Te Liu

Attenuated ability of BACE1 to cleave the amyloid precursor protein via silencing long noncoding RNA BACE1‑AS expression

Although large numbers of long noncoding RNAs (lncRNAs) expressed in the mammalian nervous system have been detected, their functions and mechanisms of regulation remain to be fully clarified. It has been reported that the lncRNA antisense transcript for β-secretase-1 (BACE1-AS) is elevated in Alzheimer’s disease (AD) and drives the rapid feed-forward regulation of β-secretase, suggesting that it is critical in AD development. In the present study, the senile plaque (SP) AD SH-SY5Y cell model was established using the synthetic amyloid β-protein (Aβ) 1–42 in vitro. Using this model, the potential of siRNA-mediated silencing of lncRNA BACE1-AS expression to attenuate the ability of β-secretase-1 (BACE1) to cleave amyloid precursor protein (APP) and to reduce the production of Aβ1–42 oligomers was investigated. MTT assays demonstrated that exogenous Aβ1–42 suppressed SH-SY5Y cell proliferation and induced APP-related factor expression and SP formation. Furthermore, quantitative polymerase chain reaction and western blot analysis revealed that the mRNA and protein expression of Aβ1–42 and Aβ1–40 was significantly increased in the AD model group, with a marked decrease in Ki-67 expression at day six. RNase protection assays (RPA) and northern blotting analysis confirmed that exogenous Aβ1–42 not only promoted the expression of the APP-cleaving enzyme BACE1, but also induced lncRNA BACE1-AS expression. Furthermore, lncRNA BACE1-AS formed RNA duplexes and increased the stability of BACE1 mRNA. Downregulation of lncRNA BACE1-AS expression in SH-SY5Y cells by siRNA silencing resulted in the attenuation of the ability of BACE1 to cleave APP and delayed the induction of SP formation in the SP AD SH-SY5Y cell model.

Induction of Estrogen-Sensitive Epithelial Cells Derived from Human-Induced Pluripotent Stem Cells to Repair Ovarian Function in a Chemotherapy-Induced Mouse Model of Premature Ovarian Failure

The incidence of premature ovarian failure (POF), a condition causing amenorrhea and hypergonadotropic hypoestrogenism in women before the age of 40, has been increasing in recent years. As an irreversible pathological change, improved treatment strategies for this disease are urgently needed. In this study, a type of microRNA (miR-17-3p) was used to guide the differentiation of human-induced pluripotent stem (iPS) cells into hormone-sensitive ovarian epithelial (OSE)-like cells in vitro. To prevent their morphological transformation into fibroblast-like cells, MiR-17-3p, a microRNA that suppresses vimentin expression, was transfected into human iPS cells. Subsequently, these cells were successfully induced into OSE-like cells in vitro after treatment with estrogen and cell growth factors. Compared with controls, iPS cells transfected with miR-17-3p expressed higher levels of epithelial markers (cytokeratin 7, AE1, AE3, and E-cadherin) and estrogen receptors (ERα and ERβ) while levels of mesenchymal markers (fibronectin, vimentin, and N-cadherin) lowered after the induction. The human iPS cell-derived OSE-like cells were then injected into cyclophosphamide-induced POF model mice to determine their potential benefit as grafts to repair ovarian tissues. The OSE-like cells survived within POF mouse ovaries for at least 14 days in vivo. Compared with the negative controls, expressions of cytokeratin 7 and ERβ proteins were elevated while fibronectin and vimentin levels in ovarian tissues were downregulated in the OSE-like cell transplantation group. Moreover, the ovarian weight and plasma E2 level increased over time in the transplantation with OSE-like cells, compared with control groups. Hence, we can draw the conclusion that iPS cells can be induced to differentiate into OSE-like cells in vitro.

Transplantation of ovarian granulosa‑like cells derived from human induced pluripotent stem cells for the treatment of murine premature ovarian failure.

Premature ovarian failure (POF) is a common cause of female infertility, for which there are currently no ideal treatments or medications. Furthermore, apoptosis of ovarian granulosa cells (OGCs) is an important mechanism underlying the decline in ovarian reserve and function. In the present study, several cellular growth factors and hormones were used to induce the differentiation of human induced pluripotent stem cells (iPSCs) into ovarian granulosa-like cells (OGLCs) in vitro. Immunohistochemical staining demonstrated that OGLCs derived from iPSCs strongly expressed granulosa cell markers, including anti-Müllerian hormone, inhibin α, inhibin β and follicle-stimulating hormone receptor, but did not express stem cell markers, including octamer-binding transcription factor 4, SRY (sex determining region Y)-box 2, Nanog and stage-specific embryonic antigen-4 12 days post-induction. In addition, a mouse model of POF was generated by cyclophosphamide treatment. Subsequently, iPSC-derived OGLCs were transplanted into the POF mice (OGLCs-iPSCs-POF group) in vivo. Results indicated that, compared with the control group (POF mice treated with phosphate-buffered saline), the growth state of OGLCs was markedly improved, and mature follicles could be detected in the ovarian tissue of the OGLCs-iPSCs-POF group. Immunohistochemical staining demonstrated that iPSC-derived OGLCs transplanted into POF mice not only exhibited substantial growth in murine ovarian tissues, but also strongly expressed OGC markers. Furthermore, enzyme-linked immunosorbent assays indicated that the levels of the hormone estradiol in peripheral blood samples were significantly enhanced following transplantation of iPSC-derived OGLCs into POF mice. Furthermore, ovarian tissue weight was significantly higher in the OGLCs-iPSCs-POF group compared with in the control group, and the number of atretic follicles in OGLCs-iPSCs-POF mice was significantly reduced, as compared with in the control mice. These results suggest that OGLCs derived from human iPSCs may not only effectively enhance OGC growth and repair damaged ovarian tissue, but may also maintain the ovarian tissue niche, promoting follicular development and maturation in a mouse model of POF.

Effect of atorvastatin on expression of TLR4 and NF-κB p65 in atherosclerotic rabbits.

OBJECTIVE To study the effect of atorvastatin on atherosclerotic rabbits. METHODS A total of 60 New Zealand male rabbits were randomly divided into the normal group, model group and atorvastatin group. The replication rabbit atherosclerotic model with immune injury combined with a high fat diet feeding was used. All rabbits were sacrificed after 3 months. TLR4 and NF-κB p65 were observed by HE staining, immunohistochemistry and western blotting. RESULTS The expression of TLR4, NF-κB p65 were significantly increased in the model group compared with the normal group. The expression of TLR4 and NF-κB p65 decreased significantly in the atorvastatin group, and there was no difference compared with the normal group. CONCLUSIONS The effect of atorvastatin on atherosclerosis may be achieved by the inhibition of the expression of TLR4 and NF-κB p65.

Induction of pancreatic β-cell-like cells from CD44+/CD105+ human amniotic fluids via epigenetic regulation of the pancreatic and duodenal homeobox factor 1 promoter.

Pancreatic and duodenal homeobox factor 1 (PDX-1) maintains β-cell function and differentiation via direct regulation of multiple islet cell genes. However, the molecular mechanisms involved in this process remain unknown. Here, we show that PDX-1 plays an important role in the induction of CD44+/CD105+ human amniotic fluid cells (HuAFCs) into functional pancreatic β-cell-like cells in vitro. CD44+/CD105+ HuAFCs were transfected with either siRNA targeting PDX-1 (siRNA-PDX-1) or mock plasmid (siRNA-MOCK). Following induction, siRNA-MOCK-transfected cells differentiated into β-cell-like cells that expressed multiple islet cell markers and produced insulin and C-peptide in a glucose-regulated manner. However, siRNA-PDX-1-transfected cells did not fully differentiate into β-cell-like cells. Further, we observed epigenetic changes at the PDX-1 gene locus in induced CD44(+)/CD105(+) HuAFCs. Therefore, CD44+/CD105+ HuAFCs could be a source of human pancreatic β-cell-like cells with potential uses in cell replacement therapy for diabetes.

Long non-coding RNA BACE1-AS is a novel target for anisomycin-mediated suppression of ovarian cancer stem cell proliferation and invasion

Human ovarian cancer stem cells (OCSCs) are one of the main factors affecting ovarian cancer cell metastasis, recurrence, prognosis and tolerance to chemotherapy drugs. However, the mechanisms of OCSC proliferation and invasion are not clear. Recent studies suggest that anisomycin can inhibit the proliferative and invasive ability of various tumor cells by increasing the production of the toxic amyloid β (Aβ1-42) peptides from the amyloid precursor protein (APP). We explored whether anisomycin could also suppress human OCSC proliferation and invasion. The CD44+/CD117+ OCSCs were enriched from human clinical ovarian tumor tissues. OCSCs treated with anisomycin showed reduced proliferation compared to controls. Moreover, anisomycin significantly suppressed the invasive capacity of OCSCs in vitro, as indicated by cell migration assays. The mRNA expression levels of long non-coding RNA (lncRNA) β-site APP cleaving enzyme 1 antisense strand (BACE1-AS) were significantly increased in anisomycin-treated OCSCs compared to controls. In addition, mRNA and protein levels of BACE1 and Aβ1-42 were increased in anisomycin-treated OCSCs compared to controls. We confirmed that anisomycin suppressed the growth of xenograft tumors formed by OCSCs in vivo. Finally, when expression of lncRNA BACE1-AS was silenced using siRNA, BACE1 expression was downregulated and the antiproliferative and anti-invasive effects of anisomycin were reduced. Overall, we identified lncRNA BACE1-AS as a novel target for anisomycin. Elevation of lncRNA BACE1-AS expression is a potential mechanism for suppressing human OCSC proliferation and invasion.

Regulation of neuronal toxicity of β‑amyloid oligomers by surface ATP synthase

Alzheimer disease (AD) is characterized by the accumulation of amyloid-β (Aβ) protein and intracellular neurofibrillary tangles. Previous studies have shown that Aβ aggregation is one of the most important initiating factors in the pathogenesis of AD. Oligomers of Aβ cause neurotoxicity, synaptic dysfunction and memory impairments that underlie AD. An increasing number of studies have shown that oligomeric Aβ may bind with a number of surface proteins to mediate its neuronal toxicity. Previously, it was shown that ATP synthase is present on the cell surface and binds with oligomeric Aβ. In the present study, ATP synthase was confirmed to be present on the surface of neurons and oligomeric Aβ was observed to induce neuron damage and expression of amyloid precursor protein (APP) and Fe65 increase. Results showed that inhibition of surface ATP synthase may reduce the neuronal damage by LDH release assay and decrease APP and Fe65 expression by immunofluorescence and western blot analysis. These results confirmed that the cell surface ATP synthase is a binding protein for Aβ on neural cells and suggested that the surface ATP synthase may be involved in the neurotoxic effects of oligomeric Aβ and may be an intervening target of pathogenesis of AD.

Telocytes as potential targets in a cyclophosphamide-induced animal model of premature ovarian failure

Premature ovarian failure (POF) refers to the presence of ovarian atrophic permanent amenorrhea in women under the age of 40. The pathogenesis of POF remains to be fully elucidated. Telocytes are a group of specialized cells with a small cell volume and very long cytoplasmic prolongations with dichotomous branching. Previous studies have indicated that telocytes function to support the trachea and serve as stem cell niches. Although it has been confirmed that telocytes are present in numerous organs in mammals, it remains to be determined whether they are present in ovarian tissues and whether they are involved in the development of POF. The present study used a cyclophosphamide-induced mouse model of POF. Hematoxylin and eosin staining and an enzyme-linked immunosorbent assay revealed that cyclophosphamide induced edema and apoptosis of ovarian stromal and granulosa cells and increased atretic follicles. In addition, cyclophosphamide induced abnormal peripheral blood FSH and E2 levels in mice. Transmission electron microscopy revealed a small number of telocyte-like cell structures in the ovarian stroma of wild-type mice. In addition, flow cytometry and immunohistochemical staining results suggested that the number of cluster of differentiation (CD)34/platelet-derived growth factor receptor (PDGFR)α, CD34/PDGFRβ and CD34/vimentin double-positive cells in the ovaries of POF mice was significantly decreased compared with wild-type mice. In conclusion, mouse ovarian tissues appear to contain telocytes, and cyclophosphamide treatment significantly reduced the number of ovarian telocytes. Therefore, telocytes may serve as a potential novel marker of POF induced by cyclophosphamide.

MicroRNA let-7b-regulated epidermal stem cell proliferation in hypertrophied anal papillae

The present study investigated the role of epidermal stem cell-expressed microRNA let-7b in the pathogenesis of hypertrophied anal papillae. Hypertrophied anal papillae were examined for the presence of epidermal stem cells. Epidermal stem cells were identified using flow cytometry and immunofluorescent staining for the cell surface markers, integrin α6 and integrin β1 subunits. Expression levels of microRNA let-7b in α6+/β1+and α6-/β1-cells were compared using reverse transcription-quantitative polymerase chain reaction and northern blotting. Lentivirus-mediated expression of microRNA let-7b in epidermal stem cells was utilized in order to study the effects of this microRNA on the cell cycle proteins, cyclin D1 (CCND1) and cyclin-dependent kinase 4 (CDK4). MicroRNA let-7b-overexpressing cells were examined using flow cytometry, in order to determine the effects of the microRNA on cell cycle progression. α6+/β1+epidermal stem cells were identified in hypertrophic anal papillae. Following isolation and enrichment of the α6+/β1+population, these cells were found to have a rapid rate of proliferation in vitro. The expression of cell cycle-related proteins was elevated in this population, compared with that in α6-/β1-cells. The expression of microRNA let-7b in α6+/β1+epidermal stem cells was significantly lower than that in α6-/β1-cells. Two microRNA let-7b target genes, CCND1 and CDK4, were found to be upregulated in α6+/β1+cells. When the exogenous precursor, microRNA let-7, was overexpressed in α6+/β1+ epidermal stem cells, the cell proliferation rate was significantly lower than that in cells expressing microRNA let-7 containing a mutated seed sequence. The addition of exogenous microRNA let-7 resulted in an increased expression level of mature microRNA let-7b, while the expression of CCND1 and CDK4 was reduced. Epidermal stem cells transfected with microRNA let-7b were arrested in the G2/M phase and the percentage of cells in S-phase was significantly reduced. In conclusion, let-7b expression results in upregulation of the cell cycle-related proteins, CCND1 and CDK4, resulting in the excessive proliferation that leads to the formation of hypertrophic anal papillae.

Growth hormone treatment of premature ovarian failure in a mouse model via stimulation of the Notch-1 signaling pathway.

Premature ovarian failure (POF) is a condition affecting 1% of women in the general population, causing amenorrhea, hypergonadotropism and hypoestrogenism before the age of 40. Currently, POF cannot be reversed and, although treatments are available, there is an urgent need for improved treatment strategies. Growth hormone (GH) is a pleiotropic hormone that affects a broad spectrum of physiological functions, from carbohydrate and lipid metabolism to the immune response. GH has previously been used to treat POF in non-transgenic preclinical trials, but the biochemical mechanism underlying these effects are unclear. In the present study, a mouse model of POF was generated using cyclophosphamide. Treatment of POF mice with recombinant mouse growth hormone (rmGH) was revealed to markedly reduce POF histopathology in ovarian tissue, relieve ovarian granulosa cell injury, reduce the number of atretic follicles and significantly increase the number of mature oocytes. Furthermore, an enzyme-linked immunosorbent assay revealed that plasma estradiol levels increased and plasma follicle stimulating hormone levels decreased with time in a group of mice treated with a medium dose of rmGH (0.8 mg/kg) when compared with the POF model group (P<0.05). In addition, reverse transcription-quantitative polymerase chain reaction and immunohistochemical analysis demonstrated elevated levels of Notch-1 signaling pathway factors (Notch1, CBF1, and HES1) in wild-type mice and those treated with medium and high doses of rmGH, but not in those treated with low doses of rmGH. In conclusion, GH may promote ovarian tissue repair, estrogen release and oocyte maturation via activation of the Notch-1 signaling pathway in ovarian tissue.