Renewal of stem cells differs from cancer cell growth in self-controlled cell division. The mir-302 microRNA (miRNA) family (mir-302s) is expressed most abundantly in slow-growing human embryonic stem (ES) cells, and quickly decreases after cell differentiation and proliferation. Therefore, mir-302s was investigated as one of the key factors essential for maintenance of ES cell renewal and pluripotency in this study. The Pol-II-based intronic miRNA expression system was used to transgenically transfect the mir-302s into several human cancer cell lines. The mir-302 -transfected cells, namely, miRNA-induced pluripotent stem (mirPS) cells, not only expressed many key ES cell markers, such as Oct3/4, SSEA-3, SSEA-4 ,Sox2, and Nanog, but also had a highly demethylated genome similar to a reprogrammed zygotic genome. Microarray analyses further revealed that genomewide gene expression patterns between the mirPS and human ES H1 and H9 cells shared over 86% similarity. Using molecular guidance in vitro, these mirPS cells could differentiate into distinct tissue cell types, such as neuron-, chondrocyte-, fibroblast-, and spermatogonia-like primordial cells. Based on these findings, we conclude that mir-302s not only function to reprogram cancer cells into an ES-like pluripotent state but also to maintain this state under a feeder-free cultural condition, which may offer a great opportunity for therapeutic intervention.

Global demethylation is required for early zygote development to establish stem cell pluripotency, yet our findings reiterate this epigenetic reprogramming event in somatic cells through ectopic introduction of mir-302 function. Here, we report that induced mir-302 expression beyond 1.3-fold of the concentration in human embryonic stem (hES) H1 and H9 cells led to reprogramming of human hair follicle cells (hHFCs) to induced pluripotent stem (iPS) cells. This reprogramming mechanism functioned through mir-302-targeted co-suppression of four epigenetic regulators, AOF2 (also known as KDM1 or LSD1), AOF1, MECP1-p66 and MECP2. Silencing AOF2 also caused DNMT1 deficiency and further enhanced global demethylation during somatic cell reprogramming (SCR) of hHFCs. Re-supplementing AOF2 in iPS cells disrupted such global demethylation and induced cell differentiation. Given that both hES and iPS cells highly express mir-302, our findings suggest a novel link between zygotic reprogramming and SCR, providing a regulatory mechanism responsible for global demethylation in both events. As the mechanism of conventional iPS cell induction methods remains largely unknown, understanding this microRNA (miRNA)-mediated SCR mechanism may shed light on the improvements of iPS cell generation.

The pattern of microRNA (miRNA) expression is associated with the degree of tumor cell differentiation in human prostate cancer. MiRNAs bind complementarily to either oncogenes or tumor suppressor genes, which are consequently silenced, resulting in alterations of tumorigenecity. We have detected eight down-regulated and three up-regulated known miRNAs in androgen-independent human prostate cancer cells compared to those in androgen-dependent cells, using miRNA microarray analyses. These identified miRNAs showed the same expression patterns in hormone-refractory prostate carcinomas (HRPC) compared to androgen-sensitive noncancerous prostate epithelium as determined by fluorescent in situ hybridization assays in human prostate cancer tissue arrays. One of the eight down-regulated miRNAs, mir-146a, was selected and constitutively expressed to examine its effects on suppression of prostate cancer transformation from androgen-dependent to -independent cells as determined by in vitro tumorigenecity assays. Transfection of mir-146a, which perpetually express the miRNA, suppressed >82% of the expression of the targeted protein-coding gene, ROCK1, in androgen-independent PC3 cells, consequently markedly reducing cell proliferation, invasion, and metastasis to human bone marrow endothelial cell monolayers. Given that ROCK1 is one of the key kinases for the activation of hyaluronan (HA)-mediated HRPC transformation in vivo and in PC3 cells, mir-146a may function as a tumor-suppressor gene in modulating HA/ROCK1-mediated tumorigenecity in androgen-dependent prostate cancer.

A ctivins and inhibins are members of the transforming growth factor-B (TGF-I) superfamily, which consists of TGF-I), activins, bone morphogenic proteins (BMPs), anti-Mullerian hormone (AMH), and others. These structurally related growth factors have essential functions in regulating tissue development, homeostasis, cell proliferation, and apoptosis. Activin and inhibin were originally isolated based on their activity in regulating follicle-stimulating hormone (FSH) release from the anterior pituitary: activin stimulates, whereas inhibin inhibits FSH secretion (1, 2). In addition to its endocrine function, activin has been found to possess various activities in different biological systems, e.g., erythroid differentiation, nerve cell survival, Xenopus laevis embryonic mesoderm induction, bone growth promotion, and somatostatin induction (3, 4). Subsequently, it was found that activin regulates a wide variety of cellular events, including cell proliferation, differentiation, and apoptosis. For example, in addition to its endocrine function in the pituitary, activin also controls the activity of hypothalamus and ovary (reviewed in Ref. 5), indicating that activin has profound paracrine and autocrine effects on the female reproductive system. In addition, activin expression has been detected in cell lines derived from various human tissues, including prostate cancer (6-8), breast cancer (9, 10), retinoblastoma (11), retinal pigment epithelium (12) placenta (13), gut (14), and bone marrow stroma (15), as well as a number of in vivo human tissues, including the testis (16), ovary (17), endometrium (18), placenta (19), oocyte (20), adrenal CELL PROLIFERATION, APOPTOSIS, AND CARCINOGENESIS 75 Research in the laboratories of Dr. Shao-Yao Ying was supported by the National Institutes of Health (grants HD 22876. HD24648. OK 47607. and CA 85772). Research in the laboratory of Dr. Ye-Guang Chen was supported by the Bugher Foundation. by the University of California Cancer Research Coordinating Committee. and by UCR Start-Up Funds. I This manuscript is an update of a previously published minireview (Proc Soc Exp Bioi Med 214: 114-122. (997).

miR-302 is the major microRNA found in human embryonic stem cells and induced pluripotent stem cells, but its function has been unclear. In mice, there is evidence that miR-302 may silence p21Cip1 (CDKN1A) to promote cell proliferation, whereas studies in human reprogrammed pluripotent stem cells suggested that elevated miR-302 expression inhibited cell cycle transit. Here, we clarify this difference, reporting that in human cells, miR-302 simultaneously suppressed both the cyclin E-CDK2 and cyclin D-CDK4/6 pathways to block >70% of the G 1 -S cell cycle transition. Concurrent silencing of BMI-1, a cancer stem cell marker targeted by miR-302, further promoted tumor suppressor functions of p16Ink4a and p14/p19Arf directed against CDK4/6-mediated cell proliferation. Among all G 1 phase checkpoint regulators, human p21Cip1 was found not to be a valid target of miR-302. Overall, our findings indicate that miR-302 inhibits human pluripotent stem cell tumorigenicity by enhancing multiple G 1 phase arrest pathways rather than by silencing p21Cip1.