MicroRNA-92a -mediated endothelial to mesenchymal transition controls vein graft neointimal lesion formation.

PURPOSE: Long-term failure of vein grafts due to neointimal hyperplasia remains an important problem in coronary artery bypass graft surgery. Endothelial to mesenchymal transition (EndMT) contributes to vein graft vascular remodeling. However, there is little study on microRNA-mediated EndMT contributions to neointimal formation in vein graft. We hypothesized that microRNA-92a (miR-92a) might play an important role in determining EndMT contributions to neointimal formation. METHODS: miR-92a and EndMT-related proteins detected by qRT-PCR and Western blot in vitro and in vivo. Adeno-associated virus 6 (AAV6) delivery gene therapy was used to inhibit neointimal formation in vivo. The intimal hyperplasia of vein grafts was measured by HE staining, the expression of EndMT-related protein in vein grafts was measured by immunofluorescence. Immunohistochemistry and luciferase assay were used to detect potential targets of miR-92a. RESULTS: The expression of miR-92a was found to be upregulated in neointimal hyperplasic lesions after vein grafting. Using cultured human umbilical vein endothelial cells (HUVECs), we show that TGF-ß1 treatment of HUVECs significantly increased miR-92a expression and induced EndMT, characterized by suppression of endothelial-specific markers (CD31 and VE-cadherin) and an increase in mesenchymal-specific markers (a-SMA and vimentin), while inhibition of miR-92a expression blunted EndMT in cultured HUVECs. Furthermore, AAV6 mediated miR-92a suppression gene therapy effectively resulted in decreased EndMT and less neointimal formation in vein grafts in vivo. We further identified that integrin alpha 5 (ITGA5) is a potential target gene involved in the development of neointima formation in these vein grafts. CONCLUSION: This data suggests that neointimal formation does not solely rely on vascular smooth muscle cell phenotypic switching but is also related to EndMT, and miR-92a-mediated EndMT is an important mechanism underlying neointimal formation in vein grafts.

Neuroprotective effect of minocycline in a rat model of branch retinal vein occlusion.

Branch retinal vein occlusion (BRVO) is the second most frequent retinal vascular disorder. Currently the first-line therapies for BRVO include anti-VEGF and dexamethasone implant treatment, however, with direct or indirect damage on retinal neurons, it has limited effect in improving patients visual acuity. Therefore, novel treatments with neuroprotective effect for BRVO retina were expected. Minocycline is a semisynthetic, broad spectrum tetracycline antibiotic with high penetration through the blood brain barrier. The neuroprotective effects of minocycline have been shown in various central nervous system (CNS) disease. Since both CNS and retina were composed of neurons and glials, it is reasonable to expect a neuroprotective effect by minocycline for BRVO retina. Therefore, the aim of the present study was to study whether minocycline has neuroprotective effect in branch retinal vein occlusion (BRVO) and the possible underlying molecular basis. We created BRVO in rats using laser photocoagulation. The animals were then randomly divided into 4 groups to evaluate the effect of minocycline: group A: minocycline 45 mg/kg intraperitoneal injection (i.p.), group B: minocycline 90 mg/kg i.p., group C: normal saline i.p., group D: sham injection. Fundus photography and fluorescein angiography (FA) were conducted. The changes in thickness of retinal layers were measured with optical coherence tomography (OCT) in vivo. We found that retinal edema occurred predominantly in the inner retinal layers. Intraperitoneal administration of minocycline significantly ameliorated retinal edema in the early stage of BRVO. We performed Full field Electroretinography (ffERG) to evaluate retinal function and found that the reduction of b wave amplitude decreased in the combined maximal response. The expressional levels of apoptosis related genes (Bax, Bcl-2) and inflammation related genes (IL-1 ß, TNF α, MCP-1 and CCR2) were measured by real-time PCR, the results showed that minocycline treatment upregulated Bcl-2 expression and inhibits TNF α expression since early stage of BRVO. We also performed Hematoxylin-Eosin (HE) and immunostaining for Iba 1 (a microgilal marker), active caspase-3, Bax, Bcl-2, IL-1 ß, TNF α and found that minocycline inhibits retinal microglial activation, prevents retinal ganglion cell loss, and inhibits retinal caspase-3 activation. Thus, our study indicates that systemic administration of minocycline ameliorates retinal edema and preserves retinal function in the early stage of BRVO possibly via inhibiting microglia activation and protecting RGC from apoptosis.

[Overexpression of high mobility group A2 and its correlation with microRNA let-7 family in serous ovarian cancers].

OBJECTIVE: To examine the expression of high mobility group A2 (HMGA2), P53 and let-7 family microRNA, to investigate the correlation of HMGA2 and let-7, and to compare the HMGA2 and P53 expressions in human serous ovarian cancer. METHODS: Immunohistochemistry assay was used to examine the expressions of HMGA2 and P53 in 50 paraffin-embedded tissue specimens of human serous ovarian cancer and 4 normal fallopian tube tissues. HMGA2 mRNA and let-7 family microRNA were detected by real time fluorescent quantitative reverse transcription polymerase chain reaction in the corresponding frozen tissues. RESULTS: HMGA2 and P53 were immuno-positive in 70% (35/50) and 78% (39/50) of the ovarian cancer tissues, respectively. HMGA2 was weakly expressed in the ciliated cells, but negative in the secretary cells of the fallopian tube. There was a tendency that the expression of HMGA2 increased with higher pathological grade of the ovarian cancer, but no correlation was observed between the HMGA2 overexpression and clinical stages. HMGA2 mRNA was detected in all the ovarian cancer samples, and its expression level was higher than that of the normal fallopian tube tissues in 72% (36/50) of the ovarian cancer samples. The expression of HMGA2 mRNA was much higher in more malignant SKOV3.ipl cells than in its corresponding SKOV3 cells. All let-7 family members were detectable in all ovarian cancer samples, and their expression were inversely correlated with HMGA2 mRNA expression (r=-0.305,P<0.05). CONCLUSION: HMGA2 can be a biomarker complement to P53, and its high expression has an inclination of more malignancy. The downregulation of let-7 is, but not the only mechanism of HMGA2 overexpression in serous ovarian cancer.

[Expression and significance of microRNAs in the p53 pathway in ovarian cancer cells and serous ovarian cancer tissues].

OBJECTIVE: The aim of this study was to investigate whether miR-449a, miR-449b and miR-192 family microRNAs play the same roles in p53 pathway as miR-34 family in ovarian cancer. METHODS: Wild-type p53 ovarian carcinoma cell line A2780 cells were treated with genotoxic agent adriamycin. The reactivation of p53 was detected by Western blot. The expression of miR-449a/b, miR-34a, miR-34b, miR-34c, miR-192 and miR-194 were detected by real-time quantitative PCR. Mutant p53 ovarian cancer cell line SKOV3.ipl cells were transfected with pre-microRNAs and the cell-cycle changes were detected. The expression level of miR-449a/b, miR-34a, miR-34b, miR-34c, miR-192 and miR-194 in serous ovarian carcinomas of varying grade and stage were compared with real-time PCR. RESULTS: The expressions of miR-449a/b, miR-34b and miR-34c were 19-fold to 21-fold elevated after p53 activation by genotoxic agent. Ectopic expression of miR-449b, as well as miR-34c, resulted in cell-cycle arrest in SKOV3.ipl cells. The expression of miR-449a/b was parallel with that of miR-34b, miR-34c, and were significantly lower in late stage and high-grade serous carcinomas than in the normal fallopian tube, early stage and low-grade serous carcinomas. The expression of miR-192, miR-194 and miR-34a did not show evident features in serous ovarian carcinomas and were much lower than miR-449a/b, miR-34b and miR-34c in normal fallopian tube. CONCLUSIONS: As tumor-suppressor microRNAs, miR-449a/b, miR-34b and miR-34c cooperate and play important roles in p53 pathway. Their inactivation may contribute to the carcinogenesis and progression of serous ovarian carcinomas.

[miR-449b and miR-34c on inducing down-regulation of cell cycle-related proteins and cycle arrests in SKOV3-ipl cell, an ovarian cancer cell line].

OBJECTIVE: To investigate the effects of miR-449 and miR-34 on cell growth, cell cycle and target gene expression based on these miRNA different expressions in ovarian cancer cell lines SKOV3 and SKOV3-ipl both with mutation of p53. METHODS: The expressions of miR-449a/b and miR-34b,c in SKOV3 and SKOV3-ipl were detected by RT-PCR. miR-449a,b and miR-34b,c were ectopically expressed by transfection of SKOV3-ipl. The cell growth rate was assayed by MTS method. The changes of cell cycle were measured by FCM. The changes of expression of cell cycle related proteins were detected by Western blot. RESULTS: Ectopic expression of miR-449b and miR-34c resulted in lowered adhesion activities by 28%-34%, and in cell cycle arrests with increased cell number of 15.62% and 15.71% in G1 and with decreased cell number of 15.96% and 16.56% in S. Cell cycle related proteins CDK6 and CDC25A were down-regulated. The decreases of CDK6 and CDC25A by miR-449b were 39% and 22% respecyively; 49% and 32% by miR-34c respectively. The more decreases were seen in co-action by miR-449b and miR-34c with decreases of 69% in CDK6, 86% in CDC25A, and 59% in CyclinA. CONCLUSION: miR-449b and miR-34c resulted in cell cycle arrests and down-regulation of CDK6, CDC25A and CyclinA in high malignant ovarian cancer cell line SKOV3-ipl.

[Abnormal expression of microRNAs in the hippocampus of Ts65Dn mice].

OBJECTIVE: To identify the differentially expressed microRNAs in the hippocampus of Down Syndrome model mouse Ts65Dn. METHODS: Low molecular weight RNA from hippocampus were tailed and reverse transcribed by extended RT-primer. MicroRNAs primers were arrayed on plates according to the Tm of each primer. PCR were carried out at different annealing temperatures using a gradient real-time PCR instrument. The relative expression level of each microRNAs was calculated using the most stable reference gene as normalizer which was selected by geNorm and Normfinder software. RESULTS: miR-27a was identified as the most stable reference gene by geNorm and Normfinder software. Among the 52 microRNAs detected by real-time PCR array, miR-33 and miR-19a were significantly down-regulated, whereas miR-130 were significantly up-regulated in the hippocampus of Ts65Dn mice as compared with the euploid control mice. The expression of miR-802, which was the trisomy chromosome 16 derived microRNAs, was very low in hippocampus with no difference between the two groups. CONCLUSION: MicroRNAs are dysregulated in the hippocampus of Ts65Dn mice, which may contribute to the reduced neurogenesis of Down syndrome.

[Profiling of microRNAs in mouse brain with real-time PCR array].

OBJECTIVE: To examine global expression levels of microRNAs(miRNAs) in mouse cerebrum and to provide an important basis for detailed studies of individual miRNAs, their target genes, the miRNA-related regulatory networks in the mammalian central nervous system, and their implications in diseases. METHODS: Low molecular weight RNA from cerebrum of five C57BL/6J mice were tailed and reverse transcribed by extended RT-primer. miRNA primers were carefully designed and arrayed on plates according to the Tm of each primer. PCR was carried out at different annealing temperatures using a gradient real-time PCR instrument. The relative expression level of each miRNA was calculated using 5sRNA for normalization. RESULTS: Among the 285 miRNAs detected, 260 were positive with varying abundance. Their frequency distribution was approximately a normal distribution. The expression levels of most miRNAs were in accordance with previously published results by microarray. However, the positive rate was higher than that detected by microarray. miRNAs originating from the same hairpin precursors expressed at similar or significantly different levels. Clusters of proximal miRNAs were similar or quite different in abundance. It is suggested that the fate of miRNA after transcription determined their abundance. CONCLUSION: Using the RNA-tailing and primer-extension PCR array method, we obtained expression profile of miRNA in mouse cerebrum, especially the relative expression data of many low abundant miRNA in mouse cerebrum, which will be of special help for studying the fine-tuning function of low-level miRNAs.

Increasing specificity of real time PCR to detect microRNA through primer design and annealing temperature increase.

OBJECTIVE: To investigate the non-specific and inaccurate amplification in cases of highly similar sequences among family members and the length heterogeneity of mature microRNA (miRNA), and find a condition that discriminates maximally among similar miRNA family members and detects the accurate expression level of miRNAs. METHODS: Primers with their mismatches and/or 3' end at different positions were designed. Amplification efficiencies were compared using matched and various mismatched primers by RNA-tailing and primer-extension RT-PCR at different annealing temperatures. Expression levels of several miRNAs in mouse brain were compared using miRNA specific primers with different termini. RESULTS: Raising annealing temperatures 12 degrees Celsius-14 degrees Celsius above the T(m) of the primers maximally increased amplification specificity without sacrificing sensitivity. Primers designed with their termini on or near variant positions could efficiently discriminate between miRNA isoforms. Using primers that terminated before the end of the mature miRNA did not miss the detection of shorter mature miRNA and provided accurate expression levels. CONCLUSION: Careful primer design and higher annealing temperature can increase specificity and accuracy of real time PCR miRNA detection.

Intra-tumor heterogeneity for endometrial cancer and its clinical significance.

BACKGROUND: Management of tumors has become more complex owing to tumor heterogeneity. Fewer studies have been performed on intra-tumor heterogeneity of endometrial cancer (EC) until now. Therefore, it is of great clinical value to explore the intra-tumor heterogeneity of EC based on clinical features and gene expression profiles. METHODS: A total of 1688 patients with EC were screened and 114 patients were finally selected, including specimens from 84 patients with primary EC without relapse (PE) and the paired metastases (P-M) specimens, as well as specimens from 30 patients with primary EC with relapse (RPE) and the paired relapsed EC (P-RE) specimens. Microarray and RNA-seq were used to detect gene expression of EC samples. Clinicopathological characteristics and molecular data were compared between PE and P-M groups and between RPE and P-RE groups to explore the intra-tumor heterogeneity of EC. RESULTS: The clinical intra-tumor spatial heterogeneity of pathological type, grade, ER status, and PR status between PE and P-M were 17.9%, 13.1%, 28.6%, and 28.6%, respectively. The clinical intra-tumor spatiotemporal heterogeneity of pathological type, grade, ER status, and PR status between RPE and P-RE were 16.7%, 33.3%, 25.0%, and 37.5%, respectively. Cluster analysis sorts EC samples based on progression type of lesion and their pathological type. There were differentially expressed genes between PE and P-M and between RPE and P-RE, of which gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis were mainly enriched in cell proliferation, the p53 signaling pathway, etc. CONCLUSIONS:: Clinical and molecular data showed that there was spatiotemporal heterogeneity in intra-tumor of EC, which may add to the complexity of diagnosis and therapeutics for EC. Considering the intra-tumor heterogeneity, sequential chemotherapy and precision medicine may be a more suitable treatment plan for EC.

[Real-time quantification of microRNAs by RNA-tailing and primer-extension RT-PCR].

OBJECTIVE: To optimize and evaluate the modified RNA-tailing and primer-extension RT-PCR method in relative quantification of microRNAs (miRNAs) in several kinds of tissues. METHODS: Small-sized RNAs (<200 bp) were extracted and polyadenylated by poly(A) polymerase. One-base anchored oligo-dT primers with 40 nt extension at their 5'-ends were used to reversely transcribe the poly(A)-tailed miRNAs. miRNAs were then amplified by SYBR Green real-time PCR using miRNA specific primers. RESULTS: This method had a high dynamic range and could detect low abundant miRNAs. The assay was capable of discriminating between related miRNA family members that differed by subtle sequence differences at 3' ends, but it could not distinguish those differed in the middle of their sequences. Fifteen mature miRNAs were differentially expressed in rat heart, liver, and cortex,which were in accordance with published results by other methods. The miRNA expression signature in hippocampus was different from that in cortex. miR-122a expression was higher and miR-124a expression was lower in hippocampus than in cortex. A single dissociation peak on the thermal melting curve and a single DNA band about 80 bp on 3.5% NuSeive agarose gel signified the amplification specificity for these miRNAs. CONCLUSION: This method enables fast and sensitive relative quantification for most miRNAs and can identify potential biomarkers specific to tissues or diseases.

Adenovirus-Mediated Gene Transfer of microRNA-21 Sponge Inhibits Neointimal Hyperplasia in Rat Vein Grafts.

Background: Vein graft failure due to neointimal hyperplasia remains an important and unresolved complication of cardiovascular surgery. microRNA-21 (miR-21) plays a major role in regulating vascular smooth muscle cell (VSMC) proliferation and phenotype transformation. Thus, the purpose of this study was to determine whether adenovirus-mediated miR-21 sponge gene therapy was able to inhibit neointimal hyperplasia in rat vein grafts. Methods: Adenovirus-mediated miR-21 sponge was used to inhibit VSMC proliferation in vitro and neointimal formation in vivo. To improve efficiency of delivery gene transfer to the vein grafts, 20% poloxamer F-127 gel was used to increase virus contact time and 0.25% trypsin to increase virus penetration. Morphometric analyses and cellular proliferation were assessed for neointimal hyperplasia and VSMC proliferation. Results: miR-21 sponge can significantly decrease the expression of miR-21 and proliferation in cultured VSMCs. Cellular proliferation rates were significantly reduced in miR-21 sponge-treated grafts compared with controls at 28 days after bypass surgery (14.6±9.4 vs 34.9±10.8%, P=0.0032). miR-21 sponge gene transfer therapy reduced the intimal/media area ratio in vein grafts compared with the controls (1.38±0.08 vs. 0.6±0.10, P<0.0001). miR-21 sponge treatment also improved vein graft hemodynamics. We further identified that phosphatase and tensin homolog (PTEN) is a potential target gene that was involved in the miR-21-mediated effect on neointimal hyperplasia in vein grafts. Conclusions: Adenovirus-mediated miR-21 sponge gene therapy effectively reduced neointimal formation in vein grafts. These results suggest that there is potential for miR-21 sponge to be used to prevent vein graft failure.

[Construction of recombinant adenoviral vector containing gene of EWS-FLI1 and antitumor immunity of its modified dentritic cell in vitro].

OBJECTIVE: To construct an adenoviral vector containing cDNA of EWS-FLI1 and detect its expression in peripheral blood mononeuclear cell(PBMC). To Investigate the antitumor immunity in vitro of the EWS-FLI1 gene modified-dendritic cells. METHODS: The EWS-FLI1 cDNA in plasmid Pec1/ EWS-FLI1 was digested and subcloned into the shuttle plasimid padtrack-cmv. The shuttle plasmid and the bone plasmid pADeasy-1 were cotransformed into BJ5183 cells. The recombinant plasmid was generated by homologous recombination in BJ5183 cells. The positive clone was obtained by digestion and electrophoresis. Transforming the recombinant plasmid into "293 cells" by lipofectamine method. Adenoviruses with high titer and purity were obtained by amplifying in the"293 cells" on a large scale and ultra-centrifugation in CsCL step gradient solutions. The cytotoxic activity of stimulated T cells to Ewing sarcoma cells was detected by (51)Cr release assay. RESULTS: PCR showed that the adenovirus contained EWS-FLI1 cDNA. After the PBMC were transfected by Ad EWS-FLI1, the EWS-FLI1 mRNA was detected by RT-PCR. The antigen-specific CTL was induced successfully by the EWS-FLI1 gene modified-DC. The vigorous antigen-specific CTL response against A673 cells was detected by (51)Cr release assay. The killing percentage was 35.18%+/-0.0128% at effector-target ratio 40:1, which was more efficient than that of the control. CONCLUSION: The recombinant adenovirus was successfully constructed and could efficient express EWS-FLI1 in PBMC. After T lymphocytes were stimulated by DCs modified with EWS-FLI1 gene, the specific CTL response against Ewing's sarcoma cell line A 673 in vitro was observed successfully. (51)Cr release assay showed that there was significant difference between the experimental group and the control group.

Aberrant Alternative Polyadenylation is Responsible for Survivin Up-regulation in Ovarian Cancer.

BACKGROUND: Survivin is an oncoprotein silenced in normal mature tissues but reactivated in serous ovarian cancer (SOC). Although transcriptional activation is assumed for its overexpression, the long 3'-untranslated region (3'-UTR) in survivin gene, which contains many alternate polyadenylation (APA) sites, implies a propensity for posttranscriptional control and therefore was the aim of our study. METHODS: The abundance of the coding region, the proximal and the distal region of survivin mRNA 3'-UTR, was evaluated by real-time polymerase chain reaction (PCR) in SOC samples, cell lines, and normal fallopian tube (NFT) tissues. The APA sites were confirmed by rapid amplification of cDNA 3' ends and DNA sequencing. Real-time PCR were used to screen survivin-targeting microRNAs (miRNAs) that were inversely correlated with survivin. The expression of an inversely correlated miRNA was restored by pre-miRNA transfection or induction with a genotoxic agent to test its inhibitory effect on survivin overexpression. RESULTS: Varying degrees of APA were observed in SOC by comparing the abundance of the proximal and the distal region of survivin 3'-UTR, and changes of 3'-UTR correlated significantly with survivin expression (r = 0.708, P< 0.01). The main APA sites are proved at 1197 and 1673 of survivin 3'-UTR by DNA sequencing. Higher level of 3'-UTR proximal region than coding region was observed in NFT, as well as in SOC and cell lines. Among the survivin-targeting miRNAs, only a few highly expressed miRNAs were inversely correlated with survivin levels, and they mainly targeted the distal part of the 3'-UTR. However, in ovarian cancer cells, restoration of an inversely correlated miRNA (miR-34c) showed little effect on survivin expression. CONCLUSIONS: In NFT tissues, survivin is not transcriptionally silenced but regulate posttranscriptionally. In SOC, aberrant APA leads to the shortening of survivin 3'-UTR which enables it to escape the negative regulation of miRNAs and is responsible for survivin up-regulation.

No association of AQP4 polymorphisms with neuromyelitis optica and multiple sclerosis.

Multiple sclerosis (MS) and neuromyelitis optica (NMO) are inflammatory demyelinating disorders of the central nervous system (CNS). Various genetic and environmental factors have been identified to contribute to etiology of MS and NMO. Aquaporin 4 (AQP4), is the most abundant water channel in CNS. AQP4 is expressed in astrocytes of the brain, spinal cord, optic nerve and supportive cells in sensory organs. In contrast to MS, immunoreactivity of AQP4 is abolished in NMO lesions. However, conflicting results have been reported regarding the association between AQP4 polymorphisms and demyelinating disorders. Considering the ethnic differences of genetic variations, replications in other cohorts are required. In this study, single nucleotide polymorphisms (SNPs) of AQP4 gene in patients with NMO/neuromyelitis optica spectrum disorders (NMOSD), and MS in the Northern Han Chinese population were examined. Six selected AQP4 SNPs were genotyped by high-resolution melting (HRM) method. Compared with healthy control (HC), there was no significant difference of AQP4 allele and genotype frequency in MS or NMO/NMOSD group. This study showed no significant association of common AQP4 SNPs with MS or NMO/NMOSD, strongly suggesting that polymorphisms of AQP4 gene are unlikely to confer MS or NMO/NMOSD susceptibility, at least in Northern Han Chinese population.

MicroRNA-221 sponge therapy attenuates neointimal hyperplasia and improves blood flows in vein grafts.

BACKGROUND: Vein graft failure due to neointimal hyperplasia remains an important and unresolved problem of cardiovascular surgery. MicroRNA-221 (miR-221) has been shown to play a major role in regulating vascular smooth muscle cell (VSMC) proliferation and phenotype transformation. Thus, the purpose of this study is to determine whether adenovirus mediated miR-221 sponge gene therapy could inhibit vein graft neointimal hyperplasia. METHODS: Adenovirus encoding miR-221 sponge (Ad-miR-221-SP) was used to inhibit VSMC proliferation in vitro and neointimal formation in vivo. Expression of miRNA-221 was evaluated in cultured VSMC and in rat vein graft models following transduction with Ad-miR-221-SP, Ad-Control-SP (without miR-221 antisense binding sites), or Ad-GFP (control). To accelerate the transfer of miR-221 sponge gene to the vein grafts, 20% poloxamer F-127 gel was used to extend virus contact time and 0.25% trypsin to increase virus penetration. RESULTS: miR-221 sponges can significantly decrease the expression of miR-221 and proliferation in cultured VSMC. Cellular proliferation rates were significantly reduced in miR-221 sponge treated grafts as compared with controls at 6 weeks after bypass surgery (19.8% versus 43.6%, P=0.0028). miR-221 sponge gene transfer reduced the neointimal area (210.75 ± 24.13 versus 67.01 ± 12.02, P<0.0001), neointimal thickness (171.86 ± 27.87 versus 64.13 ± 16.23, P<0.0001) and neointima/media ratio (0.74 ± 0.21 versus 1.95 ± 0.25, P<0.0001) in vein grafts versus controls. miR-21 sponge treatment was also improved hemodynamics in vein grafts. We have further identified that p27 (Kip1) is a potential target gene of miR-221 in vein grafts. CONCLUSION: miR-221 sponge therapy can significantly reduce miR-221 activity and inhibit neointimal hyperplasia in vein grafts. Locally adventitial delivery of adenoviruses mediated miRNA sponges may be promising gene therapies to prevent vein graft failure.

Study on the adoption of Schwann cell phenotype by bone marrow stromal cells in vitro and in vivo.

OBJECTIVE: To explore the possibilities of bone marrow stromal cells (MSCs) to adopt Schwann cell phenotype in vitro and in vivo in SD rats. METHODS: MSCs were obtained from tibia and femur bone marrow and cultured in culture flasks. Beta-mercaptoethanol followed by retinoic acid, forskolin, basic-FGF, PDGF and heregulin were added to induce differentiation of MSCs'. Schwann cell markers, p75, S-100 and GFAP were used to discriminate induced properties of MSCs' by immunofluorescent staining. PKH-67-labelled MSCs were transplanted into the mechanically injured rat sciatic nerve, and laser confocal microscopy was performed to localize the PKH67 labelled MSCs in the injured sciatic nerve two weeks after the operation. Fluorescence PKH67 attenuation rule was evaluated by flow cytometry in vitro. RESULTS: MSCs changed morphologically into cells resembling primary cultured Schwann cells after their induction in vitro. In vivo, a large number of MSCs were cumulated within the layer of epineurium around the injured nerve and expressed Schwann cell markers, p75, S-100, and GFAP. CONCLUSION: MSCs are able to support nerve fiber regeneration and re-myelination by taking on Schwann cell function, and can be potentially used as possible substitutable cells for artificial nerve conduits to promote nerve regeneration.

[Cell-cycle negative regulatory gene ANA is over-expressed in the brain tissues of patients with Down syndrome].

OBJECTIVE: To investigate the expression level of genes located in chromosome 21 in the brain tissues of Down syndrome(DS). METHODS: An optimized semi-quantitative RT-PCR method was used to evaluate the expression levels of seven genes encoded in chromosome 21 in fetal cortex brain and cerebellum of DS and the control at the end of 20 weeks of gestation. B2M was used as internal reference to normalize cell loss. RESULTS: The expression levels of 6 genes in cortex and cerebellum, including DYRK1A, SYNJ1, PCP4, C21orf5, C21orf2 and C21orf106, were comparable between DS and the control. ANA, a cell-cycle negative regulatory gene, was over-expressed dramatically in the cortex but not in the cerebellum of DS. CONCLUSION: Over-expression of ANA may contribute to the reduction of neuronal density in DS brain.

[Apoptin induces G2-M arrest in cancer cells].

OBJECTIVE: To further study the effect of apoptin in inducing cancer cell specific apoptosis and the possible applications in cancer therapy. METHODS: Apoptin gene was amplified by PCR and inserted into pcDNA3.1(+) with a FLAG tag in front of the multi-cloning-site. Apoptin gene with the FLAG tag was sub-cloned into an adenovirus vector. Several cancer cell lines were transfected with pcDNA3.1/FLAG/apoptin or infected with apoptin containing recombinant adenoviruses to study the morphologic changes. Ad/apoptin infected cells were also analyzed by flowcytometry after staining with PI. RESULTS: Expressed apoptin was localized in the nucleus of cancer cells. Chromatin condensation occurred 2 or 3 days after Ad/apoptin(+) infection. Cell number in G(2)-M phase increased dramatically after Ad/apoptin(+) infection. CONCLUSION: Apoptin can induce cell cycle G(2)-M arrest and chromatin condensation in cancer cells.

Detection and functional annotation of misregulated microRNAs in the brain of the Ts65Dn mouse model of Down syndrome.

BACKGROUND: Brain hypoplasia and mental retardation in Down syndrome (DS) can be attributed to a severe and selective disruption of neurogenesis. Secondary disruption of the transcriptome, as well as primary gene dosage imbalance, is responsible for the phenotype. MicroRNA (miRNA) expression is relatively abundant in brain tissue. Perturbed miRNA expression might contribute to the cellular events underlying the pathology in DS. METHODS: MiRNA expression profiles in the cerebrum of Ts65Dn mice, a DS model, were examined with a real-time RT-PCR array. MiRNA target gene expression was detected by real-time quantitative PCR and Western blotting. Based on the prediction of their cerebrum-specific targets, the functions of the misregulated miRNAs were annotated by Gene Ontology (GO) enrichment analysis. RESULTS: A total of 342 miRNAs were examined. Among them, 20 miRNAs showed decreased expression in the brains of Ts65Dn mice, and some of these belonged to the same family. Two known targets of the miR-200 family, Lfng and Zeb2, were specifically selected to compare their expression in the cerebrum of Ts65Dn mice with those of euploids. However, no significant difference was found in terms of mRNA and protein expression levels of these genes. By enrichment analysis of the cerebrum-specific targets of each miRNA, we found that 15 of the differential miRNAs could significantly affect target genes that were enriched in the GO biological processes related to nervous system development. CONCLUSION: Perturbed expression of multiple functionally cooperative miRNAs contributes to the cellular events underlying the pathogenesis of DS.

EGFP expression controlled by GFAP promoter in mesenchymal cells: an efficient tool for glial lineage selection and transplantation.

In order to demonstrate a new method to label and select enough glial cells from induced MSCs to provide cells for cell therapy, MSCs were induced with Beta-mercaptoethanol followed by retinoic acid, forskolin, basic-FGF, PDGF and heregulin. Induced MSCs were transfected with reconstructed vector pGFAP-EGFP by inserting GFAP promotor into pEGFP-N3 to substitute CMV promotor. Living cells against G418 were enriched and checked by flowcytometry. EGFP expressing cells were sorted and used for transplantation in vivo. Immunoelectronmicroscopy was accomplished using anti-EGFP to relocalize the transplanted cells. Almost all MSCs took on phenotypes of glial cells after induction, expressing S100 and GFAP. The EGFP expression rate of survived MSCs against G418 was 82.74%. Glial cells expressing EGFP accumulated mainly around the damaged nerve fibers. MSCs were relocalized by immunoelectronmicroscopy and remyelination was observed. EGFP expression controlled by GFAP promoter in mesenchymal cells was an efficient tool for glial lineage selection and transplantation. Induced MSCs can promote nerve regeneration by participating remyelination.