Alteration of Pituitary Tumor Transforming Gene 1 by MicroRNA-186 and 655 Regulates Invasion Ability of Human Oral Squamous Cell Carcinoma.

BACKGROUND: Pituitary tumor-transforming gene 1 (PTTG1) was recently shown to be involved in the progression as well as the metastasis of cancers. However, their expression and function in the invasion of oral squamous cell carcinoma (SCC) remain unclear. METHODS: The expressions of PTTG1 and PTTG1-targeted miRNA in oral SCC cell lines and their invasion capability depended on PTTG1 expression were analyzed by quantitative RT-PCR, Western blots, the transwell insert system and Zymography. RESULTS: Invasion abilities were decreased in oral SCC cells treated with siRNA-PTTG1. When PTTG1 were downregulated in oral SCC cells treated with microRNA-186 and -655 inhibited their invasion abilities via MMP-9 activity. CONCLUSIONS: These results indicate that alteration of expression of PTTG1 in oral SCC cells by newly identified microRNA-186 and -655 can regulate invasion activity. Therefore, these data offer new insights into further understanding PTTG1 function in oral SCC and should provide new strategies for diagnostic markers for oral SCC.

miR-373-3p Regulates Invasion and Migration Abilities of Trophoblast Cells via Targeted CD44 and Radixin.

Preterm labor (PTL) is one of the obstetric complications, and is known to be associated with abnormal maternal inflammatory response and intrauterine inflammation and/or infection. However, the expression of specific miRNAs associated with PTL is not clear. In this study, we performed combination analysis of miRNA array and gene array, and then selected one miRNA (miR-373-3p) and its putative target genes (CD44 and RDX) that exhibited large expression differences in term and PTL placentas with or without inflammation. Using qRT-PCR and luciferase assays, we confirmed that miR-373-3p directly targeted CD44 and RDX. Overexpression of miR-373-3p reduced the migration and invasion of trophoblast cells, while inhibition of miR-373-3p restored the migration and invasion abilities of trophoblast cells. Finally, we validated the expression of miR-373-3p and its target genes in clinical patients' blood. miR-373-3p was increased in PTL patients' blood, and was the most expressed in PTL patients' blood with inflammation. In addition, by targeting the miR-373-3p, CD44 and RDX was decreased in PTL patients' blood, and their expression were the lowest in PTL patients' blood with inflammation. Taken together, these findings suggest that miR-373-3p and its target genes can be potential biomarkers for diagnosis of PTL.

Rational design of small molecule RHOA inhibitors for gastric cancer.

Previously, we identified Ras homologous A (RHOA) as a major signaling hub in gastric cancer (GC), the third most common cause of cancer death in the world, prompting us to rationally design an efficacious inhibitor of this oncogenic GTPase. Here, based on that previous work, we extend those computational analyses to further pharmacologically optimize anti-RHOA hydrazide derivatives for greater anti-GC potency. Two of these, JK-136 and JK-139, potently inhibited cell viability and migration/invasion of GC cell lines, and mouse xenografts, diversely expressing RHOA. Moreover, JK-136's binding affinity for RHOA was >140-fold greater than Rhosin, a nonclinical RHOA inhibitor. Network analysis of JK-136/-139 vs. Rhosin treatments indicated downregulation of the sphingosine-1-phosphate, as an emerging cancer metabolic pathway in cell migration and motility. We assert that identifying and targeting oncogenic signaling hubs, such as RHOA, represents an emerging strategy for the design, characterization, and translation of new antineoplastics, against gastric and other cancers.

Microenvironmental Interaction Between Hypoxia and Endothelial Cells Controls the Migration Ability of Placenta-Derived Mesenchymal Stem Cells via α4 Integrin and Rho Signaling.

Mesenchymal stem cells (MSCs) are a powerful source for cell therapy in degenerative diseases. The migration ability of MSCs is an important factor that enhances the therapeutic effect of the cells when they are transplanted into target tissues or organs. Hypoxia and the endothelial barrier, which are representative migration microenvironmental factors, are known to be regulated by the integrin-mediated pathway in several cancers. However, their regulatory mechanisms in MSCs remain unclear. Here, the objectives of the study were to compare the expression of markers related to integrin-mediated signaling in placenta-derived MSCs (PDMSCs) dependent on hypoxia and co-cultured with human umbilical vein endothelial cells (HUVECs) and to evaluate their correlations between migration ability and microenvironmetal factors including hypoxia and endothelial cells. The migration abilities of PDMSCs exposed to hypoxic conditions were significantly increased compared with normal fibroblasts (WI-38) and control (P < 0.05). Interestingly, decreased integrin α4 in PDMSCs under hypoxia induce to increase migration abilities of PDMSCs. Also, Rho family-related markers were significantly increased in PDMSCs under hypoxic conditions compared with normoxia (P < 0.05). Furthermore, the migration ability of PDMSCs was decreased by Rho kinase inhibitor treatment (Y-27632) and co-culturing with HUVECs in an ex vivo system. ROCK activity was increased by inhibiting integrin α4 with HUVECs and hypoxia compared with the absence of HUVECs and under normoxia. The findings suggest microenvironment event by hypoxia and the interaction with endothelial cells may be useful as a regulator of MSC migration and provide insight into the migratory mechanism of MSCs in stem cell-based therapy.

High Expression of PRNP Predicts Poor Prognosis in Korean Patients with Gastric Cancer.

Gastric cancer (GC) has the highest occurrence and fourth-highest mortality rate of all cancers in Korea. Although survival rates are improving with the development of diagnosis and treatment methods, the five-year survival rate for stage 4 GC in Korea remains <10%. Therefore, it is important to identify candidate prognostic factors for predicting poor prognosis. PRNP is a gene encoding the prion protein PrP, which has been noted for its role in the nervous system and is known to be upregulated in various cancers and associated with both cell proliferation and metastasis. However, the value of PRNP as a prognostic factor for Korean GC patients remains unclear. Here, we analyzed the relationship between PRNP expression and survival in three independent datasets for Korean patients with GC as well as the TCGA-STAD dataset. Survival analysis indicates that high levels of PRNP expression are associated with poor overall survival of patients with GC. Gene set enrichment analysis showed that PRNP is associated with epithelial mesenchymal transition and Hedgehog signaling. In addition, proliferation of GC cell lines was inhibited after siRNA-mediated knockdown of PRNP. In conclusion, our study suggests a potential role for PRNP as a candidate prognostic factor for patients with GC.

Microenvironmental changes induced by placenta-derived mesenchymal stem cells restore ovarian function in ovariectomized rats via activation of the PI3K-FOXO3 pathway.

BACKGROUND: Translational studies have explored the therapeutic potential and feasibility of mesenchymal stem cells (MSCs) in several degenerative diseases; however, mechanistic studies of the function of these cells have been insufficient. As ovarian failure causes anovulation as well as ovarian steroid hormonal imbalances, the specific aims of this study were to analyze the therapeutic role of placenta-derived MSCs (PD-MSCs) in an ovarian failure ovariectomy (OVX) rat model and evaluate whether PD-MSC transplantation (Tx) improved folliculogenesis and oocyte maturation in the injured ovary through PI3K/Akt and FOXO signaling. METHODS: Blood and ovary tissue were collected and analyzed after various PD-MSC Tx treatments in an ovariectomized rat model. Changes in the expression of folliculogenesis- and ovary regeneration-related genes induced by PD-MSC treatments were analyzed by qRT-PCR, Western blotting, and histological analysis. RESULTS: The levels of hormones related to ovary function were significantly increased in the PD-MSC Tx groups compared with those in the nontransplantation group (NTx). The follicle numbers in the ovarian tissues were increased along with the increased expression of genes related to folliculogenesis in the PD-MSC Tx groups compared with the NTx groups. Furthermore, Tx PD-MSCs induced follicle maturation by increasing the phosphorylation of GSK3 beta and FOXO3 (p < 0.05) and shifting the balance of growth and apoptosis in oocytes. CONCLUSIONS: Taken together, these results show that PD-MSC Tx can restore ovarian function and induce ovarian folliculogenesis via the PI3K/Akt and FOXO signaling pathway.

Identification of microRNAs and their target genes in the placenta as biomarkers of inflammation.

OBJECTIVE: Recently, microRNA (miRNA) has been identified both as a powerful regulator involved in various biological processes through the regulation of numerous genes and as an effective biomarker for the prediction and diagnosis of various disease states. The objective of this study was to identify and validate miRNAs and their target genes involved in inflammation in placental tissue. METHODS: Microarrays were utilized to obtain miRNA and gene expression profiles from placentas with or without inflammation obtained from nine normal pregnant women and 10 preterm labor patients. Quantitative real-time polymerase chain reaction and Western blots were performed to validate the miRNAs and differentially-expressed genes in the placentas with inflammation. Correlations between miRNA and target gene expression were confirmed by luciferase assays in HTR-8/SVneo cells. RESULTS: We identified and validated miRNAs and their target genes that were differentially expressed in placentas with inflammation. We also demonstrated that several miRNAs (miR-371a-5p, miR-3065-3p, miR-519b-3p, and miR-373-3p) directly targeted their target genes (LEF1, LOX, ITGB4, and CD44). However, some miRNAs and their direct target genes showed no correlation in tissue samples. Interestingly, miR-373-3p and miR-3065-3p were markedly regulated by lipopolysaccharide (LPS) treatment, although the expression of their direct targets CD44 and LOX was not altered by LPS treatment. CONCLUSION: These results provide candidate miRNAs and their target genes that could be used as placental biomarkers of inflammation. These candidates may be useful for further miRNA-based biomarker development.

Alterations in IL-6/STAT3 Signaling by Korean Mistletoe Lectin Regulate the Self-Renewal Activity of Placenta-Derived Mesenchymal Stem Cells.

Korean mistletoe (Viscum album L. var. coloratum) lectin (VCA) is known as an anticancer drug. However, it is not clear whether VCA affects the self-renewal activity of mesenchymal stem cells (MSCs). Therefore, the objectives of this study were to analyze the effect of VCA on the proliferation of MSCs and expression of stemness markers. We also evaluated the usefulness of placenta-derived MSCs (PD-MSCs) as a screening tool. VCA was stably administered to MSCs, and analyzed self-renewal activities. The effect of IL-6 signaling on MSC proliferation was explored by quantitative methylation-specific PCR (qMSP) and western blot analysis. Compared with the control condition, low concentrations of VCA (10 pg/mL) induced an increase in the self-renewal activity of MSCs. Interestingly, a low concentration of VCA promoted IL-6 signaling in PD-MSCs through altered IL-6/STAT3 gene methylation. Furthermore, inhibition of IL-6 expression in PD-MSCs using an anti-IL-6 antibody caused a decrease in their self-renewal activity through IL-6/STAT3 signaling by altering IL-6/STAT3 gene methylation. These findings provide helpful data for understanding the mechanism of MSC self-renewal via VCA and show that VCA may be useful as a functional natural product for developing efficient therapies using placenta-derived stem cells.

3D-cultured human placenta-derived mesenchymal stem cell spheroids enhance ovary function by inducing folliculogenesis.

Placenta-derived mesenchymal stem cells (PD-MSCs) have numerous advantages over other adult MSCs that make them an attractive cell source for regenerative medicine. Here, we demonstrate the therapeutic effect of PD-MSCs in ovariectomized (Ovx) rats and compare their efficacy when generated via a conventional monolayer culture system (2D, naïve) and a spheroid culture system (3D, spheroid). PD-MSC transplantation significantly increased the estradiol level in Ovx rats compared with the non-transplantation (NTx) group. In particular, the estradiol level in the Spheroid group was significantly higher than that in the Naïve group at 2 weeks. Spheroid PD-MSCs exhibited a significantly higher efficiency of engraftment onto ovarian tissues at 2 weeks. The mRNA and protein expression levels of Nanos3, Nobox, and Lhx8 were also significantly increased in the Spheroid group compared with those in the NTx group at 1 and 2 weeks. These results suggest that PD-MSC transplantation can restore ovarian function in Ovx rats by increasing estrogen production and enhancing folliculogenesis-related gene expression levels and further indicate that spheroid-cultured PD-MSCs have enhanced therapeutic potential via increased engraftment efficiency. These findings improve our understanding of stem-cell-based therapies for reproductive systems and may suggest new avenues for developing efficient therapies using 3D cultivation systems.

Alteration of Pituitary Tumor Transforming Gene-1 Regulates Trophoblast Invasion via the Integrin/Rho-Family Signaling Pathway.

Trophoblast invasion ability is an important factor in early implantation and placental development. Recently, pituitary tumor transforming gene 1 (PTTG1) was shown to be involved in invasion and proliferation of cancer. However, the role of PTTG1 in trophoblast invasion remains unknown. Thus, in this study we analyzed PTTG1 expression in trophoblasts and its effect on trophoblast invasion activity and determined the mechanism through which PTTG1 regulates trophoblast invasion. Trophoblast proliferation and invasion abilities, regardless of PTTG1 expression, were analyzed by quantitative real-time polymerase chain reaction, fluorescence-activated cell sorting analysis, invasion assay, western blot, and zymography after treatment with small interfering RNA against PTTG1 (siPTTG1). Additionally, integrin/Rho-family signaling in trophoblasts by PTTG1 alteration was analyzed. Furthermore, the effect of PTTG1 on trophoblast invasion was evaluated by microRNA (miRNA) mimic and inhibitor treatment. Trophoblast invasion was significantly reduced through decreased matrix metalloproteinase (MMP)-2 and MMP-9 expression when PTTG1 expression was inhibited by siPTTG1 (p < 0.05). Furthermore, knockdown of PTTG1 increased expression of integrin alpha 4 (ITGA4), ITGA5, and integrin beta 1 (ITGB1); otherwise, RhoA expression was significantly decreased (p < 0.05). Treatment of miRNA-186-5p mimic and inhibitor controlled trophoblast invasion ability by altering PTTG1 and MMP expression. PTTG1 can control trophoblast invasion ability via regulation of MMP expression through integrin/Rho-family signaling. In addition, PTTG1 expression and its function were regulated by miRNA-186-5p. These results help in understanding the mechanism through which PTTG1 regulates trophoblast invasion and thereby implantation and placental development.