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.     

丛枝菌根真菌和糖醇螯合钙对草莓采后果实硬度和细胞壁酶活及其关键基因表达的影响

以盆栽‘红颜’草莓为试材,研究了摩西管柄囊霉（Funneliformis mosseae）和糖醇螯合钙不同浓度（0.10%、0.15%、0.20%）处理对草莓采后果实硬度和细胞壁酶活的变化,同时,初步探究了不同处理对草莓果实软化关键基因FaPG1、FaβGal4的表达水平的影响。结果表明,与对照相比,以糖醇螯合钙单独施用（0.15%和0.20%）、接种丛枝菌根真菌（Arbuscular mycorrhizal fungi, AMF）配施糖醇螯合钙均能显著提高草莓果实硬度。同时,不同处理均抑制多聚半乳糖醛酸酶（PG）、果胶甲酯酶（PME）、β-半乳糖苷酶（β-Gal）活性的上升,草莓果实软化关键基因FaPG1和FaβGal4在处理后均下调表达。在接种AMF条件下,随着糖醇螯合钙浓度的增加,果实硬度逐渐增强,其中以接种AMF联合施用0.20%糖醇螯合钙溶液效果最好。     

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.     

Mechanism of elemene enhancing radiosensitivity of human glioma U251 cells

AIM To investigate the effect of elemene on the radiosensitivity of human glioma U251 cells and its mechanism. METHODS The U251 cells were used as a glioma model in vitro, and were exposed to different concentrations of elemene and different doses of radiation. The cell viability was measured by MTT assay, the apoptosis and cell cycle distribution were analyzed by flow cytometry, and the related protein levels were determined by Western blot. RESULTS Elemene inhibited the viability of U251 cells in vitro and enhanced the radiosensitivity of the cells. The cells in radiotherapy combined with elemene group had higher rates of early apoptosis, secondary necrosis and total cell death than those in radiation group. Elemene induced G₂/M phase arrest in the U251 cells. Elemene reduced the protein expression of cell division cycle protein 2 （Cdc2）, which resulted in the decrease in cyclin B1 expression induced by radiotherapy, thereby inhibiting the formation of cyclin B-Cdc2 complex. Elemene reduced Cdc2 activity by inhibiting the phosphorylation of Cdc2 protein at threonine 161, thereby inducing G₂/M phase arrest in the cells. It also mediated apoptosis by down-regulating survivin expression. CONCLUSION Elemene may increase the sensitivity of U251 cells to radiotherapy by down-regulating Cdc2 protein, decreasing cyclin B1 expression, inhibiting the formation of cylcin B-Cdc2 complex and down-regulating the expression of survivin.     

Molecular mechanisms of Belinostat inhibiting viability of osteosarcoma cells

AIM: To observe the effect of histone deacetylase inhibitor (HDACi) Belinostat on the viability of osteosarcoma cells and to study the underlying mechanism. METHODS: Osteosarcoma cell lines SAOS-2 and U2OS were incubated with Belinostat at different concentrations in vitro. The viability of the cells was measured by MTT assay. The activity of caspase-3/-7 and the DNA fragmentation were detected by fluorescence probe and ELISA, respectively. Western blot was used to detect the levels of histone acetylation, expression of PTEN, caspase-3, Bcl-xL and Akt, and phosphorylation of glycogen synthetase kinase 3β (GSK-3β) and Akt. Finally, the cells were incubated with Belinostat and doxorubicin at different concentrations, and then the combination index (CI) was calculated by MTT. RESULTS: Belinostat at 0.5, 1, 2.5 and 5 μmol/L inhibited the viability of U2OS cells and SAOS-2 cells in a dose-dependent manner, induced DNA fragmentation, enhanced caspase-3/-7 activity, and promoted the activation of caspase-3. At the same time, in the SAOS-2 cells, the expression of Bcl-xL was reduced, and the acetylation of histones H3 and H4 was increased. The results of Western blot showed that phosphorylation levels of Akt and GSK-3β in U2OS cells and SAOS-2 cells were decreased significantly after treatment with Belinostat (P<0.05). MTT results showed that combination of Belinostat and doxorubicin further reduced the viability of U2OS and SAOS-2 cells (CI<1). CONCLUSION: Belinostat inhibits the viability of osteosarcoma cells treated with doxorubicin, and the mechanism may be related to the inhibition of Akt signaling pathway.     

mTOR kinase inhibitor CC-223 suppresses human breast cancer cell viability

AIM: To investigate the inhibitory effect of CC-223, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on the viability of human breast cancer cells and its mechanism. METHODS: The inhibitory effect of CC-223 on the viability of MCF-7 cells and MDA-MB-231 cells was measured by CCK-8 assay. The cell cycle distribution of breast cancer cells was examined by flow cytometry. The expression of cell cycle-related proteins and oncoproteins c-Myc and survivin was analyzed by Western blot. RESULTS: CC-223 significantly inhibited the viability of both MCF-7 and MDA-MB-231 cells (P<0.05). CC-223 induced cell cycle arrest in both G₁ phase and G₂/M phase in the MCF-7 cells (P<0.05). However, low concentration of CC-223 treatment resulted in the accumulation of MDA-MB-231 cell cycle in the G₂/M phase, and the cell number in G₁ phase was unaffected. Treatment with CC-223 for 24 h clearly inhibited the protein levels of cyclin B1, cyclin D1 and phosphorylated cell division cycle protein 2 in the breast cancer cells (P<0.05). CC-223 suppressed the expression of c-Myc and survivin in both MCF-7 cells and MDA-MB-231 cells (P<0.05). CONCLUSION: CC-223 inhibits cell viability by blocking cell cycle progression and down-regulating expression of c-Myc and survivin in both MCF-7 and MDA-MB-231 cells.     

Expression and function of circular RNA_0000231 in non-small-cell lung cancer

AIM: To investigate the expression and function of circular RNA_0000231 (circ_0000231) in non-small-cell lung cancer (NSCLC). METHODS: RT-qPCR was used to detect the expression of circ_0000231 in the NSCLC tissues and cell lines. circ_0000231 small interfering RNA (si-circ_0000231) or negative control siRNA of circ_0000231 (NC) was transfected into the NSCLC cells. The proliferation and apoptosis of the NSCLC cells were detected by CCK-8 assay, colony formation assay and flow cytometry, respectively. The expression of cyclin D1 (CCND1) and anti-apoptotic protein Bcl-2 were determined by RT-qPCR and Western blot. RESULTS: The expression of circ_0000231 in the NSCLC tissues and cell lines was significantly up-regulated compared with precancerous tissues and lung epithelial cells BEAS-2B (P<0.05). After transfection of NSCLC cells with si-circ_0000231, the cell viability, colony formation numbers were significantly decreased, and the apoptotic rate in si-circ_0000231 group was significantly increased as compared with NC group (P<0.01). In addition, the results of RT-qPCR and Western blot showed that transfection of si-circ_0000231 inhibited the expression of CCND1 and Bcl-2 (P<0.01). CONCLUSION: The expression of circ_0000231 is significantly increased in the NSCLC tissues and cells. Knock-down of circ_0000231 expression significantly inhibits the proliferation of NSCLC cells.     

草鱼mst2在免疫应答中的作用机制

为了初步阐明草鱼哺乳动物STE20样蛋白激酶2基因(mammalian sterile 20-like kinase 2, mst2)在机体免疫中的作用机制,实验采用RNA-Seq技术对干扰mst2后经脂多糖(lipopolysaccharide,LPS)应激的草鱼肾细胞系(Ctenopharyngodon idella kidney cell lines,CIK)进行了转录组测序与验证分析。测序原始数据经De novo拼接与组装后共获得22 374个独立功能基因(unigenes),其中已知功能基因为21 199个,预测的新基因为1 175个。干扰mst2后经LPS应激的unigenes表达差异分析表明,对照组与实验组之间共存在38个差异基因(differentially expressed genes,DEGs),其中上调基因16个,下调基因22个。利用实时荧光定量PCR技术(quantitative real-time PCR, qRT-PCR)对38个DEGs的RNASeq结果进行验证,结果显示,qRT-PCR和RNA-Seq分析一致,说明RNA-Seq分析结果可靠。采用RNA干扰技术干扰mst2后经LPS处理,CIK细胞转录组中DEGs参与免疫代谢的途径主要有MAPK信号通路、内吞作用途径、自噬途径和细胞因子受体相互作用途径。凋亡相关基因检测结果显示,干扰mst2并经LPS处理后,促凋亡基因(fas、bad1、bad2、caspase-3、caspase-8和caspase-9)转录水平上调,抗凋亡基因(bcl2)转录水平下调,证明干扰mst2后经LPS处理会诱发细胞发生凋亡。综上所述,mst2可通过调控凋亡相关过程参与机体免疫反应。本研究结果初步阐明了草鱼mst2参与机体免疫应答的分子机理,可为草鱼细菌性疾病防控提供一定的基础理论参考。     

草鱼肾组织细胞系CIK的建立及其生物学特性

作者于1982年1月开始进行草鱼肾组织单层细胞培养，至今已连续培养32个月，传至120多代，建立了细胞系，定名为草鱼肾组织细胞系(CIK)。本文介绍了原代和传代培养、细胞形态、细胞生长速度和分裂指数、细胞的保存和对温度的适应性、细胞染色体分析以及细胞系对病毒敏感性的试验和研究结果。CIK生长迅速、适应性强、以20℃-38℃生长较好，28℃左右生长稳定，pH为6．5时仍能保持致密单层，染色体数为非整倍体，众数为55。对草鱼出血病左右病毒FRV具敏感性。     

显微注射技术在制备鱼类嵌合体和转基因海水鱼上的应用

以鱼类胚胎细胞和胚胎干细胞为核供体进行细胞移植构建鱼类嵌合体研究方面,现有的成功报道均采用显微注射方法;在转基因海水鱼类研究中,显微注射也是最为常用的技术,本实验室在花鲈胚胎干细胞嵌合体构建和外源基因向花鲈胚胎的转移研究中取得的结果也充分证实,显微注射技术是开展海水鱼类细胞移植和转基因研究的首选技术。