MagT1 is essential for Drosophila development through the shaping of Wingless and Decapentaplegic signaling pathways.

Magnesium transporter subtype 1 (MagT1) is a magnesium membrane transporter with channel like properties. We have previously identified MagT1 (CG7830) in Drosophila genome and characterized its protein product by electrophysiological means. Here, we report the generation of fly MagT1 mutants and show that MagT1 is essential for early embryonic development. In wings and primordial wings, by clonal analysis and RNAi knock down of MagT1, we have found that loss of MagT1 results in enhanced/ectopic Wingless (Wg, a fly Wnt) signaling and disrupted Decapentaplegic (Dpp) signaling, indicating the crucial role of MagT1 for fly development at later stages. Finally, we demonstrate directly that magnesium transportations are proportional with the MagT1 expressional levels in Drosophila S2  cells. Taken together, these findings may suggest that MagT1 is a major magnesium transporter/channel profoundly involved in fly development by affecting developmental signaling pathways, such as Wg and Dpp signaling.

Drosophila MagT1 is upregulated by PKC activation.

Magnesium transporter subtype 1 (MagT1) is a newly discovered and evolutionarily conservative magnesium membrane transporter with channel like properties. Previous reports have demonstrated that MagT1 is important to cellular magnesium homeostasis. In this study, we investigated whether drosophila MagT1 (dMagT1) was functionally regulated by PKC activation in vitro. With patch clamping, we have observed that whole cell currents of wild type dMagT1 were magnesium selective and non-voltage dependent when expressed in a human neuroblastoma SH-SY5Y cell line. Furthermore, dMagT1 currents were significantly increased in cells treated with a non specific PKC activator PMA, but not in cells treated with the inactive form of PMA, 4α-PMA. Lastly, we have demonstrated that upregulation of dMagT1 currents by PKC activation involves specific PKC phorsphorylation sites in dMagT1. Of all three dMagT1 mutants created for testing the putative PKC phorsphorylation sites, dMagT1-S35A displayed a significant increase of whole cell currents while dMagT1-S100A and -S108A were not affected by PKC activation. Thus, we have demonstrated that dMagT1 is a magnesium selective transporter with basic biophysical characters similar to its mammalian homolog and can be functionally upregulated by PKC activation. Both dMagT1 Ser100 and Ser106 are equally important to this PKC-dependent modulation, therefore the most likely molecular sites for PKC phorsphorylation. The data presented here may establish a general regulatory mechanism for MagT1 by PKC activation.

[Effects of inhibition of ANG-2 expression in Ishikawa cell line].

OBJECTIVE: To investigate the effect of RNA interference mediated angiopoietin-2 (ANG-2) gene silencing on human endometrial carcinoma cell line Ishikawa. METHODS: Short hairpin RNA (shRNA) targeting ANG-2 gene was designed and transfected into Ishikawa cells with Lipofectamine 2000. The mRNA and protein expression level of ANG-2, proliferation, morphological changes, apoptosis, cell cycle and invasive ability of the cells after transfection were analyzed. RESULTS: The shRNA targeting the human ANG-2 gene effectively decreased the expression of ANG-2 on both mRNA and protein level, the proliferation inhibition rate of the Ishikawa cells was 63.11%, cell apoptosis was induced, and the cell cycle was arrested in G1 phase. The apoptotic rate of the Ishikawa cells in the transfected group was significantly higher, and the invasive ability was decreased markedly, than that of control groups. CONCLUSION: The shRNA targeting human ANG-2 gene could reduce ANG-2 expression, inhibit cellular growth and invasion in Ishikawa cells in vitro.

Combined silencing of VEGF-A and angiopoietin-2, a more effective way to inhibit the Ishikawa endometrial cancer cell line.

BACKGROUND: Angiogenesis is critical for the growth and metastasis of solid tumors and is, therefore, an important therapeutic target. Despite the great research advances in tumor therapies targeting vascular endothelial growth factor (VEGF), drug resistance frequently occurs, and further strategies targeting the tumor vasculature are of primary concern. PURPOSE: The present study aimed to determine whether a combination of small interfering RNAs (siRNAs) targeting VEGF-A and angiopoietin-2 (Ang-2) inhibited the biologic mechanisms of endometrial cancer more effectively compared to either one alone, in vitro and in vivo. METHODS: VEGF-A and Ang-2 were silenced by siRNA in Ishikawa endometrial cancer cells. Cell growth, apoptosis, metastasis, and tumor angiogenesis were measured in vitro and in vivo. RESULTS: There was no difference observed in cell apoptosis rate; however, combined silencing of VEGF-A and Ang-2 resulted in a stronger inhibition of cell proliferation and invasion (P<0.05). Similarly, a greater reduction of tumor size and angiogenesis was seen with the concurrent administration of siRNAs targeting VEGF-A and Ang-2 in nude mice (P<0.05). CONCLUSION: Our data indicated that simultaneous blockade of VEGF-A and Ang-2 may serve as a novel and effective therapeutic strategy in endometrial cancer.

[Mechanism of hypothalamic effect in small intestine electro-activity of rats regulated by fructus aurantii immaturus].

OBJECTIVE: To observe the effect of Fructus Aurantii Immaturus (FAI) on the electro-activity of small intestines in rats, and evaluate the interrelations between the FAI regulating effect and choecystokinin (CCK) and somatostatin (SS). METHODS: Migrating myoelectric complex (MMC) cyclic period, the ratio of the active time to the cyclic period, and the number of the fast wave within the active time per minute were observed between FAI and the normal saline group by external alimentary canal electrodes; the CCK contents in dorsomedial hypothalamic nucleus (DMH), ventromedia hypothalamic nucleus (VMH), lateral hypothalamus area (LHA) and SS in VMH, LHA, paraventricular nucleus (PVN) by using immuno-chemistry technique and micro-image pattern quantitative analysis and scanning system. RESULTS: The MMC cyclic period shortened, the ratio of the active time to the cyclic period increased and the number of the fast wave within the active time per minute increased in the FAI group, which showed significant difference from the normal saline group; CCK positive neurons were reduced in the areas of DMH, VMH and LHA, SS positive neurons were increased in the areas of VMH, LHA and PVN in the FAI gioup,which showed significant difference compared with the normal saline and the blank control group. CONCLUSION: FAI can stimulate the electro-reactivity of small intestines. The stimulative effect of FAI might be related to CCK and SS in hypothamus.

Noise induced hearing loss impairs spatial learning/memory and hippocampal neurogenesis in mice.

Hearing loss has been associated with cognitive decline in the elderly and is considered to be an independent risk factor for dementia. One of the most common causes for acquired sensorineural hearing loss is exposure to excessive noise, which has been found to impair learning ability and cognitive performance in human subjects and animal models. Noise exposure has also been found to depress neurogenesis in the hippocampus. However, the effect is mainly attributed to the oxidant stress of noise on the cognitive brain. In the present study, young adult CBA/CAJ mice (between 1.5 and 2 months of age) were briefly exposed a high sound level to produce moderate-to-severe hearing loss. In both the blood and hippocampus, only transient oxidative stress was observed after noise exposure. However, a deficit in spatial learning/memory was revealed 3 months after noise exposure. Moreover, the deficit was correlated with the degree of hearing loss and was associated with a decrease in neurogenesis in the hippocampus. We believe that the observed effects were likely due to hearing loss rather than the initial oxidant stress, which only lasted for a short period of time.

Spatial learning and memory deficits in young adult mice exposed to a brief intense noise at postnatal age.

Noise pollution is a major hazardous factor to human health and is likely harmful for vulnerable groups such as pre-term infants under life-support system in an intensive care unit. Previous studies have suggested that noise exposure impairs children's learning ability and cognitive performance and cognitive functions in animal models in which the effect is mainly attributed to the oxidant stress of noise on the cognitive brain. The potential role of noise induced hearing loss (NIHL), rather than the oxidant stress, has also been indicated by a depression of neurogenesis in the hippocampus long after a brief noise exposure, which produces only a tentative oxidant stress. It is not clear if noise exposure and NIHL during early development exerts a long term impact on cognitive function and neurogenesis towards adulthood. In the present study, a brief noise exposure at high sound level was performed in neonatal C57BL/6J mice (15 days after birth) to produce a significant amount of permanent hearing loss as proved 2 months after the noise. At this age, the noise-exposed animals showed deteriorated spatial learning and memory abilities and a reduction of hippocampal neurogenesis as compared with the control. The averaged hearing threshold was found to be strongly correlated with the scores for spatial learning and memory. We consider the effects observed are largely due to the loss of hearing sensitivity, rather than the oxidant stress, due to the long interval between noise exposure and the observations.

Estrogen regulates the tumour suppressor MiRNA-30c and its target gene, MTA-1, in endometrial cancer.

MicroRNA-30c (miR-30c) has been reported to be a tumour suppressor in endometrial cancer (EC). We demonstrate that miR-30c is down-regulated in EC tissue and is highly expressed in estrogen receptor (ER)-negative HEC-1-B cells. MiR-30c directly inhibits MTA-1 expression and functions as a tumour suppressor via the miR-30c-MTA-1 signalling pathway. Furthermore, miR-30c is decreased upon E2 treatment in both ER-positive Ishikawa and ER-negative HEC-1-B cells. Taken together, our results suggest that miR-30c is an important deregulated miRNA in EC and might serve as a potential biomarker and novel therapeutic target for EC.

Treatment with Tie2-siRNA in combination with carboplatin suppresses the growth of Ishikawa human endometrial carcinoma cell xenografts in vivo.

It is well-known that tumor angiogenesis is important in cancer development, and studies on blocking angiogenesis to treat tumors have become one of the most promising and active fields in anticancer research. The present study investigated the effect of siRNA targeting the tyrosine kinase receptor 2 (Tie2) gene in combination with carboplatin in a mouse model of endometrial carcinoma in an attempt to elucidate the role of Tie2 in the carcinogenesis and progression of endometrial carcinoma via angiogenesis, in order to establish a basis for the development of complementary molecule targeting and chemotherapeutic actions. Ishikawa cells were used to establish a human endometrial carcinoma nude mouse tumor xenograft model. Tie2-siRNA (20 µg/mouse) and/or carboplatin (25.0 mg·kg-1) were administered as the treatment strategy. Real-time PCR and western blotting were used to evaluate the expression levels of Tie2 mRNA and protein and immunohistochemistry was used to assess the vessel density of the tumor tissues. The present data demonstrated that Tie2-siRNA and/or carboplatin were able to suppress the growth of endometrial xenografts in vivo and attenuate the expression of Tie2 mRNA and protein, as assessed by real-time PCR and western blotting. Furthermore, immunohistochemical assessment showed that the vessel density of the tumors decreased with treatment. The present results suggest that treatment with Tie2-siRNA or carboplatin alone was able to inhibit the growth of human endometrial carcinoma nude mouse xenografts markedly and decrease the expression of Tie2. The combination of Tie2-siRNA and carboplatin increased the therapeutic effect of carboplatin which may eliminate the tumor microenvironment, increase the apoptosis of tumor cells, normalize the abnormal tumor vessels and increase the efficiency of chemotherapy for endometrial carcinoma with carboplatin. The synergy of Tie2-siRNA in combination with carboplatin may involve the regulation of other angiogenesis and metastasis pathways.

microRNA-103 regulates the growth and invasion of endometrial cancer cells through the downregulation of tissue inhibitor of metalloproteinase 3.

Despite improvements in treatment over the past few decades, endometrial cancer remains one of the most common causes of mortality in women and there is an urgent need for the development of targeted therapies. The aim of this study was to confirm the target gene of miR-103 in human endometrial cancer and investigate the biological functions in which miR-103 is involved through the regulation of the expression of its target gene. This study may provide useful data to gain a better understanding of the effect of miR-103 in tumor formation. miR-103 expression levels were measured using real-time quantitative PCR. The effect of miR-103 on tissue inhibitor of metalloproteinase 3 (TIMP-3) expression was assessed in endometrial cancer cell lines with a miR-103 inhibitor to decrease the level of miR-103 expression. Furthermore, the roles of miR-103 in cell growth and invasion were analyzed using miR-103 inhibitor-transfected cells. The level of expression of miR-103 decreased following transfection with the miR-103 inhibitor. miR-103 inhibitor transfection increased the activity of the luciferase reporter assay containing the TIMP-3 3'-untranslated region (UTR) construct and increased the levels of the TIMP-3 protein but not its mRNA in endometrial cancer cell lines. Finally, miR-103 inhibitor-transfected cells exhibited reduced cell growth and invasive characteristics. Our data suggested that miR-103 post-transcriptionally downregulates the expression of the tumor suppressor TIMP-3 and stimulates growth and invasion in endometrial cancer cell lines. This provides a possible therapeutic target that may upregulate TIMP-3 in endometrial cancer.

microRNA-30c negatively regulates endometrial cancer cells by targeting metastasis-associated gene-1.

It is well known that microRNAs (miRNAs) play important roles in cancer development by targeting oncogenes or tumor-suppressor genes. However, little is known regarding the mechanisms of miR-30c action in endometrial cancer. In this study, we aimed to determine whether miR-30c targets metastasis-associated gene-1 (MTA1) and acts as a tumor suppressor in endometrial cancer cell lines Ishikawa (estrogen receptor-positive, ER+) and HEC-1-B (ER-) by down-regulating MTA1. As a result, in both Ishikawa and HEC-1-B cells, real-time PCR demonstrated that overexpression of miR-30c led to the down-regulation of MTA1 mRNA (P<0.05), while Western blotting confirmed the reduced expression levels of MTA1 protein (P<0.01). A dual-luciferase reporter assay demonstrated that miR-30c was directly bound to the 3'-untranslated regions of MTA1. Then we studied the biological mechanisms of endometrial cancer cells transfected with the Pre-miR-30c plasmid. MTT assay and growth curves revealed that miR-30c inhibits both Ishikawa and HEC-1-B cell proliferation. However, we did not see obvious differences in rates of apoptosis between miR-30c-overexpressing and the negative control cells. Then using wound-healing and Matrigel invasion assays, we found that the migratory and invasive abilities of cells transfected with the Pre-miR-30c plasmid were significantly suppressed compared with the control cells (P<0.01). Overall, our study, for the first time, showed that MTA1 is negatively regulated by miR-30c and that overexpression of miR-30c inhibits the proliferative, migratory and invasive abilities of endometrial cancer cells. These results suggest that miR-30c acts as a tumor suppressor and negatively regulates endometrial cancer cells by targeting MTA1.

[Experimental study on the effect of Glycyrrhiza on small intestinal motility in rats].

AIM: To investigate the effects of glycyrrhiza decoction on migrating myoelectric complex (MMC) and gastrointestinal hormone in small intestine in rats. METHODS: We observed MMC cycle,phase Ill duration,fast wave numbers of phase III of MMC in one minute, fast wave numbers of one cluster in phase III of MMC of small intestine of glycyrrhiza group and control group rats with electrophysiology method, and immunohistochemistry to examine relative content of serotonin (5-HT), substance p(SP) and vasoactive intestinal polypeptide (VIP) in small intestinal chromophil (EC) and myenteric nerve plexus in small intestine of control group and glycyrrhiza group rats. RESULTS: Compared glycyrrhiza group with control group,we found that glycyrrhiza was able to decrease fast wave numbers in one minute and fast wave numbers in one cluster in phase III of MMC of small intestine (P < 0.05), and evidently extend small intestinal cycle of MMC (P < 0.05), it also shortened the phase III III duration (P < 0.05) or made the phase III of MMC absent. Compared glycyrrhiza group with control group it was indicated that content of 5-HT in small intestinal mucous membrane and myenteric nerve plexus was evidently decreased (P < 0.05), and content of SP in myenteric nerve plexus of small intestine of rats was evidently decreased (P < 0.05), and content of VIP in small intestine of rats was evidently increased (P < 0.05). CONCLUSION: Glycyrrhiza is able to inhibit small intestinal motility, this inhibition is related with the amount of 5-HT, SP, VIP secreted by small intestinal mucous membrane of rats.