Li-li Wang

Diagnostic role of microRNA expression profile in the serum of pregnant women with fetuses with neural tube defects

J. Neurochem. (2012) 122, 641–649.

Therapeutic potential of in utero mesenchymal stem cell (MSCs) transplantation in rat foetuses with spina bifida aperta

Neural tube defects (NTDs) are complex congenital malformations resulting from incomplete neurulation in embryo. Despite surgical repair of the defect, most of the patients who survive with NTDs have a multiple system handicap due to neuron deficiency of the defective spinal cord. In this study, we successfully devised a prenatal surgical approach and transplanted mesenchymal stem cells (MSCs) to foetal rat spinal column to treat retinoic acid induced NTDs in rat. Transplanted MSCs survived, grew and expressed markers of neurons, glia and myoblasts in the defective spinal cord. MSCs expressed and perhaps induced the surrounding spinal tissue to express neurotrophic factors. In addition, MSC reduced spinal tissue apoptosis in NTD. Our results suggested that prenatal MSC transplantation could treat spinal neuron deficiency in NTDs by the regeneration of neurons and reduced spinal neuron death in the defective spinal cord.

Semaphorin 3A expression in the colon of Hirschsprung disease.

BACKGROUND Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of intrinsic ganglion cells in the nerve plexuses of the lower colon. The Semaphorin 3A (SEMA3A) gene is involved in the migration of enteric neural precursors (ENPs). To analyze the function of SEMA3A in HSCR, the SEMA3A expression in different colon segments in HSCR was examined. METHODS The expression levels of SEMA3A in both ganglionic and aganglionic colon tissues of 32 patients with HSCR and in colon tissue of 5 newborn unaffected individuals were examined by real-time RT-PCR, Western-blot, and immunohistology. RESULTS Comparison of SEMA3A expression levels between ganglionic and aganglionic tissues in HSCR revealed upregulation of SEMA3A expression in 43.75% (14/32) of the aganglionic colons. SEMA3A was expressed in the ganglion cells of the myenteric plexus, submucosa, as well as in the longitudinal and circular muscle layer of the normal colon of both unaffected newborns and patients with HSCR. In the aganglionic segment of patients with HSCR, SEMA3A was highly expressed in the circular muscle layer and was also detected in the submucosa and in the longitudinal muscles layer. The fluorescence intensity of SEMA3A in the circular muscle layer in the aganglionic segment was much higher than that in ganglionic segment (p < .001). CONCLUSION SEMA3A expression was upregulated in the aganglionic smooth muscle layer of the colon in some patients with HSCR and our data suggest that increased SEMA3A expression may be a risk factor for HSCR pathology in a subset of patients.

Disturbed apoptosis and cell proliferation in developing neuroepithelium of lumbo-sacral neural tubes in retinoic acid-induced spina bifida aperta in rat.

Spina bifida is a complex congenital malformation resulting from failure of fusion in the spinal neural tube during embryogenesis. However, the cellular mechanism underlying spina bifida is not fully understood. Here, we investigated cell apoptosis in whole embryos and proliferation of neural progenitor cells in the spinal neural tube during neurulation in all‐trans retinoic acid (atRA)‐induced spina bifida in fetal rats. Cell apoptosis was assessed by TUNEL assay on whole‐mount and serially sectioned samples of rat embryos with spina bifida. Cell proliferation of lumbo‐sacral neural progenitor cells was assessed by staining for the mitotic marker Ki67 and pH3. We found an excess of apoptosis in the neuroepithelium of embryos with spina bifida, which became more marked as embryos progress from E11 to E13. Conversely, there was a reduction in cell proliferation in spina bifida embryos, with a progressively greater difference from controls with stage from E11 to 13. Thus, atRA‐induced spina bifida in rat shows perturbed apoptosis and proliferation of neural progenitors in the lumbo‐sacral spinal cord during embryonic development, which might contribute to the pathogenesis of spina bifida.

Role of the lncRNA ABHD11-AS1 in the tumorigenesis and progression of epithelial ovarian cancer through targeted regulation of RhoC.

BackgroundThere is increasing evidence in support of the role of lncRNAs in tumor cell proliferation, differentiation and apoptosis.MethodsWe examined the expression of the lncRNA ABHD11-AS1 in epithelial ovarian cancer (EOC) tissues and normal ovarian tissues by real-time quantitative PCR (qRT-PCR). After inducing ABHD11-AS1 downregulation by small interfering RNA (siRNA) or ABHD11-AS1 overexpression by plasmid transfection, we examined the EOC cell phenotypes and expression of related molecules.ResultsExpression of the lncRNA ABHD11-AS1 in EOC tissues was higher than that in normal ovarian tissue. It was positively associated with the tumor stage (stage I/II vs. stage III/IV), and it was lower in the well-differentiated group than in the poorly/moderately differentiated group. Overexpression of ABHD11-AS1 in the ovarian cancer cell lines A2780 and OVCAR3 promoted ovarian cancer cell proliferation, invasion and migration, and inhibited apoptosis. Silencing of ABHD11-AS1 had the opposite effect. Subcutaneous injection of tumor cells in nude mice showed that ABHD11-AS1 could significantly promote tumor growth. In addition, intraperitoneal injection of tumor cells in the nude mice resulted in an increase in the metastatic ability of the tumor. Further, overexpression of ABHD11-AS1 upregulated the expression of RhoC and its downstream molecules P70s6k, MMP2 and BCL-xL. Silencing of ABHD11-AS1 had the opposite effect. The RNA pull-down assay showed that ABHD11-AS1 can combine directly with RhoC. Silencing of RhoC was found to inhibit the cancer-promoting effects of lncRNA ABHD11-AS1. Thus, it seems that RhoC is a major target of the lncRNA ABHD11-AS1.ConclusionsThis is the first study to demonstrate the role of RhoC in the tumor-promoting effects of the lncRNA ABHD11-AS1. The present findings shed light on new therapeutic targets for ovarian cancer treatment.

Comparative proteomics of spinal cords of rat fetuses with spina bifida aperta.

Neural tube defects (NTDs) are complex congenital anomalies of the central nervous system, with a prevalence of 5 per 10,000 worldwide. However, current therapeutics for NTDs are unsatisfactory. The neurological complications remain the main problem for therapy. Neurological dysfunction could result from the primary defect or injuries to the uncovered neural tissue in the uterus. However, the pathological changes in the uncovered neural tissue have not been described. Here, we present our comparative proteomics study of the spinal cord from rat fetuses with all-trans retinoic-acid-induced spina bifida aperta. Proteins from spinal cords were subjected to 2-D gel electrophoresis, then protein identification by mass spectrometry. We identified 13 proteins with differential expression between normal spinal cords and those with spina bifida aperta. These identified proteins were reported to be involved in signal transduction, cell adhesion and migration, protein folding and apoptosis. We confirmed 4 identified proteins by immunoblot analysis and assessed their mRNA levels by quantitative real-time PCR. This is the first comparative proteomics of spinal cords from rat fetuses with spina bifida aperta. We demonstrate protein alterations that reflect the pathological situation of the uncovered neural tissue, which may help improve the treatment of NTDs.

DLEU1 contributes to ovarian carcinoma tumourigenesis and development by interacting with miR-490-3p and altering CDK1 expression

Recently, a large number of studies have focused on the important role of long non‐coding RNAs (lncRNAs) in metabolism and development and have found that abnormal lncRNA expression is associated with the pathogenesis and development of many diseases. The lncRNA DLEU1 is involved in many solid tumours and haematological malignancies. However, its role in epithelial ovarian carcinoma (EOC) and the associated molecular mechanisms has not been reported. In this study, quantitative reverse transcription–PCR (qRT–PCR) demonstrated higher lncRNADLEU1 expression in EOC tissues than in normal tissues. Plasmid transfection of DLEU1 to up‐regulate its expression in the ovarian cancer cell lines A2780 and OVCAR3 increased cell proliferation, migration, and invasion, while inhibited apoptosis. Nude mouse xenograft assay demonstrated that DLEU1 overexpression promoted tumour growth in vivo. QRT–PCR showed decreased miR‐490‐3p expression, while Western blotting demonstrated increased its target genes CDK1, cyclinD1 and SMARCD1, as well as matrix metalloproteinase‐2 (MMP2), Bcl‐xL and P70S6K protein expression, respectively. Short interfering RNA silencing of DLEU1 produced opposite results, where qRT–PCR showed increased miR‐490‐3p expression. The dual‐luciferase reporter assay revealed a direct interaction between DLEU1 and miR‐490‐3p. MiR‐490‐3p plays a tumour suppressor role in epithelial ovarian cancer by targeting CDK1 regulation and influencing SMARCD1 and cyclin D1 (CCND1) expressions. Therefore, we suggest that through interaction with miR‐490‐3p, DLEU1 may influence the expression of CDK1, CCND1 and SMARCD1 protein, subsequently promoting the development and progression of EOC.

Altered Expression of 14-3-3ζ Protein in Spinal Cords of Rat Fetuses with Spina Bifida Aperta

Background A large number of studies have confirmed that excessive apoptosis is one of the reasons for deficient neuronal function in neural tube defects (NTDs). A previous study from our laboratory used 2-D gel electrophoresis to demonstrate that 14-3-3ζ expression was low in the spinal cords of rat fetuses with spina bifida aperta at embryonic day (E) 17. As a member of the 14-3-3 protein family, 14-3-3ζ plays a crucial role in the determination of cell fate and anti-apoptotic activity. However, neither the expression of 14-3-3ζ in defective spinal cords, nor the correlation between 14-3-3ζ and excessive apoptosis in NTDs has been fully confirmed. Methodology/Principal Findings We used immunoblotting and quantitative real-time PCR (qRT-PCR) to quantify the expression of 14-3-3ζ and double immunofluorescence to visualize 14-3-3ζ and apoptosis. We found that, compared with controls, 14-3-3ζ was down-regulated in spina bifida between E12 and E15. Excessive apoptotic cells and low expression of 14-3-3ζ were observed in the dorsal region of spinal cords with spina bifida during the same time period. To initially explore the molecular mechanisms of apoptosis in NTDs, we investigated the expression of microRNA-7 (miR-7), microRNA-375 (miR-375) and microRNA-451 (miR-451), which are known to down-regulate 14-3-3ζ in several different cell types. We also investigated the expression of p53, a molecule that is downstream of 14-3-3ζ and can be down-regulated by it. We discovered that, in contrast to the reduction of 14-3-3ζ expression, the expression of miR-451, miR-375 and p53 increased in spina bifida rat fetuses. Conclusions/Significance These data suggest that the reduced expression of 14-3-3ζ plays a role in the excessive apoptosis that occurs in spina bifida and may be partly regulated by the over-expression of miR-451 and miR-375, and the consequent up-regulation of p53 might further promote apoptosis in spina bifida.

Fascaplysin inhibit ovarian cancer cell proliferation and metastasis through inhibiting CDK4

OBJECTIVE Cyclin-dependent kinases (CDKs) are important regulators of the cell cycle; previous studies have shown that misregulation of CDK4 (cyclin-dependent kinase 4) activity can lead to cancer. The present study investigated the anti-tumor effects of a highly selective CDK4 inhibitor fascaplysin in ovarian carcinoma cell lines. MATERIALS AND METHODS In our study, cell proliferation, cell cycle, cell apoptosis, cell invasion, and cell migration relative assays were performed in ovarian cancer cell lines A2780 and OVCAR3 in the presence of different concentrations of fascaplysin. The protein expression levels of CDK4, cyclin D1, Bcl-2 (B-cell lymphoma-2), and VEGFA (vascular endothelial growth factor A) were determined by western blot. RESULTS Our results showed that fascaplysin inhibited ovarian cancer cell proliferation, invasion and migration, as well as inducing S arrest and cell apoptosis. Treatment with fascaplysin also suppressed CDK4, cyclin D1, Bcl-2, and VEGFA expression at protein levels. CONCLUSIONS Above all, our results showed that fascaplysin has anti-tumor activity against ovarian cancer cell lines through inhibiting CDK4, and may be a therapeutic target for the treatment of ovarian carcinomas.

LncRNA ABHD11‐AS1 promotes the development of endometrial carcinoma by targeting cyclin D1

To investigate the expression, role and mechanism of action of long non‐coding RNA (lncRNA) ABHD11‐AS1 in endometrial carcinoma. The expression of lncRNA ABHD11‐AS1 was quantified by qRT‐PCR in human endometrial carcinoma (n = 89) and normal endometrial tissues (n = 27). LncRNA ABHD11‐AS1 was stably overexpressed or knocked‐down in endometrial carcinoma cell lines to examine the cellular phenotype and expression of related molecules. Compared to normal endometrial tissue, lncRNA ABHD11‐AS1 was significantly overexpressed in endometrial carcinoma. Overexpression of lncRNA ABHD11‐AS1 promoted the proliferation, G1‐S progression, invasion and migration of endometrial cancer cells; inhibited apoptosis; up‐regulated cyclin D1, CDK1, CDK2, CDK4, Bcl‐xl and VEGFA; and down‐regulated p16, while ABHD11‐AS1 down‐regulation has the opposite effect. RNA pull down demonstrated that lncRNA ABHD11‐AS1 binds directly to cyclin D1. Knockdown of cyclin D1 can reverse the effect of ABHD11‐AS1. Overexpression of lncRNA ABHD11‐AS1 increased the tumorigenicity and up‐regulated cyclin D1 in an in vivo model of endometrial cancer in nude mice. LncRNA ABHD11‐AS1 functions as an oncogene to promote cell proliferation and invasion in endometrial carcinoma by positively targeting cyclin D1.