved in the regulation of follicle maturation. DESIGN: FF exosomes were characterized by nanosight, flow cytometry, and exosome-specific surface markers. Expression microRNA profiles from total and exosomal FF were compared with those from plasma of the same women. SETTING: University laboratory and an IVF center. PATIENT(S): Fifteen healthy women who had undergone intracytoplasmic sperm injection. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): TaqMan low-density array to investigate the expression profile of 384 microRNAs; DataAssist and geNorm for endogenous control identification; significance analysis of microarrays to identify differentially expressed microRNAs; nanosight, flow-cytometry, and bioanalyzer for exosome characterization; bioinformatic tools for microRNAs target prediction, gene ontology, and pathway analysis. RESULT(S): We identified 37 microRNAs upregulated in FF as compared with plasma from the same women. Thirty-two were carried by microvesicles that showed the well-characterized exosomal markers CD63 and CD81. These FF microRNAs are involved in critically important pathways for follicle growth and oocyte maturation. Specifically, nine of them target and negatively regulate mRNAs expressed in the follicular microenvironment encoding inhibitors of follicle maturation and meiosis resumption. CONCLUSION(S): This study identified a series of exosomal microRNAs that are highly represented in human FF and are involved in follicular maturation. They could represent noninvasive biomarkers of oocyte quality in assisted reproductive technology.

The apoptotic transcriptome of the human MII oocyte: characterization and age-related changes.

Fully competent oocytes represent the final outcome of a highly selective process. The decline of oocyte competence with ageing, coupled to quantitative decrease of ovarian follicles has been well established; on the contrary, its molecular bases are still poorly understood. Through quantitative high throughput PCR, we investigated the role of apoptotic machinery (AM) in this process. To this aim, we determined AM transcriptome in mature MII oocyte pools from women aged more than 38 years (cohort A), and compared to women aged up to 35 years (cohort B). Subsequently, 10 representative AM genes were selected and analyzed in 33 single oocytes (15 from cohort A and 18 from cohort B). These investigations led us to identify: (1) the significant upregulation of proapoptotic genes such us CD40, TNFRSF10A, TNFRSF21 and the downregulation of antiapoptotic genes such as BCL2 and CFLAR in cohort A respect to cohort B; (2) AM transcripts that have not previously been reported in human oocytes (BAG3, CD40, CFLAR, TNFRSF21, TRAF2, TRAF3). Our results demonstrated that during maturation the oocytes from older women selectively accumulate mRNAs that are able to trigger the extrinsic apoptotic pathway. These data contribute to clarify the molecular mechanisms of AM involvement in the natural selection strategy of removing low quality oocytes and preventing unfit or poorly fit embryos.

TAp73 is downregulated in oocytes from women of advanced reproductive age.

Studies on oocyte transcriptome are important to understand the biological pathways involved in oogenesis, totipotence and early embryonic development. Moreover, genes regulating physiological pathways in gametes could represent potential candidates for reproductive disorders. In addition to oocyte specific transcription factors, also the members of the p53 family could be etiologically involved due to their biological functions. In fact, their role in the control of cell cycle, apoptosis, and germ-line genome stability is well known. Female reproductive aging is one of the causes of fertility reduction and it is often associated with egg aneuploidy increase. In order to verify the potential involvement of p73 in reproductive aging, we determined its expression in single mature MII oocytes from two groups of women, younger than 35 or older than 38 years, respectively. We found that TAp73 isoforms are down regulated in oocytes from women older than 38 years. We confirmed these data in pools of mouse oocytes. TAp73 down regulation in oocytes from women of advanced reproductive age could explain both the reduction of fertility and the increase of newborns with chromosomal abnormalities.

Specific alterations of microRNA transcriptome and global network structure in colorectal carcinoma after cetuximab treatment.

The relationship between therapeutic response and modifications of microRNA (miRNA) transcriptome in colorectal cancer (CRC) remains unknown. We investigated this issue by profiling the expression of 667 miRNAs in 2 human CRC cell lines, one sensitive and the other resistant to cetuximab (Caco-2 and HCT-116, respectively), through TaqMan real-time PCR. Caco-2 and HCT-116 expressed different sets of miRNAs after treatment. Specifically, 21 and 22 miRNAs were differentially expressed in Caco-2 or HCT-116, respectively (t test, P < 0.01). By testing the expression of differentially expressed miRNAs in CRC patients, we found that miR-146b-3p and miR-486-5p are more abundant in K-ras-mutated samples with respect to wild-type ones (Wilcoxon test, P < 0.05). Sixty-seven percent of differentially expressed miRNAs were involved in cancer, including CRC, whereas 19 miRNA targets had been previously reported to be involved in the cetuximab pathway and CRC. We identified 25 transcription factors putatively controlling these miRNAs, 11 of which have been already reported to be involved in CRC. On the basis of these data, we suggest that the downregulation of let-7b and let-7e (targeting K-ras) and the upregulation of miR-17* (a CRC marker) could be considered as candidate molecular markers of cetuximab resistance. Global network functional analysis (based on miRNA targets) showed a significant overrepresentation of cancer-related biological processes and networks centered on critical nodes involved in epidermal growth factor receptor internalization and ubiquitin-mediated degradation. The identification of miRNAs, whose expression is linked to the efficacy of therapy, should allow the ability to predict the response of patients to treatment and possibly lead to a better understanding of the molecular mechanisms of drug response.

Expression profile and specific network features of the apoptotic machinery explain relapse of acute myeloid leukemia after chemotherapy.

BACKGROUND: According to the different sensitivity of their bone marrow CD34+ cells to in vitro treatment with Etoposide or Mafosfamide, Acute Myeloid Leukaemia (AML) patients in apparent complete remission (CR) after chemotherapy induction may be classified into three groups: (i) normally responsive; (ii) chemoresistant; (iii) highly chemosensitive. This inversely correlates with in vivo CD34+ mobilization and, interestingly, also with the prognosis of the disease: patients showing a good mobilizing activity are resistant to chemotherapy and subject to significantly higher rates of Minimal Residual Disease (MRD) and relapse than the others. Based on its known role in patients' response to chemotherapy, we hypothesized an involvement of the Apoptotic Machinery (AM) in these phenotypic features. METHODS: To investigate the molecular bases of the differential chemosensitivity of bone marrow hematopoietic stem cells (HSC) in CR AML patients, and the relationship between chemosensitivity, mobilizing activity and relapse rates, we analyzed their AM expression profile by performing Real Time RT-PCR of 84 AM genes in CD34+ pools from the two extreme classes of patients (i.e., chemoresistant and highly chemosensitive), and compared them with normal controls. RESULTS: The AM expression profiles of patients highlighted features that could satisfactorily explain their in vitro chemoresponsive phenotype: specifically, in chemoresistant patients we detected up regulation of antiapoptotic BIRC genes and down regulation of proapoptotic APAF1, FAS, FASL, TNFRSF25. Interestingly, our analysis of the AM network showed that the dysregulated genes in these patients are characterized by high network centrality (i.e., high values of betweenness, closeness, radiality, stress) and high involvement in drug response. CONCLUSIONS: AM genes represent critical nodes for the proper execution of cell death following pharmacological induction in patients. We propose that their dysregulation (either due to inborn or de novo genomic mutations selected by treatment) could cause a relapse in apparent CR AML patients. Based on this, AM profiling before chemotherapy and transplantation could identify patients with a predisposing genotype to MRD and relapse: accordingly, they should undergo a different, specifically tailored, therapeutic regimen and should be carefully checked during the post-treatment period.

Molecular profiling of human oocytes after vitrification strongly suggests that they are biologically comparable with freshly isolated gametes.

To assess the effects of vitrification on the biomolecular profile of oocytes, we analyzed through real-time reverse transcriptase-polymerase chain reaction eight genes encoding critically important proteins for embryo development and compared this partial transcriptome with that of freshly collected gametes isolated from the same women. The comparison of the molecular profiles demonstrated that our vitrification protocol does not alter the biomolecular quality of oocytes: in fact, between the two groups we found the absence of statistically significant variations. Accordingly, this cryopreservation technique might be helpful in preserving women's fertility.

MIR152, MIR200B, and MIR338, human positional and functional neuroblastoma candidates, are involved in neuroblast differentiation and apoptosis.

MicroRNAs (MIRs) perform critical regulatory functions within cell networks, both in physiology as well as in pathology. Through the positional gene candidate approach, we have identified three MIRs (MIR152, MIR200B, and MIR338) that are located in regions frequently altered in neuroblastoma (NB) and target mRNAs encoding proteins involved in cell proliferation, neuroblast differentiation, neuroblast migration, and apoptosis. Expression analysis in NB biopsies and NB cell lines showed that these MIRs are dysregulated. We have characterized a CpG island, close to the gene encoding MIR200B and hypermethylated in NB samples, that explains its negative regulation. Expression of MIR152, MIR200B, and MIR338 is specifically modulated in NB cell lines during differentiation and apoptosis. Functional genomic experiments through enforced expression of MIR200B and knockdown of MIR152 resulted in a significant decrease of the invasion activity of SH-SY5Y cells. Reconstruction of a NB network comprising MIR152, MIR200B, and MIR338 allowed us to confirm their role in the control of NB cell stemness and apoptosis: This suggests that altered regulation of these MIRs could have a role in NB pathogenesis by interfering with the molecular mechanisms, which physiologically control differentiation and death of neuroblasts. Accordingly, they could be considered as new NB biomarkers and potential targets of antagomirs or epigenetic therapies.

Genomics, evolution, and expression of TBPL2, a member of the TBP family.

TBPL2 is the most recently discovered and less characterized member of the TATA box binding protein (TBP) family that also comprises TBP, TATA box binding protein-like 1 (TBPL1), and Drosophila melanogaster TBP related factor (TRF). In this paper we report our in silico and in vitro data on (i) the genomics of the TBPL2 gene in Homo sapiens, Pan troglodytes, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus tropicalis, and Takifugu rubripes; (ii) its evolution and phylogenetic relationship with TBP, TBPL1, and TRF; (iii) the structure of the TBPL2 proteins that belong to the recently identified group of the intrinsically unstructured proteins (IUPs); and (iv) TBPL2 expression in different organs and cell types of Homo sapiens and Rattus norvegicus. Similar to TBP, both the TBPL2 gene and protein are bimodular. The 3' region of the gene encoding the DNA binding domain (DBD) was well conserved during evolution. Its high homology to vertebrate TBP suggests that TBPL2 also should bind to the TATA box and interact with the proteins binding to TBP carboxy-terminal domain, such as the TBP associated factors (TAFs). As already demonstrated for TBP, TBPL2 amino-terminal segment is intrinsically unstructured and, even though variable among vertebrates, comprises a highly conserved motif not found in any other known protein. Absence of TBPL2 from the genome of invertebrates and plants demonstrates its specific origin within the subphylum of vertebrates. Our RT-PCR analysis of human and rat RNA shows that, similar to TBP, TBPL2 is ubiquitously synthesized even though at variable levels that are at least two orders of magnitude lower. Higher expression of TBPL2 in the gonads than in other organs suggests that it could perform important functions in gametogenesis. Our genomic and expression data should contribute to clarify why TBP has a general master role within the transcription apparatus (TA), whereas both TBPL1 and TBPL2 perform tissue-specific functions.