Effect of miR-19a on lipid catabolism in hepatocyte LO2

AIM: To observe the effect of microRNA-19a (miR-19a) on the lipid catabolism of hepatocyte LO2, and to explore the potential mechanism. METHODS: miR-19a was over-expressed or silenced by transfection of miR-19a mimics or miR-19a inhibitor into LO2 cells, then the mRNA level of miR-19a was detected by real-time PCR. The potential target of miR-19a was found by the method of bioinformatics through internet website. The effect of miR-19a on the 3' UTR of peroxisome proliferator-activated receptor α (PPARα) was measured by dual luciferase reporter assay, and the protein level of PPARα and its 2 major downstream rate-limiting enzymes involved in lipid catabolism, acyl-coenzyme a dehydrogenase (ACADM) and carnitine palmitoyltransferase 1A (CPT1A), were detected by Western blotting. Meanwhile, the effect of miR-19a on the generation of ketone body was measured by beta-hydroxybutyric acid (β-OHB) detection assay. RESULTS: The mRNA level of miR-19a was dramatically elevated by the transfection of miR-19a mimics, and sharply decreased by the transfection of miR-19a inhibitor (P<0.05). PPARα was found as a potential target of miR-19a, and dual luciferase reporter assay and Western blotting confirmed the regulatory effect of miR-19a on the expression of PPARα, with the protein level changes of ACADM and CPT1A. miR-19a mimics down-regulated, while miR-19a inhibitor up-regulated the concentration of β-OHB in LO2 cells (P<0.05). CONCLUSION: miR-19a regulates the lipid catabolism of hepatocytes by targeting the PPARα and its 2 downstream rate-limiting enzymes.     

MicroRNA-9-5p mediates sympathetic overactivity by targeting KCNN3 in paraventricular nucleus of rats with T2D

AIM To investigate whether microRNA-9-5p （miR-9-5p） mediates sympathetic overactivity by targeting KCNN3 （potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3） gene,which encoded small-conductance calcium-activated potassium channel 3 （SK3） protein, in paraventricular nucleus （PVN） of rats with type 2 diabetes mellitus （T2D）. METHODS A rat model of T2D was established by high-fat diet combined with intraperitoneal injection of 30 mg/kg streptozotocin. The levels of miR-9-5p and KCNN3 mRNA in PVN were detected by real-time PCR. The relationship between KCNN3 and miR-9-5p was predicted by TargetScan. Recombinant adeno-associated virus （rAAV）-miR-9-5p or KCNN3 were bilaterally microinjected into the PVN to observe the changes in plasma glucose levels and sympathetic drive indicators. The number of FosB and SK3 positive cells was measured by immunofluorescence staining. The protein expression of SK3 was determined by Western blot. The relationship between KCNN3 and miR-9-5p were confirmed by cell transfection and dual-luciferase reporter assay. RESULTS Compared with the rats in diabetes control （DC） group, the blood glucose, sympathetic drive indexes and the level of miR-9-5p in PVN were significantly increased, while the SK3 expression in PVN was obviously reduced in the diabetes mellitus （DM） rats. After microinjecion of rAAV-miR-9-5p in PVN, the sympathetic drive indexes, blood glucose, and the number of FosB-positive cells were increased significantly, but the SK3 protein expression was significantly reduced （P<0.05）. However, up-regulation of KCNN3 in PVN had the opposite effect. These responses were obviously enhanced in DM rats compared with DC rats. The results of cell transfection and dual-luciferase reporter assay demonstrated that miR-9-5p bound to the 3’-UTR of KCNN3 and inhibit its expression. CONCLUSION miR-9-5p was up-regulated in PVN of the rats with T2D, and it may mediate sympathoexcitation by targeting KCNN3.     

Effect of Linc00152 targeting miR-376c-3p on viability,apoptosis and radiosensitivity of cervical cancer cells

AIM: To investigate the effect of Linc00152 on the viability, apoptosis and radiosensitivity of cervical cancer cells. METHODS: RT-qPCR was used to detect the expression levels of Linc00152 and microRNA-376c-3p(miR-376c-3p) in human cervical cancer HeLa cells and SiHa cells, and normal cervical Ect1/E6E7 cells. The cervical cancer HeLa cells with low Linc00152 expression or miR-376c-3p over-expression were established. MTT assay, flow cytometry, colony formation assay and Western blot were used to determine the cell viability, apoptosis, radiosensitivity and related protein expression. The dual-luciferase reporter assay was used to verify the regulatory relationship between Linc00152 and miR-376c-3p in the HeLa cells. RESULTS: Compared with the Ect1/E6E7 cells, Linc00152 was up-regulated in the HeLa cells and SiHa cells, and miR-376c-3p was down-regulated (P < 0.05). Low expression of Linc00152 or over-expression of miR-376c-3p inhibited the viability of HeLa cells, induced apoptosis, enhanced the radiosensitivity, inhibited the protein expression of cyclin D and Bcl-2, and promoted the protein expression of P21 and Bax (P < 0.05). Linc00152 negatively regulated miR-376c-3p expression in the HeLa cells, and inhibition of miR-376c-3p expression reversed the effect of low expression of Linc00152 on HeLa cell viability, apoptosis and radiosensitivity. CONCLUSION: Linc00152 is highly expressed in the cervical cancer cells. Linc00152 affects the viability, apoptosis and radiosensitivity of HeLa cells by targeting miR-376c-3p, which is a potential diagnosis and treatment target for cervical cancer.     

Down-regulation of miR-153-3p attenuates H2O2-induced H9C2 cell injury by promoting Nrf2 expression

AIM To study the effect of microRNA-153-3p (miR-153-3p) knock-down on oxidative injury of H9C2 cells induced by H₂O₂ and its specific mechanism. METHODS The oxidative stress injury of H9C2 cell model was induced by H₂O₂, and then the cell viability and the expression of miR-153-3p were detected by MTT assay and RT-qPCR, respectively. The effects of miR-153-3p knock-down on the H9C2 cell injury under oxidative stress were studied by RNA interference technology. The targets of miR-153-3p were identified by Western blot and dual-luciferase reporter assay. RESULTS MTT assay showed that the viability of H9C2 cells was decreased with the increase in H₂O₂ concentration (P<0.05). The results of RT-qPCR showed that the expression of miR-153-3p was increased with the increase in H₂O₂ concentration (P<0.05). Knock-down of miR-153-3p increased the viability of H9C2 cells under oxidative stress, decreased the cell apoptosis and the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). The expression of nuclear factor E2-related factor 2（Nrf2） and antioxidant response element（ARE） activity were increased with the increase in H₂O₂ concentration (P<0.01). TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3p. The results of Western blot further showed that over-expression of miR-153-3p inhibited the expression of Nrf2 (P<0.01), while down-regulation of miR-153-3p increased the expression of Nrf2 (P<0.01). Dual interference with Nrf2 and miR-153-3p significantly reduced H9C2 cell viability, promoted the apoptosis, increased MDA content, and decreased SOD activity in the presence of H₂O₂ (P<0.01). CONCLUSION Inhibition of miR-153-3p expression attenuates the injury of H9C2 cells induced by H₂O₂ through up-regulating Nrf2/ARE signaling pathway.     

Resveratrol protects cortical neurons in infant rats with bacterial meningitis through miR-223-3p/NLRP3 pathway

AIM To investigate the protective effect of resveratrol (Res) on cortical neurons in rat bacterial meningitis (BM) model. METHODS Group B hemolytic Streptococcus was injected via the posterior cistern to establish a BM model. Resveratrol was administered intranasally and microRNA-223-3p (miR-223-3p) antagomir was administered by intracerebroventricular injection. HE staining was used to observe the pathological changes of the brain tissue. Loeffler scoring method was used to evaluate the neurobehavioral functions. TUNEL staining was used to detect neuronal apoptosis. The expression of interleukin-1β (IL-1β), IL-18, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba1) was detected by immunofluorescence staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3）, cleaved caspase-1, IL-1β and IL-18 were determined by Western blot. The expression level of miR-223-3p was detected by RT-qPCR. Online software TargetScan was used to search for the complementary nucleotide sequences between miR-223-3p and NLRP3 mRNA. RESULTS Compared with sham group, the thickness of meninges in BM model was increased, the neurological score was decreased (P<0.05), and the number of TUNEL positive neurons was increased significantly (P<0.05). Astrocytes and microglia were activated, the fluorescence intensity of IL-1β and IL-18 was increased (P<0.05), and the expression levels of NLRP3, cleaved caspase-1, IL-1β, IL-18 and miR-223-3p were increased (P<0.05). Compared with BM group, after treatment with resveratrol, the neurological score was increased (P<0.05), the number of TUNEL positive neurons was decreased significantly (P<0.05), and the inflammatory response of astrocytes and microglia was suppressed. The fluorescence intensity of IL-1β and IL-18 was decreased (P<0.05), the protein levels of NLRP3, cleaved caspase-1, IL-1β and IL-18 were decreased (P<0.05), and the expression level of miR-223-3p was increased (P<0.05). A nucleotide sequence in the 3'-UTR of NLRP3 mRNA might be targeted by miR-223-3p. In the brain of rat BM model, compared with antagomir control group, the expression of NLRP3 was increased in miR-223-3p antagomir group with resveratrol treatment (P<0.05). CONCLUSION Resveratrol may reduce the inflammatory death of cortical neurons in BM model of infant rats through miR-223-3p/NLRP3 pathway, thus playing a protective role for the neurons.     

MS-222对鳊鱼麻醉保活运输效果的研究

为研究MS-222对鳊鱼保活运输的影响,分析比较了在不同质量浓度MS-222(0、20、40、60、80、100 mg/L和120 mg/L)、不同水温(2、7、15、20℃)和不同鱼水质量比(2∶1、1∶1、1∶3、1∶5)下鳊鱼的存活率,从而确定及筛选最佳的保活运输条件,并检测和分析在保活运输试验中水质指标和鱼体生化指标的变化和影响。试验结果显示,当MS-222质量浓度为60 mg/L、水温7℃、鱼水比1∶3时,保活时间最长,鱼体存活率最高;随着保活时间的延长,水体指标中氨氮含量、微生物、pH均呈上升趋势,而溶解氧水平则呈下降趋势;在鱼体指标中,肌肉中糖原、pH下降,乳酸含量上升;在血液指标中乳酸脱氢酶、谷草转氨酶、血糖、尿素和肌酐均显著增加(P<0.05),说明随着保活时间的延长,鱼体肝脏和肾脏组织代谢水平受到一定的影响,但其变化幅度均小于对照组。研究结果表明,在鳊鱼保活过程中可适当使用MS-222麻醉剂以延长存活时间和提高存活率。     

鱼体组织中MS-222的高效液相色谱分析方法

建立了高效液相色谱（HPLC）分析鱼体组织中MS－222含量的方法。样品用重蒸水匀浆,离心（12000r/min)15min,取上清液过膜（0.45μm),用YWG－C18分析柱,流动相为0.2mol/L,磷酸缓冲液（pH值3.1):甲醇＝85：15,紫外检测波长223nm,此法回收率较高,重现性好。     

大白菜细胞质雄性不育的分子鉴定及序列分析

为了获得3种大白菜细胞质雄性不育系Ogu CMS,Pol CMS,CMS96和保持系间的多态性以及定位大白菜CMS96不育系所属的不育类型,利用设计的atp6,orf222单一和混合引物PCR扩增3组11份同核异质大白菜细胞质雄性不育系和保持系mtDNA。结果表明,atp6引物在大白菜Ogu CMS不育系扩增的200 bp片段为其特异带;orf222引物仅在大白菜Pol CMS和CMS96不育系有扩增产物,但二者有3点完全不同:大白菜Pol CMS不育系扩增产物为675bp,CMS96不育系扩增产物为669 bp,二者相差6个核苷酸,后者定名为大白菜CMS96-orf222。大白菜CMS96-orf222与甘蓝型油菜Nap CMS的nad5c基因和Nap-orf222基因同源性均为99%,E值为0.0;大白菜Pol CMS的675 bp序列具有ORF224开放阅读框,没有保守结构域,而大白菜CMS96的669 bp序列具有ORF222开放阅读框和保守结构域YMF19。另外,atp6和orf222混合引物多重PCR扩增产物存在明显多态性:800 bp为大白菜保持系的差异带型;2 300 bp和1 500 bp为大白菜Pol CMS不育系特异带型;200 bp为大白菜Ogu CMS不育系特异带型;690 bp为大白菜CMS96不育系特异带型。该方法仅用一次PCR反应快速地将3种大白菜细胞质雄性不育系和保持系一次性全部区分开,为大白菜分子育种和常规育种更好地相结合提供了简单、快速、准确和重复性好的方法和手段。     

miR-140-3p inhibits viability,invasion and migration of non-small-cell lung cancer A549 cells by targeting PD-L1

AIM: To explore the target relationship between microRNA-140-3p (miR-140-3p) and programmed cell death ligand 1 (PD-L1) and their effect on the viability, migration and invasion of non-small-cell lung cancer A549 cells.METHODS: RT-qPCR was used to detect the miR-140-3p expression in HLF-1, A549 and H1299 cells, and then the A549 cells with the most significant difference were selected as the subsequent research object. TargetScan software and dual-luciferase reporter assay were performed to predict and confirm the target relationship between miR-140-3p and PD-L1. RT-qPCR and Western blot were used to determine the effects of miR-140-3p mimic and inhibitor on PD-L1 expression level. MTT assay was used to detect the viability of A549 cells. Transwell assay was performed to detect the migration and invasion abilities of the A549 cells.RESULTS: miR-140-3p was significantly down-regulated in the A549 cells and H1299 cells (P<0.05). Transfection with miR-140-3p mimic decreased the expression of PD-L1 and inhibited the viability, migration and invasion of the A549 cells. Transfection with pcDNA3.0-PD-L1 reversed the inhibitory effect of miR-140-3p on the viability, migration and invasion of the A549 cells.CONCLUSION: miR-140-3p inhibits the viability, migration and invasion of A549 cells by targeting PD-L1.