Importance of the putative furin recognition site 742RNRR745 for antiangiogenic Sema3C activity in vitro

Angiogenesis is one of the key processes in the growth and development of tumors. Class-3 semaphorins (Sema3) are characterized as axon guidance factors involved in tumor angiogenesis by interacting with the vascular endothelial growth factor signaling pathway. Sema3 proteins convey their regulatory signals by binding to neuropilins and plexins receptors, which are located on the effector cell. These processes are regulated by furin endoproteinases that cleave RXRR motifs within the Sema, plexin-semaphorins-integrin, and C-terminal basic domains of Sema3 protein. Several studies have shown that the furin-mediated processing of the basic domain of Sema3F and Sema3A is critical for association with receptors. It is unclear, however, if this mechanism can also be applied to other Sema3 proteins, including the main subject of this study, Sema3C. To address this question, we generated a variant of the full-length human Sema3C carrying point mutation R745A at the basic domain at the hypothetical furin recognition site 742RNRR745, which would disable the processing of Sema3C at this specific location. The effects produced by this mutation were tested in an in vitro angiogenesis assay together with the wild-type Sema3C, Sema3A, and Sema3F proteins. Our results showed that the inhibitory effect of Sema3C on microcapillary formation by human umbilical vein endothelial cells could be abrogated upon mutation at the Sema3C basic domain within putative furin cleavage site 742RNRR745, indicating that this site was essential for the Sema3 biological activity.

Regulation of semaphorin 4D expression and cell proliferation of ovarian cancer by ERalpha and ERbeta

Ovarian cancer is one of the most common malignancies in women. Semaphorin 4D (sema 4D) is involved in the progress of multiple cancers. In the presence of estrogen-like ligands, estrogen receptors (ERα and ERβ) participate in the progress of breast and ovarian cancers by transcriptional regulation. The aim of the study was to investigate the role of sema 4D and elucidate the regulatory pattern of ERα and ERβ on sema 4D expression in ovarian cancers. Sema 4D levels were up-regulated in ovarian cancer SKOV-3 cells. Patients with malignant ovarian cancers had significantly higher sema 4D levels than controls, suggesting an oncogene role of sema 4D in ovarian cancer. ERα expressions were up-regulated in SKOV-3 cells compared with normal ovarian IOSE80 epithelial cells. Conversely, down-regulation of ERβ was observed in SKOV-3 cells. Forced over-expression of ERα and ERβ in SKOV-3 cells was manipulated to establish ERα+ and ERβ+ SKOV-3 cell lines. Incubation of ERα+ SKOV-3 cells with ERs agonist 17β-estradiol (E2) significantly enhanced sema 4D expression and rate of cell proliferation. Incubated with E2, ERβ+ SKOV-3 cells showed lower sema 4D expression and cell proliferation. Blocking ERα and ERβ activities with ICI182-780 inhibitor, sema 4D expressions and cell proliferation of ERα+ and ERβ+ SKOV-3 cells were recovered to control levels. Taken together, the data showed that sema 4D expression was positively correlated with the progress of ovarian cancer. ERα positively regulated sema 4D expression and accelerated cell proliferation. ERβ negatively regulated sema 4D expression and inhibited cell multiplication.

Suppression of miR-181 a attenuates H2O2-induced death of mesenchymal stem cells by maintaining hexokinase II expression

BACKGROUND: Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated. RESULTS: H2O2 (600 µM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2. CONCLUSIONS: These findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.

Suppression of miR-221 inhibits glioma cells proliferation and invasion via targeting SEMA3B

BACKGROUND: Gliomas are the most common primary tumors in the central nervous system. Due to complicated signaling pathways involved in glioma progression, effective targets for treatment and biomarkers for prognosis prediction are still scant. RESULTS: In this study we revealed that a new microRNA (miR), the miR-221, was highly expressed in the glioma cells, and suppression of miR-221 resulted in decreased cellular proliferation, migration, and invasion in glioma cells. Mechanistic experiments validated that miR-221 participates in regulating glioma cells proliferation and invasion via suppression of a direct target gene, the Semaphorin 3B (SEMA3B). The rescue experiment with miR-221 and SEMA3B both knockdown results in significant reversion of miR-221 induced phenotypes. CONCLUSION: Taken together, our findings highlight an unappreciated role for miR-221 and SEMA3B in glioma.