Chun Peng

MicroRNA miR-93 promotes tumor growth and angiogenesis by targeting integrin-β8

It has been reported that the miR-106b∼25 cluster, a paralog of the miR-17∼92 cluster, possesses oncogenic activities. However, the precise role of each microRNA (miRNA) in the miR-106b∼25 cluster is not yet known. In this study, we examined the function of miR-93, one of the microRNAs within the miR-106b∼25 cluster, in angiogenesis and tumor formation. We found that miR-93 enhanced cell survival, promoted sphere formation and augmented tumor growth. Most strikingly, when miR-93-overexpressing U87 cells were co-cultured with endothelial cells, they supported endothelial cell spreading, growth, migration and tube formation. In vivo studies revealed that miR-93-expressing cells induced blood vessel formation, allowing blood vessels to extend to tumor tissues in high densities. Angiogenesis promoted by miR-93 in return facilitated cell survival, resulting in enhanced tumor growth. We further showed that integrin-β8 is a target of miR-93. Higher levels of integrin-β8 are associated with cell death in tumor mass and in human glioblastoma. Silencing of integrin-β8 expression using small interfering RNA promoted cell proliferation, whereas ectopic expression of integrin-β8 decreased cell growth. These findings showed that miR-93 promotes tumor growth and angiogenesis by suppressing, at least in part, integrin-β8 expression. Our results suggest that inhibition of miR-93 function may be a feasible approach to suppress angiogenesis and tumor growth.

MicroRNA miR-378 Regulates Nephronectin Expression Modulating Osteoblast Differentiation by Targeting GalNT-7

MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3′-untranslated region (UTR) of mRNAs, resulting in translational repression. We have developed a system to study the role of miRNAs in cell differentiation. We have found that one of the miRNAs tested in our system (miR-378, also called miR-378*) plays a role in modulating nephronectin-mediated differentiation in the osteoblastic cell line, MC3T3-E1. Nephronectin is an extracellular matrix protein, and we have demonstrated that its over-expression enhanced osteoblast differentiation and bone nodule formation. Furthermore, we found that the nephronectin 3′-untranslated region (3′UTR) contains a binding site for miR-378. Stable transfection of MC3T3-E1 cells with miR-378 inhibited cell differentiation. MC3T3-E1 cells stably transfected with nephronectin exhibited higher rates of differentiation and nodule formation as compared with cells transfected with nephronectin containing the 3′UTR in the early stages of development, suggesting that endogenous miR-378 is present and active. However, in the later stages of MC3T3-E1 development, the differentiation rates were opposite, with higher rates of differentiation and nodule formation in the cells over-expressing the 3′UTR of nephronectin. This appeared to be the consequence of competition between nephronectin and UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GalNAc-T7 or GalNT7) for miR-378 binding, resulting in increased GalNT7 activity, which in turn lead to increased nephronectin glycosylation and product secretion, thereby resulting in a higher rate of osteoblast differentiation.

Placental trophoblast cell differentiation: physiological regulation and pathological relevance to preeclampsia.

The placenta is a transient organ that forms during pregnancy to support the growth and development of the fetus. During human placental development, trophoblast cells differentiate through two major pathways. In the villous pathway, cytotrophoblast cells fuse to form multinucleated syncytiotrophoblast. In the extravillous pathway, cytotrophoblast cells acquire an invasive phenotype and differentiate into either (1) interstitial extravillous trophoblasts, which invade the decidua and a portion of the myometrium, or (2) endovascular extravillous trophoblasts, which remodel the maternal vasculature. These differentiation events are tightly controlled by the interplay of oxygen tension, transcription factors, hormones, growth factors, and other signaling molecules. More recently, microRNAs have been implicated in this regulatory process. Abnormal placental development, particularly the limited invasion of trophoblast cells into the uterus and the subsequent failure of the remodeling of maternal spiral arteries, is believed to cause preeclampsia, a severe pregnancy related disorder characterized by hypertension and proteinuria. Oxidative stress, the abnormal production and/or function of signaling molecules, as well as aberrant microRNAs expression have been suggested to participate in the pathogenesis of preeclampsia. Several potential biomarkers for preeclampsia have been identified, creating new opportunities for the development of strategies to diagnose, prevent, and treat this disorder.

Endocrine/paracrine control of zebrafish ovarian development

Ovarian differentiation and the processes of follicle development, oocyte maturation and ovulation are complex events, requiring the coordinated action of regulatory molecules. In zebrafish, ovarian development is initiated at 10 days after hatching and fish become sexually mature at 3 months. Adult zebrafish have asynchronous ovaries, which contain follicles of all stages of development. Eggs are spawned daily under proper environmental conditions in a population of zebrafish, with individual females spawning irregularly every 4-7 days in mixed sex conditions. Maximal embryo viability is achieved when sexually isolated females are bred in 10-day intervals [Niimi, A.J., LaHam, Q.N., 1974. Influence of breeding time interval on egg number, mortality, and hatching of the zebra fish Brachydanio verio. Can. J. Zool. 52, 515-517]. Similar to other vertebrates, hormones from the hypothalamus-pituitary-gonadal axis play important roles in regulating follicle development. Follicle stimulating hormone (FSH) stimulates estradiol production, which in turn, promotes viteollogenesis. Luteinizing hormone (LH) stimulates the production of 17,20beta-dihydroxy-4-pregnen-3-one (17,20betaP) or maturation inducing hormone (MIH) which acts through membrane progestin receptors to activate maturation promoting factor, leading to oocyte maturation. Recent studies in zebrafish have also provided novel insights into the functions of ovary-derived growth factors in follicle development and oocyte maturation. The present review summarizes the current knowledge on how endocrine and paracrine factors regulate ovarian development in zebrafish. Special emphasis is placed on how follicle development and oocyte maturation in adult females is regulated by gonadotropins, ovarian steroids and growth factors produced by the ovary.

MiR-93 enhances angiogenesis and metastasis by targeting LATS2

Here we report that miR-93, a miRNA in the miR-106B~25 cluster, a paralog of the miR-17–92 cluster, was significantly upregulated in human breast carcinoma tissues. We stably expressed miR-93 in the MT-1 human breast carcinoma cell line and found that tumors formed by the miR-93 cells contained more blood vessels than those formed by the control cells. Co-culture experiments indicated that the MT-1 cells displayed a high activity of adhesion with endothelial cells and could form larger and more tube-like structures with endothelial cells. Lung metastasis assays were performed in a mouse metastatic model, and it was found that expression of miR-93 promoted tumor cell metastasis to lung tissue. In cell culture, expression of miR-93 enhanced cell survival and invasion. We examined the potential target that mediated miR-93’s effects and found that the large tumor suppressor, homology 2 (LATS2) was a target of miR-93. Higher levels of LATS2 were associated with cell death in the tumor mass. Silencing LATS2 expression promoted cell survival, tube formation and invasion, while ectopic expression of LATS2 decreased cell survival and invasion. These findings demonstrated that miR-93 promoted tumor angiogenesis and metastasis by suppressing LATS2 expression. Our results suggest that the inhibition of miR-93 function may be a feasible approach to repress tumor metastasis.

MicroRNA 376c enhances ovarian cancer cell survival by targeting activin receptor-like kinase 7: implications for chemoresistance.

MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in gene regulation. We have previously reported that activin receptor-like kinase 7 (ALK7) and its ligand, Nodal, induce apoptosis in human epithelial ovarian cancer cells. In this study, we examined the regulation of ALK7 by miRNAs and demonstrate that miR-376c targets ALK7. Ectopic expression of miR-376c significantly increased cell proliferation and survival, enhanced spheroid formation and blocked Nodal-induced apoptosis. Interestingly, overexpression of miR-376c blocked cisplatin-induced cell death, whereas anti-miR-376c enhanced the effect of cisplatin. These effects of miR-376c were partially compensated by the overexpression of ALK7. Moreover, in serous carcinoma samples taken from ovarian cancer patients who responded well to chemotherapy, strong ALK7 staining and low miR-376c expression was detected. By contrast, ALK7 expression was weak and miR-376c levels were high in samples from patients who responded poorly to chemotherapy. Finally, treatment with cisplatin led to an increase in expression of mRNA encoding Nodal and ALK7 but a decrease in miR-376c levels. Taken together, these results demonstrate that the Nodal–ALK7 pathway is involved in cisplatin-induced cell death in ovarian cancer cells and that miR-376c enhances proliferation, survival and chemoresistance by targeting, at least in part, ALK7.

MicroRNA miR-24 enhances tumor invasion and metastasis by targeting PTPN9 and PTPRF to promote EGF signaling

Summary MicroRNAs are known to play regulatory roles in gene expression associated with cancer development. We analyzed levels of the microRNA miR-24 in patients with breast carcinoma and found that miR-24 was higher in breast carcinoma samples than in benign breast tissues. We generated constructs expressing miR-24 and studied its functions using both in vitro and in vivo techniques. We found that the ectopic expression of miR-24 promoted breast cancer cell invasion and migration. In vivo experiments in mice indicated that the expression of miR-24 enhanced tumor growth, invasion into local tissues, metastasis to lung tissues and decreased overall mouse survival. In the miR-24-expressing cells and tumors, EGFR was highly phosphorylated, whereas expression of the phosphatases tyrosine-protein phosphatase non-receptor type 9 (PTPN9) and receptor-type tyrosine-protein phosphatase F (PTPRF) were repressed. We confirmed that miR-24 could directly target both PTPN9 and PTPRF. Consistent with this, we found that the levels of phosphorylated epidermal growth factor receptor (pEGFR) were higher whereas the levels of PTPN9 and PTPRF were lower in the patients with metastatic breast carcinoma. Ectopic expression of PTPN9 and PTPRF decreased pEGFR levels, cell invasion, migration and tumor metastasis. Furthermore, we found that MMP2, MMP11, pErk, and ADAM15 were upregulated, whereas TIMP2 was downregulated; all of which supported the roles of miR-24 in tumor invasion and metastasis. Our results suggest that miR-24 plays a key role in breast cancer invasion and metastasis. miR-24 could potentially be a target for cancer intervention.

MicroRNAs in Human Placental Development and Pregnancy Complications

MicroRNAs (miRNAs) are small non-coding RNAs, which function as critical posttranscriptional regulators of gene expression by promoting mRNA degradation and translational inhibition. Placenta expresses many ubiquitous as well as specific miRNAs. These miRNAs regulate trophoblast cell differentiation, proliferation, apoptosis, invasion/migration, and angiogenesis, suggesting that miRNAs play important roles during placental development. Aberrant miRNAs expression has been linked to pregnancy complications, such as preeclampsia. Recent research of placental miRNAs focuses on identifying placental miRNA species, examining differential expression of miRNAs between placentas from normal and compromised pregnancies, and uncovering the function of miRNAs in the placenta. More studies are required to further understand the functional significance of miRNAs in placental development and to explore the possibility of using miRNAs as biomarkers and therapeutic targets for pregnancy-related disorders. In this paper, we reviewed the current knowledge about the expression and function of miRNAs in placental development, and propose future directions for miRNA studies.

DEHP Impairs Zebrafish Reproduction by Affecting Critical Factors in Oogenesis

Public concerns on phthalates distributions in the environment have been increasing since they can cause liver cancer, structural abnormalities and reduce sperm counts in male reproductive system. However, few data are actually available on the effects of Di-(2-ethylhexyl)-phthalate (DEHP) in female reproductive system. The aim of this study was to assess the impacts of DEHP on zebrafish oogenesis and embryo production. Female Danio rerio were exposed to environmentally relevant doses of DEHP and a significant decrease in ovulation and embryo production was observed. The effects of DEHP on several key regulators of oocyte maturation and ovulation including bone morphogenetic protein-15 (BMP15), luteinizing hormone receptor (LHR), membrane progesterone receptors (mPRs) and cyclooxygenase (COX)-2 (ptgs2) were determined by real time PCR. The expressions of BMP15 and mPR proteins were further determined by Western analyses to strengthen molecular findings. Moreover, plasma vitellogenin (vtg) titers were assayed by an ELISA procedure to determine the estrogenic effects of DEHP and its effects on oocyte growth. A significant reduction of fecundity in fish exposed to DEHP was observed. The reduced reproductive capacity was associated with an increase in ovarian BMP15 levels. This rise, in turn, was concomitant with a significant reduction in LHR and mPRβ levels. Finally, ptgs2 expression, the final trigger of ovulation, was also decreased by DEHP. By an in vitro maturation assay, the inhibitory effect of DEHP on germinal vesicle breakdown was further confirmed. In conclusion, DEHP affecting signals involved in oocyte growth (vtg), maturation (BMP15, LHR, mPRs,) and ovulation (ptgs2), deeply impairs ovarian functions with serious consequences on embryo production. Since there is a significant genetic similarity between D.rerio and humans, the harmful effects observed at oocyte level may be relevant for further molecular studies on humans.

MicroRNA-376c Impairs Transforming Growth Factor-β and Nodal Signaling to Promote Trophoblast Cell Proliferation and Invasion

Preeclampsia is a major disorder of pregnancy and a leading cause of maternal and perinatal morbidity and mortality. MicroRNAs are small noncoding RNAs that regulate gene expression posttranscriptionally. In this study, we examined the expression of miR-376c and found that miR-376c levels were downregulated in both placental and plasma samples collected from preeclamptic patients, when compared with the normal pregnant women at the same gestational stage. Overexpression of miR-376c induced trophoblast cell proliferation, migration, and invasion in HTR8/SVneo cells and promoted placental explant outgrowth. In contrast, inhibition of endogenous miR-376c resulted in a decrease in trophoblast cell invasion and placental explant outgrowth. We identified activin receptor-like kinase 5 (ALK5), a type I receptor for transforming growth factor-&bgr;, and ALK7, a type I receptor for Nodal, as targets of miR-376c. Overexpression of miR-376c repressed transforming growth factor-&bgr; and Nodal functions, whereas overexpression of ALK5 and ALK7 reversed the effects of miR-376c. These results demonstrate that miR-376c inhibits both ALK5 and ALK7 expression to impair transforming growth factor-&bgr;/Nodal signaling, leading to increases in cell proliferation and invasion. An unbalanced Nodal/transforming growth factor-&bgr; and miR-376c expression may lead to the development of preeclampsia.