MiR-296-5p suppresses papillary thyroid carcinoma cell growth via targeting PLK1.

OBJECTIVE: Our study aimed to explore the effects of miRNA-296-5p on the biological behaviors of papillary thyroid carcinoma (PTC) cells and its potential mechanism. PATIENTS AND METHODS: Twenty-eight PTC tissues and the corresponding non-cancerous tissues were collected. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) analysis was performed to detect the expression levels of miR-296-5p in PTC tissues and the adjacent non-cancerous tissues. Besides, the different endogenous expression levels of miR-296-5p in PTC cell line (K1) and normal thyroid gland cell line (Nthy-ori3-1) were also detected by RT-qPCR. Bioinformatics analysis, Western blot and Dual-Luciferase reporter gene assay were performed to demonstrate whether polo-like kinase 1 (PLK-1) was a downstream target of miR-296-5p. Subsequently, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, flow cytometry analysis, colony formation assay and TUNEL assay were performed to estimate whether PLK1 down-regulation could attenuate the malignant behaviors of PTC cells in vitro. RESULTS: RT-qPCR results showed that the expression level of miR-296-5p was significantly down-regulated in PTC tissues and cells, indicating that miR-296-5p may participate in PTC development. We predicted target genes of miR-296-5p by bioinformatics and identified PLK1 as a target gene of miR-296-5p. By Western blot and Dual-Luciferase reporter gene assay, we confirmed that miR-296-5p was partially complement to PLKl mRNA 3'UTR sequence and inhibited PLK1 expression at the post-transcriptional level. In vitro experiments suggested that the transfection of miR-296-5p mimics into K1 cells suppressed cell proliferation, inhibited cell clone formation, arrest the cell cycle in G2/M phase and induced apoptosis. Importantly, PLK1 reversed the inhibitory effects of miR-296-5p on biological behaviors of PTC. CONCLUSIONS: MiR-296-5p influences the biological behaviors of PTC by regulating PLK1. These findings provide a new perspective for the molecular mechanism of PTC pathogenesis and also contribute to developing new targets and methods for PTC treatment.

[Impact of novel P2Y12 receptor inhibitors on platelet reactivity in acute coronary syndrome patients undergoing percutaneous coronary intervention].

OBJECTIVE: To investigate the impact of novel P2Y(12) receptor inhibitors including prasugrel or ticagrelor on platelet reactivity in patients with acute coronary syndrome (ACS) receiving percutaneous coronary intervention (PCI), and provide clinical data for novel oral P2Y(12) receptor inhibitors use among Chinese patients. METHODS: Between October 2011 to February 2014, 174 consecutive patients (135 males; (67.8±11.8) years old) with ACS undergoing PCI in Kiang Wu Hospital, Macau were prospectively enrolled in this study. Oral aspirin and one P2Y(12) receptor inhibitor were administered for 5 days or above after PCI, patients were divided into clopidogrel, prasugrel and ticagrelor groups in accordance with the agent administered. Platelet reactivity of the patients was detected by VerifyNow P2Y(12) reaction unit (PRU); and the high on-treatment platelet reactivity (HPR) and non-HPR were defined as PRU≥208 and PRU<208 respectively. Patients with HPR during clopidogrel therapy were switched either to prasugrel or ticagrelor, or continued the same treatment; and then the platelet reactivity was monitored again. RESULTS: There were 113 clopidogrel cases (64.9%), 20 prasugrel cases (11.5%) and 41 ticagrelor cases (23.6%). Fifty-seven cases (32.8%) were defined as HPR post P2Y(12) receptor inhibitor use, in which 55 cases (55/113, 48.7%) were treated with clopidogrel. The degree of inhibition of platelet reactivity was significantly different in patients on clopidogrel, prasugrel and ticagrelor therapy, percent inhibition assayed by the VerifyNow P2Y(12) system was 28.2%±23.5%, 61.4%±26.7% and 81.3%±19.8% respectively (P<0.05). Different degree of platelet reactivity was achieved by the 3 P2Y(12) receptor inhibitors at multiple time points. The among-group differences in platelet reactivity became apparent at the early treatment stage (P<0.05). Platelet aggregation decreased significantly in patients switched from clopidogrel to prasugrel or ticagrelor (P<0.05). CONCLUSION: Novel oral P2Y(12) receptor inhibitors are more effective in inhibiting platelet reactivity in ACS patients, and our results show that novel oral P2Y(12) receptor inhibitors provide a new option for ACS patients with HPR post clopidogrel or high-risk features of ischemic complications, including stent thrombosis and post-PCI ischemic events.

Pravastatin and C reactive protein modulate protease- activated receptor-1 expression in vitro blood platelets.

Protease-activated receptor-1 (PAR-1) plays an important role in mediating activation of human platelets by thrombin. However, mechanism of statin in ADP-induced platelet PAR-1 expression is also unknown. Aggregometry, flow cytometry, immunoblotting and ELISA were used to determine role of pravastatin participating in ADP-induced platelet activation and PAR-1 expression. ADP stimulation significantly increased PAR-1 expression on platelets. PAR-1 antagonist SCH-79797 inhibited platelet aggregation as well as decreased platelet P-selectin expression induced by ADP. CRP inhibited PAR-1 expression induced by ADP in a concentration-dependent manner. Pravastatin treatment reduced PAR-1 expression in a concentration-dependent manner. Combination treatment of CRP and Pravastatin significantly reduced platelet PAR-1 expression induced by ADP. By western-blot analysis, pravastatin treatment did not influence total PAR-1 after ADP treatment. CRP decreased platelet total PAR-1 expression induced by ADP. Pravastatin and CRP reduced TXB2 formation by ADP significantly. CRP decreased thrombin fragment F1+2 level with ADP treatment. Pravastatin, in contrast, did not influence F1+2 level. Upon treatment with Pravastatin reduced platelet LOX-1 expression induced by ADP. In conclusion, PAR-1 served as a critical mechanism to relay platelet activation process induced by ADP. CRP and pravastatin reduce PAR-1 expression in platelet by ADP pathway.

Serum levels of calcitonin gene-related peptide and substance P are decreased in patients with diabetes mellitus and coronary artery disease.

OBJECTIVE: This study evaluated serum levels of the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) in coronary artery disease (CAD) patients with and without a history of diabetes mellitus (DM). METHODS: Patients undergoing coronary angiography for suspected myocardial ischaemia were divided into four groups depending on their clinical status: control group (no CAD or DM; n=44), DM group (DM without CAD; n=46), CAD group (stable CAD without DM; n=44) and DM+CAD group (stable CAD with DM; n=50). Serum levels of CGRP and SP were determined using radioimmunoassays. RESULTS: CGRP and SP levels in the DM and CAD groups were significantly lower than in the control group. The lowest levels of CGRP and SP were observed in the DM+CAD group. There were no significant differences in CGRP and SP levels between the DM group and the CAD group. CONCLUSION: CGRP and SP may have a role in the pathogenesis of CAD in patients with diabetes.

Glucometabolic state of in-hospital patients with primary hypertension in sub-population of partial regions in China.

Abnormal glucose metabolism (AGM) is common but underestimated in patients with coronary heart disease (CHD). Here, we reported 898 in-hospital patients with primary hypertension (PH) at the university hospitals in developed regions of China. Oral glucose tolerance test (OGTT) was performed in those without known type-2 diabetes mellitus (2-DM). A total of 158 patients had known 2-DM and 32 were newly diagnosed as 2-DM by fasting blood glucose (FBG). OGTT revealed that 83 had 2-DM and 296 had impaired glucose tolerance (IGT). The proportion of 2-DM and AGM increased from 21.2 to 30.4% and from 57.5 to 68.7% upon OGTT. Prevalence of AGM and 2-DM increased with the increase of age, and incidence of AGM and 2-DM was significant higher in patients with risk factors (including CHD, overweight, hyperlipidaemia, proteinuria) than those without risk factors mentioned above. Glucose was not sufficiently controlled in 55.1% of the patients with 2-DM upon treatment, well controlled in 35.4% and not controlled in 9.5%. So AGM is also prevalent in PH patients especially the elders and those with risk factors, which was underestimated in most cases. Moreover, much lower awareness, treatment and control of 2-DM occurred in some regions of China, thus strengthening health education for patients and heightening consciousness of doctor are very important and eminent. Except for FBG, more attention should be paid to postprandial blood glucose ignored before, and OGTT should be a routine procedure in PH patients, especially in older patients and those with the factors mentioned above.

Prognostic value of dobutamine stress test in patients with septic shock.

AIM: To evaluate the prognostic effect of dobutamine stress test in patients with septic shock. METHODS: Patients with septic shock received intravenous infusion of dobutamine at 10 microg . kg-1 . min-1 for 1 h. Hemodynamics and oxygen metabolism was observed. A patient who was able to increase oxygen consumption index (VO2) by >15 % was designated as a responder to the test. RESULTS: In 47 patients with septic shock, twenty one responders and twenty six nonresponders were identified, and mortality was 33.3 % and 76.9 % respectively. After the dobutamine infusion, the responders showed increases in cardiac index (18.1 %), oxygen delivery index (12.7 %), VO2 (38.6 %), and oxygen extraction ratio (18.0 %), and reductions in systemic vascular resistance index (18.5 %). The nonresponders demonstrated increases in cardiac index (11.5 %), but no change in oxygen delivery, VO2, and oxygen extraction ratio. CONCLUSION: Dobutamine stress test might be a good predictor of outcome in patients with septic shock.

The allocation and differentiation of mouse primordial germ cells.

Analysis of the lineage potency of epiblast cells of the early-streak stage mouse embryo reveals that the developmental fate of the cells is determined by their position in the germ layer. Epiblast cells that are fated to become neuroectoderm can give rise to primordial germ cells (PGCs) and other types of somatic cells when they were transplanted to the proximal region of the epiblast. On the contrary, proximal epiblast cells transplanted to the distal region of the embryo do not form PGCs. Therefore, the germ line in the mouse is unlikely to be derived from a predetermined progenitor population, but may be specified as a result of tissue interactions that take place in the proximal epiblast of the mouse gastrula. The initial phase of the establishment of the PGC population requires, in addition to BMP activity emanating from the extraembryonic ectoderm, normal Lim1 and Hnf3beta activity in the germ layers. The entire PGC population is derived from a finite number of progenitor cells and there is no further cellular recruitment to the germ line after gastrulation. The XX PGCs undergo X-inactivation at the onset of migration from the gut endoderm and re-activate the silenced X-chromosome when they enter the urogenital ridge. Germ cells that are localised ectopically in extragonadal sites do not re-activate the X-chromosome, even when nearly all germ cells in the fetal ovary have restored full activity of both X-chromosomes. XXSxr germ cells can re-activate the X-chromosome in the sex-reversed testis, suggesting that the regulation of X-chromosome activity is independent of ovarian morphogenesis.

Effects of tripcholorolide on inflammatory reaction of mouse alveolar macrophages in vitro.

AIM: To observe the effects of tripcholorolide (T4) on inflammatory reaction of mouse alveolar macrophages. METHODS: RT-PCR was used to investigate tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), IL-6, IL-10, and inducible nitric-oxide synthase (iNOS) mRNA expression in alveolar macrophages after LPS 10 mg/L and T4 500 micrograms/L treatment. ELISA was used to detect TNF alpha, IL-1 beta, IL-6, and IL-10 protein expression. Nitrite was measured by Griess reaction. RESULTS: TNF alpha, IL-1 beta, IL-6, IL-10, and nitrite increased in supernatants, when alveolar macrophages were stimulated by LPS 10 mg/L at 24 h. Both T4 500 micrograms/L and dexamethasone 100 mumol/L had inhibitory effects on the production of TNF alpha, IL-1 beta, IL-6, IL-10, and nitric oxide. The mRNA expression of TNF alpha, IL-6, IL-10, and iNOS increased at 5 h after LPS stimulation which was decreased on addition of T4 500 micrograms/L or dexamethasone 100 mumol/L. T4 had no effect on stability of LPS-induced mRNA expression in TNF alpha, IL-6, and IL-10. CONCLUSION: T4 had inhibitory effects on the expression of proinflammatory and antiinflammatory mediators.

Expression of vascular endothelial growth factor during the early stage of maxillofacial blast injury.

OBJECTIVE: To determine the expression and the change of vascular endothelial growth factor (VEGF) and its acceptor KDR during the early stage of maxillofacial blast injury. To evaluate the effect of VEGF on traumatic wound healing. METHODS: The rabbit model of maxillofacial blast injury was made by KTY-04 blasting cap. The expression of VEGF and KDR in wound tissue was determined by ABC immunohistochemistry of 6 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after injury. The results were compared with preinjury groups. RESULTS: In the wound tissue of the maxillofacial blast injury, the expression of VEGF rose steadily during the first week after the injury. Compared with normal tissue, the wound tissue showed differences by the first day after injury, and showed significant differences by the third day (P < 0.01). The differences reached a peak at the seventh day after injury. The expression of KDR showed no difference within 3 days after injury when compared to normal tissue. Between 5 and 7 days after injury, the protein was more strongly expressed. CONCLUSION: The stage of the VEGF expression at the maxillofacial blast injury site is similar to the angiopoietic stage during would healing, and KDR expression also occurs during that period. VEGF takes part in angiogenic cascades of traumatic wound healing and is produced as an auxiliary action to the regeneration of blood vessels.

Sertoli cell differentiation and Y-chromosome activity: a developmental study of X-linked transgene activity in sex-reversed X/XSxra mouse embryos.

The requirement of Y-chromosome activity for the differentiation of somatic cells and germ cells was studied in the fetal gonads of X/XSxra mouse embryos where the activity of the Sxra fragment of the Y chromosome is influenced by the inactivation and reactivation of the X chromosome. In the interstitial somatic cells, random inactivation of the X and the XSxra chromosomes took place which was revealed by the mosaic expression of an X-linked lacZ transgene. The Sertoli cells, however, displayed a preferentially active XSxra chromosome and the presence of Sxra-active Sertoli cells was associated with the morphogenesis of testicular tubules in the sex-reversed gonads. The activity of the Y-chromosome fragment is therefore necessary for the differentiation of the Sertoli cells which may direct the development of the testis. The expression pattern of the X-linked transgene in X/XSxra germ cells suggests that both the X and the XSxra chromosomes are active. This finding suggests that the presence of Sxra has no impact on the reactivation of the X chromosome in the germ cells and that the X chromosome can be reactivated even though the germ cells are found in the testicular environment. Our results are consistent with the concept that the activity of genes on the XSxra fragment is essential for the differentiation of Sertoli cells and the morphogenesis of the testis, but not for premeiotic differentiation of germ cells in sex-reversed mice.

Effects of amiodarone on cardiac electrophysiology in right ventricular rapid pacing-induced heart failure dogs.

AIM: To study the effects of amiodarone (Ami) on cardiac electrophysiologic properties and ventricular fibrillation threshold (VFT) in right ventricular rapid pacing-induced congestive heart failure (CHF) dogs. METHODS: Dogs (n = 25) were randomly allocated into 3 groups: A) control group; B) CHF group induced by right ventricular rapid pacing (4 pulses.s-1) for 4-5 wk; C) CHF models p Ami 300 mg.d-1 for 4-5 wk. The electrophysiologic parameters and VFT were evaluated by electric stimulation and monophasic action potential (MAP) recording. RESULTS: In CHF models, ventricular MAP duration (MAPD90), ventricular late repolarization duration (VLRD), and intra-ventricular conduction time (IVCT) were prolonged by 43%, 318%, and 19%, respectively; the ratio of ventricular effective refractory period (VERP) to MAPD90 (VERP/ MAPD90) and VFT were decreased by 13% and 48% respectively; the dispersion of ventricular recovery time (RT-D) was increased by 185%. In CHF models, Ami had no effects on ventricular MAPD90, but increased VERP/ MAPD90, IVCT, and VFT by 15%, 10%, and 67%, respectively, shortened VLRD by 87%; and decreased RT-D by 87%. Ami had no significant influences on the hemodynamic parameters of the CHF dogs. CONCLUSION: Ami normalizes the cardiac electrophysiologic properties in CHF dogs.

X-chromosome inactivation during the development of the male urogenital ridge of the mouse.

In the mouse, the activity of Sry (sex-determining gene on the Y chromosome) initiates the transformation of the indifferent gonad into a testis. In humans, a partial Xp21 duplication leads to the development of ovaries instead of testes in XY individuals. This observation indicates that sex determination might also be influenced by a gene-dosage compensation mechanism, in addition to a dominant action of the Sry gene. In female mammals, the regulation of X-linked gene dosage at early embryogenesis is achieved through the inactivation of one of the two X chromosomes. Here we have investigated the possibility that inactivation of the X chromosome may play a role in male sex determination. We have shown, using an X-linked lacZ transgenic mouse line, that loss of beta-galactosidase activity occurs in certain somatic cells of the developing male urogenital ridge. When changes associated with apoptosis of mesonephric tubules in the developing urogenital ridges are taken into account, expression of the Xist (X inactive specific transcript) gene correlates with X inactivation revealed by loss of beta-galactosidase activity in very early mesonephric tubule epithelial cells, gonadal interstitial mesenchymal cells and coelomic epithelial cells.

The allocation of epiblast cells to ectodermal and germ-line lineages is influenced by the position of the cells in the gastrulating mouse embryo.

The developmental potency of cells in the proximal and distal regions of the epiblast of pre- and early-primitive-streak-stage mouse embryos was assessed by their differentiation in the host embryo following orthotopic and heterotopic cell transplantation. Normally, cells in the distal epiblast differentiate predominantly into neuroectoderm and surface ectoderm. However, when they were transplanted to proximal regions of the epiblast, distal epiblast cells behaved like proximal epiblast cells: they colonised the extraembryonic mesoderm and other mesodermal tissues in the posterior region of the host embryo. In addition, about 3.7% of the transplanted distal epiblast cells differentiated into primordial germ cells. This proportion is comparable to the 3.9% of orthotopically transplanted proximal epiblast cells that became primordial germ cells. When proximal epiblast cells were transplanted heterotopically to distal sites, their descendants were generally absent from the extraembryonic mesoderm and the germ cell population of the host embryo. Like cells in the distal epiblast, they mostly colonised the neural plate and surface ectoderm. This plasticity of cell fate suggests that the epiblast cells are not irreversibly allocated to any specific lineages, including the germ line. The adoption of developmental fate that is typical of the cell population at the site of transplantation suggests that the specification of cell lineages is subject to certain site-specific influences in the epiblast. Allocation of cells to the ectodermal and germ cell lineages may be subject to local tissue interactions and the restriction of morphogenetic tissue movement of different epiblast cell populations during gastrulation.

[Prospective study on the treatment of facial palsy with dynamic free muscle transplantation].

In this prospective study, patients were randomly divided into 3 groups according to 3 different operation procedures used to correct facial palsy with microneurovascular anastomosed muscle transplantation: In group 1, 9 patients accepted cross face transplantation of segmental latissimus dorsi muscle flap with super-long neurovascular pedicle, through upper labial tunnel, anastomosed in normal face (one stage method); In group 2, 7 patients accepted cross face nerve graft and secondary free muscle transplantation by neurovascular anastomoses in paralyzed face (two stage method); In group 3, 5 patients, free muscle transplantation by microneurovascular anastomosis in paralyzed face. The appreciation for the results was carried out in 16 patients who had been followed-up more than 1 year. In terms of functional recovery, the success rate ranged in excellent and good reached 93.8%. There were not significant differences among groups. The patients of less than 40-year-old got better results and the correlation between operative effects and both cause and course of disease could not be found. It was considered that the nerve regeneration speed and quality in one stage method were better than sural nerve graft with small saphenous vein anastomosis, because physiological blood supply was kept well along the nerve pedicle and regenerating nerve fibers need not pass through two suture sites. Patients satisfied the results after the swelling was gone because one stage method had the advantages of face lift and selective neurotomy at the same time. The course of treatment was shortened significantly as well.

X-chromosome activity of the mouse primordial germ cells revealed by the expression of an X-linked lacZ transgene.

We have determined the timing of the inactivation and reactivation of the X chromosome in the mouse primordial germ cells (PGCs) by monitoring the expression of an X-linked HMG-lacZ reporter gene. PGCs were identified by their distinct alkaline phosphatase activity and they were first localised in the primitive streak and allantoic bud of the 7.5-day gastrulating embryo. Although inactivation of the transgene was found in some PGCs at these sites, at least 85% of the population were still expressing the lacZ gene. This suggests that, although X-inactivation has commenced during gastrulation, the majority of PGCs still possess two active X chromosomes. Transgene activity remained unchanged during the relocation of PGCs to the hindgut endoderm, but decreased abruptly when PGCs left the hindgut and migrated through the mesentery. X-inactivation was completed during the migration of PGCs, but not simultaneously for the whole population. The first wave of PGCs entering the genital ridge at 9.5 days did not immediately re-activate the silent transgene until about 24 hours later. Re-activation of the transgene took place in over 80% of PGCs entering the genital ridge at 10.5-13.5 days p.c., preceding the entry into meiosis. About 90% of the meiotic germ cells in the 14.5-15.5 day fetal ovary expressed the transgene. Similar profiles of transgene activity were observed in PGCs of embryos that have inherited the lacZ transgene from different parents, showing unequivocally that X-inactivation in the germ cell lineage is not related to parental legacy.(ABSTRACT TRUNCATED AT 250 WORDS)