lncRNA GHET1 has effects in development of pre-eclampsia.

To explain long noncoding RNA (lncRNA) gastric carcinoma high expressed transcript 1 (GHET1) affects the mechanism in development of pre-eclampsia. The pathological changes of normal, mild, and severe pre-eclampsia were evaluated by hematoxylin and eosin staining and measured the lncRNA GHET1 expression in different tissues by reverse-transcription polymerase chain reaction. In the cell experiment, the BeWo cells were randomly divided into three groups: normal control (NC) group, model group, and lncRNA group. The JEG3 cells of the model and lncRNA groups were cultured in the hypoxia condition. The JEG3 cells invasion and migration abilities were measured by Tanswell and wound-healing assays. The relative protein expressions of different groups were evaluated by Western blot (WB) assay. Compared with normal puerperal, the lncRNA GHET1 gene expression of pre-eclampsia was significantly downregulated (P < 0.05, respectively). In the cell experiment, the invasion cell number and wound-healing rate of the model group were significantly suppressed compared with the NC group (P < 0.05, respectively). However, the invasion cell number and wound-healing rate of lncRNA group were enhanced by lncRNA GHET1 overexpression (P < 0.05, respectively). In WB assay, the E-cadherin, fibronectin, and vimentin proteins expression showed significant differences between the model and lncRNA groups (P < 0.05, respectively). lncRNA GHET1 overexpression had restored cell invasion and migration abilities reduced by hypoxia in pre-eclampsia.

Multiple steps determine CD73 shedding from RPE: lipid raft localization, ARA1 interaction, and MMP-9 up-regulation.

Physiologically, retinal pigment epithelium (RPE) expresses high levels of CD73 in their membrane, converting AMP to immune suppressive adenosine, mediates an anti-inflammatory effect. However, after being exposed to inflammatory factors, RPE rapidly becomes CD73-negative cells, which render RPE's immune suppressive function and accelerate local inflammation. Here, we investigated the mechanism leading to the loss of membrane CD73 in RPE. We found the controversy that when membrane CD73 was significantly diminished in inflammatory RPE, Cd73 mRNA levels were not changed at all. It was further verified that, matrix metalloproteinase-9 (MMP-9) mediated the shedding of CD73 from the cell membrane of inflammatory RPE by catalyzing its K547/F548 site. However, MMP-9 could not catalyze uncomplexed CD73, the interaction of CD73 with adenosine receptor A1 subtype (ARA1) is necessary for being catalyzed by MMP-9. After being treated by LPS and TNF-α, the formation of CD73/ARA1 complex in RPE was verified by co-immunoprecipitation and FRET-based assays. It was also revealed that CD73 need to be localized in lipid rafts to be capable of interacting with ARA1, since CD73/ARA1 interaction and CD73 shedding were completely blocked by the addition of lipid raft synthesis inhibitor. As a conclusion, multiple steps are involved in CD73 shedding in RPE, including up-regulation of MMP-9 activity, localization of CD73 in lipid rafts, and the formation of CD73/ARA1 complex. Lipid rafts committed CD73 with high mobility, shuttled CD73 to ARA1 to form a complex, which was capable of being recognized and catalyzed by MMP-9.

The Association of CYP1A1 Gene With Cervical Cancer and Additional SNP-SNP Interaction in Chinese Women.

AIMS: The aim of this study was to investigate the association between CYP1A1 gene polymorphism and cervical cancer risk, and the impact of SNP-SNP interaction on cervical cancer risk in Chinese women. METHODS: A total of 728 females with a mean age of 60.1 ± 14.5 years old were selected, including 360 cervical cancer patients and 368 normal controls. Logistic regression was performed to investigate association between single-nucleotide polymorphisms (SNP) and cervical cancer risk. Generalized multifactor dimensionality reduction (GMDR) was used to analyze the SNP-SNP interaction. RESULTS: Logistic analysis showed a significant association between rs4646903 and increased cervical cancer risk. The carriers of homozygous mutant of rs4646903 polymorphism revealed increased cervical cancer risk than those with wild-type homozygotes, OR (95%CI) were 1.45 (1.20-1.95). There was a significant two-locus model (P = 0.0107) involving rs4646903 and rs1048943, indicating a potential SNP-SNP interaction between rs4646903 and rs1048943. Overall, the two-locus models had a cross-validation consistency of 10 of 10, and had the testing accuracy of 60.72%. Subjects with TC or CC of rs4646903 and AG or GG of rs1048943 genotype have the highest cervical cancer risk, compared to subjects with TT of rs4646903 and AA of rs1048943 genotype, OR (95%CI) was 2.03 (1.42-2.89). CONCLUSIONS: rs4646903 minor alleles and interaction between rs4646903 and rs1048943 were associated with increased cervical cancer risk.

CD73 expression in RPE cells is associated with the suppression of conventional CD4 cell proliferation.

CD73 is intensively involved in the regulation of immune responses through the conversion of pro-inflammatory ATP to immunosuppressive adenosine. Herein, we clarified whether cells in the retina express CD73 and participate in the regulation of inflammatory eye diseases such as experimental autoimmune uveitis (EAU). First, immunofluorescence staining was performed to compare the distribution of CD73(+) cells in the retinas of EAU-induced and normal B10RIII mice. The results revealed that a layer of cells in the normal retina that was consistent with the location of retinal pigment epithelial (RPE) cells strongly expressing CD73, and the expression was markedly reduced in the presence of EAU. Thereafter, EAU was also induced in C57BL/6 mice by active immunization or adoptive transfer. CD73 expression in isolated RPE cells was assessed by real-time RT-PCR and western blotting, and the catalytic abilities of the cells to convert AMP to adenosine were determined using HPLC analyses. Compared to the normal control, significantly decreased CD73 expression and AMP catalytic ability were found in the RPE cells isolated from inflamed eyes. CD73 expression and activity were also studied in cultured RPE cells treated with different stimuli, such as Toll-like receptor ligands and cytokines. Highly varied functional CD73 expression was observed in RPE cells through cytokines or Toll-like receptor agonist treatments. Finally, whether RPE cells could regulate the immune response, particularly the proliferation of CD4 cells, through surface-expressed CD73 was determined using a two-chamber assay. The robust inhibition of conventional T-cell proliferation was uniquely observed when CD73(+) RPE cells in the upper chamber were in the presence of AMP. To further confirm the function of CD73 in RPE cells, Cd73(-/-) RPE cells were isolated, and CD73-rescued control cells were constructed. CD73(+)Cd73(-/-) RPE, not Cd73(-/-) RPE, significantly suppressed interacted CD4 cells proliferation and cytokine production. Taken together, these data suggest that naive RPE cells suppressed the immune response through their high expression of CD73. The expression of CD73 in RPE cells could be regulated through many factors, and down-regulated CD73 expression attenuated the suppressive effect of RPE on the proliferation of conventional CD4 cells.

Evaluation of preoperative coagulation function changes and deep vein thrombosis incidence in elderly patients with hip fractures.

OBJECTIVE: This study involved an analysis of preoperative deep vein thrombosis (DVT) incidence and changes in coagulation function among elderly patients suffering from hip fractures. The objective was to offer guidance on the prevention and management of preoperative DVT in the lower extremities of elderly individuals with hip fractures. METHODS: A total of 282 elderly individuals with a hip fracture were enrolled and divided into two groups based on the location of the fracture: femoral intertrochanteric fracture (FIF, 161 individuals) and femoral neck fracture (FNF, 121 individuals). The two groups were compared with respect to baseline characteristics, including gender, age, and comorbid chronic diseases. Furthermore, the analysis encompassed the incidence of preoperative DVT in both lower extremities, along with seven coagulation parameters and platelet count before the surgical procedure. RESULTS: There was no significant difference in baseline information between the two groups. The incidence of preoperative DVT in the FIF group was higher than that in the FNF group, along with a significantly higher percentage of patients exhibiting increased levels of D-dimer and fibrinogen/fibrin degradation products (FDPs). CONCLUSION: Preoperative hypercoagulability and a greater prevalence of DVT were observed in elderly individuals with FIF compared to individuals with FNF. This indicates that clinicians should pay attention to elderly patients with FIFs, especially those with increased D-dimer and FDP levels.

Cromolyn prevents cerebral vasospasm and dementia by targeting WDR43.

Background: Cerebral vasospasm (CV) can cause inflammation and damage to neuronal cells in the elderly, leading to dementia. Purpose: This study aimed to investigate the genetic mechanisms underlying dementia caused by CV in the elderly, identify preventive and therapeutic drugs, and evaluate their efficacy in treating neurodegenerative diseases. Methods: Genes associated with subarachnoid hemorrhage and CV were acquired and screened for differentially expressed miRNAs (DEmiRNAs) associated with aneurysm rupture. A regulatory network of DEmiRNAs and mRNAs was constructed, and virtual screening was performed to evaluate possible binding patterns between Food and Drug Administration (FDA)-approved drugs and core proteins. Molecular dynamics simulations were performed on the optimal docked complexes. Optimally docked drugs were evaluated for efficacy in the treatment of neurodegenerative diseases through cellular experiments. Results: The study found upregulated genes (including WDR43 and THBS1) and one downregulated gene associated with aneurysm rupture. Differences in the expression of these genes indicate greater disease risk. DEmiRNAs associated with ruptured aortic aneurysm were identified, of which two could bind to THBS1 and WDR43. Cromolyn and lanoxin formed the best docking complexes with WDR43 and THBS1, respectively. Cellular experiments showed that cromolyn improved BV2 cell viability and enhanced Aß42 uptake, suggesting its potential as a therapeutic agent for inflammation-related disorders. Conclusion: The findings suggest that WDR43 and THBS1 are potential targets for preventing and treating CV-induced dementia in the elderly. Cromolyn may have therapeutic value in the treatment of Alzheimer's disease and dementia.

CircHIPK3 promotes proliferation and metastasis of villous trophoblasts through miR-30a-3p/Wnt2 axis.

The aim of this paper was to explore the role and mechanism of circHIPK3 in unexplained recurrent spontaneous abortion (URSA). The expression of circHIPK3 and miR-30a-3p mRNA in URSA villous tissues was detected by quantitative polymerase chain reaction. The effects of circHIPK3 on the proliferation and migration of villous trophoblasts were analysed by MTTassay and scratch assay. Hoechst/PI staining was used to detect the effect of circHIPK3 on villous trophoblast apoptosis. The binding of circHIPK3 to miR-30a-3p and miR-30a-3p to Wnt2 was analysed by dual-luciferase assay. When URSA occurred, the expression level of circHIPK3 was downregulated, while the expression level of miR-30a-3p was upregulated in villous trophoblasts. Inhibition of circHIPK3 in villous trophoblasts can reduce the proliferation and migration of villous trophoblasts while promoting their apoptosis. The dual-luciferase assay showed that circHIPK3 was able to interact with miR-30a-3p and increased the miR-30a-3p expression after inhibition of circHIPK3, and if miR-30a-3p was also inhibited it was able to reverse the effect of circHIPK3 on villous trophoblast proliferation and migration. It was demonstrated by prediction and dual-luciferase assay that miR-30a-3p binds to Wnt2, and when miR-30a-3p and Wnt2 are inhibited simultaneously, it has an inhibitory effect on the proliferation and migration process of villous trophoblasts. Downregulation of circHIPK3 expression in URSA leads to increased expression of miR-30a-3p, which in turn inhibits the expression of target gene Wnt2 and exerts a weakening effect on the proliferation and migration process of trophoblasts, thereby decreasing trophoblast invasiveness and shallow placental implantation, which in turn leads to recurrent spontaneous abortion.

MicroRNA-15a-5p promotes the proliferation and invasion of T98G glioblastoma cells via targeting cell adhesion molecule 1.

Glioblastoma (GBM) is a type of malignant tumor occurring in the brain that severely influences the life of affected individuals. GBM cells are highly infiltrative, which is one of the main obstacles in the treatment of the disease. Numerous microRNAs (miRNAs/miRs) are associated with the development of GBM. However, the effects of miR-15a-5p on GBM remain elusive. In the present study, reverse transcription-quantitative PCR and western blot analysis were applied for the detection of RNA and protein levels, respectively. Cell Counting Kit-8 and Transwell assays were performed to examine cell proliferation and invasion, respectively. TargetScan 7.1 and dual-luciferase reporter assay were utilized for the prediction and verification of the association between miRNAs and mRNAs. The present study revealed that miR-15a-5p expression was upregulated in the GBM T98G cell line. The results further demonstrated that, through the inhibition of cell adhesion molecule 1 expression and the promotion of Akt phosphorylation, miR-15a-5p was able to promote GBM cell proliferation and invasion. Overall, the present findings revealed a novel mechanism responsible for the development of GBM and provided an experimental basis for the diagnosis and treatment of GBM.

Pro-inflammatory Effect of Downregulated CD73 Expression in EAE Astrocytes.

CD73, an ectonucleotidase, participates in the regulation of immune responses by controlling the conversion of extracellular AMP to adenosine. In this study, we investigated whether any type of brain cells, especially neuroglia cells, exhibit altered CD73 expression, localization or activity upon experimental autoimmune uveitis (EAU) induction and whether altered CD73 manipulates the activation of effector T cells that interact with such cell types. First, the amount of cell membrane-exposed CD73 was detected by flow cytometry in various types of brain cells collected from either naïve or EAE mice. Compared to that in astrocytes from naïve control mice, the amount of membrane-bound CD73 was significantly decreased in astrocytes from EAE mice, while no significant differences were detected in other cell types. Thereafter, wild-type and CD73-/- astrocytes were used to study whether CD73 influences the function of inflammatory astrocytes, such as the production of cytokines/chemokines and the activation of effector T cells that interact with astrocytes. The results indicated that the addition of exogenous AMP significantly inhibited cytokine/chemokine production by wild type astrocytes but had no effect on CD73-/- astrocytes and that the effect of AMP was almost completely blocked by the addition of either a CD73 inhibitor (APCP) or an adenosine receptor A1 subtype (ARA1) antagonist (DPCPX). Although the addition of AMP did not affect CD73-/- astrocytes, the addition of adenosine successfully inhibited their cytokine/chemokine production. The antigen-specific interaction of astrocytes with invading CD4 cells caused CD73 downregulation in astrocytes from mice that underwent EAE induction. Collectively, our findings support the conclusion that, upon EAE induction, likely due to an interaction with invading CD4+ cells, astrocytes lose most of their membrane-localized CD73; this inhibits the generation of adenosine in the local microenvironment. As adenosine has anti-inflammatory effects on astrocytes and CNS-infiltrating effector T cells in EAE, the downregulation of CD73 in astrocytes may be considered a pro-inflammatory process for facilitating the pathogenesis of EAE.

Adenosine binds predominantly to adenosine receptor A1 subtype in astrocytes and mediates an immunosuppressive effect.

The four kinds of adenosine receptor subtypes (ARs), named as ARA1, ARA2A, ARA2B and ARA3, have multiple biological functions. ARs are differently distributed across the body and have distinguished ability of binding adenosine. We try to figure out how these ARs were expressed in astrocytes and which one has the first priority of utilizing adenosine. Firstly, mRNA expressions and membrane localization of all ARs were evaluated by qPCR and western blot. After the membrane localization of all ARs in astrocytes was being confirmed their individual adenosine binding ability was determined by radio-active ligand binding assay respectively. It was revealed that ARA1 had much superior adenosine binding ability than other AR subtypes. Functional study demonstrated that ARA1 potentially mediated an immune suppressive effect in astrocytes. The activation of ARA1 signaling lead to decreased IL-12 and IL-23 production, and decreased chemokine production, including CCL2, CXCL8 and IP-10. When interacted with CD4 cells ARA1 agonist pre-treated astrocytes showed hindered ability of stimulating CD4 cells to secret IL-17 and IFN-γ and inducing CD4 cells' chemo taxi. Finally, in vivo experiment confirmed that local administration of ARA1agonist ameliorated EAE in wild type B6 recipients, but not Ara1-/- recipients. As a conclusion, this paper suggested that adenosine receptor A1 subtype predominantly binds adenosine in astrocytes and mediates an immunosuppressive effect.

Superior transfection efficiency of phagocytic astrocytes by large chitosan/DNA nanoparticles.

PURPOSE: Mechanism study of why astrocytes isolated from experimental autoimmune encephalomyelitis (EAE)-induced B6 mice or after being exposed to inflammatory factors had the highest transfection efficiency to larger-sized, but not compacted, pspCS/pDNA particles. METHODS: Phosphorylatable short peptide conjugated chitosan (pspCS) was compounded with plasmid DNA (pDNA) at different N:P ratios to form pspCS/pDNA particles of different size and zeta potentials. These pspCS/pDNA particles were used for the transfection of astrocytes isolated from either EAE induced or healthy B6 mice. Transfection efficiency and cell permeability of the particles were determined by the internalization of radio [H3]-labeled plasmid and the expression of a luciferase reporter gene respectively. Phagocytosis of EAE-astrocytes was determined by the internalization of FITC labeled dextran beads. By comparing the transfection efficiency of differently-sized pspCS/pDNA particles to normal and phagocytic astrocytes, with or without cytochalasin D, a phagocytosis inhibitor, in the presence, the contribution of phagocytosis to cell permeability and transfection efficiency was evaluated. RESULTS: In vivo EAE-induction or in vitro inflammatory factors treatment transferred normal astrocytes to be phagocytic astrocytes which underwent phagocytosis, had the highest cell permeability and transfection efficiency to larger-sized pspCS/pDNA particles formed at lower N:P ratios. When phagocytosis was inhibited by cytochalasin D, both cell permeability and transfection efficiency of phagocytic astrocytes to larger were significantly decreased. Thereafter, particle size, not zeta potential, was verified as the key factor for determining whether the particles could be phagocytosed. In addition phagocytosis was successfully induced in ARPE-19 cells as well, which also improved the transfection efficiency of larger pspCS/pDNA particles. CONCLUSION: A generally accepted concept is that the internalization of cationic polymer/pDNA particles, chitosan-DNA complex for instance, is mainly through the procedure of endocytosis of the transfected cells. More compacted particles with higher zeta potential were used to be considered had higher cell permeability and transfection efficiency. However, here we reported that phagocytosis is another important procedure for determining internalization and transfection efficiency of cationic polymer/pDNA nanoparticles, especially for advanced transfection efficiency of large pspCS/pDNA particles. Thus, for gene delivery applications, the environmental condition of the target cells should be seriously considered for selecting an appropriate gene transfer strategies.

Suppression of PDCD4 mediated by the long non-coding RNA HOTAIR inhibits the proliferation and invasion of glioma cells.

Programmed cell death protein 4 (PDCD4) has recently been demonstrated to be implicated in translation and transcription, and the regulation of cell growth. However, the mechanisms underlying PDCD4 function in glioma cells remain to be elucidated. The current study investigated the function and regulation of PDCD4 and the results demonstrated that the expression of PDCD4 was significantly reduced in glioma cells compared with normal cells. When PDCD4 was overexpressed in glioma cells, the proliferation rate and invasive capability of the cells greatly decreased, suggesting that PDCD4 functions as a tumor suppressor in this cell type. In addition, the histone modification status of the PDCD4 gene was analyzed, and chromatin immunoprecipitation assay identified a high density of histone 3 lysine 27 trimethylation on the promoter of PDCD4, which was associated with the long non-coding RNA, homeobox transcript antisense RNA (HOTAIR). The expression of HOTAIR was significantly increased in glioma cells compared with normal cells, and it exerted its function in a polycomb repressive complex 2-dependent manner. These results may provide novel approaches to therapeutically target PDCD4 and HOTAIR in patients with gliomas.

Phosphorylatable short peptide conjugated low molecular weight chitosan for efficient siRNA delivery and target gene silencing.

Small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for diseases with genetic defects. However, its application was greatly hampered by the rapid degradation and poor cellular uptake. Recently, chitosan (CS) and its derivant have been considered as a promising siRNA transporter with the advantages of low toxicity, good biodegradability and biocompatibility. Chitosan of different molecular weight (Mw) and degrees of deacetylation (DD) showed significantly varied target gene silencing efficacy, and it is still not well clarified how these characteristics influence CS mediated siRNA transfection. To compare the aspects of cell permeability and intracellular unpacking of CS/siRNA complex on the effect of CS/siRNA transfection. A radiolabeled siRNA, targeting firefly luciferase gene, was loaded by chitosan of different molecular weight (varying from 2000 to 800,000 Da) and subjected to the transfection against MDA-MB-231/Luc human breast cancer cell line which stably expressed knocked in firefly Luciferase reporter gene. Following transfection intracellular radioactivity was measured to represent cell entrance ability of the CS/siRNA, while, luciferase activity in the cell lysate was also determined to reflect target gene silencing effect. The results revealed that although low molecular weight chitosan (LMWC) condensed siRNA has the highest cell permeability of almost two folds of medium molecular weight chitosan and lipofactamine, its target gene silencing effect is really low of almost eight times less than lipofectamine. This conspicuous contradiction gave us the hypothesis that LMWC generated more condensed CS/siRNA complex to facilitate cell entrance but the tight electrostatic interaction probably limited intracellular siRNA unpacking as well and unfavorably hindered target gene silencing as the final consequence. To approve this hypothesis a phosphorylatable short peptide conjugated LMWC was adopt to promote intracellular siRNA unpacking. Which was demonstrated of perfect target gene knock down ability to the extent of being even superior to lipofactamine 2000. In a conclusion, low molecular weight chitosan has the great potential to be an ideal siRNA vehicle if the issue of siRNA unpacking could be properly resolved.

Phosphorylatable short peptide conjugation for facilitating transfection efficacy of CS/DNA complex.

Previously, we had demonstrated that enhancing intracellular unpacking of exogene from its chitosan carrier by promoting chitosan degradation could markedly improve transfection efficiency of the CS/DNA complex. In this article we addressed a novel strategy of phosphorylatable short peptide modification for further facilitating intracellular DNA unpacking and optimizing transfection efficiency of the CS/DNA complex. A short peptide (SP) with the amino acid composition of "LLLRRRDNEY*FY*VRRLL" containing two potentially phosphorylatable tyrosine residues was synthesized. The short peptide could be phosphorylated by constitutively expressed cytoplasmic protein kinase Jak2. The SP was conjugated to chitosan and combined with GFP/luciferase reporter gene plasmid DNA to form SP-CS/DNA complex. In vitro phosphorylation and DNA releasing assays verified that mammalian cell lysate could effectively phosphorylate SP and hence promote plasmid DNA unpacking from the SP-CS carrier. Thereafter, C2C12 myoblast cells were transfected by SP-CS/DNA and the transfection efficiency was presented by the expression of GFP and luciferase reporters. Further more, multiple cell lines were transfected by SP-CS/DNA complexes loading luciferase reporter gene. Results revealed that, compared with CS, SP-CS could intensively augment the transfection efficiency to the level of near lipofectamine 2000.