All experiments were performed in triplicate. were associated with the high levels of cervical cancer-derived exosomes. In conclusion, we demonstrated the abundance of exosomes in the cervicovaginal lavage specimens of women with cervical cancer. Furthermore, our results indicated that abnormally Rabbit Polyclonal to OR1E2 high levels of microRNA-21 and microRNA-146a existed in the cervical cancer-derived exosomes and the two microRNAs were functional in 293T cells. and invasive) . Despite the robust carcinogenic potential, HPV infection alone is not sufficient for the development of cervical cancer, because only a minor fraction of patients infected with HPV develop cervical cancer . Indeed, several cofactors, including dysregulation of microRNAs, have been implicated in the genesis of HPV-associated cervical cancer [9,10]. MicroRNAs (miRNAs) are non-coding RNA molecules of approximately 22 nucleotides that regulate gene expression in organisms ranging from nematodes to humans , and in a broad array of cell processes in mammals [12C14]. Recent years, numerous oncogenic microRNAs have been reported to be associated with cervical cancer tumorigenesis [15,16]. MicroRNA-10a, microRNA-21, microRNA-19, and microRNA-146a promote cell growth, migration and invasion in human cervical cancer cells [9,10,17]. Conversely, tumor suppressive microRNAs have been down-regulated in human cervical cancer, such as microRNA-372, Fabomotizole hydrochloride microRNA-214, and microRNA-218 [18C20]. In addition, there are numerous other microRNA dysregulation in the human cervical cancer. MicroRNA-34a suppresses invasion through down-regulation of Notch1 and Jagged1 in cervical carcinoma and choriocarcinoma cells . The microRNA-302C367 cluster suppresses the proliferation of cervical carcinoma cells through the novel target AKT1 . miRNA expression profiles have been shown to be promising biomarkers for the diagnosis, classification or outcome prediction of a wide array of human cancers (reviewed in refs. [23,24]). The clinical value of the above-mentioned miRNAs as markers for cervical cancer needs to be further investigated. The majority of microRNAs are present within the cell, and only a smaller number of them have been detected outside cells, including various body fluids [25C27]. Surprisingly, extracellular microRNAs are remarkably stable despite high extracellular RNase activity , indicating that these microRNAs are likely packaged to avoid RNase digestion. Recent studies have indeed confirmed that extracellular microRNAs are shielded against degradation by packaging in one type of extracellular vesicles, exosomes [25,28]. Exosomes are first discovered in the maturing mammalian reticulocyte , and are later Fabomotizole hydrochloride found to be present in many and perhaps all biological fluids, including urine, blood, ascites, cervical secretions, etc., with the diameter between 30 and 110 nm and the density ranging from 1.13 to 1 1.19 g/mL [30,31]. The exosome has been implicated in intercellular communication via cargos of proteins, mRNAs, and miRNAs . More recently, the exosomal microRNAs are used as diagnostic biomarkers for lung cancer, ovarian cancer, and cardiovascular diseases [33C36]. The present study is to determine whether the cervical cancer-derived exosomes contain abnormally high levels of microRNA-21 and microRNA-146a, which are up-regulated in cervical cancers, and to evaluate whether the exosomal microRNA-21 and microRNA-146a can potentially serve as useful biomarkers for Fabomotizole hydrochloride cervical cancer diagnosis. 2.?Results 2.1. Abundance of Exosomes in the Cervicovaginal Lavage Specimens of Women with Cervical Cancer Cervicovaginal lavages specimens were collected from 45 patients with cervical cancers, 25 HPV-positive subjects and 32 normal HPV-negative subjects. Exosomes were isolated from the specimens by ultracentrifugation. The exosomes were examined and confirmed by electron microscopy. Under electron microscopy, the pellets were spherical in shape, with an average diameters varying between 30 and 110 nm (Figure 1A), consistent with the previously reported characteristics of exosomes . The exosomal vesicles were further confirmed by western blot analysis using antibodies against two common exosomal markers, the tetraspanin molecules CD63 and CD9 [38,39] (Figure 1B,C). The expression level of CD9 was quantitatively measured in the exosomes (Figure 1D,E). Open in a separate window Figure 1. Confirmation of the exosomal vesicles in the cervicovaginal lavage specimens. (A) Electron microscopy of the vesicles from cervicovaginal lavage specimens revealed the characteristic spherical shape and the size (40C100 nm; sized from 10 vesicles) of exosomes; (B,C) Western blot showed strong staining of the ultracentrifugation pellets with the antibodies against the exosomal membrane markers, CD63 and CD9; (D,E) Dose-dependent correlation between the CD9 expression level and the presence of exosomes isolated from the cervicovaginal.