Zhongwei Lv

MiR-138 Inhibits Tumor Growth Through Repression of EZH2 in Non-Small Cell Lung Cancer

Background/Aims: MicroRNAs (miRNAs) play important roles in tumorigenesis. We investigated the roles and mechanisms of miR-138 in human non-small cell lung cancer (NSCLC). Methods: The expression of miR-138 was first examined in NSCLC cell lines and tumourtissues by real-time PCR The in vitro and in vivo functional effect of miR-138 was examined further. A luciferase reporter assay was conducted to confirm target association between miR-138 and the enhancer of zeste homolog 2 (EZH2). Results: miR-138 was frequently downregulated in NSCLC cells and tissues. Overexpression of miR-138 inhibited proliferation of NSCLC cells in vitro and tumor growth in vivo. The EZH2 oncogene, which is often overexpressed in various human cancers and acts as an important regulator of cell growth and tumor invasion, was identified as a novel target of miR-138. miR-138 can bind to the 3′ untranslated region (3′ UTR) of EZH2 and suppress the expression of EZH2 at both mRNA and protein levels. Furthermore, knockdown of EZH2 phenocopied the tumor suppressive effects of miR-138 in cell models, whereas ectopic expression of EZH2 rescued the suppressive effects of miR-138. Conclusion: These findings define a tumor suppressor function for miR-138 in NSCLC and further suggest that miR-138 may represent a potential therapeutic target for NSCLC patients.

Adult rat mesenchymal stem cells differentiate into neuronal-like phenotype and express a variety of neuro-regulatory molecules in vitro.

Bone marrow stromal stem cells (MSCs), which normally differentiate into mesenchymal derivatives, have recently reported to trans-differentiate into neurons. However, the findings from different groups and interpretations have been challenged. The purpose of this paper is to re-evaluate the phenomenon of neuronal trans-differentiation of MSCs and compare the expression levels of neurotrophins in rMSCs and neuronal-like phenotypes derived from rMSCs. We put rMSCs in 2-mercaptoethanol and 2% dimethylsulfoxide for 5h. Then, the cells were transferred to neuronal induction media composed of DMEM+10%FBS, 10ug/L basic fibroblast growth factor, 10ug/L human epidermal growth factor, 1mmol dibutyryl cyclicn AMP and 0.5mmol isobutylmethylxanthine for 7 days and 14 days. The study demonstrated that the level of BDNF, NGF, NT3, CNTF and GDNF of rMSCs is remarkably higher in rMSCs than the neuronal-like phenotypes, especially CNTF. The expression level of these neurotrophins did not change significantly after enduring induction. We believed that rMSCs can trans-differentiate into neuronal-like phenotype under certain conditions. The non-induced rMSCs has a dynamic expression profile of neurotrophins and may serves as a better tool than the trans-differentiated rMSCs for transplant therapy.

Downregulation of MicroRNA-130a Contributes to Endothelial Progenitor Cell Dysfunction in Diabetic Patients via Its Target Runx3

Dysfunction of endothelial progenitor cells (EPCs) contributes to diabetic vascular disease. MicroRNAs (miRs) have emerged as key regulators of diverse cellular processes including angiogenesis. We recently reported that miR-126, miR-130a, miR-21, miR-27a, and miR-27b were downregulated in EPCs from type II diabetes mellitus (DM) patients, and downregulation of miR-126 impairs EPC function. The present study further explored whether dysregulated miR-130a were also related to EPC dysfunction. EPCs were cultured from peripheral blood mononuclear cells of diabetic patients and healthy controls. Assays on EPC function (proliferation, migration, differentiation, apoptosis, and colony and tubule formation) were performed. Bioinformatics analyses were used to identify the potential targets of miR-130a in EPCs. Gene expression of miR-103a and Runx3 was measured by real-time PCR, and protein expression of Runx3, extracellular signal-regulated kinase (ERK), vascular endothelial growth factor (VEGF) and Akt was measured by Western blotting. Runx3 promoter activity was measured by luciferase reporter assay. A miR-130a inhibitor or mimic and lentiviral vectors expressing miR-130a, or Runx3, or a short hairpin RNA targeting Runx3 were transfected into EPCs to manipulate miR-130a and Runx3 levels. MiR-130a was decreased in EPCs from DM patients. Anti-miR-130a inhibited whereas miR-130a overexpression promoted EPC function. miR-130a negatively regulated Runx3 (mRNA, protein and promoter activity) in EPCs. Knockdown of Runx3 expression enhanced EPC function. MiR-130a also upregulated protein expression of ERK/VEGF and Akt in EPCs. In conclusion, miR-130a plays an important role in maintaining normal EPC function, and decreased miR-130a in EPCs from DM contributes to impaired EPC function, likely via its target Runx3 and through ERK/VEGF and Akt pathways.

Long noncoding RNA H19 competitively binds miR‐17‐5p to regulate YES1 expression in thyroid cancer

The long noncoding RNA H19 is overexpressed in many cancers and acts as an oncogene. Here, we investigated the role of H19 in thyroid carcinogenesis and its relation to microRNA miR‐17‐5p and its target gene YES1. H19 expression was higher in tumor samples and in thyroid cancer cell lines than nontumor tissues and normal thyroid cells. H19 knockdown and ectopic expression in the TPC‐1 and NIM thyroid cancer cell lines showed that overexpression of H19 promoted proliferation, migration, and invasion, whereas H19 knockdown reduced cell viability and invasion and induced growth arrest in vitro and in vivo. H19 was identified as a target of miR‐17‐5p, by Dual‐Luciferase Reporter assays and RNA‐binding protein immunoprecipitation assays. H19 antagonized the function of miR‐17‐5p on upregulation of its target YES1 and inhibited miR‐17‐5p‐induced cell cycle progression. Our results suggest that H19 functions as a competitive endogenous RNA (ceRNA) by acting as a sink for miR‐17‐5p, revealing a potential ceRNA regulatory network involving H19 and miR‐17‐5p with a role in the modulation of YES1 expression. This mechanism may contribute to a better understanding of thyroid cancer pathogenesis and provide new insights into the treatment of this disease.

RLIP76 is overexpressed in human glioblastomas and is required for proliferation, tumorigenesis and suppression of apoptosis

The guanosine triphosphatase-activating protein RLIP76 is overexpressed in many malignant tumor cells, but it is unclear if RLIP76 overexpression contributes to the high proliferative potential of glioma cells. We demonstrate that RLIP76 messenger RNA and protein expression are positively correlated with glioma grade and that higher RLIP76 expression correlates with shorter patient survival. Immunohistochemical staining revealed that RLIP76 expression was positively correlated with the expression of Ki-67, a biomarker for cell proliferation. Inhibition of RLIP76 expression in U87 and U251 glioma cell lines by stable transfection of a targeted siRNA suppressed anchorage-independent growth and enhanced apoptosis in vitro. Conversely, overexpression of RLIP76 in SW1088 and U251 cell lines enhanced proliferation and reduced apoptosis. Inhibition of RLIP76 in U251 cells also significantly suppressed tumorigenicity and induced apoptosis in an endotopic xenograft mouse model. Moreover, we demonstrate that knockdown of RLIP76 increases apoptosis in different human gliomas independently of p53 status. In addition, a constitutively active Rac1 reversed both the suppression of proliferation and the promotion of apoptosis induced by the RLIP76-targeted siRNA, indicating that RLIP76 is an upstream activator of Rac1. Rac1-mediated suppression of apoptosis and promotion of proliferation were dependent on intact c-jun N-terminal kinase (JNK) signaling. Furthermore, we demonstrate that RLIP76 promotes proliferation and suppresses glioma cell apoptosis through a mechanism independent of Rho-selective GTPase-activating protein. Instead, we found that the adenosine triphosphatase function of Rlip76 modulates Rac1 activity by regulating Rac1 protein ubiquitylation and degradation. These data demonstrate that RLIP76 may suppress apoptosis and promote the proliferation of glioma cells by direct adenosine triphosphate-dependent xenobiotic transport and by activating the Rac1-JNK signaling pathway. Inhibition of RLIP76 signaling is a potential treatment for malignant glioma.

MicroRNA-34a suppresses cell proliferation by targeting LMTK3 in human breast cancer mcf-7 cell line.

Breast cancer remains the leading cause of cancer mortality in females, and about 70% of the primary breast cancer patients are diagnosed ERα-positive, which is the most common type of breast cancer. MicroRNA-34a (miR-34a) has been shown to be a master regulator of tumor suppression in many types of cancers including breast cancer. However, the role of miR-34a in ERα-positive breast cancer has not been elucidated. Here, we find that in MCF-7, which is an ERα-positive breast cancer cell line, miR-34a is remarkably downregulated after E2 treatment. Overexpression of miR-34a by lentivirus suppresses cell proliferation, S phase ratio, and tumor formation in an E2-dependent manner in vitro. According to the mRNA sequence, lemur tyrosine kinase 3 (LMTK3), which is an important regulator of estrogen receptor alpha (ERα), is a predicted target of miR-34a. This is confirmed by dual luciferase reporter assay and the decrease of LMTK3 mRNA and protein levels after overexpression of miR-34a. Moreover, miR-34a overexpression decreases AKT signaling pathway and increases ERα phosphorylation status. Taken together, these results suggest that miR-34a inhibits breast cancer proliferation by targeting LMTK3 and might be used as an anti-ERα agent in breast cancer therapy.

Theranostic nanoparticles based on bioreducible polyethylenimine-coated iron oxide for reduction-responsive gene delivery and magnetic resonance imaging.

Theranostic nanoparticles based on superparamagnetic iron oxide (SPIO) have a great promise for tumor diagnosis and gene therapy. However, the availability of theranostic nanoparticles with efficient gene transfection and minimal toxicity remains a big challenge. In this study, we construct an intelligent SPIO-based nanoparticle comprising a SPIO inner core and a disulfide-containing polyethylenimine (SSPEI) outer layer, which is referred to as a SSPEI-SPIO nanoparticle, for redox-triggered gene release in response to an intracellular reducing environment. We reveal that SSPEI-SPIO nanoparticles are capable of binding genes to form nano-complexes and mediating a facilitated gene release in the presence of dithiothreitol (5–20 mM), thereby leading to high transfection efficiency against different cancer cells. The SSPEI-SPIO nanoparticles are also able to deliver small interfering RNA (siRNA) for the silencing of human telomerase reverse transcriptase genes in HepG2 cells, causing their apoptosis and growth inhibition. Further, the nanoparticles are applicable as T2-negative contrast agents for magnetic resonance (MR) imaging of a tumor xenografted in a nude mouse. Importantly, SSPEI-SPIO nanoparticles have relatively low cytotoxicity in vitro at a high concentration of 100 μg/mL. The results of this study demonstrate the utility of a disulfide-containing cationic polymer-decorated SPIO nanoparticle as highly potent and low-toxic theranostic nano-system for specific nucleic acid delivery inside cancer cells.

miR-26a and its target CKS2 modulate cell growth and tumorigenesis of papillary thyroid carcinoma.

Background While many studies have shown that levels of miR-26a are lower in papillary thyroid carcinoma (PTC), the role and mechanism of miR-26a in PTC are unclear. Method We used database searches to select potential mRNA targets of miR-26a. Anti-miR-26a, miR-26a mimic, siRNA for CKS2 and their effects on cell growth, cell-cycle distribution and colony formation were evaluated. We also evaluate the over-expressed miR-26a in TPC-1 cells in severe combined immune-deficient mice. We used luciferase reporter assays, real-time PCR and western blot analysis to measure the expression and activity of miR-26a, CKS2, and related factors such as cyclin B1, cyclin A, cdk1, bcl-xl and Akt. Finally, we measured the relationship between the levels of miR-26a and CKS2 in PTC and normal thyroid tissues. Results Relative to normal thyroid tissues, miR-26a is consistently down-regulated in TPC specimens, and CKS2 was identified as a potential target. Up-regulated miR-26a expression or down-regulated CKS2 expression in TPC-1 and CGTH W3 cells lines caused G2 phase-arrest. Decreased miR-26a expression or increased CKS2 expression could have inverse function on PTC cell lines. CyclinB1, cyclinA, bcl-xl and AKt are indirectly regulated by miR-26a in a CKS2-dependent manner. Finally, CKS2 is overexpressed in PTC specimens relative to normal thyroid tissue, and a significant inverse correlation exists between miR-26a and CKS2 expression in clinical PTC specimens. Conclusion Our data indicate that miR-26a functions as a growth-suppressive miRNA in PTC, and that its suppressive effects are mediated mainly by repressing CKS2 expression.

Adipose tissue-derived stem cells embedded with eNOS restore cardiac function in acute myocardial infarction model

This study assessed the potential therapeutic efficacy of endothelial NO syntheses (eNOS)-expressing adipose tissue-derived stem cells (ADSCs) on infarcted hearts. We isolated CD29+, CD44+, CD45- cells from adipose tissue. Multipotent property of ADSCs was characterized by induction to differentiate into myogenic, neurogenic, and endothelic lineages. We hypothesized that combination of eNOS over-expression and transplantation of ADSCs could restore NO bioavailability and improve cardiac function in infarcted hearts. Here with several lines of experimental evidences, we demonstrated that ADSCs with eNOS overexpression induced eNOS expression in host endothelial cells and vascular smooth muscle cells, both in vitro and in vivo. This effect was possibly mediated by calcium signal. Transplantation of ADSCs with eNOS embedded showed great therapeutic efficacy in reduction of infarcted size, compared with normal ADSC. Results of this study suggest that ADSCs could be an attractive vehicle for the exogenous eNOS expression into heart after infarction, which is beneficial to restoration of cardiac function. Paracrine effect by mobilizing the host endothelial cells and smooth muscle cells may be the mechanism underlying the therapeutic effect.

MicroRNA-130a Targets MAP3K12 to Modulate Diabetic Endothelial Progenitor Cell Function.

Aims: The aim of the present study was to explore the influence of microRNA (miR)-130a dysregulation on the JNK signal pathway through its target MAP3K12 in diabetic endothelial progenitor cells (EPCs). Methods: The expression of miR-130a was compared between diabetic and normal EPCs. Computational target prediction was performed to identify MAP3K12 as a functionally relevant target of miR-130a in EPCs. The role of miR-130a was investigated regarding its anti-apoptotic effects and its role on the regulation of EPC function was evaluated through the negative regulation of the JNK signal pathway Results: MiR-130a expression was significantly downregulated in diabetic EPCs, and cell proliferation was reduced in EPCs under high glucose condition. miR-130a inhibited the JNK pathway by targeting MAP3K12, contributing to its anti-apoptotic effect and the maintenance of EPC function. In diabetic EPCs, high glucose affects the expression of miR-130a, inducing sustained JNK activation and promoting EPC apoptosis and dysfunction. Conclusions: Downregulation of miR-130a may underlie endothelial dysfunction in diabetes through the activation of JNK signal pathway.