miR-222 enhances HBx-HepG2 cell growth via regulation of BCL2L13 gene

AIM: To investigate the regulation of miR-222 on BCL2L13 gene and its effect on the growth and apoptosis of HBx-HepG2 cells, and to explore the underlying molecular mechanisms. METHODS: The expression level of miR-222 was detected by RT-qPCR. The HBx-HepG2 cell growth was examined by MTT and colony formation assays. The cell cycle and apoptosis were analyzed by flow cytometry. The recombination vector pmirGLO-BCL2L13 was constructed, and dual-luciferase reporter experiment was performed to validate the target of miR-222. RESULTS: The expression level of miR-222 in the HBx-HepG2 cells was significantly higher than that in the L02 cells (P<0.05). Over-expression of miR-222 enhanced HBx-HepG2 cell growth, changed cell cycle, and inhibited apoptosis (P<0.05). Knockdown of miR-222 reduced HBx-HepG2 cell growth, changed cell cycle, and increased cell apoptotic rate (P<0.05). BCL2L13 was down-regulated in the HBx-HepG2 cells as compared with L02 cells (P<0.05), and knockdown of miR-222 in the HBx-HepG2 cells increased the expression level of BCL2L13 (P<0.05). The results of dual-luciferase reporter assay and restore experiment showed that miR-222 negatively regulated the expression of BCL2L13 via targeting 3'UTR of BCL2L13, resulting in the promotion of HBx-HepG2 cell growth. CONCLUSION: miR-222 enhances HBx-HepG2 cell growth via down-regulation of BCL2L13.     

Anaesthesia of Atlantic halibut (Hippoglossus hippoglossus) Effect of pre‐anaesthetic sedation,and importance of body weight and water temperature

The efficacy of the anaesthetic agents benzocaine, metacaine (MS‐222), metomidate, 2‐phenoxyethanol, quinaldine and isoeugenol was studied in Atlantic halibut (Hippoglossus hippoglossus). Fish with an average body weight of 33 g were anaesthetized at 8 °C and fish with an average body weight of 1243 g were anaesthetized at 8 and 15 °C. Agents were tested individually and as combination anaesthesia comprising pre‐anaesthetic sedation, followed by anaesthesia. Induction and recovery times varied in relation to the body weight and water temperature. Large fish had longer induction times and shorter recovery times, and displayed reduced responsiveness to handling compared with small fish. A higher temperature resulted in shorter induction times, longer recovery times and increased responsiveness to handling. Lower dosages were used for all agents in combination anaesthesia. In small fish, this had no effect on the induction times but resulted in shorter recovery times and reduced responsiveness to handling. In large fish, combination anaesthesia resulted in shorter induction times whereas no uniform trend in recovery times and no differences in responsiveness to handling were observed. Neither individual agents nor combinations blocked all reflex reactions to external stimulation in all fish of any treatment group. MS‐222 and benzocaine, used separately or in combination anaesthesia, were the most effective agents in reducing reflex reactions.     

MS-222不同处理方式对史氏鲟和中华鲟麻醉效果的影响

MS-222的药液浸浴和药液鳃部喷洒可作为史氏鲟（Acipenser schrenckii）和中华鲟（Acipenser sinensis）的麻醉方式。在水温16～18℃的条件下,史氏鲟的浸浴有效浓度为90～110 mg/L,浸浴安全时间不超过30 min;中华鲟的浸浴有效浓度为80～100 mg/L,浸浴安全时间不超...     

Simultaneous effect of sex and dose on efficacy of clove oil,tricaine methanesulfonate, 2‐phenoxyethanol and propofol as anaesthetics in guppies,Poecilia reticulata (Peters)

We studied the simultaneous effect of sex and dose on anaesthesia efficacy to estimate, if possible, the lowest effective dose (LED) for clove oil, tricaine methanesulphonate (MS‐222), 2‐phenoxyethanol (2‐PE) and propofol in mature guppies. LED is the lowest dose needed to reach A5 stage in a mean time of 3 min, with mean recovery (R5) time of 5 min. We used four doses/anaesthetic: 25, 50, 75 and 100 mg/L for clove oil; 120,140,160 and 180 mg/L for MS‐222; 800, 1,000, 1,200 and 1,400 mg/L for 2‐PE, and 7.50, 8.25, 10.00 and 11.25 mg/L for propofol. Each dose was tested on 10 females and 10 males. Morbidity, mortality and behavioural changes were checked on two control groups (10 males and 10 females/group). Sedation (A3), A5 and R5 times were recorded. Significant interactive effect dose*sex on A5 time was found for each anaesthetic agent (p_dose*sex < .05). Except for MS‐222 (p_dose*sex = .284), significant interactive effect dose * sex on R5 time was found (p_dose*sex < .05). A5 time in females tended to be greater than in males, but, in general, R5 times were longer in males. Body size differences between males and females could explain these differences in MS‐222 on A5 time and for clove oil, 2‐PE and propofol on R5 time. No dose simultaneous meet LED′s conditions relating to both A5 and R5 times; therefore the lowest doses inducing A5 in a mean time of 3 min could be a safe guideline for anaesthetic procedure in both male and females.     

血浆miR-200c作为肺癌早期诊断的临床价值研究

目的 分析血浆miR-200c的表达与肺癌患者临床病理特征的相关性,并探讨miR-200c能否作为肺癌早期诊断的潜在肿瘤标志物的可能性。方法 收集55例肺癌患者和53例健康体检者,采用实时荧光定量PCR检测受检者血浆miR-200c的表达情况,对检测结果进行统计学分析。结果 miR-200c在肺癌患者组血浆中表达水平均高于正常对照组,差异有统计学意义（P<0.01）。受试者工作特征曲线显示miR-200c能够将肺癌与正常对照组区分出来（AUC=0.676）。结论 血浆miR-200c的表达可能与肺癌的发生有关,提示miR-200c可作为特异性生物标志物对肺癌患者进行早期诊断的可能性。     

miR-210-5p对南极独角雪冰鱼心脏发育的作用机制研究

microRNA为短链非编码RNA,通过与靶基因3'UTR序列互补在转录后水平发挥作用。已有研究表明,microRNA在心脏发育过程中起着重要的调控作用。南极冰鱼因体内缺乏功能性血红细胞,其心脏出现了补偿性增生。前期的研究提示,独角雪冰鱼心脏中特异表达的microRNAs可能与冰鱼心脏的补偿性增生相关。本研究针对南极冰鱼心脏中高表达的miR-210-5p,运用斑马鱼显微注射、靶基因预测等手段研究了miR-210-5p对心脏发育的作用机制。结果表明:斑马鱼胚胎注射miR-210-5p后,出现心包膜水肿,心脏发育畸形等现象。qRT-PCR分析显示,过表达miR-210-5p的斑马鱼胚胎中,心脏发育相关的标志性基因bmp4、smad1、gata6以及tbx2b的表达水平下调。Western blotting分析发现,bmp/smad通路中的BMP2、BMP4、SMAD1、GATA6以及TBX2B的蛋白表达水平也显著下调。通过对tbx20基因3'UTR的靶基因生物信息学预测,以及对其进行GFP荧光表达分析,发现tbx20基因可能是miR-210-5p的一个靶基因。由此推测,miR-210-5p可能通过抑制tbx20基因的表达,并调控bmp/smad通路以抑制冰鱼心脏发育。     

Silver catfish Rhamdia quelen immersion anaesthesia with essential oil of Aloysia triphylla (L'Hérit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status

Responses to anaesthesia with essential oil (EO) of Aloysia triphylla (135 and 180 mg L^?1) and tricaine methanesulfonate (MS222) (150 and 300 mg L^?1) were assessed in silver catfish. Exposure to the anaesthetics elicited a stress response in the species. In the case of MS222, it was displayed as a release of cortisol into bloodstream, elevation in hematocrit and plasma ion loss. The EO presented cortisol‐blocking properties, but increased haematocrit and disturbances of hydromineral balance were observed. Liver antioxidant/oxidant status of EO and MS222‐anaesthetized silver catfish was also estimated. The synthetic anaesthetic induced lipoperoxidation, notwithstanding increased catalase contents, whereas the naturally occurring product was capable of preventing the formation of lipid peroxides, possibly due to combined actions of catalase and glutathione‐S‐transferase. Anaesthetic efficacy was also tested via induction and recovery times. Overall, the promising results obtained for the physiological parameters of the EO‐treated fish counterbalanced the slight prolonged induction time observed for 180 mg L^?1. As for 135 mg L^?1, both induction and recovery times were lengthy; despite that, the EO was able to promote oxidative protection and mitigate stress. None of the MS222 concentrations prompted such responses concomitantly.     

斑点叉尾 ipu-miR-143靶基因EB1的鉴定

采用生物信息学方法对斑点叉尾 (Ictalurus punctatus)ipu-miR-143的靶基因进行预测, 并对预测到的ipu-miR-143靶基因进行生物学鉴定。生物信息学预测结果显示, 斑点叉尾 微管相关蛋白基因RP/EB家族EB1基因的3′-UTR区具有ipu-miR-143的潜在作用位点。结合对几种近缘物种中RP/EB家族EB1基因和miR-143的进化保守性进行分析, 推测ipu-miR-143在斑点叉尾 中可以通过靶向EB1基因的3′-UTR区而发挥其调控功能。因此, 本研究将斑点叉尾 EB1基因的3′-UTR区(含有miR-143靶位点)构建到pMIR-REPORTTM Luciferase载体的下游, 通过双荧光素酶报告基因检测系统对ipu-miR-143的靶基因进行鉴定。采用HEK293和CCK两种细胞进行细胞转染, 两种细胞中转染miR-143 mimics组荧光相对活性与对照组相比均表现为显著性降低(P<0.05)。共转染miR-143 inhibitors后, CCK细胞中荧光素酶相对活性(8.27±1.02)与对照组相比(5.44±1.55)显著上调(P<0.05); HEK293细胞中荧光素酶相对活性(6.30±1.19)与对照组(4.26±0.84)相比有所上升, 但无显著性差异(P>0.05)。初步研究结果提示, miR-143可以通过靶向EB1基因的3′-UTR区而发挥其调控功能。本研究旨为后续深入研究斑点叉尾 EB1基因的转录后调控机制提供基础依据。

     

miR-193a在MDBK细胞中诱导细胞凋亡的研究

本试验旨在研究miR-193a在致细胞病变型牛病毒性腹泻病毒(cp BVDV)感染MDBK细胞过程中诱导细胞凋亡的分子机制。试验利用TargetScan和Microcosm Targets等生物信息学在线软件预测凋亡相关的miR-193a靶基因Bax,并利用双荧光素酶报告基因系统验证miR-193a的靶基因;用过表达miR-193a的慢病毒pre-miR-193a-lv和抑制miR-193a表达的慢病毒pre-miR-193a-inhibitor-lv分别侵染MDBK细胞,48 h后收集细胞,然后用实时荧光定量PCR、Western blotting和流式细胞仪检测凋亡通路中Bax的表达水平及MDBK细胞凋亡率。结果预测并验证了miR-193a的靶基因为Bax;双荧光素酶报告基因分析结果显示miR-193a能直接结合到Bax 3'UTR区域中的miRNA反应位点,并极显著下调Bax的表达水平(P<0.01);流式细胞仪检测凋亡率结果显示,感染pre-miR-193a-lv慢病毒的MDBK细胞凋亡率极显著升高(P<0.01)。结果表明miR-193a能直接靶向凋亡通路中Bax基因,从而促进凋亡的发生。     

Anaesthetic efficacy of clove oil,benzocaine, 2‐phenoxyethanol and tricaine methanesulfonate in juvenile marbled spinefoot (Siganus rivulatus)

Anaesthetics are used in aquaculture and fisheries to facilitate routine procedures, such as capture, handling, transportation, tagging, grading and measurements that can often cause injury or induce physiological stress. Two experiments were performed to assess the efficacies of four anaesthetic agents, clove oil, benzocaine, 2‐phenoxyethanol and MS‐222 on juvenile marbled spinefoot rabbitfish (Siganus rivulatus). In the first experiment we tested the lowest effective doses that produced induction and recovery times in 3 min or less and 5 min or less respectively. Dosages were 70 mg L^?1 for clove oil, 60–70 mg L^?1 for benzocaine, 400 μL L^?1 for 2‐phenoxyethanol and 100–125 mg L^?1 for MS‐222. In the second experiment, we determined optimal concentrations of the four anaesthetics if they were to be used to transport rabbitfish fry. Anaesthetic concentrations suitable for handling and transport were: 10–15 mg L^?1 of MS‐222, 5–10 mg L^?1 of benzocaine, 5 mg L^?1 of clove oil and 50–100 μL L^?1 of 2‐phenoxyethanol. All anaesthetic agents are acceptable for use on S. rivulatus, however, 2‐phenoxyethanol, MS‐222 and clove oil appear to be more suitable than benzocaine. Further studies need to be conducted on effects of high and low doses of anaesthetic agents on physiology of marbled spinefoot.