Yongyi Huang

Transplantation of Human Menstrual Blood Stem Cells to Treat Premature Ovarian Failure in Mouse Model

The incidence of premature ovarian failure (POF), also known as ovarian insufficiency, has been increasing in recent years. Although some treatments are currently available, improved treatment strategies are urgently required. Many researchers have reported that human endometrial stem cells (HuMenSCs), which exhibit stem/progenitor cell properties in vitro repaired damaged cells in vivo. Thus, we aimed to determine whether HuMenSCs can serve as cell therapy tools and be used for the treatment of POF. After treating with cyclophosphamide, on the first estrus period (we predicted mouse estrus cycle was generally 5 days), HuMenSCs were injected into a cyclophosphamide-induced mouse model of POF. The results revealed that the HuMenSCs could survive within POF mouse ovaries for at least 14 days in vivo; further, ovaries of the HuMenSCs-transplanted group expressed higher levels of ovarian markers [AMH, inhibin α/β, and follicle-stimulating hormone receptor (FSHR)], and the proliferative marker Ki67. In addition, the ovarian weight, plasma E2 level, and the number of normal follicles increased over time in the HuMenSC group compared with the control group. Further, microarray analysis of cDNA expression patterns revealed that, after HuMenSC transplantation, the gene mRNA expression patterns in the ovarian cells following stimulation of the host ovarian niche became increasingly similar to those observed in human ovarian tissue compared with the pretransplantation mRNA expression pattern in HuMenSCs. Hence, we can safely conclude that the mesenchymal stem cell properties and in vivo survival of HuMenSCs make them ideal seed cells for stem cell transplantation in the treatment of POF.

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.

MicroRNA‐134 suppresses endometrial cancer stem cells by targeting POGLUT1 and Notch pathway proteins

We aimed to ascertain the role of microRNAs (miRNAs) in regulating human endometrial cancer stem cells (HuECSCs). The expression level of miRNA‐134 (miR‐134), a member of the DLK1‐DIO3 genomic imprinted miRNA cluster, differed significantly between HuECSCs and human endometrial cancer cells (HuECCs). miR‐134 inhibited HuECSCs proliferation and migration by targeting protein O‐glucosyltransferase 1 (POGLUT1) expression. Exogenous miR‐134 overexpression downregulated POGLUT1 and Notch pathway proteins in HuECSCs in vitro. miR‐134 overexpression affected the G2/M phase of HuECSCs and suppressed the growth of xenograft tumours formed. Thus, endogenous miR‐134 regulation in HuECSCs may suppress tumourigenesis in human endometrial carcinoma.

Enterovirus 71 induces apoptosis of SH‑SY5Y human neuroblastoma cells through stimulation of endogenous microRNA let-7b expression.

Enterovirus 71 (EV71) is a pathogenic microorganism that causes hand, foot and mouth disease. However, the epigenetic mechanisms behind how EV71 regulates host cell proliferation and apoptosis are unclear. In the present study, the ability of EV71 to induce apoptosis was analyzed in the SH-SY5Y human neuroblastoma cell line and the effect of this virus on the mRNA expression levels of various apoptotic markers, miRNA let-7b and cyclin D1 (CCND1), was also investigated. The results demonstrated that EV71 induced SH-SY5Y cell apoptosis. An MTT assay revealed a significant inhibitory effect of EV71 on cell proliferation between 12–72 h post injection, compared with the control group. Furthermore, quantitative polymerase chain reaction and western blot analyses demonstrated that expression level of the apoptosis inhibitor Bcl-2 was markedly reduced, but the expression levels of the apoptosis-promoting factors Bax, caspase-7, caspase-3 and active caspase-3 were markedly higher in the SH-SY5Y cells 12–48 h after EV71 infection, compared with the non-infected cells. In addition, flow cytometric assays revealed that EV71 arrested the cell cycle of host SH-SY5Y cells. Northern blot analysis revealed a marked miRNA let-7b hybridization signal in the EV71 virus-infected group compared with the non-infected group. Furthermore, western blotting confirmed that the CCND1 protein expression levels were significantly reduced in EV71-infected SH-SY5Y cells. EV71-inhibited SH-SY5Y proliferation was abrogated using let-7b specific 2′-O-Methyl-RNA, which inhibited endogenous miRNA let-7b expression. Thus, EV71 regulated the host SH-SY5Y cell cycle and cell proliferation via stimulating endogenous miRNA let-7b and directly targeting CCND1, therefore EV71 is a potential candidate for antiviral therapy.

MicroRNA-17 promotes normal ovarian cancer cells to cancer stem cells development via suppression of the LKB1-p53-p21/WAF1 pathway

The mechanism underlying the development of human ovarian cancer is poorly understood. The liver kinase protein, LKB1, is hypothesized to play a pivotal role in tumor cell proliferation and invasion capacity through regulation of p53 and p21/WAF1 expression. Previous studies suggest LKB1 may, in turn, be regulated by microRNA-17. Here, we examined the role of miR-17 in the expression of LKB1 and the downstream effects on proliferation and invasion capacity of normal ovarian cancer cells (OCCs) and ovarian stem cells. In this study, both the mRNA and protein expression levels of LKB1, p53, and p21 decreased in OCCs following transfection with a miR-17 expression plasmid. MiR-17 expression affected cell cycle regulation and stimulated the proliferation and invasion capacity of OCCs in vitro. ChIP assays indicated that the binding efficiency of p53 to the p21/WAF1 gene promoter was much lower in miR-17 transfected OCCs than in OCCs transfected with a mutated miR-17. Co-immunoprecipitation and western blotting showed significantly lower levels of p53 and p53 Ser15-pho in the miR-17 transfected OCCs as compared to the mutant miR-17 transfected OCCs. Xenograft experiments confirmed that suppression of tumor growth in vivo occurred in the absence of functional miR-17. These findings suggest that mature miR-17 expression may have an important role in the pathogenesis of human ovarian tumors through its interference with the LKB1-p53-p21/WAF1 pathway expression by epigenetic modification. These findings are of potential importance in the identification of novel therapeutic targets in human ovarian cancer.

Curcumin suppresses proliferation and in vitro invasion of human prostate cancer stem cells by ceRNA effect of miR-145 and lncRNA-ROR

Many studies have demonstrated that curcumin can effectively inhibit the proliferation, invasion, and tumorigenesis of prostate cancer cells in vitro and in vivo. In this study, CD44+/CD133+ human prostate cancer stem cells (HuPCaSCs) were isolated from the prostate cancer cell lines Du145 and 22RV1. Curcumin treatment of these cells resulted in the inhibition of in vitro proliferation and invasion, and cell cycle arrest. The expression levels of cell cycle proteins (Ccnd1 and Cdk4) and stem cell markers (Oct4, CD44, and CD133) were decreased in curcumin-treated HuPCaSCs. Microarray analysis and northern blotting assays indicated that miR-145 was overexpressed in curcumin-treated HuPCaSCs. Insights of the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatic analysis, bioinformatics analysis and luciferase activity assays showed that the lncRNA-ROR and Oct4 mRNA both contain miR-145 binding sites, and Oct4 and lncRNA-ROR directly compete for microRNA binding. Curcumin induced high miR-145 expression and inhibited the expression of lncRNA-ROR. The tumorigenicity of curcumin- treated HuPCaSCs in nude mice was significantly reduced. In summary, reducing the expression of endogenous lncRNA-ROR could effectively increase the available concentration of miR-145 in HuPCaSCs, where miR-145 prevents cell proliferation by decreasing Oct4 expression. In particular, we hypothesized that lncRNA-ROR may act as a ceRNA, effectively becoming a sink for miR-145, thereby activating the derepression of core transcription factors Oct4. Thus, curcumin suppresses the proliferation, in vitro invasion, and tumorigenicity of HuPCaSCs through ceRNA effect of miR-145 and lncRNA-ROR caused.

Cisplatin targets the stromal cell-derived factor-1-CXC chemokine receptor type 4 axis to suppress metastasis and invasion of ovarian cancer-initiating cells

In ovarian cancer, CD44+/CD117+ stem cells, also known as cancer-initiating cells (CICs), are highly proliferative and invasive. Therefore, the CD44+/CD117+ subpopulation is thought to be an important target for novel therapeutic strategies. In this study, we investigated the effects of cisplatin (CDDP) on metastasis and invasion suppression of ovarian CICs by targeting the CXC chemokine receptor-4 (CXCR4) signaling pathway in vitro and in vivo. CD44+/CD117+ ovarian CICs were enriched from human primary ovarian tumor tissues and confirmed by flow cytometry sorting. A 3-(4,5-dimethylthiazol-2-yl)-2.5-dipheny-tetrazolium bromide (MTT) assay revealed significant inhibition of proliferation of ovarian CICs with increasing CDDP drug concentrations. Moreover, colony formation and transwell migration assays indicated that CDDP significantly suppressed the invasive capacity of ovarian CICs in vitro. The expression levels of stromal cell-derived factor (SDF)-1, CXCR4, matrix metalloproteinase (MMP) 2, and MMP9 mRNA and protein levels were significantly reduced in CDDP-treated cells compared to untreated ovarian CICs. Furthermore, xenograft experiments confirmed that CDDP suppressed the growth of xenograft tumors formed by ovarian CICs in vivo. In addition, CXCR4 agonist (diprotin A) treatment of ovarian CICs weakened the effects of CDDP and enhanced SDF-1-CXCR4 axis expression in ovarian CICs. Thus, the SDF-1-CXCR4 axis is an important mediator of proliferation and invasion in CXCR4-overexpressing ovarian cancer-initiating cells (OCICs). Furthermore, CDDP inhibits invasion and metastasis of OCICs by targeting SDF-1-CXCR4 axis expression.

miR-145 modulates lncRNA-ROR and Sox2 expression to maintain human amniotic epithelial stem cell pluripotency and β islet-like cell differentiation efficiency.

In this study, we observed a great reduction in the expression of the endogenous long noncoding RNA ROR (lncRNA-ROR) and the stem cell transcription factor Sox2, in contrast to a marked increase in miR-145 expression, during the course of in vitro induced differentiation of human amniotic epithelial stem cells (HuAECs). Bioinformatics analysis and the luciferase reporter assay revealed binding of miR-145 to specific sites in lncRNA-ROR and Sox2, silencing their expression. Overexpression of a lncRNA-ROR-specific siRNA effectively downregulated the expression levels of Sox2 and other stem cell markers in HuAECs while weakening the efficiency of HuAEC differentiation into β islet-like cells. Moreover, the in vitro response of HuAEC-derived β islet-like cells to extracellular stimuli and C-peptide release by these cells were markedly weakened in the siRNA-ROR transfection group. Furthermore, the in vivo expression of β islet-like cell biomarkers was substantially reduced in HuAECs in the siRNA-ROR transfection group, and their in vivo β islet-like cell differentiation and insulin release capacities were reduced in a streptozocin-induced diabetic rat model. The experimental results indicate that lncRNA-ROR effectively maintains Sox2 gene expression through competitive binding to miR-145, achieving pluripotency maintenance in HuAECs and regulation of their directed β islet-like cell differentiation efficiency.

Human amniotic epithelial cells inhibit CD4+ T cell activation in acute kidney injury patients by influencing the miR-101-c-Rel-IL-2 pathway

HighlightsHuAECs reduced proliferation rate of patient‐derived CD4+ T cells.HuAECs reduced IL‐2 released from patient‐derived CD4+ T cells.Overexpressed miR‐101 reduced expression c‐Rel and IL‐2 in AKI patient‐derived CD4+ T cells.Overexpressed miR‐101 decreased binding capacities of ‘c‐Rel‐NF&kgr;B’ complex on IL‐2 promoter.HuAECs stimulate miR‐101 expression to inhibit AKI patient‐derived CD4+ T cells activation. ABSTRACT In the pathogenesis of acute kidney injury (AKI), the release of multiple interleukins can lead to increased kidney damage. Human amniotic epithelial cells (HuAECs) can inhibit immune cell activation in vivo and in vitro. We hypothesized that HuAECs could weaken patient‐derived peripheral blood CD4+ T‐cell activation and decreasing the ability of these cells to express and release IL‐2. −Cell proliferation assay revealed that under the same culture conditions, activated AKI patient‐derived CD4+ T cells had a significantly reduced proliferation rate when were co‐cultured with HuAECs. And the level of IL‐2 released was also significantly reduced. Western blot and qRT‐PCR assays showed that the expression of c‐Rel in the CD4+ T cells was also significantly reduced. However, the expression level of endogenous miR‐101 in the CD4+ T cells co‐cultured with HuAECs was significantly increased. Luciferase reporter assay results suggested that miR‐101 could bind to a specific site in the c‐Rel 3′ UTR and induce the post‐transcriptional silencing of c‐Rel. Subsequently, we over‐expressed miR‐101 in AKI patient‐derived CD4+ T cells. The qRT‐PCR and western blot assay results revealed that the expression of endogenous c‐Rel was significantly reduced, while the ELISA results indicated that the level of IL‐2 released was also significantly decreased. Finally, ChIP‐PCR assay results showed that the miR‐101‐overexpressing CD4+ T‐cell group and the HuAEC co‐culture CD4+ T‐cell group exhibited significantly decreased binding capacities between the ‘c‐Rel‐NF&kgr;B’ complex and the IL‐2 gene promoter, and the transcriptional activity of IL‐2 was also significantly decreased. Therefore, we confirmed that HuAECs can stimulate miR‐101 expression in AKI patient‐derived peripheral blood CD4+ T cells, thus inhibiting the expression of the miR‐101 target gene c‐Rel and leading to a reduction in IL‐2 expression and release.

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.