Transfer learning for clustering single-cell RNA-seq data crossing-species and batch, case on uterine fibroids.

Due to the high dimensionality and sparsity of the gene expression matrix in single-cell RNA-sequencing (scRNA-seq) data, coupled with significant noise generated by shallow sequencing, it poses a great challenge for cell clustering methods. While numerous computational methods have been proposed, the majority of existing approaches center on processing the target dataset itself. This approach disregards the wealth of knowledge present within other species and batches of scRNA-seq data. In light of this, our paper proposes a novel method named graph-based deep embedding clustering (GDEC) that leverages transfer learning across species and batches. GDEC integrates graph convolutional networks, effectively overcoming the challenges posed by sparse gene expression matrices. Additionally, the incorporation of DEC in GDEC enables the partitioning of cell clusters within a lower-dimensional space, thereby mitigating the adverse effects of noise on clustering outcomes. GDEC constructs a model based on existing scRNA-seq datasets and then applying transfer learning techniques to fine-tune the model using a limited amount of prior knowledge gleaned from the target dataset. This empowers GDEC to adeptly cluster scRNA-seq data cross different species and batches. Through cross-species and cross-batch clustering experiments, we conducted a comparative analysis between GDEC and conventional packages. Furthermore, we implemented GDEC on the scRNA-seq data of uterine fibroids. Compared results obtained from the Seurat package, GDEC unveiled a novel cell type (epithelial cells) and identified a notable number of new pathways among various cell types, thus underscoring the enhanced analytical capabilities of GDEC. Availability and implementation: https://github.com/YuzhiSun/GDEC/tree/main.

Heterogeneous graph framework for predicting the association between lncRNA and disease and case on uterine fibroid.

Long non-coding RNAs (lncRNAs) play crucial regulatory roles in various cellular processes, including gene expression, chromatin remodeling, and protein localization. Dysregulation of lncRNAs has been linked to several diseases, making it essential to understand their functions in disease mechanisms and therapeutic strategies. However, traditional experimental methods for studying lncRNA function are time-consuming, expensive, and offer limited insights. In recent years, computational methods have emerged as valuable tools for predicting lncRNA functions and their associations with diseases. However, many existing methods focus on constructing separate networks for lncRNA and disease similarity, resulting in information loss and insufficient processing capacity for isolated nodes. To address this, we developed 'RGLD' by combining Random Walk with restarting (RWR), Graph Neural Network (GNN), and Graph Attention Networks (GAT) to predict lncRNA-disease associations in a heterogeneous network. RGLD achieved an impressive AUC of 0.88, outperforming other methods. It can also predict novel associations between lncRNAs and diseases. RGLD identified HOTAIR, MEG3, and PVT1 as lncRNAs associated with uterine fibroids. Biological experiments directly or indirectly verified the involvement of these three lncRNAs in uterine fibroids, validating the accuracy of RGLD's predictions. Furthermore, we extensively discussed the functions of the target genes regulated by these lncRNAs in uterine fibroids, providing evidence for their role in the development and progression of the disease.

Small incision lenticule extraction and femtosecond-assisted laser in situ keratomileusis in patients with deep corneal opacity: case series.

AIM: To report the safety, efficacy, and accuracy of small-incision lenticule extraction (SMILE) or femtosecond-assisted laser in situ keratomileusis (FS-LASIK) for the correction of myopia or myopic astigmatism in patients with deep corneal opacity denoted by anterior segment optical coherence tomography (AS-OCT). METHODS: Four patients with monocular corneal opacity (3 due to mechanical injury, 1 due to a firecracker wound) were recruited and treated with refractive surgery (3 for SMILE, 1 for FS-LASIK combined with limbal relaxing incision (LRI). Preoperative ocular manifestations, surgical details, postoperative visual outcomes, corneal opacity parameters, and corneal topography were analyzed. RESULTS: Preoperatively, spherical diopter ranged from -3.0 D to -4.75 D with cylinder ranging from -0.75 to -5.0 D, and corrected distance visual acuity (CDVA) ranging from 20/25 to 20/20. One eye's corneal opacity was located in the central zone and three were in the mid-peripheral optical zone. Three patients underwent uneventful SMILE in both eyes, whilst one patient underwent FS-LASIK for high astigmatism in both eyes and LRI in the right eye. CDVA of the eye with corneal opacity ranged from 20/22 to 20/20 one to six weeks postoperatively. Two patients achieved better CDVA and no patients lost Snellen lines. The postoperative diopter was within ±0.75 D for all eyes. Significant edema existed above the corneal opacity in one eye and dissipated soon. No eccentric corneal topography or morphological anomaly of the corneal cap or flap was observed. CONCLUSION: The cases demonstrate that SMILE or FS-LASIK is safe and effective to treat myopic astigmatism combined with deep corneal opacity lesions after comprehensive preoperative evaluation and appropriate candidate selection. FS-LASIK combined with LRI is also sufficient for correcting high astigmatism due to corneal scarring.

Triplet pregnancy with hydatidiform mole: A report of two cases with literature review.

AIM: We present two cases of triplet pregnancy with complete hydatidiform mole (CHM) in contrasting outcomes and discuss the complications of mothers and outcomes of fetuses through a literature review, raising an important issue on the management of this special pregnancy. METHODS: We share our manage experience for two cases of triplet pregnancy with CHM and retrospectively analyze 18 similar pregnancies reported previously with different pregnancy outcomes. RESULTS: In our cases, one case receiving Clomiphene ovulation induction delivered two live fetuses by cesarean section at 30+ weeks without GTN (gestational trophoblastic neoplasia), unfortunately, the other case following ICSI-ET terminated the pregnancy in the setting of complications at 18+ weeks without GTN. No severe complications were detected during pregnancy and no pGTD was developed after delivery in neither of the pregnant. CONCLUSIONS: Co-existing complete hydatidiform mole in multiple pregnancies may become more common owing to the spreading use of ART. The decision for whether continue pregnancy depending on the personalized conditions including the complications of the pregnancy, the outcomes of the fetuses, the gestational age for delivery, and the potential progression of persistent gestational trophoblastic disease (pGTD). Furthermore, close monitor is necessary for the pregnant with triplet pregnancy with CHM who want to continue pregnancy.

Chlorin e6-1,3-diphenylisobenzofuran polymer hybrid nanoparticles for singlet oxygen-detection photodynamic abaltion.

A dual-functional nanosysterm is developed by means of Chlorin e6 (Ce6) as photosensitizer and 1,3-Diphenylisobenzofuran (DPBF) as fluorescent singlet oxygen (1O2) probe. Under 660 nm laser irradiation, Ce6 exhibites efficient 1O2 generation, and subsequently the production of 1O2 is assessed by the ratiometric fluorescence of PFO and DPBF under one-photon and two-photon excitation mode. The nanoparticles with excellent biocompatibility can be internalized into Hela cells and applied for tumor treatment. For intracellular PDT, the nanoparticles perform a high phototoxicity, while the PDT proccess can be evaluated in time by monitoring fluorescence signals of DPBF. This theranostic nanosysterm provides a facile strategy to fabricate 1O2-detection PDT, which can realize accurate and efficient photodynamic therapy based on singlet oxygen detection.

Dengzhan shengmai capsule combined with donepezil hydrochloride in the treatment of Alzheimer's disease: preliminary findings, randomized and controlled clinical trial

SUMMARY OBJECTIVE: To observe the effects of Dengzhan Shengmai capsule combined with donepezil hydrochloride on cognitive function, daily living ability, and safety in patients with Alzheimer's disease. METHODS: A total of 294 patients with Alzheimer's disease were randomly divided into a treatment group and a control group, 147 cases each group. The control group was given oral donepezil hydrochloride 5 mg once a day, and the treatment group was given oral Dengzhan Shengmai capsule 0.36 g three times a day, based on the control group. RESULTS: At 3 and 6 months of treatment, the ADAS-cog score of the treatment group was 48.69±6.23 and 44.24±5.53; for the control group, 45.48±5.94 and 41.57±5.10. The difference between the two groups is statistically significant (p<0.05). At 3 and 6 months of treatment, the NO level in the treatment group was (46.28±6.68) umol/l, (43.55±7.92) umol/l, and the control group was (42.95±7.92) umol/l, (38.89±5.93) umol/l. The differences between both groups were statistically significant (p<0.05). At 3 and 6 months of treatment, ET levels in the treatment group were (156.08±17.39) ng/l, (144.91±17.60) ng/l, and the control group was (150.48±22.94) ng/l, (135.04±10.08) ng/l. Correlation analysis showed that ADAS-cog score was negatively correlated with NO and ET (p<0.001). CONCLUSIONS: Dengzhan Shengmai capsule combined with donepezil hydrochloride can improve cognitive function and the living capacity of patients with Alzheimer's disease, reduce the production of neurotoxic substances NO and ET, and provide higher safety.

Correlation of Vitamin D3 with the Expression of RORγt and Foxp3 mRNAs in the Peripheral Blood of Myasthenia Gravis Patients.

OBJECTIVES: to investigate the expression levels of 1,25(OH)2D3 in the peripheral blood from patients with myasthenia gravis (MG) and to correlate levels with retinoid-related orphan receptor γt (RORγt) and forkhead or winged-helix transcription factor 3 (Foxp3) mRNA expression. METHODS: Sixty-seven patients with MG were enrolled in the experimental group, and 50 normal subjects were selected as the control group. The expression levels of 1,25(OH)2D3 and RORγt and Foxp3 mRNAs were measured in the serum of the 2 patient groups and the relationship between factors were correlated with the severity score of MG. The relationship between the levels of 1,25(OH)2D3 and the relative expressions of RORγt and Foxp3 mRNAs was determined. RESULTS: There were no differences between groups regarding patient's baseline data. 1,25(OH)2D3 and RORγt and Foxp3 mRNAs are differentially expressed in the MG group and the control group (p < 0.05). QMG score is negatively correlated with the expression level of peripheral blood 1,25(OH)2D3 and Foxp3 mRNA (r = -0.797, -0.543; p < 0.01) and positively correlated with the relative expression level of RORγt mRNA (r = 0.539; p < 0.01). 1,25(OH)2D3 expression level was negatively correlated with the relative expression of RORγt mRNA (r = -0.559; p < 0.01) and positively correlated with the relative expression of Foxp3 mRNA (r = 0.390; p < 0.01). CONCLUSIONS: The levels of 1,25(OH)2D3 were shown to be lower in patients with MG compared to normal controls. The observed low levels of 1,25(OH)2D3 may lead to changes in the expression of RORγt and Foxp3 mRNAs involved in MG.

Effects of Carotid Artery Stent and Carotid Endarterectomy on Cognitive Function in Patients with Carotid Stenosis.

BACKGROUND: Carotid artery stenosis is closely related to cognitive dysfunction, in which decreased cerebral perfusion is one of the important factors. Both carotid artery stent implantation and carotid endarterectomy can relieve stenosis and increase cerebral perfusion. In this study, we aimed to compare the effects of carotid artery stent implantation and endarterectomy on cognitive function. METHODS: A total of 98 patients with carotid artery stenosis hospitalized in our hospital from July 2015 to January 2017 were included. Among them, 50 cases underwent carotid artery stent implantation treatment as stent implantation group (CAS group), and 48 cases underwent carotid artery endarterectomy treatment as carotid endarterectomy group (CEA group). Using the Mini-Mental State Examination Scale (MMSE Scale) and the Montreal Cognitive Assessment Scale (MoCA Scale), the cognitive function scores of the two groups of patients before and after 3 and 6 months of operation were measured, and the patients were also measured before and after surgery, after the serum NSE, hs-CRP content. RESULTS: The serum NSE, hs-CRP content, MMSE score, and MoCA score of the two groups before treatment were not statistically significant (P > 0.05). The MMSE score and MoCA score of the two groups of patients before treatment were lower than the normal value, suggesting carotid artery stenosis combined with different degrees of cognitive dysfunction. Carotid artery stenosis is different, and patients' cognitive function is also different. The MMSE score and MoCA score of the two groups at 3 and 6 months after operation were higher than before treatment, and there was a statistically significant difference between 6 and 3 months after operation (P < 0.05), but at each time There was no statistically significant difference between the two groups (P > 0.05). The NSE content of the two groups of patients after operation decreased compared with that before treatment, and the decrease in 6 months after operation was more obvious than that in March (P < 0.05). However, the difference between the two groups at each time point was not statistically significant (P > 0.05). The content of hs-CRP in the two groups of patients was higher than that before the operation, and the CAS group was significantly higher than the CEA group; the difference was statistically significant (P < 0.05). CONCLUSION: Carotid artery stent and carotid endarterectomy are effective in improving the cognitive function of patients with carotid stenosis, but there is no significant difference between the two.

The Role of miRNA-146a and Proinflammatory Cytokines in Carotid Atherosclerosis.

The aim of this study was to investigate the expression and significance of miRNA-146a in peripheral blood of patients with carotid atherosclerosis (CAS). Patients with CAS were selected as the stenosis (CAS) group (n = 180). According to the degree of stenosis, they were divided into the mild stenosis group (MS group, n = 64), moderate stenosis group (M group, n = 62 cases), and severe stenosis group (SS group, n = 54). According to the plaque type, patients were divided into a stable plaque group (SP group, n = 96) and a vulnerable plaque group (VP group, n = 84). Patients without CAS represented the normal group (n = 90). The expression levels of miRNA-146a as well as IL-6 and TNF-α in peripheral blood were detected by RT-PCR and ELISA, respectively. The expression levels of miRNA-146a, IL-6, and TNF-α in the CAS group were higher than those in the normal group and positively correlated with the degree of stenosis and plaque vulnerability. The expression levels of miRNA-146a, IL-6, and TNF-α in the stable plaque group were lower than those in the vulnerable plaque group. The area under the curve of miRNA-146a predicting the vulnerability of plaques was significant at 0.641. The expression level of miRNA-146a in the CAS group was positively correlated with IL-6 and TNF-α levels. Our results indicate that miRNA-146a may participate in the occurrence and development of CAS by regulating the expression of IL-6 and TNF-α, which may be potential biomarkers of CAS.

A novel mitochondria-targeted ratiometric fluorescent probe for endogenous sulfur dioxide derivatives as a cancer-detecting tool.

A new mitochondria-targeted fluorescent probe RBC, constructed using a coumarin moiety which was selected as the donor and a benzothiazole derivative as the acceptor, for SO2 derivatives (HSO3-/SO32-) was presented. The probe designed on a new FRET platform showed high selectivity and a low detection limit. Importantly, the probe could respond to HSO3-/SO32- within 35 s. Furthermore, the probe could target mitochondria and was successfully used for fluorescence imaging of endogenous bisulfite in HepG2 with low cytotoxicity, which significantly assisted in cancer diagnosis.

Estrogen-ERα signaling and DNA hypomethylation co-regulate expression of stem cell protein PIWIL1 in ERα-positive endometrial cancer cells.

BACKGROUND: We previously identified PIWIL1 as an oncogene involved in endometrial carcinogenesis. However, the mechanism of Piwil1 mediated regulation of tumorigenesis remains poorly understood. METHODS: The expression levels of target genes in endometrial cancer cells were detected by quantitative reverse transcription-PCR (RT-qPCR) and western blotting. Up- or down-regulation of ERα or PIWIL1 was achieved by transient transfection with expressing plasmids or short hairpin RNA (shRNA). Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) were used to demonstrate the ERα bound to the half estrogen response element (half-ERE) located in PIWIL1 promoter. The expression of PIWIL1 and ERα in endometrial carcinoma tissues were investigated using immunohistochemistry and RT-qPCR. The proliferation ability of cancer cells were evaluated by MTT. Methylation status of the PIWIL1 promoter was detected by bisulfite sequencing PCR (BSP). RESULTS: In the present study, we found that PIWIL1 mediated E2-stimulated cancer cell proliferation. In ERα-positive endometrial cancer cells, we demonstrated that estrogen-ERα signaling significantly up-regulated the expression of PIWIL1, which was mediated by binding of the ERα onto the PIWIL1 promoter. Furthermore, we found that a half-ERE in the PIWIL1 promoter was essential for ERα binding. The PIWIL1 promoter was hypomethylated in ERα-positive endometrial cancer cells. Treatment with 5-aza-deoxycytidine (5-aza-dC) could up-regulate PIWIL1 expression. CONCLUSIONS: These findings uncover a novel molecular mechanism by which estrogen-ERα signaling and DNA hypomethylation co-regulate PIWIL1 expression. These findings provide novel insights into the hormonal regulation of PIWIL1 in endometrial cancer and the PIWIL1's role in estrogen-stimulated endometrial carcinogenesis. Video Abstract. (MP4 41319 kb).

A near-infrared and mitochondria-targeted fluorescence probe for ratiometric monitoring of sulfur dioxide derivatives in living cells.

A promising near-infrared emissive and mitochondria-targeted fluorescence probe (SNB) for the ratiometric detection of sulfur dioxide derivatives with a novel reaction mechanism was developed on the basis of FRET and the ICT platform. Probe SNB showed favorable fluorescence properties, including an active NIR emission signal (671 nm), ultra-large Stokes shift (251 nm) and an ultra-broad emission band gap (193 nm). Furthermore, SNB was applied to detect sulfur dioxide derivatives with a low detection limit (17 nM), excellent sensitivity and exceptional selectivity. Of greater significance, SNB also successfully implemented the real-time and ratiometric surveillance for exogenous or endogenous sulfur dioxide derivatives in living HeLa, L-O2 and HepG2 cells. Interestingly, SNB possessed a subcellular organelle targeting ability with a preferred co-localization coefficient of 0.91 for mitochondria, which revealed its promising potential in the detection of subcellular sulfur dioxide derivatives.

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model.

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.

The effect of two nucleoside antitumor drugs on the proliferation and DNA methylation of human gastric cancer cells.

Fluorouracil (5-Fu) and 5-azacitidine (5-aza) are two types of nucleoside analog, which have been widely applied in the treatment of several types of cancer. However, the effect of these two types of drug on the proliferation and DNA methylation of cancer cells has not been compared in a single study. In the present study, in vitro cultured human gastric cancer cells (hGCCs) were treated with various concentrations of 5-Fu and 5-aza, and cell counting, MTT assay and methyl-sensitive amplified polymorphism were used to evaluate the resulting levels of proliferation and DNA methylation of hGCCs. The results revealed that the two drugs were able to inhibit the proliferation of hGCCs, but that the effect of 5-aza was weaker than that of 5-Fu. However, 5-aza decreased the level of DNA methylation in hGCCs, whereas 5-Fu did not alter DNA methylation. These results indicated that 5-Fu was able to more efficiently inhibit the proliferation of hGCCs than 5-aza, and that this difference may be due to differences in the anticancer mechanism of these two types of drug.

Multiple low doses of erythropoietin delay the proliferation of hepatocytes but promote liver function in a rat model of subtotal hepatectomy.

PURPOSE: The impact of various doses of erythropoietin (EPO) on liver regeneration after partial hepatectomy (PH) in different animal models is still under debate. We investigated the impact of low doses of EPO on liver regeneration in a rat model of subtotal hepatectomy. METHODS: We established a 90 % PH rat model with perioperative injections of low-dose EPO (1,000 IU/kg). We analyzed survival and hepatocyte proliferation in animals treated with or without EPO and assessed liver function by blood ammonia measurement and the indocyanine green 15-min retention test. RESULTS: Low doses of EPO treatment improved the survival of rats after 90 % PH. Unexpectedly, during the first 24 h after the operation, liver regeneration in the EPO-treated rats was inhibited. DNA synthesis, cell proliferation, and the expression of cyclins and p-STAT3 peaked 48 h after PH, which was delayed by about 24 h vs. the control rats. Furthermore, EPO treatment increased the serum level of IL-6 and protected the hepatocytes from apoptosis. CONCLUSION: Low doses of EPO do not stimulate early hepatocyte proliferation in the regenerating liver, but contribute to liver protection by inducing IL-6 and inhibiting apoptosis.

The development and expression of pluripotency genes in embryos derived from nuclear transfer and in vitro fertilization.

Somatic cell nuclear transfer can be used to produce embryonic stem (ES) cells, cloned animals, and can even increase the population size of endangered animals. However, the application of this technique is limited by the low developmental rate of cloned embryos, a situation that may result from abnormal expression of some zygotic genes. In this study, sheep-sheep intra-species cloned embryos, goat-sheep inter-species cloned embryos, or sheep in vitro fertilized embryos were constructed and cultured in vitro and the developmental ability and expression of three pluripotency genes, SSEA-1, Nanog and Oct4, were examined. The results showed firstly that the developmental ability of in vitro fertilized embryos was significantly higher than that of cloned embryos. In addition, the percentage of intra-species cloned embryos that developed to morula or blastocyst stages was also significantly higher than that of the inter-species cloned embryos. Secondly, all three types of embryos expressed SSEA-1 at the 8-cell and morula stages. At the 8-cell stage, a higher percentage of in vitro fertilized embryos expressed SSEA-1 than occurred for cloned embryos. However, at the morula stage, all detected embryos could express SSEA-1. Thirdly, the three types of embryos expressed Oct4 mRNA at the morula and blastocyst stages, and embryos at the blastocyst stage expressed Nanog mRNA. The rate of expression of Oct4 and Nanog mRNA at these developmental stages was higher in in vitro fertilized embryos than in cloned embryos. These results indicated that, during early development, the failure to reactivate some pluripotency genes maybe is a reason for the low cloning efficiency found with cloned embryos.

Activation of a positive feedback loop involving IL-6 and aromatase promotes intratumoral 17ß-estradiol biosynthesis in endometrial carcinoma microenvironment.

Tumor-stroma interactions contribute greatly to intratumoral estrogen biosynthesis in endometrial carcinoma, but the mechanisms involved remain largely unknown. Previous study demonstrated that intratumoral aromatase upregulation in stromal cells participated in this process, but the specific aromatase-regulators have not been reported. In the present study, we found that aromatase expression in intratumoral stroma, but not in tumor epithelium, correlated positively with interleukin 6 (IL-6) expression in cancer epithelial cells by immunohistochemistry, which was confirmed using laser capture microdissection/real-time reverse transcription-PCR. With stimulation by exogenous IL-6, aromarase expression was increased in stromal cells not but not in cancer cells. Aromatase mRNA levels in endometrial cancer cells were not influenced by cocultivation with intratumoral stromal cells. When cocultured with 17ß-estradiol (E2 )-treated cancer cells, aromatase mRNA in stromal cells was significantly elevated and increased IL-6 protein levels were detected in E2 -treated culture medium. Next, we demonstrated that E2 -induced IL-6 production was through cooperation between estrogen receptor α and nuclear factor-kappa B. Furthermore, an IL-6 receptor blocking antibody could attenuate the upregulation of aromatase expression in stromal cells and the E2 concentration in coculture systems of cancer and stromal cells. The results were confirmed by an orthotopic nude endometrial carcinoma model in vivo. These studies elucidated the activation of a positive feedback loop, that is, IL-6 stimulated by E2 in endometrial cancer cells induced aromatase expression in stromal cells, promoting enhanced intratumoral E2 synthesis. Blocking of this tumor-stroma interaction may be a therapeutic strategy to overcome in situ estrogen biosynthesis in endometrial carcinoma.

A TrkB-STAT3-miR-204-5p regulatory circuitry controls proliferation and invasion of endometrial carcinoma cells.

BACKGROUND: We previously identified TrkB as an oncogene involved in promoting metastasis in endometrial carcinoma (EC). Here, we sought to delineate the effect of changes in TrkB expression on the global profile of microRNAs (miRNAs) in EC cells and further investigated the correlation between the expression of certain miRNA and TrkB in the clinicopathologic characteristics of EC patients. METHODS AND RESULTS: Using quantitative reverse transcription-PCR (qRT-PCR), we found that expression of TrkB mRNA has no significant difference in transcript levels between normal endometrium and EC cells captured by laser capture microdissection, while immunohistochemistry results demonstrated a markedly higher expression of TrkB protein in EC tissues. The microRNA array showed that ectopic overexpression and knockdown of TrkB expression caused global changes in miRNA expression in EC cells. qRT-PCR results showed that elevated TrkB repressed miR-204-5p expression in EC cells. Furthermore, immunoblotting assays revealed that TrkB overexpression in IshikawaTrkB cells noticeably increased JAK2 and STAT3 phosphorylation, which, however, was aborted by TrkB knockdown in HEC-1BshTrkB cells. Moreover, ChIP assays showed that phospho-STAT3 could directly bind to STAT3-binding sites near the TRPM3 promoter region upstream of miR-204-5p. Interestingly, using bioinformatics analysis and luciferase assays, we identified TrkB was a novel target of miR-204-5p. Functionally, the MTT assays, clonogenic and Transwell assays showed that miR-204-5p significantly suppressed the clonogenic growth, migration and invasion of EC cells. Furthermore, miR-204-5p also inhibited the growth of tumor xenografts bearing human EC cells. Importantly, we found lower miR-204-5p expression was associated with advanced FIGO stages, lymph node metastasis and probably a lower chance for survival in EC patients. CONCLUSIONS: This study uncovers a new regulatory loop involving TrkB/miR-204-5p that is critical to the tumorigenesis of EC and proposes that reestablishment of miR-204-5p expression could be explored as a potential new therapeutic target for this disease.

Multipotent differentiation of the EGFP gene transgenic stem cells derived from amniotic fluid of goat at terminal gestational age.

We have isolated stem cells from amniotic fluid of goat at terminal gestational age and transferred the EGFP (enhanced green fluorescent protein) gene into the stem cells previously. The aim of this study was to determine whether the transgenic stem cells have the capability of multipotent differentiation. The transgenic stem cells were induced to differentiate into neurogenic, adipogenic, osteogenic and endothelial cells in vitro. Markers associated with AFS (amniotic fluid-derived stem) cells and the differentiated cells were tested by RT-PCR (reverse transcription-PCR). The results demonstrated that the transgenic AFS cells were capable of self-renewal, a defining property of stem cells. AFS cells were positive for the undifferentiated cell markers, Oct4, Nanog, Sox2 and Hes1, while following differentiation cells expressed markers for neurogenic cells such as astrocyte [GFAP (glial fibrillary acidic protein)] and NSE (neuron-specific enolase), adipogenic cells [LPL+ (lipoprotein lipase+)], osteogenic cells (osteocalcin+ and osteonectin+) and endothelium [CD34+ and eNOS+ (endothelial nitric oxide synthase)]. The results demonstrated that the EGFP gene transgenic AFS cells have the capability of multipotent differentiation, which means that the transgenic AFS cells may be useful in cell-transplantation studies in future.