microRNA-377 acts as a suppressor in esophageal squamous cell carcinoma through CBX3-dependent P53/P21 pathway.

Stem cells play pivotal roles in esophageal squamous cell carcinoma (ESCC) recurrence and metastasis. The self-renewal ability of stem cells was associated with specific microRNAs (miRs). Herein, we identified the effects of miR-377 on ESCC stem cell activities. First, the expression of miR-377 in ESCC and adjacent normal tissues was determined. The relationship between miR-377 and chromobox protein homolog 3 (CBX3) was assessed by a dual-luciferase reporter gene assay. miR-377 was overexpressed or inhibited in ESCC stem cells to explore its role in ESCC. To further investigate the mechanism of miR-377 in ESCC, cells were introduced with short hairpin RNA against CBX3 or pifithrin-α (inhibitor of P53 pathway). Besides, the expression of P21, P53, CD133, CD13, Nanog, sex determining region Y-Box 2 (Sox2), and octamer-binding transcription factor 4 (Oct4), cell sphere formation, colony formation, and proliferation were evaluated respectively. Finally, limiting dilution assay in vivo and tumor xenograft in nude mice were conducted to confirm the roles of miR-377 in vivo. miR-377 was poorly expressed in ESCC. Overexpression of miR-377 could suppress the stem-like trait of ESCC as well as the tumor growth in vivo. miR-377 targeted CBX3 to activate the P53/P21 pathway. Besides, the expression of stem-like markers including CD133, CD13, Oct4, Sox2, and Nanog was decreased, and the abilities of cell sphere formation, colony formation, proliferation, and tumorigenicity were significantly reduced by overexpressing miR-377 or silencing CBX3. The results were reversed after inactivating the P53/P21 pathway. In summary, upregulation of miR-377 inhibits the self-renewal of ESCC stem cells by inhibiting CBX3 expression and promoting activation of the P53/P21 pathway.

Elevated microRNA-130b-5p or silenced ELK1 inhibits self-renewal ability, proliferation, migration, and invasion abilities, and promotes apoptosis of cervical cancer stem cells.

Cervical cancer (CC) is the most familiar gynecological malignancy. With the poor prognosis of CC patients, this study explored the effect of microRNA (miR)-130b-5p targeting ELK1 expression on self-renewal ability and stemness of CC stem cells. The tissues of patients with CC or cervical benign lesions were collected. MiR-130b-5p and ELK1 expression was detected by reverse transcription quantitative polymerase chain reaction and western blot analysis. Human CC cell line Hela was cultured and the induced CC stem cells were introduced with miR-130b-5p mimic or silenced ELK1 to figure their roles in self-renewal ability, stemness, colony formation, proliferation, migration, invasion abilities, and apoptosis of CC stem cells. Tumor growth was detected in nude mice in vivo. The targeting relationship between miR-130b-5p and ELK1 was analyzed using bioinformatic prediction and dual luciferase reporter gene assay. Decreased miR-130b-5p and elevated ELK1 existed in CC tissues of patients. Up-regulated miR-130b-5p decreased ELK1 expression in CC stem cells. Elevated miR-130b-5p or silenced ELK1 inhibited self-renewal ability and stemness, colony formation, proliferation, migration and invasion abilities, promoted apoptosis of CC stem cells, as well as decreased the weight and volume of tumor in nude mice. ELK1 was found to be targeted by miR-130b-5p. Overexpression ELK1 effectively reversed the cellular phenotypic changes and tumor formation in vivo caused by up-regulation of miR-130b-5p. We conclude that up-regulated miR-130b-5p or silenced ELK1 inhibits CC stem cell growth.

Downregulation of microRNA-196a inhibits stem cell self-renewal ability and stemness in non-small-cell lung cancer through upregulating GPX3 expression.

Studies have reported a high expression profile of microRNA-196a (miR-196a) in many cancers, which potently plays important roles in carcinogenesis. However, the involvement of miR-196a in affecting non-small cell lung cancer (NSCLC) carcinogenesis still remains uncertain. NSCLC-related differentially expressed genes were retrieved for this study according to the microarray-based analysis, which demonstrated that miR-196a may be involved in NSCLC progression via regulation of the Jun N-terminal kinase (JNK) pathway by targeting glutathione peroxidase 3 (GPX3). Hence, this study aimed to explore the relationship among miR-196a, GPX3, and the JNK pathway and to investigate its functional regulations in NSCLC. Initially, highly-expressed miR-196a and lowly-expressed GPX3 were determined in NSCLC tissues and cells. Next, the NSCLC cells were manipulated with a series of mimic, inhibitor or shRNA to investigate the impact of miR-196a and GPX3 on CSC viability, proliferation, self-renewal ability and stemness. The in vivo effect of miR-196a was measured in nude mice xenografted with NSCLC cells. The results demonstrated that downregulation of miR-196a and restoration of GPX3 inhibited CSC viability, proliferation, self-renewal ability, stemness and tumorigenicity. Meanwhile, the underlying relationship among miR-196a, GPX3 and JNK pathway was explored by treatment with the JNK pathway inhibitor (SP600125), or sh-GPX3. Downregulated miR-196a and upregulated GPX3 could elevate the GPX3 protein expression and reduce the extent of JNK and c-Jun phosphorylation. Taken together, miR-196a promotes the development of NSCLC via activation of the JNK pathway through down-regulation of GPX3 and serve as a potential therapeutic target in NSCLC.

Myocardial infarction does not affect circulating haematopoietic stem and progenitor cell self-renewal ability in a rat model.

NEW FINDINGS: What is the central question of this study? Although peripheral blood haematopoietic stem and progenitor cells are potentially important in regeneration after acute myocardial infarction, their self-renewal ability in the post-acute phase has not yet been addressed. What is the main finding and its importance? In rat peripheral blood, we show that myocardial infarction does not negatively affect circulating haematopoietic stem and progenitor cell self-renewal ability 2 weeks after acute infarction, which suggests a constant regenerative potential in the myocardial infarction post-acute phase. Given the importance of peripheral blood haematopoietic stem and progenitor cells (HPCs) in post-acute regeneration after acute myocardial infarction (MI), the aim of the present study was to investigate the number and secondary replating capacity/self-renewal ability of HPCs in peripheral blood before and 2 weeks after MI. In female Lewis inbred rats (n = 9), MI was induced by ligation of the left coronary artery, and another nine underwent sham surgery, without ligation, for control purposes. Myocardial infarction was confirmed by troponin I concentrations 24 h after surgery. Peripheral blood was withdrawn and fractional shortening and ejection fraction of the left ventricle were assessed before (day 0) and 14 days after MI or sham surgery (day 14). After mononuclear cell isolation, primary and secondary functional colony-forming unit granulocyte-macrophage (CFU-GM) assays were performed in order to detect the kinetics of functional HPC colony counts and cell self-renewal ability in vitro. The CFU-GM counts and cell self-renewal ability remained unchanged (P > 0.05) in both groups at day 14, without interaction between groups. In the intervention group, higher day 0 CFU-GM counts showed a relationship to lower fractional shortening on day 14 (ρ = -0.82; P < 0.01). Myocardial infarction did not negatively affect circulating HPC self-renewal ability, which suggests a constant regenerative potential in the post-acute phase. A relationship of cardiac contractile function 14 days after MI with circulating CFU-GM counts on day 0 might imply functional colony count as a predictive factor for outcome after infarction.

Phenotype change of glioma stem cells is related with the RNA binding proteins in hypoxia / 基础医学与临床

Objective To explore the relationship between the change of phenotype of glioma stem cells and expres-sion of RNA binding proteins in hypoxia. Methods Glioma stem cells(U87MG-SLC and GSC5) were cultured un-der hypoxia (1% O2) and normoxia(20% O2). Cell proliferation was measured by MTS assay and self-renewal ability was determined by tumorsphere formation assay. The expression of RNA binding protein and stemness mark-ers protein were examined by Western blot and statistics was carried out. Results The proliferation of glioma stem cells was inhibited and the self-renewal ability was promoted in hypoxia. Meanwhile,hypoxia significantly promoted the expression of HIF-1α and stemness markers.Under hypoxia, the expression of RNA binding protein was changed. The expression of hnRNPF, UNRIP and HuD increased. Meanwhile the expression of PCBP2 and UNR was downregulated. But,other RNA binding proteins(hnRNPK,ADAR1,PCBP1,CIRP,EBP1,eEF1A,PTBP1,PTBP2) had no significant change. Conclusions The change of phenotype of glioma stem cells in hypoxia is relat-ed with the RNA binding proteins (hnRNPF,UNRIP,HuD,PCBP2 and UNR).

Enhancement of Neural Stem Cell Survival, Proliferation, Migration, and Differentiation in a Novel Self-Assembly Peptide Nanofibber Scaffold.

Considerable efforts have been made to combine biologically active molecules into the self-assembling peptide in order to improve cells growth, survival, and differentiation. In this study, a novel three-dimensional scaffold (RADA4GGSIKVAV; R-GSIK) was designed by adding glycine and serine between RADA4 and IKVAV to promote the strength of the peptide. The cell adhesion, viability, proliferation, migration, and differentiation of rat embryonic neural stem cells (NSCs) in R-GSIK were investigated and compared to laminin-coated, two-dimensional, and Puramatrix cultures. The scanning electron microscopy studies of the R-GSIK showed an open porous structure and a suitable surface area available for cell interaction. R-GSIK promoted the cell adhesion, viability, proliferation, and migration compared to the other cultures. In addition, the R-GSIK enhanced NSCs differentiation into neuronal cells. The NSCs injected in R-GSIK had a lower glial differentiation rate than in the Puramatrix. The results suggest that R-GSIK holds great promise for cell therapies and neuronal tissue repair.

MicroRNA-203 As a Stemness Inhibitor of Glioblastoma Stem Cells.

Glioblastoma stem cells (GBM-SCs) are believed to be a subpopulation within all glioblastoma (GBM) cells that are in large part responsible for tumor growth and the high grade of therapeutic resistance that is so characteristic of GBM. MicroRNAs (miR) have been implicated in regulating the expression of oncogenes and tumor suppressor genes in cancer stem cells, including GBM-SCs, and they are a potential target for cancer therapy. In the current study, miR-203 expression was reduced in CD133(+) GBM-SCs derived from six human GBM biopsies. MicroRNA-203 transfected GBM-SCs had reduced capacity for self-renewal in the cell sphere assay and increased expression of glial and neuronal differentiation markers. In addition, a reduced proliferation rate and an increased rate of apoptosis were observed. Therefore, miR-203 has the potential to reduce features of stemness, specifically in GBM-SCs, and is a logical target for GBM gene therapy.

TMPRSS4 correlates with colorectal cancer pathological stage and regulates cell proliferation and self-renewal ability.

Transmembrane protease/serine 4 (TMPRSS4) is a member of the type II transmembrane serine protease (TTSP) family and it was found highly expressed in several cancers. This study aims to evaluate the expression of TMPRSS4 in colorectal cancer (CRC) and investigate its role in proliferation and self-renewal of colon cancer cells. qRT-PCR and immunohistochemistry were used to detect the mRNA and protein expression level of TMRPSS4 in CRC samples respectively. Loss of function assay was conducted with RNAi technique. Cell proliferation was done with WST-8 assay; cell apoptosis and cell cycle analysis were performed with flow cytometry; invasion and migration were done with transwell assay. Plate and soft agarose clonogenic assays were used to detect clone-formation ability. CD44 and CD133 expressions were analyzed by flow cytometry and western blot. We found that TMPRSS4 was highly expressed in CRC tissues both at mRNA and protein level and correlated with pathological stage. Knockdown of TMPRSS4 in highly expressed colon cancer cell line HCT116 resulted in inhibition of cell proliferation, induction of cell apoptosis and suppression of invasion and migration; moreover, knockdown of TMPRSS4 suppressed the in vitro clone-formation ability of HCT116 and reduced the expressions of CD44 and CD133. The findings in this research showed that TMPRSS4 was associated with CRC stage and regulated the proliferation and self-renewal ability of colon cancer cells; TMRPSS4 was involved in the development and progression of CRC.