MicroRNA-199a-3p inhibits angiogenesis by targeting the VEGF/PI3K/AKT signalling pathway in an in vitro model of diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a major inducer of blindness and visual impairment. As a critical cause for DR, hyperglycaemia is able to trigger multiple biochemical alterations. MiRNAs, which contain various functions, can effectively regulate blood glucose levels. This research aims to confirm the roles of miRNA-199a-3p in the progression of angiogenesis in an in vitro model of DR. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was carried to determine the expression levels of miR-199a-3p and VEGF in both hRMECs and APRE-19 cells. The luciferase reporter assay was used to study the interaction between miR-199a-3p and VEGF. Western blot assay was conducted to examine the expression levels of VEGF and the PI3K/AKT signalling pathway. The cell proliferation capacity was detected via the CCK-8 test. The impact of miR-199a-3p on migration was determined using Transwell and wound healing assays. A Matrigel tube formation assay was employed to determine the vascular formation of hRMECs. Flow cytometry was used to determine cell apoptosis in the presence of LY294002 as a PI3K inhibitor. RESULTS: Our results showed that high glucose (HG) decreased the relative expression level of miR-199a-3p but increased VEGF expression in hRMECs and APRE-19 cells. MiR-199a-3p inhibitor augmented cell growth, migration and angiogenesis of hRMECs. Moreover, upregulation of miR-199a-3p evidently alleviated the increases in cell proliferation, migration and angiogenesis caused by HG. In addition, the luciferase reporter assay indicated that miR-199a-3p directly targeted VEGF. The overexpression of miR-199a-3p obviously restrained the HG-stimulated PI3K/AKT signalling pathway and angiogenesis, which could be further inhibited by LY294002. Moreover, LY294002 could slightly ameliorate the miR-199a-3p inhibitor-stimulated PI3K/AKT signalling pathway and angiogenesis. CONCLUSION: MiR-199a-3p upregulation ameliorated HG-stimulated angiogenesis of hRMECs by modulating the PI3K/AKT pathway through inhibiting VEGF. Although retinal neovascularization in vivo has not been studied, these in vitro findings provide more evidence for the role of miR-199a-3p upregulation against HG-induced angiogenesis.

Expression of overall survival-EMT-immune cell infiltration genes predict the prognosis of glioma.

This study investigates the crucial role of immune- and epithelial-mesenchymal transition (EMT)-associated genes and non-coding RNAs in glioma development and diagnosis, given the challenging 5-year survival rates associated with this prevalent CNS malignant tumor. Clinical and RNA data from glioma patients were meticulously gathered from CGGA databases, and EMT-related genes were sourced from dbEMT2.0, while immune-related genes were obtained from MSigDB. Employing consensus clustering, novel molecular subgroups were identified. Subsequent analyses, including ESTIMATE, TIMER, and MCP counter, provided insights into the tumor microenvironment (TIME) and immune status. Functional studies, embracing GO, KEGG, GSVA, and GSEA analyses, unraveled the underlying mechanisms governing these molecular subgroups. Utilizing the LASSO algorithm and multivariate Cox regression, a prognostic risk model was crafted. The study unveiled two distinct molecular subgroups with significantly disparate survival outcomes. A more favorable prognosis was linked to low immune scores, high tumor purity, and an abundance of immune infiltrating cells with differential expression of non-coding RNAs, including miRNAs. Functional analyses illuminated enrichment of immune- and EMT-associated pathways in differentially expressed genes and non-coding RNAs between these subgroups. GSVA and GSEA analyses hinted at abnormal EMT status potentially contributing to glioma-associated immune disorders. The risk model, centered on OS-EMT-ICI genes, exhibited promise in accurately predicting survival in glioma. Additionally, a nomogram integrating the risk model with clinical characteristics demonstrated notable accuracy in prognostic predictions for glioma patients. In conclusion, OS-EMT-ICI gene and non-coding RNA expression emerges as a valuable indicator intricately linked to immune microenvironment dysregulation, offering a robust tool for precise prognosis prediction in glioma patients within the OBMRC framework.

Whole-genome sequencing and intensive analysis of the undomesticated soybean (Glycine soja Sieb. and Zucc.) genome.

The genome of soybean (Glycine max), a commercially important crop, has recently been sequenced and is one of six crop species to have been sequenced. Here we report the genome sequence of G. soja, the undomesticated ancestor of G. max (in particular, G. soja var. IT182932). The 48.8-Gb Illumina Genome Analyzer (Illumina-GA) short DNA reads were aligned to the G. max reference genome and a consensus was determined for G. soja. This consensus sequence spanned 915.4 Mb, representing a coverage of 97.65% of the G. max published genome sequence and an average mapping depth of 43-fold. The nucleotide sequence of the G. soja genome, which contains 2.5 Mb of substituted bases and 406 kb of small insertions/deletions relative to G. max, is ∼0.31% different from that of G. max. In addition to the mapped 915.4-Mb consensus sequence, 32.4 Mb of large deletions and 8.3 Mb of novel sequence contigs in the G. soja genome were also detected. Nucleotide variants of G. soja versus G. max confirmed by Roche Genome Sequencer FLX sequencing showed a 99.99% concordance in single-nucleotide polymorphism and a 98.82% agreement in insertion/deletion calls on Illumina-GA reads. Data presented in this study suggest that the G. soja/G. max complex may be at least 0.27 million y old, appearing before the relatively recent event of domestication (6,000∼9,000 y ago). This suggests that soybean domestication is complicated and that more in-depth study of population genetics is needed. In any case, genome comparison of domesticated and undomesticated forms of soybean can facilitate its improvement.

Vascular endothelial growth factor-165b protects the blood-retinal barrier from damage after acute high intraocular pressure in rats.

AIM: To elucidate the role of vascular endothelial growth factor-165b (VEGF-165b) in blood-retinal barrier (BRB) injury in the rat acute glaucoma model. METHODS: In this study, the rat acute high intraocular pressure (HIOP) model was established before and after intravitreous injection of anti-VEGF-165b antibody. The expression of VEGF-165b and zonula occludens-1 (ZO-1) in rat retina was detected by double immunofluorescence staining and Western blotting, and the breakdown of BRB was detected by Evans blue (EB) dye. RESULTS: The intact retina of rats expressed VEGF-165b and ZO-1 protein, which were mainly located in the retinal ganglion cell layer and the inner nuclear layer and were both co-expressed with vascular endothelial cell markers CD31. After acute HIOP, the expression of VEGF-165b was up-regulated; the expression of ZO-1 was down-regulated at 12h and then recovered at 3d; EB leakage increased, peaking at 12h. After intravitreous injection of anti-VEGF-165b antibody, the expression of VEGF-165b protein was no significantly changed; and the down-regulation of the expression of ZO-1 was more obvious; EB leakage became more serious, peaking at 3d. EB analysis also showed that EB leakage in the peripheral retina was greater than that in the central retina. CONCLUSION: The endogenous VEGF-165b protein may protect the BRB from acute HIOP by regulating the expression of ZO-1. The differential destruction of BRB after acute HIOP may be related to the selective loss of retinal ganglion cells.

[Effect of HBXIP on biological function and PI3K/Akt signaling pathway of adenoid cystic carcinoma cell line ACC-M].

PURPOSE: To study the effect of hepatitis B virus X protein binding protein (HBXIP) on proliferation, migration and invasion of adenoid cystic carcinoma cell line ACC-M, and the possible mechanism of PI3K/Akt signaling pathway. METHODS: HBXIP plasmid was transfected into ACC-M. The cells were divided into experimental group (transfected with plasmid pEGFP-N1-HBXIP) control group (non-transfected group) and blank control group (vector group, pEGFP-N1). RT-PCR was used to detect the expression HBXIP in ACC-M; MTT assay, transwell chamber experiments and scratches over the proliferation of HBXIP were utilized individually to evaluate the influence of HBXIP on ACC-M expression, migration and invasion; Western blotting was used to detect the protein expression of Akt, p-Akt, PI3K, p-PI3K and S100A4 after overexpression of HBXIP. Statistical analysis was performed using SPSS 18.0 software package. RESULTS: MTT results showed that the number of surviving cells of experimental group was significantly higher than the control group (P<0.05); Scratch test results showed that the cell mobility of the experimental group was significantly higher than the control group (P<0.01); Transwell chamber experiments showed that the number of cell invasion of the experimental group was significantly higher than the control group (P<0.01); Western blotting results showed that compared with the control group, the expression of p-Akt, p-PI3K and S100A4 in the experimental group with overexpressed HBXIP was relatively increased. CONCLUSIONS: Overexpression of HBXIP gene promotes ACC-M proliferation, invasion and migration. Further, ACC-M proliferation, invasion and migration may be promoted by increased Akt, PI3K phosphorylation and S100A4 protein expression.

[Expressions of discoidin domain receptor 1 and matrix metalloproteinase 2 in salivary gland adenoid cystic carcinoma and clinical significance].

PURPOSE: To investigate the expressions of discoidin domain receptor 1(DDR1) and matrix metalloproteinase 2(MMP2) in salivary gland adenoid cystic carcinoma (SACC) and discuss their significance and correlations with clinicopathological parameters. METHODS: SP immunohistochemical method was used to detect the expressions of DDR1 and MMP2 in 54 samples of SACC and 30 normal salivary gland samples. SPSS 13.0 software package was used for statistical analysis. RESULTS: The positive expression rate of DDR1 in SACC was 87.0%, which was significantly higher than that in normal salivary gland tissues (10.0%, P<0.01). The positive expression of DDR1 was related with neural invasion and lymph node metastasis (P<0.05). The positive expression rate of MMP2 was 68.5% in SACC, which was significantly higher than that in normal group(13.3%, P<0.01). The expression of DDR1 was positively correlated with MMP2 by Spearman rank analysis(r=0.332, P<0.05). CONCLUSIONS: High expressions of DDR1 and MMP2 correlate with carcinogenesis and progress of SACC. Supported by Research Fund of Science and Technology Project of Liaoning Province (2012225100).

Pure subdural haematoma caused by rupture of middle cerebral artery aneurysm: Case report and literature review.

Pure subdural haematoma (occurring without detectable subarachnoid haemorrhage) caused by intracranial aneurysm rupture is uncommon and is usually associated with delayed diagnosis and treatment. We describe the case of a 43-year-old man who presented with ongoing headache. Computed tomography and magnetic resonance imaging of the brain revealed subdural haematoma in the left fronto-temporo-parietal region, without subarachnoid haemorrhage. Digital subtraction angiography showed an aneurysm measuring ≤ 5 mm in diameter, arising from the distal region of the left middle cerebral artery. During hospitalization, an acute change in mental status accompanied by slurred speech and narcolepsy prompted an emergency CT scan. This revealed an enlargement of the subdural haematoma. The patient underwent an emergency craniotomy, during which a large amount of bloody fluid was evacuated, and the aneurysm was coagulated and resected. The patient had a good outcome without neurological deficit. The incidence, mechanisms and treatment of this condition are discussed.

[DNA repair gene polymorphisms in ERCC4 rs6498486 and ERCC5 rs751402 and risk of salivary gland tumors].

PURPOSE: To investigate the association between ERCC4 and ERCC5 polymorphisms and risk of salivary gland tumors. METHODS: Case-control study was carried out in 133 cases of histologically confirmed salivary gland tumors and 142 age and gender matched healthy control cases. Polymorphisms of ERCC4 rs6498486 and ERCC5 rs751402 were detected by PCR-RFLP. Multiple factors logistic regression analysis was conducted to investigate the association between gene polymorphisms and risk of salivary gland tumors using SPSS 18.0 software package. RESULTS: The genotype distribution of each polymorphism was found to be of Hardy-Weinberg equilibrium in the study. ERCC5 rs751402 TT genotype was associated with risk of salivary gland tumors (TT vs. CC+CT: OR=0.45, 95% CI: 0.21-0.98, P=0.046). No significant association was found in ERCC4 rs6498486. CONCLUSIONS: The results suggest that ERCC5 rs751402 polymorphism may be associated with risk of salivary gland tumors.

[Expression and significance of pericytes and TGF-ß in infantile parotid hemangioma].

PURPOSE: To study the expression and significance of pericytes and TGF-ß in infantile parotid hemangioma. METHODS: The expressions of pericytes and TGF-ß at protein level were examined in 76 cases of infant parotid hemangioma by strep avidin-biotin complex (SABC) immunohistochemical technique. All statistical analysis were performed using SPSS 13.0 statistical package. The relationship between the expression of pericytes and TGF-ß and clinical phase of hemangioma was analyzed by using Chi-square test. Kappa test was used to determine the relationship between pericytes and TGF-ß expression. RESULTS: The rates of positive expression of pericytes were 86.7%(13/15), 45.5%(10/22) and 51.3%(20/39) in early, middle and advanced stage of hemangioma, respectively. The rates of positive expression of TGF-ß were 33.3%(5/15), 40.9%(9/22) and 76.9%(30/39) in early, middle and advanced stage of hemangioma, respectively. There was significantly close correlation between the level of pericytes and clinical phase of hemangioma, as well as between TGF-ß expression and clinical phase of hemangioma(P<0.05). CONCLUSION: Pericytes and TGF-ß may significantly contribute to the proliferation of infantile parotid hemangioma.

[Expression of ERK and nm23-H1 in tongue squamous cell carcinoma and its relation with invasion and metastasis].

PURPOSE: To reveal the relations between expression of extracellular signal-regulated kinase (ERK) and nm23-H1 and tumor invasion and metastasis in tongue squamous cell carcinoma (TSCC). METHODS: The expression of ERK and nm23-H1 at protein level was examined in 74 cases of TSCC by strep avidin-biotin complex (SABC) immunohistochemical technique. SPSS10.0 software package was used for data analysis. RESULTS: The expression of ERK was positively related to TNM clinical stage and cervical lymph node metastasis. The expression of nm23-H1 was negatively related to TNM clinical stage and cervical lymph node metastasis. The expression of ERK had a negative correlation with the expression of nm23-H1. TSCC cases with ERK(+)/nm23-H1(-) revealed significant higher tendency to cervical lymph node metastasis than those with ERK(-)/nm23-H1(+). CONCLUSION: The expression of ERK and nm23-H1 is correlated with invasion and cervical lymph node metastasis in TSCC.

[Experimental study on osteoinduction active material in repairing the peri-implant bone defect].

PURPOSE: To explore the potential of using osteoinduction active material (OAM) for reconstruction of bone defects of peri-implant by animal experiment, and provide experimental evidence for clinical application. METHODS: Four Beagle dogs underwent extraction of the mandibular first and second premolar to create edentulous regions. 2 groups were divided randomly. Three months later, titanium implants were inserted and peri-implant bone defect models were established. The bone defects were repaired with OAM in the experimental group and repaired with tricalcium phosphate (TCP) in the control group. Two animals each were sacrificed at the end of 8 weeks and 16 weeks respectively. The specimens were evaluated with histological examination, scanning electric microscope, bone density examination, and energy disperse analysis of Ca(2+). The data was analyzed with SPSS13.0 software package for Student's t test. RESULTS: At 8-week,some parts of implants in the experimental group were directly contacted with new bone. There were fibers between the bone and implant in the control group. There was no significant difference between the experimental group and the control group in bone density (P>0.05). The percentage of Ca(2+) of the experimental group(22.16+/-3.33) was significantly greater than that of the control group(3.13+/-2.44) (P<0.05). At 16-week, there was good osseointegration between the bone and implant in the experimental group but no new bone formation in the control group. The bone density of the experimental group was significantly greater than that of the control group (P<0.05). The percentage of Ca(2+) of the experimental group(42.23+/-6.20) was significantly greater than that of the control group(10.40+/-3.12)(P<0.05). CONCLUSION: The results suggest that OAM can effectively accelerate the reconstruction of peri-implant bone defects and improve the osseointegration in the interface between the implant and bone.

[Repairing of peri-implant bone defect with platelet-rich plasma and platelet-rich plasma/ osteoinduction active material composite: an experimental study in dogs].

PURPOSE: To investigate the effect of platelet-rich plasma (PRP) and osteoinduction active material(OAM) compounded with PRP in the repair of bone defects of peri-implant and evaluate the feasibility of PRP as a material for repair of peri-implant bone defects. METHODS: Four Beagle dogs underwent extraction of the mandibular first, second and fourth premolars to create edentulous regions. Three months later, titanium implants were inserted and bone defects around implants were created. The bone defects were repaired with PRP/OAM compounds in experimental group A, repaired with PRP/ tricalcium phosphate in experimental group B, and repaired with tricalcium phosphate in the control group. The animals were sacrificed at the end of 8-week and 16-week, respectively. The specimens were observed with histological examination and energy disperse analysis of Ca(2+). The data were analyzed with SPSS13.0 software package for one-way ANOVA. RESULTS: At 8-week, in experimental group A, some parts of implants were directly contacted with new bone. Some new bones formed in the experimental group B. There was fibrous connection between the bone and implant in the control group. At 16-week, there was mature Haversian system in the experimental group A. There was good osseointegration between the bone and implant in the experimental groups but only was fibrous connection in the control group. At 8-and 16-week, there was significant difference between there groups in the percentage of Ca(2+) (P<0.05). CONCLUSIONS: PRP and PRP/OAM composite can effectively accelerate the regeneration of peri-implant bone defects and improve the osseointegration in the interface between the implant and bone.

[Effect of implant junction on bone growth by X-ray analysis].

OBJECTIVE: To explore the effects of mandibular implant junction on the growth of immature bone. METHODS: Eight Beagle dogs were randomly divided into three groups: control, unjunction and junction. Twelve implants were produced on the mandible of unjunction experimental group and junction experimental group. At three months after implanting, radiographic examination was performed. RESULTS: Three months after implanting, all implants were integrated with bone. None implants was mobile or got off. Radiographic examination demonstrated that the bone lose difference was no significant in junction and unjunction group. CONCLUSION: Mandibular implant connection wasn't effect on the growth of bone.

[Effect of implantation and implant connection on craniofacial bone growth in Beagle dogs].

PURPOSE: To study the effects of implantation and implant connection on Beagle craniofacial bone growth. METHODS: Eight Beagle dogs were randomly divided into two groups: unconnection group (4 dogs) and connection group (4 dogs). Two implant were implanted respectively in the lateral maxilla and zygomatic bone in each dog, the contra lateral side was used as normal control. Two implant was connected in the connection group 1 month later. Student's t test was used to analyze the data using SPSS11.5 software package. RESULTS: In the unconnection group, the distance increased between implants along with craniofacial bone growth; In the connection group, the distance fixed between implants along with craniofacial bone growth. The craniofacial values revealed no significant differences in the two groups (P > 0.05). CONCLUSION: The effect of implantation is localized and no inhibition in development of craniofacial bone is noted.

[The study on expression of bFGF and quantity of mast cell in infant hemangiomas of grandulae parotid gland].

The qualitative and quantitative study on mast cells and bFGF in 40 cases of infant hemangiomas of the grandulae parotid and 5 cases of normal grandulae parotid tissues were investigated by special mast cell staining methods, immunohistochemistry and image analysis technique. The quantity, area and average density of MC remarkably increased, there were significant differences. The mast cells mainly distributed around premature blood vessels and in parotid gland leaves; bFGF was highly expressed in infant hemangiomas of grandulae parotid. Its immuno-reactivity was present in the cytoplasm of endotheliocytes,mainly in extracellular matrix, distributed in light reticulation. It is concluded that there was a close relationship between bFGF and mast cell in proliferation of infant hemangiomas of grandulae parotid.