Immune pathway activation in neurons triggers neural damage after stroke.

Ischemic brain injury is a severe medical condition with high incidences in elderly people without effective treatment for the resulting neural damages. Using a unilateral mouse stroke model, we analyze single-cell transcriptomes of ipsilateral and contralateral cortical penumbra regions to objectively reveal molecular events with single-cell resolution at 4 h and 1, 3, and 7 days post-injury. Here, we report that neurons are among the first cells that sense the lack of blood supplies by elevated expression of CCAAT/enhancer-binding protein ß (C/EBPß). To our surprise, the canonical inflammatory cytokine gene targets for C/EBPß, including interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α), are subsequently induced also in neuronal cells. Neuronal-specific silencing of C/EBPß or IL-1ß and TNF-α substantially alleviates downstream inflammatory injury responses and is profoundly neural protective. Taken together, our findings reveal a neuronal inflammatory mechanism underlying early pathological triggers of ischemic brain injury.

Comprehensive RNA-Seq Data Analysis Identifies Key mRNAs and lncRNAs in Atrial Fibrillation.

Long non-coding RNAs (lncRNAs) are an emerging class of RNA species that may play a critical regulatory role in gene expression. However, the association between lncRNAs and atrial fibrillation (AF) is still not fully understood. In this study, we used RNA sequencing data to identify and quantify the both protein coding genes (PCGs) and lncRNAs. The high enrichment of these up-regulated genes in biological functions concerning response to virus and inflammatory response suggested that chronic viral infection may lead to activated inflammatory pathways, thereby alter the electrophysiology, structure, and autonomic remodeling of the atria. In contrast, the downregulated GO terms were related to the response to saccharides. To identify key lncRNAs involved in AF, we predicted lncRNAs regulating expression of the adjacent PCGs, and characterized biological function of the dysregulated lncRNAs. We found that two lncRNAs, ETF1P2, and AP001053.11, could interact with protein-coding genes (PCGs), which were implicated in AF. In conclusion, we identified key PCGs and lncRNAs, which may be implicated in AF, which not only improves our understanding of the roles of lncRNAs in AF, but also provides potentially functional lncRNAs for AF researchers.

MCL1 gene silencing promotes senescence and apoptosis of glioma cells via inhibition of the PI3K/Akt signaling pathway.

Glioma is known to be the most prevalent primary brain tumor. In recent years, there has been evidence indicating myeloid cell leukemia-1 (MCL1) plays a role in brain glioblastoma. Therefore, the present study was conducted with aims of exploring the ability of MCL1 silencing to influence glioma cell senescence and apoptosis through the mediation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Glioma and tumor-adjacent tissues were collected in order to detect the presence of higher levels of MCL1 protein expression. Next, the mRNA and protein expression of MCL1, PI3K, Akt, B cell lymphoma 2 (Bcl2), Bcl2-associated X (Bax), B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), and phosphatase and tensin homolog (PTEN) were determined. Cell counting kit-8 assay was applied to detect cell proliferation, ß-galactosidase staining for cell senescence, and flow cytometry for cell cycle entry and apoptosis. Initially, the results revealed higher positive expression rate of MCL1 protein, increased mRNA and protein expression of MCL1, PI3K, Akt, Bmi-1, and Bcl-2 and decreased that of Bax and PTEN in human glioma tissues. The silencing of MCL1 resulted in a decrease in mRNA and protein expression of PI3K, Akt, Bmi-1, and Bcl-2 and an increase in Bax and PTEN expressions in glioma cells. Moreover, silencing of MCL1 also inhibited cell proliferation and cell cycle entry in glioma cells, and promoted glioma cell senescence and apoptosis. In conclusion, the aforementioned results collectively suggested that the silencing of MCL1 promotes senescence and apoptosis in glioma cells through inhibiting the PI3K/Akt signaling pathway. Thus, decreasing the expression of MCL1 might have therapeutic functions in glioma. © 2018 IUBMB Life, 71(1):81-92, 2019.

MicroRNA-17 inhibition overcomes chemoresistance and suppresses epithelial-mesenchymal transition through a DEDD-dependent mechanism in gastric cancer.

MicroRNAs (miRNAs), a novel class of important gene-regulatory molecules, correlates with tumor growth, invasion, metastasis, and chemo resistance in gastric cancer (GC). Microarray analysis revealed that aberrant expressed microRNA-17 (miR-17) and DEDD were identified in GC. DEDD has been found to act as an endogenous suppressor of tumor growth and metastasis through epithelial-mesenchymal transition (EMT) process. However, the role of miRNA-17 (miR-17) has not been clearly evaluated in GC, thereby a series of in vitro experiments were performed in this study. The levels of miR-17 and DEDD in GC tissues from patients diagnosed with GC and in five GC cell lines (SGC-7901, MKN-45, HGC-27, BGC823, and AGS) were detected. It was found that miR-17 up-regulated and DEDD down-regulated in GC, and SGC-7901 and AGS cells were adopted for the in vitro cell experiments, in which the expression of miR-17 or DEDD was regulated by transfection. DEDD was validated to be a target gene of miR-17. Inhibition of miR-17 impaired EMT in GC cells. In addition, transwell assay and scratch test results revealed that inhibition of miR-17 hindered GC cell invasion and migration. Moreover, inhibition of miR-17 reduced resistance to cisplatin- or 5-Fu in GC cells and induced cisplatin- or 5-Fu-treated GC cell apoptosis, which evaluated by using CCK-8 and flow cytometry assays. From the short review above, the key findings emerge that inhibition of miR-17 may have tumor suppressive effects on GC and enhance its chemosensitivity by promoting DEDD, highlighting a novel target for GC therapy.

Lipoprotein (a) as a Predictor of Early Stroke Recurrence in Acute Ischemic Stroke.

Inflammation plays a crucial role in the pathogenesis of stroke. This study aims to determine lipoprotein (a) [Lp(a)] levels in serum and to investigate their associations with stroke recurrence events in a 3-month follow-up study in patients with acute ischemic stroke (AIS). Serum Lp(a) levels were determined in 203 ischemic stroke patients and 120 normal controls at admission. The severity and clinical outcome of ischemic stroke patients were evaluated by the National Institutes of Health Stroke Scale (NIHSS). We followed the participants for a median of 3 months using a standard questionnaire to determine the stroke recurrence events. The correlation analysis and multiple linear regression analysis were performed. Compared with controls, serum Lp(a) levels were significantly increased in ischemic stroke patients than in controls. NIHSS scores and infarct volume were positively correlated with Lp(a) (P < 0.001). Finally, 34 patients (16.7%; 95% CI, 11.6-21.9%) had a stroke recurrence. Serum Lp(a) levels in patients with recurrent stroke were significantly higher as compared with those in patients without recurrent stroke (P < 0.001). In multivariate analysis, there was an increased risk of stroke recurrence associated with Lp(a) levels ≥300 mg/l (OR, 2.87; 95% CI, 1.98-4.32; P = 0.009) after adjusting for possible confounders. With an AUC of 0.872 (95% CI, 0.816-0.927), Lp(a) showed a significantly greater discriminatory ability to predict stroke recurrence as compared with NIHSS score (AUC, 0.782; 95% CI, 0.704-0.859; P < 0.01). Our findings suggest that elevated serum Lp(a) levels can predict the risk of early stroke recurrence in patients with first-ever ischemic stroke. Further research is needed to replicate these findings.

Associations of TGFBR1 and TGFBR2 gene polymorphisms with the risk of hypospadias: a case-control study in a Chinese population.

This case-control study investigated the association of transforming growth factor-ß (TGF-ß) receptor type I and II (TGFBR1 and TGFBR2) gene polymorphisms with the risk of hypospadias in a Chinese population. One hundred and sixty two patients suffering from hypospadias were enrolled as case group and 165 children who underwent circumcision were recruited as control group. Single nucleotide polymorphisms (SNPs) in TGFBR1 and TGFBR2 genes were selected on the basis of genetic data obtained from HapMap. PCR-restriction fragment length polymorphism (PCR-RFLP) was performed to identify TGFBR1 and TGFBR2 gene polymorphisms and analyze genotype distribution and allele frequency. Logistic regression analysis was conducted to estimate the risk factors for hypospadias. No significant difference was found concerning the genotype and allele frequencies of TGFBR1 rs4743325 polymorphism between the case and control groups. However, genotype and allele frequencies of TGFBR2 rs6785358 in the case group were significantly different in contrast with those in the control group. Patients carrying the G allele of TGFBR2 rs6785358 polymorphism exhibited a higher risk of hypospadias compared with the patients carrying the A allele (P<0.05). The TGFBR2 rs6785358 genotype was found to be significantly related to abnormal pregnancy and preterm birth (both P<0.05). The frequency of TGFBR2 rs6785358 GG genotype exhibited significant differences amongst patients suffering from four different pathological types of hypospadias. Logistic regression analysis revealed that preterm birth, abnormal pregnancy, and TGFBR2 rs6785358 were the independent risk factors for hypospadias. Our study provides evidence that TGFBR2 rs6785358 polymorphism might be associated with the risk of hypospadias.

[Construction of recombinant plasmid pIRES-GM-CSF-IL-21 and its therapeutic effect on orthotopically transplanted liver cancer in mice].

OBJECTIVE: To construct a recombinant plasmid pIRES-GM-CSF-IL-21, and to investigate its antitumor effect on tumors in the mice. METHODS: Fifty Bal b/c mice were included in this study. Cultured hepatoma H22 cells were inoculated in the left lobe of the liver to develop orthotopically transplanted liver tumor models. The mice with orthotopically transplanted liver tumor were randomly divided into 5 groups (n = 10): (1) Each mouse received injection of recombinant plasmid pIRES-GM-CSF-IL-21; (2) Received injection of plasmid pIRES-GM-CSF; (3) pIRES-IL-21; (4) Received injection of ampty plasmid pIRES (H22/neo group); (5) Received injection of PBS (H22 group) via the tail vein, respectively. Therefore, the anti-tumor effect was induced by both GM-CSF and IL-21, or by either of them alone. The serum levels of IFN-γ and IL-2 were detected by ELISA, and the cytotoxicity of spleen NK and CTL cells were tested by MTT colorimetry. RESULTS: Comparing with the H22 and H22/Neo groups, the tumor weight in the mice of H22/GM-CSF group was (0.603 ± 0.223) g, H22/IL21-treated group (0.583 ± 0.290) g and H22/GM-CSF-IL21-treated group (0.303 ± 0.323) g, significantly lower than that in the H22 group [(1.591 ± 0.280) g] and H22/Neo group [(1.489 ± 0.155) g]. Among them the tumor growth was most significantly inhibited in the H22/GM-CSF-IL-21 group (0.303 ± 0.323) g, compared with that of H22 and H22/neo groups (P < 0.01). But there was no significant difference between the tumor weights of the H22/GM-CSF group and H22/IL-21 group, and between the tumor weights of the H22 and H22/Neo groups (P > 0.05). The levels of IFN-γ and IL-2 in peripheral blood of mouse models treated with H22/GM-CSF-IL-21 were significantly increased than that in the H22/GM-CSF group and H22/IL-21 group (all P < 0.01), but significantly decreased in the H22group and H22/Neo group (P < 0.01). The anti-tumor activity of splenic NK cells and CTLs in the H22/GM-CSF-IL21 group was significantly enhanced (P < 0.01), compared with the significantly decreased in the H22 and H22/Neo groups. CONCLUSIONS: Our results demonstrate apparent antitumor effect of the plasmid pIRES-GM-CSF-IL-21 on the orthotopically transplanted liver tumor in mice. The combination of both pIRES-GM-CSF and IL-21 is more effective than that of pIRES/IL21 or pIRES/GM-CSF treatment alone. In addition, the plasmid pIRES-GM-CSF-IL-21 can also promote the secretion of IFN-γ and IL-2 in vivo, and enhance the cytotoxic activity of splenic NK and CTLs against the transplanted liver tumor.

Mutation analysis in a Chinese family with multiple endocrine neoplasia type 1.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome which is caused by germline mutations of the tumor suppressor gene MEN1. This study aimed to identify mutations in a Chinese pedigree with MEN1. METHODS: A large Chinese family with MEN1 was collected. All of the coded regions and their adjacent sequences of the MEN1 gene were amplified and sequenced. RESULTS: In this family, a heterozygous cytosine insertion in exon 10 (c.1546_1547insC) inducing a frame shift mutation of MEN1 was found in the proband and the other two suffering members of his family. This mutation was linked to a novel single nucleotide polymorphism (SNP) in intron 3 (IVS3 + 18C > T). CONCLUSIONS: The mutation in exon 10 of MEN1 gene might induce development of parathyroid hyperplasia and pituitary adenoma and cosegregate with MEN1 syndrome. The significance of the new found IVS3 + 18C > T of MEN1 needs a further investigation.

[Complete sequence determination and phylogenetic analysis of FKN among seven higher primates including homonids and Old World Monkeys].

To obtain full-length FKN nucleotide sequences of homonids including human, chimpanzee, gorilla, orangutan and gibbon, and Old World Monkeys including macaque and leaf monkey and make phylogenetic analysis, three exons of FKN were amplified by degenerated PCR using obtained peripheral blood cells DNA as template which was extracted from homonids and Old World Monkeys. After extracting and purifying from agarose gels, PCR products were sequenced and then spliced by using BioEdit. All the FKN sequences were aligned and compared the percent identity by using DNAStar. The phylogenetic tree was constructed using maximum evolution approach in Mega. The negative selection sites were analyzed by using Datamonkey. There is an apparent 30 bp nucleotides deletion mutation in homonids FKN comparing to that of Old World Monkeys besides other point mutations. Homology of nucleotide sequence between human and chimpanzee, gorilla, orangutan, gibbon, macaque and leaf monkey is 99.2%, 98.4%, 98.1%, 96.5%, 95.9% and 93.8%, respectively. Homology of amino acid sequence of them is 98.5%, 98.0%, 97.7%, 94.7%, 93.7% and 90.5%, respectively. In the same time, the genealogical relationship of human is a lot closer to chimpanzee than it is to gorilla and other apes. It is generally agreed that the evolution rule of FKN gene is in accord with that of the higher primates. In addition, Datamonkey shows that there are 3 negative selection sites 53Q, 84D and 239N in FKN. The full-length FKN gene of human, chimpanzee, gorilla, orangutan, gibbon, macaque and leaf monkey were sequenced successfully, and the FKN sequences analysis lays the foundation for further studying its evolution in immunological function in higher primates and the relation between its structure and function.

[Effects and mechanism of hyperglycemia on development and maturation and immune function of human monocyte derived dendritic cells].

OBJECTIVE: Dendritic cells play an important role in the pathogenesis of atherosclerosis. To explore the effects of hyperglycemia on the maturation and immune function of human monocyte derived dendritic cells (MDCs). METHODS: Immature MDCs were cultured in RPMI1640 medium with either 5.5 mmol/L D-glucose (NG), 25 mmol/L D-glucose (HG) or 5.5 mmol/L D-glucose + 19.5 mmol/L mannitol (HM) in the absence or presence of 30 mmol/L N-acetylcysteine [NAC, a reactive oxygen species inhibitor (ROS)] for 48 hours. FACS was used to investigate the MDCs immunophenotypic expression. Immune function was evaluated by allogeneic mixed T lymphocyte reaction and measurement of cytokine levels from culture supernatants. Intracellular ROS production in MDCs was also measured by 2', 7'-dichlorodihydrofluorescein (DCF, 10 micromol/L) fluorescence using confocal laser-scanning microscopy techniques. RESULTS: Compared with NG and HM treated MDCs, the expression of maturation markers such as CD1a, HLA-DR, CD83, CD86 were significantly upregulated, allogeneic T cells proliferation as well as the cytokines secretions (IL-2, IL-12, IL-10 and IFN-gamma) significantly increased in HG treated MDCs. Intracellular ROS production in MDCs was also significantly increased and all these stimulatory effects of HG could be partially attenuated by NAC. CONCLUSION: High glucose promote the maturation of MDCs and augment their capacity to stimulate T-cell proliferation and cytokine secretions at least in part through enhancing intracellular ROS generation. These stimulating effects of high glucose on MDCs maturation may be one of the mechanisms of accelerated atherosclerosis found in patients with diabetes.

[Cloning and eukaryotic expression of mouse interleukin 21].

AIM: To clone mouse interleukin 21(mIL-21) gene and construct its eukaryotic expression vector. METHODS: mIL-21 cDNA was amplified from ConA-activated mouse T cells by RT-PCR. And then the cDNA was cloned into eukaryotic expression plasmid pcDNA3.1 to construct recombinant plasmid pcDNA3.1/mIL-21 which was introduced into Sp2/0 cells by lipofectin. Expression of mIL-21 gene was detected by RT-PCR and NK cytotoxicity assay. RESULTS: Recombinant plasmid pcDNA3.1/mIL-21 was constructed correctly and mIL-21 gene was expressed in transfected Sp2/0 cells. CONCLUSION: mIL-21 gene was cloned and expressed successfully, which lays the foundation for further studying anti-tumor effect of mIL-21 gene therapy in animal models.