SVPath: an accurate pipeline for predicting the pathogenicity of human exon structural variants.

Although there are a large number of structural variations in the chromosomes of each individual, there is a lack of more accurate methods for identifying clinical pathogenic variants. Here, we proposed SVPath, a machine learning-based method to predict the pathogenicity of deletions, insertions and duplications structural variations that occur in exons. We constructed three types of annotation features for each structural variation event in the ClinVar database. First, we treated complex structural variations as multiple consecutive single nucleotide polymorphisms events, and annotated them with correlation scores based on single nucleic acid substitutions, such as the impact on protein function. Second, we determined which genes the variation occurred in, and constructed gene-based annotation features for each structural variation. Third, we also calculated related features based on the transcriptome, such as histone signal, the overlap ratio of variation and genomic element definitions, etc. Finally, we employed a gradient boosting decision tree machine learning method, and used the deletions, insertions and duplications in the ClinVar database to train a structural variation pathogenicity prediction model SVPath. These structural variations are clearly indicated as pathogenic or benign. Experimental results show that our SVPath has achieved excellent predictive performance and outperforms existing state-of-the-art tools. SVPath is very promising in evaluating the clinical pathogenicity of structural variants. SVPath can be used in clinical research to predict the clinical significance of unknown pathogenicity and new structural variation, so as to explore the relationship between diseases and structural variations in a computational way.

D3AI-CoV: a deep learning platform for predicting drug targets and for virtual screening against COVID-19.

Target prediction and virtual screening are two powerful tools of computer-aided drug design. Target identification is of great significance for hit discovery, lead optimization, drug repurposing and elucidation of the mechanism. Virtual screening can improve the hit rate of drug screening to shorten the cycle of drug discovery and development. Therefore, target prediction and virtual screening are of great importance for developing highly effective drugs against COVID-19. Here we present D3AI-CoV, a platform for target prediction and virtual screening for the discovery of anti-COVID-19 drugs. The platform is composed of three newly developed deep learning-based models i.e., MultiDTI, MPNNs-CNN and MPNNs-CNN-R models. To compare the predictive performance of D3AI-CoV with other methods, an external test set, named Test-78, was prepared, which consists of 39 newly published independent active compounds and 39 inactive compounds from DrugBank. For target prediction, the areas under the receiver operating characteristic curves (AUCs) of MultiDTI and MPNNs-CNN models are 0.93 and 0.91, respectively, whereas the AUCs of the other reported approaches range from 0.51 to 0.74. For virtual screening, the hit rate of D3AI-CoV is also better than other methods. D3AI-CoV is available for free as a web application at http://www.d3pharma.com/D3Targets-2019-nCoV/D3AI-CoV/index.php, which can serve as a rapid online tool for predicting potential targets for active compounds and for identifying active molecules against a specific target protein for COVID-19 treatment.

Zika Virus Infection in the Ovary Induces a Continuously Elevated Progesterone Level and Compromises Conception in Interferon Alpha/Beta Receptor-Deficient Mice.

Zika virus (ZIKV) belongs to mosquito-borne flaviviruses. Unlike other members in the family, ZIKV can be sexually transmitted, and the female genital tracts are susceptible to ZIKV. However, the impact of ZIKV infection on nonpregnant female reproductive health is not understood. In this study, we investigated the effects of ZIKV infection on the ovary by using nonpregnant female interferon α/ß receptor-deficient (Ifnar1-/-) mice. The results showed that the ovary supported ZIKV replication, and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly reduced the numbers of antral follicles, aggravated follicular atresia, and disrupted folliculogenesis. Notably, ZIKV replication in the ovary caused disordered ovarian steroidogenesis manifested by decreased expression of key enzymes linked to sex hormone synthesis, including the cytochrome P450 17A1 (CYP17A1) and aromatase (CYP19A1). Further, we observed that ZIKV infection disrupted the estrous cycle and thus prolonged the time to conceive. More importantly, although ZIKV RNA could not be detected at 3 months postinfection, damaged ovarian structure and dysfunction were also observed. Taken together, our study demonstrates that ZIKV infection in nonpregnant female mice cause ovarian damage and dysfunction, even long after ZIKV clearance. These data provide important information to understand the effects of ZIKV infection in female reproductive tissues and basic evidence for further studies. IMPORTANCE Zika virus (ZIKV), a flavivirus, is primarily transmitted by mosquito bites. But it can also be transmitted vertically and sexually. Although ZIKV-associated Guillain-Barré syndrome and microcephaly have drawn great attention, there have been few studies on the potential effects of ZIKV on the genital tract of nonpregnant females. This study investigated the effects of ZIKV on the ovaries in mice. We found that ZIKV replicated in the ovary and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly damaged ovarian structure and function and disrupted folliculogenesis. Notably, ZIKV infection further disrupted the estrous cycle and prolonged the time to conceive in mice by causing disordered ovarian steroidogenesis. These effects were observed in both the acute phase and the recovery phase after viral elimination. Overall, the new findings provide important additions to make out the potential adverse impacts of ZIKV on reproductive health in females.

MultiDTI: drug-target interaction prediction based on multi-modal representation learning to bridge the gap between new chemical entities and known heterogeneous network.

MOTIVATION: Predicting new drug-target interactions is an important step in new drug development, understanding of its side effects and drug repositioning. Heterogeneous data sources can provide comprehensive information and different perspectives for drug-target interaction prediction. Thus, there have been many calculation methods relying on heterogeneous networks. Most of them use graph-related algorithms to characterize nodes in heterogeneous networks for predicting new drug-target interactions (DTI). However, these methods can only make predictions in known heterogeneous network datasets, and cannot support the prediction of new chemical entities outside the heterogeneous network, which hinder further drug discovery and development. RESULTS: To solve this problem, we proposed a multi-modal DTI prediction model named 'MultiDTI' which uses our proposed joint learning framework based on heterogeneous networks. It combines the interaction or association information of the heterogeneous network and the drug/target sequence information, and maps the drugs, targets, side effects and disease nodes in the heterogeneous network into a common space. In this way, 'MultiDTI' can map the new chemical entity to this learned common space based on the chemical structure of the new entity. That is, bridging the gap between new chemical entities and known heterogeneous network. Our model has strong predictive performance, and the area under the receiver operating characteristic curve of the model is 0.961 and the area under the precision recall curve is 0.947 with 10-fold cross validation. In addition, some predicted new DTIs have been confirmed by ChEMBL database. Our results indicate that 'MultiDTI' is a powerful and practical tool for predicting new DTI, which can promote the development of drug discovery or drug repositioning. AVAILABILITY AND IMPLEMENTATION: Python codes and dataset are available at https://github.com/Deshan-Zhou/MultiDTI/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Intense endoplasmic reticulum stress (ERS) / IRE1α enhanced Oxaliplatin efficacy by decreased ABCC10 in colorectal cancer cells.

BACKGROUND: Attenuated Oxaliplatin efficacy is a challenge in treating colorectal cancer (CRC) patients, contributory to the failure in chemotherapy and the risks in relapse and metastasis. However, the mechanism of Oxaliplatin de-efficacy during CRC treatment has not been completely elucidated. METHODS: Microarray screening, western blot and qPCR on clinic CRC samples were conducted to select the target gene ABCC10 transporter. The Cancer Genome Atlas data was analyzed to figure out the correlation between the clinical manifestation and ABCC10 expression. ABCC10 knock-down in CRC cells was conducted to identify its role in the Oxaliplatin resistance. Cell counting kit-8 assay was conducted to identify the CRC cell viability and Oxaliplatin IC50. Flow cytometry was conducted to detect the cell apoptosis exposed to Oxaliplatin. The intracellular Oxaliplatin accumulation was measured by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. RESULTS: CRC patients with higher ABCC10 were prone to relapse and metastasis. Differential ABCC10 expression in multiple CRC cell lines revealed a strong positive correlation between ABCC10 expression level and decreased Oxaliplatin response. In ABCC10 knock-down CRC cells the Oxaliplatin sensitivity was evidently elevated due to an increase of intracellular Oxaliplatin accumulation resulted from the diminished drug efflux. To explore a strategy to block ABCC10 in CRC cells, we paid a special interest in the endoplasmic reticulum stress (ERS) / unfolded protein response (UPR) that plays a dual role in tumor development. We found that neither the inhibition of ERS nor the induction of mild ERS had anti-CRC effect. However, the CRC cell viability was profoundly decreased and the pro-apoptotic factor CHOP and apoptosis were increased by the induction of intense ERS. Significantly, the Oxaliplatin sensitivity of CRC cells was enhanced in response to the intense ERS, which was blocked by inhibiting IRE1α branch of UPR. Finally, we figured out that the intense ERS down-regulated ABCC10 expression via regulated IRE1-dependent decay activity. CONCLUSION: Oxaliplatin was a substrate of ABCC10 efflux transporter. The intense ERS/IRE1α enhanced Oxaliplatin efficacy through down-regulating ABCC10 in addition to inducing CHOP. We suggested that introduction of intense ERS/UPR could be a promising strategy to restore chemo-sensitivity when used in combination with Oxaliplatin or other chemotherapeutic drugs pumped out by ABCC10.

Scavenger receptor MARCO contributes to macrophage phagocytosis and clearance of tumor cells.

Macrophage receptor with collagenous structure (MARCO) is a member of the class A scavenger receptor family which is expressed on the cell surface of macrophages. It is well known that MARCO avidly binds to unopsonized pathogens, leading to its ingestion by macrophages. However, the role of MARCO in the recognition and phagocytosis of tumor cells by macrophages remains poorly understood. In this study, we used lentiviral technology to knockdown and overexpress MARCO and investigated the ability of macrophages to phagocytose tumor cells. Our results showed that MARCO expression was correlated with the ability of macrophages to carry on phagocytosis. MARCO knockdown led to significant decreases in the number of engulfment pseudopodia of macrophages and inhibition of the phagocytosis of tumor cells. On the other hand, MARCO overexpression elevated activity of SYK, PI3K and Rac1 in macrophages, which led to changes in macrophage morphology and enhanced phagocytosis by promoting formation of stress fibers and pseudopodia. By Co-IP analysis we showed that MARCO directly binds to the ß5 integrin of SL4 tumor cells. In summary, these results demonstrated the important role for MARCO in demonstrated tumor cells uptake and clearance by macrophages.

c-KIT-ERK1/2 signaling activated ELK1 and upregulated carcinoembryonic antigen expression to promote colorectal cancer progression.

Carcinoembryonic antigen (CEA) is highly expressed in embryo and colorectal cancer (CRC) and has been widely used as a marker for CRC. Emerging evidence has demonstrated that elevated CEA levels promote CRC progression. However, the mechanism of the increased CEA expression in patients with primary and recurrent CRC is still an open question. In this study, we showed that c-KIT, ELK1, and CEA were hyperexpressed in patients with CRC, especially patients with recurrent disease. From bioinformatics analysis, we picked ELK1 as a candidate transcription factor (TF) for CEA; the binding site of ELK1 within the CEA promoter was confirmed by chromatin immunoprecipitation and dual luciferase reporter assays. Overexpression of ELK1 increased CEA expression in vitro, while knockdown of ELK1 decreased CEA. Upregulated ELK1 promoted the adhesion, migration, and invasion of CRC cells, however knockdown of CEA blocked the activities of ELK1-overexpressed CRC cells. Furthermore, we explored the role of c-KIT-ERK1/2 signaling in activation of ELK1. Blocking c-KIT signaling using Imatinib or ISCK03 reduced p-ELK1 expression and consequently decreased CEA levels in CRC cells, as did blocking the ERK1/2 pathway by U0126. Compared with wild type littermates, the c-kit loss-of-functional Wadsm/m mice showed lowered c-KIT, ELK1, and CEA expression. In conclusion, our study revealed that ELK1, which was activated by c-KIT-ERK1/2 signaling, was a key TF for CEA expression. Blocking ELK1 or its upstream signaling could be an alternative way to decelerate CRC progression. Besides being a biomarker for CRC, CEA could be used for guiding targeted therapy.

SCF/c-KIT Signaling Increased Mucin2 Production by Maintaining Atoh1 Expression in Mucinous Colorectal Adenocarcinoma.

Mucinous colorectal adenocarcinoma (MCA) patients often a show high risk of malignant potential and a poorer survival rate. Given that the pathological feature and oncobiological characteristics of MCA are correlated with its abundant extracellular mucin2 (MUC2), we paid interest toward investigating the key factor that promotes MUC2 production exposure to highly-activated stem cell factor (SCF)/c-KIT signaling, which we believed to contribute to MCA formation. Long-term azoxymethane and dextran sodium sulfate treatment successfully induced MCA only in wild-type (WT) mice at week 37 and 43, while all c-kit loss-of-function mutant mice (Wadsm/m) developed non-MCA. Significantly, MUC2 and its key transcriptional factor Atonal homologue 1 (Atoh1) were remarkably expressed in MCA mice compared with non-MCA mice. Atoh1 was significantly elevated in colorectal cancer (CRC) cells stimulated by exogenous SCF or overexpressing c-KIT in vitro, while decreased by the blockage of SCF/c-KIT signaling with Imatinib. Furthermore, the maintained Atoh1 protein level was due to the inactive glycogen synthase kinase 3ß (p-GSK3ß) by virtue of the activated SCF/c-KIT-Protein Kinase B (AKT) signaling. Similar results were obtained from the ONCOMINE database and CRC patients. In conclusion, we suggested that SCF/c-KIT signaling promoted MUC2 production and MCA tumorigenesis by maintaining Atoh1 expression. Therefore, targeting the related key molecules might be beneficial for treating MCA patients.

Persistent distention of colon damages interstitial cells of Cajal through Ca2+ -ERK-AP-1-miR-34c-SCF deregulation.

Gastrointestinal motility disorders (GMDs) are attributed to loss of interstitial cells of Cajal (ICC), whose survival and function are deeply dependent on the activation of KIT/SCF signalling. Based on the facts that gastrointestinal distention is common in GMD patients and SCF produced by smooth muscle cells (SMCs) is usually decreased before ICC loss, we considered a possible contribution of persistent gastrointestinal distention/stretch to SCF deficiency. In this study, chronic colonic distention mouse model, diabetic gastrointestinal paresis mouse model, cultured mouse colonic SMCs and colon specimens from Hirschsprung's disease patients were used. The results showed that SCF was clearly decreased in distent colon of mice and patients, and microRNA array and real-time PCR indicated a concomitant increase of miR-34c in distent colon. A negative regulation of miR-34c on SCF expression was confirmed by luciferase reporter assays together with knock-down and overexpression of miR-34c in cultured colonic SMCs. Using EMSA and ChIP assays, we further consolidated that in response to persistent stretch, the transcription factor AP-1/c-Jun was highly activated in colonic SMCs and significantly promoted miR-34c transcription by binding to miR-34c promoter. Knock-down or overexpression of AP-1/c-Jun in cultured colonic SMCs leads to down- or up-regulation of miR-34c, respectively. In addition, the activation of AP-1/c-Jun was through ERK1/2 signalling provoked by Ca2+ overload in colonic SMCs that were subject to persistent stretch. In conclusion, our data demonstrated that persistent distention/stretch on colonic SMCs could suppress SCF production probably through Ca2+ -ERK-AP-1-miR-34c deregulation, resulting in ICC loss or impairment and GMD progress.

Phosphorylation of ETV4 at Ser73 by ERK kinase could block ETV4 ubiquitination degradation in colorectal cancer.

It was reported that Src-mediated and RTK-dependent accumulation of key transcription factor, ETV4, which played an important role in the migration of embryonic cells and tumor cells, were regulated by their common downstream MAPK molecules. However, the detailed mechanism was not completely clear. In the present study, we revealed that ETV4 protein was significantly enhanced by ERK kinase activation in the colorectal cancer (CRC) patients and mouse models as well as in the CRC cell lines. It was further confirmed that the activation of ERK kinase led to the phosphorylation of ETV4 at Ser73 and the ETV4 phosphorylation could block its binding to COP1, thereby stabilized ETV4 via avoiding its ubiquitination degradation. In addition, this effect was not due to altering an E3 ubiquitin ligase, COP1 amount or p-COP1/COP1 ratio. Our results will help understand the mechanism of ETV4 overexpression in CRC patients and provide a clue to search new therapeutic target to treat the related tumors in clinical practice.

Persistent mechanical stretch-induced calcium overload and MAPK signal activation contributed to SCF reduction in colonic smooth muscle in vivo and in vitro.

Gastrointestinal (GI) distention is a common pathological characteristic in most GI motility disorders (GMDs), however, their detail mechanism remains unknown. In this study, we focused on Ca2+ overload of smooth muscle, which is an early intracellular reaction to stretch, and its downstream MAPK signaling and also reduction of SCF in vivo and in vitro. We successfully established colonic dilation mouse model by keeping incomplete colon obstruction for 8 days. The results showed that persistent colonic dilation clearly induced Ca2+ overload and activated all the three MAPK family members including JNK, ERK and p38 in smooth muscle tissues. Similar results were obtained from dilated colon of patients with Hirschsprung's disease and stretched primary mouse colonic smooth muscle cells (SMCs). Furthermore, we demonstrated that persistent stretch-induced Ca2+ overload was originated from extracellular Ca2+ influx and endoplasmic reticulum (ER) Ca2+ release identified by treating with different Ca2+ channel blockers, and was responsible for the persistent activation of MAPK signaling and SCF reduction in colonic SMCs. Our results suggested that Ca2+ overload caused by smooth muscle stretch led to persistent activation of MAPK signaling which might contribute to the decrease of SCF and development of the GMDs.

Evidence of alterations in the expression of orphan receptors GPR26 and GPR39 due to the etiology of the metabolic syndrome.

AIMS: Metabolic syndrome (MS) is composed of several metabolic abnormalities that increase the risk of cardiovascular diseases and diabetes. Although there are treatments for the components of MS, this pathology maintains a high mortality, suggesting that there are other mechanisms in which orphan receptors such as GPR26 and GPR39 may be involved. For this reason, the aim of this work was to evaluate the expression of GPR26 and GPR39 orphan receptors in two models of MS (diet and genetics). MATERIALS AND METHODS: We used male Wistar rats, which received 70% fructose in drinking water for 9 weeks, and obese Zucker rats. We measured weight, blood pressure, glucose, triglycerides, total cholesterol, HDL cholesterol, LDL cholesterol to determine the MS and the expression of the orphan receptors GPR26 and GPR39 in brain, heart, aorta, liver, and kidney by RT-PCR. RESULTS: The analysis of the expression of the orphan receptors GPR26 and GPR39 showed that the receptors are expressed in some tissues, but the expression of the GPR26 tends to decrease in the heart and aorta, whereas in the brain, no changes were observed, this receptor is not expressed in the liver and kidney of both strains. The expression of GPR39 isoforms depends on the tissue and MS model. CONCLUSIONS: We conclude that the orphan receptors GPR26, GPR39v1, and GPR39v2 are expressed in different tissues and their profile expression is dependent on the etiology of the MS.

SCF/C-Kit/JNK/AP-1 Signaling Pathway Promotes Claudin-3 Expression in Colonic Epithelium and Colorectal Carcinoma.

Claudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and c-kit loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with CLDN-3 promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the c-Kit loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development.

Inhibition of miR-222-3p activity promoted osteogenic differentiation of hBMSCs by regulating Smad5-RUNX2 signal axis.

miRNAs are recently found playing important roles in osteogenesis. In this study, we identified that miR-222-3p decreased during osteogenic differentiation of human mesenchymal stem cells (hBMSCs) using Quantitative Real-Time Reverse Transcription PCR (qRT-PCR). Furthermore, we investigated the effect of miR-222-3p on osteogenic differentiation of hBMSCs. Inhibition of miR-222-3p function in hBMSCs using infection of lentiviruses carrying miR-222-3p specific inhibitor promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Whereas, overexpression of miR-222-3p inhibited osteoblast differentiation of hBMSCs in vitro. Moreover, Smad5 and RUNX2, which are the critical transcription factors in osteogenic differentiation, were predicted to be targets of miR-222-3p by bioinformatic analysis. Overexpression of miR-222-3p in hBMSCs significantly suppressed the protein levels of Smad5 and RUNX2, while inhibition of miR-222-3p increased their protein levels. Furthermore, inhibition of miR-222-3p increased phosphorylation of Smad1/5/8, which regulated the expression of osteogenic genes. Our findings suggest that suppression of miR-222-3p activity promoted osteogenic differentiation hBMSCs through regulating Smad5-RUNX2 signaling axis.

Resveratrol elicits anti-colorectal cancer effect by activating miR-34c-KITLG in vitro and in vivo.

BACKGROUND: Silence of the tumor suppressor miR-34c is implicated in the development of colorectal cancer (CRC). For the past few years, Resveratrol (Res) has been introduced to oncotherapies alone or with traditional chemotherapeutic drugs. However, the study of molecular mechanism involved in the anti-CRC effect of Res is still ongoing. METHODS: The anti-CRC effect of Res alone or with Oxaliplatin (Oxa) was determined by cell viability assay, soft agar colony formation assay, flow cytometry and real-time cellular analyzer in HT-29 (p53+) and HCT-116 (p53-) CRC cell lines. Expressions of miR-34c and its targets were detected by qPCR and/or western blot. To evaluate the role of miR-34c in anti-CRC effect by Res alone or with Oxa, miR-34c was up or down-regulated by lentiviral mediation or specific inhibitor, respectively. To investigate how miR-34c was increased by Res, the methylation status of miR-34c promoter was detected by MSP. The tumor bearing mouse model was established by subcutaneous injection of HCT-116 cells to assess anti-CRC effect of Res alone or with Oxa in vivo. IL-6 and TNF-α in xenografts were detected by ELISA. RESULTS: Res inhibited cell viability, proliferation, migration and invasion as well as promoted apoptosis both in HT-29 and HCT-116 CRC cells. The anti-CRC effect of Res was partially but specifically through up-regulating miR-34c which further knocked down its target KITLG; and the effect was enhanced in the presence of p53 probably through inactivating PI3K/Akt pathway. Besides, Res sensitized CRC cells to Oxa in a miR-34c dependent manner. The xenograft experiments showed that exposure to Res or Oxa suppressed tumor growth; and the efficacy was evidently augmented by the co-treatment of Res and Oxa. Likewise, miR-34c level was elevated in xenografts of Res-treated mice while the KITLG was decreased. Finally, Res clearly reduced IL-6 in xenografts. CONCLUSION: Res suppressed CRC by specifically activating miR-34c-KITLG in vitro and in vivo; and the effect was strengthened in the presence of p53. Besides, Res exerted a synergistic effect with Oxa in a miR-34c dependent manner. We also suggested that Res-increased miR-34c could interfere IL-6-triggered CRC progression.

Hypoxia enhances glucocorticoid-induced apoptosis and cell cycle arrest via the PI3K/Akt signaling pathway in osteoblastic cells.

Although osteonecrosis of the femoral head is a known primary limitation of long-term or high-dose clinical administration of glucocorticoids, the mechanisms underlying this side effect remain unclear. Hypoxia is an important biological state under numerous pathological conditions. In this study, we investigated glucocorticoid-induced osteonecrosis under hypoxic conditions in the MC3T3-E1 osteoblast cell line using a cell cytotoxicity assay, flow cytometry, and western blotting. 6α-Methylprednisolone sodium succinate (MPSL) more effectively induced apoptosis and G0/G1 arrest of MC3T3-E1 osteoblasts under hypoxic conditions than under normoxic conditions. Correspondingly, MPSL more effectively upregulated cellular levels of cleaved caspase 3, p53, and its target p21, and downregulated cyclin D1 levels in hypoxia. Moreover, overexpression of Akt abrogated the MPSL activation of p53, p21, and cleaved caspase 3 and the attenuation of cyclin D1 expression and rescued osteoblasts from MPSL-induced cell cycle arrest and apoptosis, indicating that phosphatidylinositol 3-kinase (PI3K)/Akt signaling might play an essential role in MPSL-induced inhibition of osteoblasts. Furthermore, the suppression of PI3K/Akt signaling and upregualtion of cellular p85α monomer levels by MPSL were more pronounced under hypoxic conditions than under normoxic conditions. Finally, we found that the enhancement of the effects of MPSL under hypoxic conditions was attributed to hypoxia-upregulated glucocorticoid receptor activity. In conclusion, our results demonstrate that MPSL, a synthetic glucocorticoid receptor agonist, promotes the level of p85α and inhibits PI3K/Akt signaling to induce apoptosis and cell cycle arrest in osteoblasts, and that this effect is enhanced under hypoxic conditions.

KITLG is a novel target of miR-34c that is associated with the inhibition of growth and invasion in colorectal cancer cells.

MiR-34c is considered a potent tumour suppressor because of its negative regulation of multiple target mRNAs that are critically associated with tumorigenesis and metastasis. In the present study, we demonstrated a novel target of miR-34c, KITLG, which has been implicated in colorectal cancer (CRC). First, we found a significant negative relationship between miR-34c and KITLG mRNA expression levels in CRC cell lines, including HT-29, HCT-116, SW480 and SW620 CRC cell lines. In silico analysis predicted putative binding sites for miR-34c in the 3' untranslated region (3'UTR) of KITLG mRNA. A dual-luciferase reporter assay further confirmed that KITLG is a direct target of miR-34c. Then, the cell lines were infected with lentiviruses expressing miR-34c or a miR-34c specific inhibitor. Restoration of miR-34c dramatically reduced the expression of KITLG mRNA and protein, while silencing of endogenous miR-34c increased the expression of KITLG protein. The miR-34c-mediated down-regulation of KITLG was associated with the suppression on proliferation, cellular transformation, migration and invasion of CRC cells, as well as the promotion on apoptosis. Knockdown of KITLG by its specific siRNA confirmed a critical role of KITLG down-regulation for the tumour-suppressive effects of miR-34c in CRC cells. In conclusion, our results demonstrated that miR-34c might interfere with KITLG-related CRC and could be a novel molecular target for CRC patients.

Alterations of the interstitial cells of Cajal and the microstructure of the gastrointestinal tract in KIT distal kinase mutant mice.

The development and maintenance of interstitial cells of Cajal (ICC) are closely associated with SCF/KIT signal activity. In this study, we evaluate the distribution of ICC in KIT distal kinase domain mutant mice (Wads) and determine whether the loss-of-function mutations in KIT easily lead to gastrointestinal (GI) disorders. ICC were examined by anti-KIT immunohistochemistry and western blotting. The GI microstructure of wild-type (WT) and Wads mice in normal intestines and incomplete intestinal obstruction was evaluated by hematoxylin and eosin staining. The results in Wads(m/m) mice were as follows. Myenteric ICC were obviously decreased in the stomach and colon and were totally absent in the small intestine. Intramuscular ICC were nearly absent in the stomach and irregularly distributed in the colon. Moreover, the smooth muscle thickness of the small intestine was increased 1.3-fold in Wads(m/m), compared to WT and Wads(m/+) mice and the diameter of the intestinal lumen was also enlarged in Wads(m/m) mice. When constructing an incomplete intestinal obstruction model, the extent of distention involved was greater in Wads mice (1.6-fold in Wads(m/+) mice and 1.8-fold in Wads(m/m) mice vs. WT mice). Meanwhile, the intestinal lumen expansion and decrease in ICC were more pronounced in Wads mice than in WT mice. Our results suggest that the KIT distal kinase domain mutation leads to an ICC loss in a subtype and location-specific pattern in Wads(m/m) mice. The injury of the KIT signaling in mutant mice results in more serious pathological manifestations after being exposed to pathogenic factors.

Conditioned medium from human amniotic epithelial cells may induce the differentiation of human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells.

Dopaminergic (DA) neuron therapy has been established as a new clinical tool for treating Parkinson's disease (PD). Prior to cell transplantation, there are two primary issues that must be resolved: one is the appropriate seed cell origin, and the other is the efficient inducing technique. In the present study, human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were used as the available seed cells, and conditioned medium from human amniotic epithelial cells (ACM) was used as the inducing reagent. Results showed that the proportion of DA neuron-like cells from hUCB-MSCs was significantly increased after cultured in ACM, suggested by the upregulation of DAT, TH, Nurr1, and Pitx3. To identify the process by which ACM induces DA neuron differentiation, we pretreated hUCB-MSCs with k252a, the Trk receptor inhibitor of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and found that the proportion of DA neuron-like cells was significantly decreased compared with ACM-treated hUCB-MSCs, suggesting that NGF and BDNF in ACM were involved in the differentiation process. However, we could not rule out the involvement of other unidentified factors in the ACM, because ACM + k252a treatment does not fully block DA neuron-like cell differentiation compared with control. The transplantation of ACM-induced hUCB-MSCs could ameliorate behavioral deficits in PD rats, which may be associated with the survival of engrafted DA neuron-like cells. In conclusion, we propose that hUCB-MSCs are a good source of DA neuron-like cells and that ACM is a potential inducer to obtain DA neuron-like cells from hUCB-MSCs in vitro for an ethical and legal cell therapy for PD.