miR-34a-5p might have an important role for inducing apoptosis by down-regulation of SNAI1 in apigenin-treated lung cancer cells.

Apigenin is a flavonoid with antioxidant and anticancer effects. It has been reported that apigenin inhibits proliferation, migration, and invasion and induces apoptosis in cultured lung cancer cells. However, there is little information on the involvement of microRNAs (miRNAs) in its effects. miRNA microarray analysis and polymerase-chain-reaction analysis of miRNAs revealed that treatment of human lung cancer A549 cells with apigenin up-regulated the level of miR-34a-5p. Furthermore, mRNA microarray analysis and the results of three microRNA target prediction tools showed that Snail Family Transcriptional Repressor 1 (SNAI1), which inhibits the induction of apoptosis, had its mRNA expression down-regulated in A549 cells treated with apigenin. Our findings suggest that apigenin might induce apoptosis by down-regulation of SNAI1 through up-regulation of miR-34a-5p in A549 cells.

MicroRNA-493-5p-mediated repression of the MYCN oncogene inhibits hepatic cancer cell growth and invasion.

Primary hepatic tumors mainly include hepatocellular carcinoma (HCC), which is one of the most frequent causes of cancer-related deaths worldwide. Thus far, HCC prognosis has remained extremely poor given the lack of effective treatments. Numerous studies have described the roles played by microRNAs (miRNAs) in cancer progression and the potential of these small noncoding RNAs for diagnostic or therapeutic applications. The current consensus supports the idea that direct repression of a wide range of oncogenes by a single key miRNA could critically affect the malignant properties of cancer cells in a synergistic manner. In this study, we aimed to investigate the oncogenes controlled by miR-493-5p, a major tumor suppressor miRNA that inactivates miR-483-3p oncomir in hepatic cancer cells. Using global gene expression analysis, we highlighted a set of candidate genes potentially regulated by miR-493-5p. In particular, the canonical MYCN protooncogene (MYCN) appeared to be an attractive target of miR-493-5p given its significant inhibition through 3'-UTR targeting in miR-493-5p-rescued HCC cells. We showed that MYCN was overexpressed in liver cancer cell lines and clinical samples from HCC patients. Notably, MYCN expression levels were inversely correlated with miR-493-5p in tumor tissues. We confirmed that MYCN knockdown mimicked the anticancer effect of miR-493-5p by inhibiting HCC cell growth and invasion, whereas MYCN rescue hindered miR-493-5p activity. In summary, miR-493-5p is a pivotal miRNA that modulates various oncogenes after its reexpression in liver cancer cells, suggesting that tumor suppressor miRNAs with a large spectrum of action could provide valuable tools for miRNA replacement therapies.

The clinical relevance of the miR-197/CKS1B/STAT3-mediated PD-L1 network in chemoresistant non-small-cell lung cancer.

Programmed cell death ligand-1 (PD-L1) has recently gained considerable attention for its role in tumor immune escape. Here, we identify a miR-197/CKS1B/STAT3-mediated PD-L1 network in chemoresistant non-small-cell lung cancer (NSCLC), independent of immunoinhibitory signals. miR-197 is downregulated in platinum-resistant NSCLC specimens, resulting in the promotion of chemoresistance, tumorigenicity, and pulmonary metastasis in vitro and in vivo. Mechanistic investigations reveal that a miR-197-mediated CKS1B/STAT3 axis exerts tumor progression regulated by various oncogenic genes (Bcl-2, c-Myc, and cyclin D1), and PD-L1 is a putative biomarker of this axis. Furthermore, we demonstrate that a miR-197 mimic sensitizes PD-L1(high) drug-resistant cells to chemotherapy. These results indicate that the biological interaction between PD-L1 and chemoresistance occurs through the microRNA regulatory cascade. More importantly, expression levels of miR-197 are inversely correlated with PD-L1 expression (n = 177; P = 0.026) and are associated with worse overall survival (P = 0.015). Our discoveries suggest that the miR-197/CKS1B/STAT3-mediated network can drive tumor PD-L1 expression as a biomarker of this cascade, and miR-197 replacement therapy may be a potential treatment strategy for chemoresistant NSCLC.

Clinical relevance and therapeutic significance of microRNA-133a expression profiles and functions in malignant osteosarcoma-initiating cells.

Novel strategies against treatment-resistant tumor cells remain a challenging but promising therapeutic approach. Despite accumulated evidence suggesting the presence of highly malignant cell populations within tumors, the unsolved issues such as in vivo targeting and clinical relevance remain. Here, we report a preclinical trial based on the identified molecular mechanisms underlying osteosarcoma-initiating cells and their clinical relevance. We identified key microRNAs (miRNAs) that were deregulated in a highly malignant CD133(high) population and found that miR-133a regulated the cell invasion that characterizes a lethal tumor phenotype. Silencing of miR-133a with locked nucleic acid (LNA) reduced cell invasion of this cell population, and systemic administration of LNA along with chemotherapy suppressed lung metastasis and prolonged the survival of osteosarcoma-bearing mice. Furthermore, in a clinical study, high expression levels of CD133 and miR-133a were significantly correlated with poor prognosis, whereas high expression levels of the four miR-133a target genes were correlated with good prognosis. Overall, silencing of miR-133a with concurrent chemotherapy would represent a novel strategy that targets multiple regulatory pathways associated with metastasis of the malignant cell population within osteosarcoma.

Neutral sphingomyelinase 2 (nSMase2)-dependent exosomal transfer of angiogenic microRNAs regulate cancer cell metastasis.

The release of humoral factors between cancer cells and the microenvironmental cells is critical for metastasis; however, the roles of secreted miRNAs in non-cell autonomous cancer progression against microenvironmental cells remain largely unknown. Here, we demonstrate that the neutral sphyngomyelinase 2 (nSMase2) regulates exosomal microRNA (miRNA) secretion and promotes angiogenesis within the tumor microenvironment as well as metastasis. We demonstrate a requirement for nSMase2-mediated cancer cell exosomal miRNAs in the regulation of metastasis through the induction of angiogenesis in inoculated tumors. In addition, miR-210, released by metastatic cancer cells, was shown to transport to endothelial cells and suppress the expression of specific target genes, which resulted in enhanced angiogenesis. These findings suggest that the horizontal transfer of exosomal miRNAs from cancer cells can dictate the microenviromental niche for the benefit of the cancer cell, like "on demand system" for cancer cells.

RPN2-mediated glycosylation of tetraspanin CD63 regulates breast cancer cell malignancy.

BACKGROUND: The tetraspanin CD63 is a highly N-glycosylated protein that is known to regulate cancer malignancy. However, the contribution of glycosylation of CD63 to cancer malignancy remains unclear. Previously, we reported that ribophorin II (RPN2), which is part of an N-oligosaccharyle transferase complex, is responsible for drug resistance in breast cancer cells. In this study, we demonstrate that cancer malignancy associated with the glycosylation of CD63 is regulated by RPN2. RESULTS: Inhibition of RPN2 expression led to a reduction in CD63 glycosylation. In addition, the localization of CD63 was deregulated by knockdown of RPN2. Interestingly, multidrug resistance protein 1 (MDR1) localization was displaced from the cell surface in CD63-silenced cells. CD63 silencing reduced the chemoresistance and invasion ability of malignant breast cancer cells. Furthermore, the enrichment of CD63/MDR1-double positive cells was associated with lymph node metastasis. Taken together, these results indicated that high glycosylation of CD63 by RPN2 is implicated in clinical outcomes in breast cancer patients. CONCLUSIONS: These findings describe a novel and important function of RPN2-mediated CD63 glycosylation, which regulates MDR1 localization and cancer malignancy, including drug resistance and invasion.

miR-148a plays a pivotal role in the liver by promoting the hepatospecific phenotype and suppressing the invasiveness of transformed cells.

UNLABELLED: MicroRNAs (miRNAs) are evolutionary conserved small RNAs that post-transcriptionally regulate the expression of target genes. To date, the role of miRNAs in liver development is not fully understood. By using an experimental model that allows the induced and controlled differentiation of mouse fetal hepatoblasts (MFHs) into mature hepatocytes, we identified miR-148a as a hepatospecific miRNA highly expressed in adult liver. The main finding of this study revealed that miR-148a was critical for hepatic differentiation through the direct targeting of DNA methyltransferase (DNMT) 1, a major enzyme responsible for epigenetic silencing, thereby allowing the promotion of the "adult liver" phenotype. It was also confirmed that the reduction of DNMT1 by RNA interference significantly promoted the expression of the major hepatic biomarkers. In addition to the essential role of miR-148a in hepatocyte maturation, we identified its beneficial effect through the repression of hepatocellular carcinoma (HCC) cell malignancy. miR-148a expression was frequently down-regulated in biopsies of HCC patients as well as in mouse and human HCC cell lines. Overexpressing miR-148a led to an enhancement of albumin production and a drastic inhibition of the invasive properties of HCC cells, whereas miR-148a silencing had the opposite consequences. Finally, we showed that miR-148a exerted its tumor-suppressive effect by regulating the c-Met oncogene, regardless of the DNMT1 expression level. CONCLUSION: miR-148a is essential for the physiology of the liver because it promotes the hepatospecific phenotype and acts as a tumor suppressor. Most important, this report is the first to demonstrate a functional role for a specific miRNA in liver development through regulation of the DNMT1 enzyme.

Dark side of the exosome: the role of the exosome in cancer metastasis and targeting the exosome as a strategy for cancer therapy.

Cell-cell communication is essential for the regulation of various biological phenomena in multicellular organisms, including development and homeostasis. Deregulation of these interactions leads to inappropriate cell-cell communication, resulting in disease development. Cancer cells communicate closely with the cells in their microenvironment, and this communication promotes malignancy via abnormal growth, invasion, drug resistance and metastasis. Understanding cell-cell interactions in cancer is essential for the development of novel anticancer agents. As a result, discovering the communication tools used by cancer cells is important to understanding these interactions. In this review, we summarize the recent findings regarding exosome-mediated cancer development. In addition, we propose that targeting the exosome represents a novel strategy for cancer therapy.

Competitive interactions of cancer cells and normal cells via secretory microRNAs.

Normal epithelial cells regulate the secretion of autocrine and paracrine factors that prevent aberrant growth of neighboring cells, leading to healthy development and normal metabolism. One reason for tumor initiation is considered to be a failure of this homeostatic cell competitive system. Here we identify tumor-suppressive microRNAs (miRNAs) secreted by normal cells as anti-proliferative signal entities. Culture supernatant of normal epithelial prostate PNT-2 cells attenuated proliferation of PC-3M-luc cells, prostate cancer cells. Global analysis of miRNA expression signature revealed that a variety of tumor-suppressive miRNAs are released from PNT-2 cells. Of these miRNAs, secretory miR-143 could induce growth inhibition exclusively in cancer cells in vitro and in vivo. These results suggest that secretory tumor-suppressive miRNAs can act as a death signal in a cell competitive process. This study provides a novel insight into a tumor initiation mechanism.

4-Hydroxyderricin inhibits osteoclast formation and accelerates osteoblast differentiation.

4-Hydroxyderricin (4-HD) is a major polyphenol of Angelica keiskei (Japanese name Ashitaba), exhibiting anti-allergic, anti-diabetic, anti-oxidant, and antitumor effects. The present study was designed to evaluate the effects of 4-HD on bone formation and maintenance by using cultured osteoclasts and osteoblasts. 4-HD did not affect cell proliferation of stromal ST2 cells and preosteoblast MC3T3-E1 cells at concentrations of 1-10 µM. This compound inhibited the formation of multinucleated osteoclasts from mouse splenic cells, and we identified a molecular pathway of osteoclast differentiation mediated by 4-HD, which led to inhibition of the expression of receptor activator of nuclear factor-κB ligand and macrophage-colony stimulating factor in ST2 cells. By contrast, 4-HD enhanced indices of osteoblast differentiation, such as alkaline phosphatase activity and calcium deposition by osteoblastic MC3T3-E1 cells, at concentrations of 1-10 µM. Furthermore, we found that 4-HD at 1 µM attenuated H2O2 levels in MC3T3-E1 cells. Our findings indicate that 4-HD may have critical effects on bone formation and maintenance.

Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity.

A challenge for advancing approaches to liver regeneration is loss of functional differentiation capacity when hepatocyte progenitors are maintained in culture. Recent lineage-tracing studies have shown that mature hepatocytes (MHs) convert to an immature state during chronic liver injury, and we investigated whether this conversion could be recapitulated in vitro and whether such converted cells could represent a source of expandable hepatocytes. We report that a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, can convert rat and mouse MHs in vitro into proliferative bipotent cells, which we term chemically induced liver progenitors (CLiPs). CLiPs can differentiate into both MHs and biliary epithelial cells that can form functional ductal structures. CLiPs in long-term culture did not lose their proliferative capacity or their hepatic differentiation ability, and rat CLiPs were shown to extensively repopulate chronically injured liver tissue. Thus, our study advances the goals of liver regenerative medicine.

Commitment of Annexin A2 in recruitment of microRNAs into extracellular vesicles.

Extracellular vesicles (EVs) contain microRNAs (miRNAs). However, the exact molecular mechanisms of the recruitment of miRNAs in EVs are not well characterized. Based on proteomic analysis, we identified that silencing of Annexin A2 (ANXA2) significantly decreased the amount of miRNAs in EVs. In addition, microarray analysis revealed that ANXA2 regulated the loading of miRNAs into EVs in a sequence independent manner. Lastly, immunoprecipitation analysis confirmed that ANXA2 could bind miRNAs in EVs in the presence of Ca(2+). These observations demonstrate that ANXA2 plays an important role in the packaging process of miRNAs into EVs.

A robust screening method for dietary agents that activate tumour-suppressor microRNAs.

Certain dietary agents, such as natural products, have been reported to show anti-cancer effects. However, the underlying mechanisms of these substances in human cancer remain unclear. We recently found that resveratrol exerts an anti-cancer effect by upregulating tumour-suppressor microRNAs (miRNAs). In the current study, we aimed to identify new dietary products that have the ability to activate tumour-suppressor miRNAs and that therefore may serve as novel tools for the prevention and treatment of human cancers. We describe the generation and use of an original screening system based on a luciferase-based reporter vector for monitoring miR-200c tumour-suppressor activity. By screening a library containing 139 natural substances, three natural compounds - enoxolone, magnolol and palmatine chloride - were identified as being capable of inducing miR-200c expression in breast cancer cells at 10 µM. Moreover, these molecules suppressed the invasiveness of breast cancer cells in vitro. Next, we identified a molecular pathway by which the increased expression of miR-200c induced by natural substances led to ZEB1 inhibition and E-cadherin induction. These results indicate that our method is a valuable tool for a fast identification of natural molecules that exhibit tumour-suppressor activity in human cancer through miRNA activation.

Apigenin inhibits osteoblastogenesis and osteoclastogenesis and prevents bone loss in ovariectomized mice.

Polyphenol have been reported to have physiological effects with respect to alleviating diseases such as osteoporosis and osteopetrosis. We recently reported that the olive polyphenol hydroxytyrosol accelerates bone formation both in vivo and in vitro. The present study was designed to evaluate the in vivo and in vitro effects of apigenin (4',5,7-trihydroxyflavone), one of the major polyphenols in olives and parsley, on bone formation by using cultured osteoblasts and osteoclasts and ovariectomized (OVX) mice, respectively. Apigenin markedly inhibited cell proliferation and indices of osteoblast differentiation, such as collagen production, alkaline phosphatase activity, and calcium deposition in osteoblastic MC3T3-E1 cells at concentrations of 1-10 µM. At 10 µM, apigenin completely inhibited the formation of multinucleated osteoclasts from mouse splenic cells. Moreover, injection of apigenin at 10 mg kg(-1) body weight significantly suppressed trabecular bone loss in the femurs of OVX mice. Our findings indicate that apigenin may have critical effects on bone maintenance in vivo.

Human induced hepatic lineage-oriented stem cells: autonomous specification of human iPS cells toward hepatocyte-like cells without any exogenous differentiation factors.

Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs) using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs) were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG), conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5), transporters (SULT2A1, SLC13A5, and SLCO2B1), and urea cycle-related enzymes (ARG1 and CPS1). In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density) in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the necessity of optimizing culture conditions to generate other specific lineage-oriented hiPSCs, allowing for a very simple differentiation.

A paradigm shift for extracellular vesicles as small RNA carriers: from cellular waste elimination to therapeutic applications.

RNA interference (RNAi) is an important avenue for target-specific gene silencing that is mainly performed by either small interfering RNAs (siRNAs) or microRNAs (miRNAs). This novel method is rapidly becoming a powerful tool for gene therapy. However, the rapid degradation of siRNAs and miRNAs and the limited duration of their action in vivo call for an efficient delivery technology. Recently, increasing attention has been paid to the use of extracellular vesicles (EVs) as delivery systems. The use of EVs as small RNA carriers has multiple advantages over conventional delivery systems. In this review, we summarize recent findings regarding the potential application of EVs as small RNA delivery systems. Moreover, we focus on some of the obstacles to EV-based therapeutics.

Functional analysis of exosomal microRNA in cell-cell communication research.

Circulating microRNAs (miRNAs), also known as secretory miRNAs, are packaged in small membrane vesicles called exosomes. These exosomal miRNAs are secreted from various cell types and incorporated inside the recipient cells. The functions of exosomal miRNAs are poorly understood, but some reports have shown their essential roles in cancer development. Therefore, methods to study the function of exosomal miRNAs not only in vitro but also in vivo might be essential. We have analyzed the function of exosomal miRNAs by miRNA-enriched exosomes both in vitro and in vivo. In this chapter, the methods to concentrate the targeted miRNAs are provided. This simple and useful method enables the study of the precise mechanisms of exosomal miRNAs under physiological and pathological conditions.

Exosomal tumor-suppressive microRNAs as novel cancer therapy: "exocure" is another choice for cancer treatment.

MicroRNAs (miRNAs) act to fine-tune cellular responses in a variety of biological circumstances such as development, organogenesis, and homeostasis. The dysregulation of miRNA expression accelerates disease progression, including metabolic disease, immunological disease and cancer, through the gene network disorder. Therefore, understanding the miRNA maturation process may unravel the mechanisms of cancer malignancy; however, the life of miRNA has not been clarified. In this article, we summarize the recent findings regarding the novel forms of miRNA, especially secretory miRNAs, focusing on exosomal miRNAs. Recent research has revealed that exosomal miRNAs affect many aspects of physiological and pathological conditions, and may be useful as novel therapy. Here, we propose a method for the delivery of tumor-suppressive miRNAs to desired sites using exosomes, and we named this method "exocure".

Trash or Treasure: extracellular microRNAs and cell-to-cell communication.

Circulating RNAs in human body fluids are promising candidates for diagnostic purposes. However, the biological significance of circulating RNAs remains elusive. Recently, small non-coding RNAs, microRNAs (miRNAs), were isolated from multiple human body fluids, and these "circulating miRNAs" have been implicated as novel disease biomarkers. Concurrently, miRNAs were also identified in the extracellular space associated with extracellular vesicles (EVs), which are small membrane vesicles secreted from various types of cells. The function of these secreted miRNAs has been revealed in several papers. Circulating miRNAs have been experimentally found to be associated with EVs; however, other types of extracellular miRNAs were also described. This review discusses studies related to extracellular miRNAs, including circulating miRNAs and secreted miRNAs, to highlight the importance of studying not only secreted miRNAs, but also circulating miRNAs to determine the contribution of extracellular miRNAs especially in cancer development.