Intermedin Enlarges the Vascular Lumen by Inducing the Quiescent Endothelial Cell Proliferation.

OBJECTIVE: Intermedin plays an important role in vascular remodeling and significantly improves blood perfusion, but the precise mechanism remains unclear. Herein, we aimed to define whether vascular lumen enlargement is responsible for the intermedin-increased blood perfusion and explore the underlying cellular and molecular mechanisms. APPROACH AND RESULTS: To study the role of intermedin, we generated the IMD-KO (Adm2-/-) mice using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system. Intermedin significantly promoted vascular lumen enlargement in vitro (fibrin beads assay) and in vivo (murine retinas), which contributed to the improved blood perfusion in both physiological (retinal) and pathological (tumor) angiogenic models. We designed experiments to calculate the endothelial cell (EC) size and found that the lumen enlargement is because of EC proliferation but not because of a change in cell shape. ECs that construct vessel walls are considered quiescent cells because they are in a state of contact inhibition and show reduced responsiveness to VEGF (vascular endothelial growth factor). Using immunoprecipitation, Western blot assay, and fluorescent microscopy, we found that intermedin induced the formation of a signaling complex containing CRLR (calcitonin receptor-like receptor)/ß-arr1 (ß-arrestin1)/Src in ECs and promoted it internalizing into cytoplasm in a clathrin-dependent manner to activate downstream ERK1/2 (extracellular signal-regulated kinase 1/2). Importantly, this effect was not abrogated by cell-cell contacts of ECs. Through this mechanism, intermedin could reactivate the quiescent ECs to proliferate, resulting in continuous lumen expanding and a more effective blood perfusion. CONCLUSIONS: Our findings suggest a novel mechanism that may explain how quiescent ECs overcome the contact inhibition and regain the ability to proliferate for continuous vascular lumen enlargement.

MicroRNA Expression Profile on Solid Subtype of Invasive Lung Adenocarcinoma Reveals a Panel of Four miRNAs to Be Associated with Poor Prognosis in Chinese Patients.

According to the reclassification of lung adenocarcinoma (LAC) proposed in 2011, solid predominant lung adenocarcinoma (SPA) has been associated with poor outcomes for LAC patients. However, the prognostic value of the presence of solid subtype remains unclear. Besides, there is little data about the roles of microRNA (miRNA) in solid subtype of LAC. In this study, 243 LAC patients were classified into solid subtype positive and negative groups (S+ LAC, n=134 and S- LAC, n=109) according to whether the solid subtype was more than 5% of the tumor component or not. We analyzed the relationship between solid subtype and patients' outcome by univariate and multivariate analyses. Solid subtype was proved to be significantly associated with the 5-year overall survival and played as an independent prognostic factor for stage I-III invasive LAC patients. Then miRNA microarray was used to identify differentially expressed miRNAs in solid subtype, resulting in 31 differential miRNAs. Quantitative reverse transcription-PCR (QRT-PCR) was used to validate 4 key miRNAs (miR-133b, miR-155-5p, miR-124-3p, miR-145-5p). Further, CCK-8 and transwell assays were performed to validate the impact of one dysregulated miRNA (miR-133b) on LAC cell function. Interestingly, while miR-133b could significantly inhibit the proliferation of A549 and SPC-A1, it showed no effect on the migration or invasion of LAC cell lines. These results suggest that solid subtype can exert independent prognostic impact on LAC patients, and 4 important dysregulated miRNAs in solid subtype of LAC may be involved in the malignancy of S+LAC, thus may further have clinical perspective for S+ LAC in the future.

Identify the signature genes for diagnose of uveal melanoma by weight gene co-expression network analysis.

AIM: To identify and understand the relationship between co-expression pattern and clinic traits in uveal melanoma, weighted gene co-expression network analysis (WGCNA) is applied to investigate the gene expression levels and patient clinic features. Uveal melanoma is the most common primary eye tumor in adults. Although many studies have identified some important genes and pathways that were relevant to progress of uveal melanoma, the relationship between co-expression and clinic traits in systems level of uveal melanoma is unclear yet. We employ WGCNA to investigate the relationship underlying molecular and phenotype in this study. METHODS: Gene expression profile of uveal melanoma and patient clinic traits were collected from the Gene Expression Omnibus (GEO) database. The gene co-expression is calculated by WGCNA that is the R package software. The package is used to analyze the correlation between pairs of expression levels of genes. The function of the genes were annotated by gene ontology (GO). RESULTS: In this study, we identified four co-expression modules significantly correlated with clinic traits. Module blue positively correlated with radiotherapy treatment. Module purple positively correlates with tumor location (sclera) and negatively correlates with patient age. Module red positively correlates with sclera and negatively correlates with thickness of tumor. Module black positively correlates with the largest tumor diameter (LTD). Additionally, we identified the hug gene (top connectivity with other genes) in each module. The hub gene RPS15A, PTGDS, CD53 and MSI2 might play a vital role in progress of uveal melanoma. CONCLUSION: From WGCNA analysis and hub gene calculation, we identified RPS15A, PTGDS, CD53 and MSI2 might be target or diagnosis for uveal melanoma.

[Effect of ligand pioglitazone activating PPAR-gamma on the invasiveness of cholangiocarcinoma cell in vitro and the expression regulation of related genes].

OBJECTIVE: To explore the effect and mechanism of ligand pioglitazone (PGZ) activating PPAR-gamma on the invasiveness of cholangiocarcinoma cell in vitro. METHODS: Human hilar cholangiocarcinoma cell line QBC939 was selected and cultured in vitro for this research. The rate of QBC939 cell growth inhibition was detected by MTT, and the influence of PGZ on the expression of MMP-7 mRNA and TIMP-1 mRNA was measured by using FQ-PCR. The in vitro invasiveness and mobility of QBC939 were quantified by Matrigel invasion assay and crossing-river test. RESULTS: Among the low concentration (5 micromol/L-40 micromol/L) groups at the point of 12-hours, PGZ did not show to inhibit significantly the growth of QBC939 cells (P>0. 05). However, the PGZ could down-regulate the expression of MMP-7 mRNA in QBC939 cells (P<0. 01), and up-regulate the TIMP-1 mRNA expression to be without obvious statistics significance (P>0. 05). At last, PGZ could reduce the number of QBC939 cell breaking through the matrigel and prolonging the time of crossing-river significantly (P< 0. 01) in a dose-dependent manner. CONCLUSION: For ligand PGZ to activate PPAR-gamma can inhibit the invasiveness of QBC939 cells in vitro via regulating the expression of MMP-7 and the mobility of QBC939 cells probably.

[Expression of glucocorticoid receptor alpha and beta mRNA in orbital tissues in thyroid associated ophthalmopathy].

OBJECTIVE: To evaluate the expression of glucocorticoid receptor alpha (GRalpha) and beta (GRbeta) messenger RNA (mRNA) in orbital tissues from thyroid associated ophthalmopathy (TAO). METHODS: Samples of extraocular muscle and orbital fat were obtained from 17 patients with TAO and 10 healthy individuals. Total RNA was extracted and reversely transcripted into cDNA. The expression of GRalpha and GRbeta mRNA was detected by means of fluorescent quantitative polymerase chain reaction (PCR). RESULTS: Expression of GRalpha mRNA was much higher than GRbeta mRNA in all extraocular muscle and orbital fat biopsies. The relative copy of GRalpha was 40.15 +/- 11.37 in TAO patients and 20.64 +/- 7.07 in the controls. GRalpha: GRbeta mRNA ratio of these two groups was 77.76 +/- 18.77 and 148.34 +/- 23.86, respectively. There was significant difference between these two groups (P < 0.05). No significant difference was noted between extraocular muscle and orbital fat biopsies, between glucocorticoid-treated and non-treated patients or among hyperthyroidism, hypothyroidism and euthyroidism (P > 0.05). CONCLUSIONS: The increased expression of GRalpha mRNA and decreased GRalpha: GRbeta ratio in orbital tissues may play an important role in the pathogenesis of TAO and the effects of glucocorticoid treatment.

[Gene mutation analysis of coagulation factor VIII from a female patient with hemophilia A].

Hemophilia A affects male, whereas females are carriers and generally spared from this disease. However, we here reported a 65-year-old female with Hemophilia A while screening the gene mutation of coagulation factor VIII. The female went to hospital because of tripping to lead her right chest to be injured with subcutaneous hematoma. She had historically a hemorrhagic diathesis. The physical examination discovered her hip limited to bend and move, but no discrepancy length between her two legs. The initial laboratory tests showed that the activated partial thromboplastin time (APTT) was 61. 3 seconds (20-40 seconds), and the APTT corrected by mixing with normal plasma was 41.3 s, but the levels of PT, FIB and TT were normal. The plain radiographs revealed the hip joints to suffer from the acetabular dysplasia and osteoarthritis. The level of FVIII:C was 2%, F IX:C 200%, vWF:Ag 120%, vWF:Rcof 100%, vWF:CBA 128%, and the F VIII binding assay to vWF was normal. The primers for exon 14 of F VIII gene were designed according to the NM - 000132 gene sequence. DNA was abstracted from the patient blood. PCR were carried out and the DNA sequence was followed. A new mutation of 4111A-->C was discovered, which caused the amino acid sequence changed (T 1314 P). The mutation of T 1314 P may be the cause of this female patient to get the hemophilia A. This mutation was a novel one which has never been reported before.

Toll-like receptor 4 detected in exocrine pancreas and the change of expression in cerulein-induced pancreatitis.

OBJECTIVES: To detect Toll-like receptor 4 (TLR4) expression and distribution in rat pancreas and the change of TLR4 expression in cerulein-induced pancreatitis (CIP). METHODS: Acute pancreatitis was induced by subcutaneous injections of cerulein at a total dose of 20 microg/kg. Immunohistochemistry (IHC) was used to detect and localize TLR4 in rat pancreas, and real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to quantitatively determine the expression of TLR4 mRNA in CIP. RESULTS: IHC showed the presence of TLR4 in rat pancreas, and its distribution was specifically localized to pancreatic ductal epithelium, vascular endothelium, and islet. No TLR4 staining was detected in exocrine acinar cells. Real-time RT-PCR results revealed low-level TLR4 mRNA expression in the rat pancreas, and the change of TLR4 in CIP only developed within the first 4 hours, which is a rapid up-regulation process that peaks at the first hour. TLR4 mRNA was sustained at baseline level from 4 to 24 hours. CONCLUSIONS: TLR4 protein was expressed in pancreas and localized to epithelial (pancreatic duct) or endothelial (vessels) tissues; TLR4 responded favorably to the inflammatory process, and the change of expression was characterized as a rapid up-regulation in the early stage of CIP.

[Applying competitive polymerase chain reaction to the detection of hepatitis B virus DNA].

OBJECTIVE: To reduce the rate of accidental false negative result in the HBV DNA PCR test on clinical serum samples. METHODS: A competitive polymerase chain reaction (C-PCR) was used to decrease the false negative ratio. In the C-PCR, a constructed inner control DNA was added for co-amplification with the HBV target DNA. RESULTS: In a 20 microl C-PCR system, about 60 to 200 copies of inner control DNA could give apparent co-amplification signal band after electrophoresis on a 2% agarose gel. Five of 120 samples of clinical serum (4.2%) could not be amplified. CONCLUSION: C-PCR has the advantage of yielding information on false negative in the HBV DNA PCR assay of clinical serum samples.