Xueting Su

MicroRNA-200b Is Overexpressed in Endometrial Adenocarcinomas and Enhances MMP2 Activity by Downregulating TIMP2 in Human Endometrial Cancer Cell Line HEC-1A Cells

MicroRNAs (miRNAs) play important roles in tumorigenesis and metastasis. In this study, we investigated miR-200b expression in endometrial adenocarcinomas and normal adjacent tissues and found that miR-200b is more highly expressed in cancer tissues than in normal adjacent tissues. A novel target of miR-200b, tissue inhibitor of metalloproteinase 2 (TIMP2), was predicted using a bioinformatics approach and was confirmed in human endometrial cancer cell line HEC-1A cells by luciferase assay, quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. We found that miR-200b repressed TIMP2 expression at both the messenger RNA and protein levels, although a family member, miR-200a, had no such effect. Using reverse gelatin zymography, we showed that miR-200b enhances matrix metallopeptidase 2 (MMP2) activity by downregulating TIMP2 expression in HEC-1A cells. These data suggest that miR-200b may play an important role in the metastasis of endometrial adenocarcinomas.

MiR-138 downregulates miRNA processing in HeLa cells by targeting RMND5A and decreasing Exportin-5 stability

MicroRNAs (miRNAs) are a class of non-coding small RNAs that consist of ∼22 nt and are involved in several biological processes by regulating target gene expression. MiR-138 has many biological functions and is often downregulated in cancers. Our results showed that overexpression of miR-138 downregulated target RMND5A (required for meiotic nuclear division 5 homolog A) and reduced Exportin-5 stability, which results in decreased levels of pre-miRNA nuclear export in HeLa cells. We also found that miR-138 could significantly inhibit HeLa cell migration by targeting RMND5A. Our study therefore identifies miR-138–RMND5A–Exportin-5 as a previously unknown miRNA processing regulatory pathway in HeLa cells.

MiR‐152 reduces human umbilical vein endothelial cell proliferation and migration by targeting ADAM17

As a cleavage enzyme of precursor TNF‐α, the high expression level of ADAM17 in endothelial cells is an important factor in atherosclerosis. In this study, we demonstrate that ADAM17 is the target of miR‐152. We found that miR‐152 could reduce TNF precursor cleavage and inhibit cell proliferation and migration by targeting ADAM17 in human umbilical vein endothelial cells (HUVECs). Furthermore, the expression pattern of miR‐152 and corresponding target ADAM17 was opposite in HUVECs under hypoxic conditions. The levels of circulating miR‐152 in AS patient sera were lower than those detected in the sera of normal individuals. Our results indicate that miR‐152 may be involved in the development of human atherosclerosis and could be used as diagnostic biomarker or therapeutic target in atherosclerosis.

Exosomal transfer of vasorin expressed in hepatocellular carcinoma cells promotes migration of human umbilical vein endothelial cells.

Vasorin (VASN) is a type I transmembrane protein that plays important roles in tumor development and vasculogenesis. In this paper, we showed that VASN could be a key mediator of communication between tumor cells and endothelial cells. We confirmed for the first time that HepG2-derived VASN can be transferred to human umbilical vein endothelial cells (HUVECs) via receptor mediated endocytosis of exosomes, at least in part through HSPGs. The HepG2-derived VASN containing exosomes promote migration of recipient HUVECs cells. Our results identify a novel pathway by which a functional protein expressed in tumor cells affects the biological fate of endothelial cells via exosomes.

Nanoparticle-conjugated aptamer targeting hnRNP A2/B1 can recognize multiple tumor cells and inhibit their proliferation.

In this study, we further investigated a previously developed aptamer targeting ROS 17/2.8 (rat osteosarcoma) cells. We found that this C6-8 aptamer specifically binds to heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 and that it specifically labeled multiple tumor-cell lines as effectively as hnRNP A2/B1 monoclonal antibodies. When conjugated with fluorescent carbon nanodots (CDots) it could freely enter multiple living tumor cell lines (HepG2, MCF-7, H1299, and HeLa), whose growth it inhibited by targeting hnRNP A2/B1. Similar inhibitory effects were observed when the GFP-HepG2 hepatocarcinoma cells treated with C6-8-conjugated CDots were implanted in nude mice. Our work provides a new aptamer for targeting/labeling multiple tumor cell types, and its nanoparticle conjugates bring further advantages that increase its potential for use in cancer diagnosis and therapy.

Identification and expression of small non-coding RNA, L10-Leader, in different growth phases of Streptococcus mutans.

Streptococcus mutans is one of the major cariogenic bacteria in the oral environment. Small non-coding RNAs (sRNAs) play important roles in the regulation of bacterial growth, stress tolerance, and virulence. In this study, we experimentally verified the existence of sRNA, L10-Leader, in S. mutans for the first time. Our results show that the expression level of L10-Leader was growth-phase dependent in S. mutans and varied among different clinical strains of S. mutans. The level of L10-Leader in S. mutans UA159 was closely related to the pH value, but not to the concentrations of glucose and sucrose in culture medium. We predicted target mRNAs of L10-Leader bioinformatically and found that some of these mRNAs were related to growth and stress response. Five predicted mRNA targets were selected and detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and we found that the expression levels of these mRNAs were closely related to the level of L10-Leader at different growth phases of the bacteria. Our results indicate that L10-Leader may play an important role in the regulation of responses in S. mutans, especially during its growth phase and acid adaption response.

A strategy to rapidly identify the functional targets of microRNAs by combining bioinformatics and mRNA cytoplasmic/nucleic ratios in culture cells

MicroRNAs are approximately 22nt non‐coding RNAs that are present in a broad range of multicellular organisms. MicroRNAs play important roles in many biological or pathological processes by regulating the expression of their target genes. The fast and accurate identification of miRNA targets is a bottleneck in the clarification of the function of miRNAs. Here, we established a rapid and accurate strategy to identify miRNA functional target genes by combination of bioinformatic prediction with Cytoplasmic/Nuclear (C/N) ratios of mRNAs. The strategy comprises three steps: bioinformatic prediction, determination of mRNA C/N ratios, and confirmation by Western blotting, and might be suitable to most miRNAs. Our method will make a significant contribution to the study of the biological functions of miRNAs.

Aptamer BC15 against heterogeneous nuclear ribonucleoprotein A1 has potential value in diagnosis and therapy of hepatocarcinoma.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was reported to be participated in tumor development. The association between hnRNP A1 and liver cancer and the functional role of hnRNP A1 in liver cancer have never been reported. Herein, hnRNP A1-specific single-stranded DNA aptamer, BC15, was used to (a) evaluate hnRNP A1 expression in liver cancer, and (b) treat hepatocarcinoma by acting as an inhibitor of hnRNP A1. Results showed that there is high hnRNP A1 expression in liver cancer including serum α-fetoprotein-negative liver cancer tissues compared with either para-cancer or benign controls. Down regulation of hnRNP A1 expression by RNA interference inhibits the proliferation and migration of cancerous HepG2 cells, while overexpression of hnRNP A1 in normal HL-7702 cells increased the proliferation and migration of the cells. Importantly, BC15 showed a stronger inhibiting effect on the proliferation of cultured hepatoma cells than hnRNP A1 small interfering RNA, strongly suggesting that BC15 could also be a potential drug candidate for an hnRNP A1 inhibitor besides its prospect utility in in situ histological examination.

Species-specific mutual regulation of p53 and miR-138 between human, rat and mouse.

In recent years, p53 was identified to regulate the expression of many miRNAs and was also regulated by miRNAs. In this paper, we found that miR-138 showed a pronounced increase after p53 activation in human non-small cell lung cancer (NSCLC) cells, which is mediated by p53 binding sites in the promoter region of its host gene, but this did not happen with rat and mouse cells. More interestingly, we found that p53 could be also regulated by miR-138 in mouse and rat cells, but not in the human NSCLC cells. Our results suggest the existence of species-specific differences of the regulations of miRNA against its targets and the regulations of miRNA itself by other proteins.

MiR-200b modulates the properties of human monocyte-derived dendritic cells by targeting WASF3

AIMS The aim of the study was to explore the effect of miR-200b on the development of human peripheral blood monocyte-deriveddendritic-cell (DC) and its mechanisms. MAIN METHODS Expression levels of miR-200b and its predicted targets were measured by real time-PCR. Protein expression of WASF3 was determined by Western blot and immunohistochemistry. Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation from the buffy coat fraction of anticoagulated blood. Monocytes were purified from PBMCs using anti-CD14 microbeads. The immunophenotypes of DCs were tested by flow cytometry. KEY FINDINGS A strong reduction in miR-200b expression was associated with human peripheral blood monocyte-derivedDC differentiation. The overexpression of miR-200b significantly reduced the numbers of protruding veils in mature DCs (mDCs) that are critical for promoting antigen-specificT-cell activation. Further experiments showed that miR-200b could regulate the function of DCs by targeting WASF3, a protein involved in cell movement and invasion. SIGNIFICANCE Our results define an important function of miR-200b in the negative regulation of DC development and provide a potential form of miRNA-mediated cell therapy for diseases that range from auto-immunity to graft-versus-host disease.