反义miR-30a-5p抑制人脑胶质瘤细胞U87生长的体外实验研究

目的 前期发现miR-30a-5p在胶质瘤中表达明显上调,本实验探究敲低miR-30a-5p对人脑胶质瘤细胞U87细胞生物学特征的影响.方法 real-time PCR检测转染miR-30a-5p I(miR-30a-5p抑制物)后U87细胞的miR-30a-5p表达水平,流式细胞术、MTT、Transwell、Annexin V法检测细胞周期、生长、侵袭及凋亡的改变;Western blot检测相关蛋白.结果 real-time PCR结果显示miR-30a-5p I可有效降低U87细胞中miR-30a-5p表达,抑制细胞增殖活性,使细胞周期阻滞在G0/G1期,侵袭能力明显受抑,凋亡增加,促增殖蛋白PCNA、促细胞周期进展蛋白Cyclin D1及促侵袭蛋白MMP-9的表达明显下调,而抑制侵袭蛋白TIMP-1及凋亡相关蛋白p53表达明显上调.结论 敲低U87细胞的miR-30a-5p表达可抑制胶质瘤的增殖及侵袭,诱导凋亡;miR-30a-5p可成为人脑胶质瘤基因治疗的潜在候选靶点.

Abstract：

Objective Our previous study had shown that there was overexpression of miR- 30a- 5p in malignant glioma cell lines.In the present study, we aim to investigate the effect of knocking -down miR -30a -5p on the biological characteristics of U87 glioblastoma cells.Method The U87 cells were divided into three groups:control cells,cells transfected with scramble oligonucleotides and cells transfected with miR -30a -5p inhibitors(miR-30a-5p I).Oligonucleotides mediated by lipofectamine were transfected to U87 cells.Real -time PCR was conducted to detect the expression of miR- 30a -5p in transfected cells.The cell proliferation was determined by 3- (4,5- Dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide(MTT) assay and flow cytometry.The cell invasion was evaluated by Transwell assay and cell apoptosis was detected with Annexin V staining Moreover, the relevant molecules regulating proliferation, invasion, cell cycle progression and apoptosis were examined by Western blot analysis.Results The expression of miR - 30a - 5p in the cells transfected with miR - 30a - 5p I was significantly reduced.The cell proliferation activity of U87 cell was inhibited.The cell cycle was arrested in G0/G1 phase, cell invasive ability was attenuated and apoptotic cells were increased in cells transfected with miR -30a -5pI as compared to those of the cells transfected with scrambled siRNA and control cells.The expression of proliferating cell nuclear antigen (PCNA), matrix metallopeptidase 9 ( MMP - 9) and Cyclin D1 were downregulated while the tissue inhibitor of metalloproteinase1 (TIMP - 1 ) and p53 were upregulated.Conclusions Transfection of miR - 30a-5p I into glioma cells could inhibit the proliferation activity and invasive ability of U87 cell and induce cell apoptosis, miR -30a -5p is a potential target of gene therapy for glioma.     

RNA干扰技术阻断α-synuclein过表达诱导的细胞凋亡

BACKGROUND： Overexpression of α-synuclein can induce cell apoptosis. RNA interference （RNAi） may block specific gene function and cause gene silencing. OBJECTIVE： To construct a specific and effective RNAi plasmid for the α-synuclein gene and investigate if RNAi can block apoptosis in HEK293 cells, induced by overexpression of wild-type α-synuclein.
DESIGN, TIME AND SETTING： A contrast experiment based on genetically engineered cytobiology was performed at the State Key Lab of Medical Genetics of China, Xiangya Medical College of Central South University, between October 2004 and October 2008.
MATERIALS： HEK293 cells and pBSHH1 plasmid were provided by the State Key Lab of Medical Genetics of China; OligDNA sequence by Sagon Bioengineering Company, Shanghai; Lipofectamine 2000 by Invitrogen, USA; α-synuclein monoclonal antibody, Hoechst 33258, and MTT by Sigma, USA; Horseradish peroxidase-coupled goat anti-rat IgG by KPL, USA; FACSan flow cytometry by BD, USA.
METHODS： Four target sites were used to construct hairpin RNA pBSHH1 vectors - pSYNi-1, pSYNi-2, pSYNi-3 and pSYNi-4 - which were cloned in the pBSHH1 plasmid. HEK293 cells were transfected using Lipofectamine 2000. In addition, a non-transfect group and a negative plasmid transfect group were established. The cultured HEK293 cells were processed as follows： transfection of blank plasmid （blank control group）, transfection of α-synuclein-pEGFP and RNAi negative vector （negative control group）, and transfection of α-synuclein-pEGFP and pSYNi-1 （transfection group）. Cells in all groups were transfected with Lipofectamine 2000 for 48 hours.
MAIN OUTCOME MEASURES： Expression of α-synuclein mRNA and protein were detected by RT-PCR and Western blot. Cell morphology was observed under an inverted fluorescence microscope; cell viability was measured using MTT method; and cell apoptosis was determined with Annexin V-PE flow cytometry.
RESULTS： α-synuclein mRNA and protein expressions were significantly decreased in the pSYNi-1 group when compared with the non-transfect and negative plasmid transfect groups （P 〈 0.05）. The expressions were partially decreased in the pSYNi-2 group, but there was no significant difference in the pSYNi-3 and pSYNi-4 groups. Hoechst staining indicated that cell nuclei were enlarged in the negative control group, coloring was not uniform, and chromatin was accumulated and appeared spot-like. The nucleus coloring was uniform in the transfection group compared to negative control group. Cell viability in the negative control group was significantly lower than blank control group with cell apoptosis being significantly increased （P 〈 0.05）. In comparison with negative control group, cell viability was significantly increased in the transfection group and cell apoptosis was significantly decreased （P 〈 0.05）.
CONCLUSION： pSYNi-1 can inhibit α-synuclein gene expression and block apoptosis of HEK293 cells induced by overexpression of wild-type α-synuclein.     

HER-2/neu siRNA对人胶质瘤细胞增殖及相关基因表达的影响

目的 探讨HER-2/neu特异性小干扰核糖核酸(siRNA)对高表达HER-2/neu的人胶质瘤细胞系U251MG和T98G增殖的影响及其可能机制.方法 脂质体介导HER-2/neu siRNA转染体外常规培养的U251MG和T98G细胞,同时设脂质体为对照组.转染后3 d实时定量PCR和免疫印迹实验检测HER-2/neu mRNA和蛋白的表达;四甲基偶氮唑盐(MTT)比色法检测转染后3、4d细胞增殖率的变化;免疫印迹实验检测转染后3 d细胞蛋白激酶B(AKT)、磷酸化AKT、磷酸化叉头转录因子(FOXO1)、p27、Cyclin D1蛋白的表达.结果 与脂质体组比较,HER-2/neu siRNA组U251MG、T98G细胞转染后3 d HER-2/neu mRNA和蛋白的表达均下降,转染后3、4 d细胞增殖率均下降,转染后3 d细胞磷酸化AKT和磷酸化FOXO1水平降低、p27蛋白表达增多、Cyclin D1蛋白表达减少,差异均有统计学意义(P＜0.05).结论 HER-2/neu siRNA转染人胶质瘤细胞系U251MG和T98G后明显抑制细胞增殖,可能与抑制AKT/FOXO1信号通路,调控下游基因p27、Cyclin D1蛋白的表达有关.

Abstract：

Objective To investigate the effect of HER-2/neu siRNA on proliferation of human glioma cell lines U251MG and T98G which over-express HER-2/neu, and explore its mechanism.Methods Liposome-mediated HER-2/neu siRNA was transfected into human glioma cell lines U251MG and T98G;lipofectin group was established as controls. The mRNA and protein levels of HER-2/neu were detected by real-time PCR and Western blotting 3 d after the transfection. The proliferation of glioma cells was investigated using methyl thiazolyl tetrazolium (MTT) assay 3 and 4 d after the transfection. The effects of HER-2/neu siRNA on AKT/FOXO1 pathway and protein expression of p27 and Cyclin D1 were studied using Western blotting. Results HER-2/neu mRNA and protein expressions in the transfected U251MG cells were decreased to (28.833±4.174)% and (22.167±1.955)% while those in cells of the lipofectin group were (92.067±5.698)% and (96.100±1.682)%, respectively,with significant differences (P=0.000, 0.001). HER-2/neu mRNA and protein expressions of the transfected T98G cells were decreased to (28.067 ±6.165)% and (12.433 ±8.864)% while those in the untransfected cells were (96.000 ±5.110)% and (94.333 ±3.215)%, respectively, with significant differences (P=0.001, 0.008). Three d after the transfection, the rates of proliferation in the transfected T98G and U251MG cells were (58.467±5.561)% and (63.933±5.363)%, respectively;4 d after the transfection, the rates of proliferation in the transfected T98G and U251MG cells were (57.500±4.770)% and (60.167±3.253)%, respectively;an obvious decrease was noted as compared them with cells of the lipofectin group (P=0.020, 0.023, 0.021, 0.008). Cyclin Dl expression was decreased, while p27 protein expression was up-regulated in the transfected cells as compared with those in cells of the lipofectin group (P<0.05). Moreover, the levels of phosphorylated AKT and phosphorylated FOXO1 were decreased in the transfected cells as compared with those in cells of the lipofectin group (P<0.05).Conclusion The specific siRNA targeting HER-2/neu in human glioma cell lines U251MG and T98G could inhibit the cell proliferation, which might relate to the suppression of AKT/FOXO1 pathway and the regulation of expresion of thier downstream molecules such as p27 and Cyclin D1.     

Claudin-15 overexpression inhibits proliferation and promotes apoptosis of Schwann cells in vitro

Our previous experiments have discovered that Claudin-15 was up-regulated in Schwann cells of the distal nerve stumps of rat models of sciatic nerve injury.However,how Claudin-15 affects Schwann cell function is still unknown.This study aimed to identify the effects of Claudin-15 on proliferation and apoptosis of Schwann cells cultured in vitro and explore the underlying mechanisms.Primary Schwann cells were obtained from rats.Claudin-15 in Schwann cells was knocked down using siRNA(siRNA-1 group)compared with the negative control siRNA transfection group(negative control group).Claudin-15 in Schwann cells was overexpressed using pGV230-Claudin-15 plasmid(pGV230-Claudin-15 group).The pGV230 transfection group(pGV230 group)acted as the control of the pGV230-Claudin-15 group.Cell proliferation was analyzed with EdU assay.Cell apoptosis was analyzed with flow cytometric analysis.Cell migration was analyzed with Transwell inserts.The mRNA and protein expressions were analyzed with quantitative polymerase chain reaction assay and western blot assay.The results showed that compared with the negative control group,cell proliferation rate was up-regulated;p-AKT/AKT ratio,apoptotic rate,p-c-Jun/c-Jun ratio,mRNA expression of protein kinase C alpha,Bcl-2 and Bax were down-regulated;and mRNA expression of neurotrophins basic fibroblast growth factor and neurotrophin-3 were increased in the siRNA-1 group.No significant difference was found in cell migration between the negative control and siRNA-1 groups.Compared with the pGV230 group,the cell proliferation rate was down-regulated;apoptotic rate,p-c-Jun/c-Jun ratio and c-Fos protein expression increased;mRNA expression of protein kinase C alpha and Bax decreased;and mRNA expressions of neurotrophins basic fibroblast growth factor and neurotrophin-3 were up-regulated in the pGV230-Claudin-15 group.The above results demonstrated that overexpression of Claudin-15 inhibited Schwann cell proliferation and promoted Schwann cell apoptosis in vitro.Silencing of Claudin-15 had the reverse effect and provided neuroprotective effect.This study was approved by the Experimental Animal Ethics Committee of Jilin University of China(approval No.2016-nsfc001)on March 5,2016.     

反义miR-21通过RECK抑制胶质瘤细胞侵袭性生长的体内外研究

目的 探讨胶质瘤细胞LN229中RECK作为miR-21的调控靶点,在胶质瘤侵袭性生长中的作用.方法 将反义miR-21(AS-miR-21)寡核苷酸转染至人脑胶质瘤细胞LN229中.Real-time PCR检测LN229细胞中miR-21的表达量.荧光素酶实验检测miR-21对RECK的调控关系.Transwell实验评价LN229细胞侵袭能力的变化,应用Western blot检测细胞内MMP2/9和RECK蛋白水平的变化,ELISA实验检测培养基中活性MMP2/9的表达量,动物实验评价体内条件下肿瘤侵袭性的变化.结果 Real-time PCR显示转染组中miR-21的表达量与对照组相比下调60%.荧光素酶实验证明RECK是miR-21的靶点.Transwell实验证实胶质瘤细胞侵袭能力下降,Western blot和ELISA实验证实MMP2/9表达降低,动物实验及免疫荧光反映肿瘤侵袭性生长受抑制.结论 反义miR-21通过上调RECK的表达而抑制恶性胶质瘤细胞的侵袭性生长.

Abstract：

Objective To investigate the regulation of miR - 21 on invasion growth of human glioma cells by RECK.Method The human glioma LN229 cells were transfected with AS - miR - 21 or scrambled sequences by Lipofectamine2000.Real time PCR was conducted to detect the expression of miR-21.Luciferase experiment was performed to detect the relationship between miR-21 and RECK.The expression of RECK was evaluated by Western blot.The invasion ability was evaluated by transwell assay and subcutaneous models.Western Blot, ELISA and immunofluorescence were used to estimate the changes of MMP2/9.Results The expression of miR - 21 in LN229 cells decreased after transfection with AS-miR-21. It was proved that RECK was a direct target of miR -21 by luciferase experiment.Meanwhile, the high expression of RECK protein in AS - miR -21 group conformed its important function in this mechanism.Transwell assay demonstrated decreased invasion capability of LN229 cell lines transfected with AS- miR- 21.Western blot, ELISA, and immunofluorescence demonstrated the levels of MMP2/9 were down -regulated in AS -miR -21 group compared with control and scrambled group.Conclusions AS - miR -21 could depress the invasion of glioma cells owing to up - regulating the level of RECK which could inhibit MMP2/9 activities both in vitro and vivo.     

Expression of galectin-3 correlates with apoptosis in pituitary adenoma cells

Objective To investigate the expressions of Galectin-3 （Gal-3）, Bcl-2 and Bax in human pituitary adenomas, and to explore the interrelation among them. Methods RT-PCR and immunohistochemistry were applied to detect the mRNA and protein expressions of Gal-3, Bcl-2 and Bax in surgically excised human pituitary adenoma tissues, including invasive and non-invasive pituitary adenomas, and the correlation analysis was performed. Results The Gal-3 and Bcl-2 expressions in the invasive pituitary group were significantly higher than those in the non-invasive group, and the expression of Bax had no significant difference between the two groups. Pearson＇s correlation analyses showed that the Gal-3 expression was posi- tively correlated with Bcl-2, but was not correlated with Bax, which was inversely correlated with expression of Bcl-2. Conclusion Gal-3 may function through a cell death inhibition pathway involving Bcl-2 to enhance cell proliferation, which result in the invasive growth of pituitary adenoma. These results indicate that Gal-3 has an important role in pituitary tumor cell proliferation and may serves as a possible therapeutic target in treatment of pituitary tumors.     

沉默HIF-1α基因对U87细胞增殖、侵袭及转移能力的影响

目的 观察沉默低氧诱导因子1α(HIF-1a)基因后对胶质母细胞瘤U87细胞增殖、侵袭及转移能力的影响.方法 实验分3组,干扰组(转染表达HIF-1α-shRNA的质粒)、对照干扰组(转染阴性对照shRNA序列)及未处理组.干扰组利用前期构建的HIF-1α基因的短发夹RNA(shRNA)沉默HIF-1α基因,构建HIF-1α-shRNA慢病毒表达载体,在脂质体介导下转染人胶质母细胞瘤细胞U87.采用RT-PCR和Western blotting检测HIF-1α基因干扰效率,MTT法检测细胞生长增殖能力,迁移实验检测细胞的体外迁移能力,Transwell小室模型检测细胞的体外侵袭及转移能力.结果 RT-PCR和Western blotting实验证实,与对照干扰组及未处理组比较,干扰组细胞HIF-1αmRNA表达水平明显下降,蛋白条带明显减弱.MTT细胞增殖实验显示,干扰组细胞增殖水平较其他2组明显降低,差异有统计学意义(P<0.05).Transwell小室侵袭实验中,未处理组、对照干扰组、干扰组穿膜细胞数分别为(125.2±10.8)个、(118.3±8.3)个、(60.9±5.4)个,差异有统计学意义(P<0.05).结论 HIF-1α-shRNA能有效抑制U87细胞株HIF-1α mRNA及蛋白的表达,并能抑制U87细胞的增殖、侵袭及转移能力.

Abstract：

Objective To observe the influence of silencing hypoxia-inducible factor-1α(HIF-1α)gene on the proliferation, invasion and metastasis of glioblastoma U87 cells. Methods The samples were divided into 3 groups: blank group: samples without giving any treatments, control group: cells with empty shRNA vector, and experimental group: cells with HIF-1α-shRNA transfection complex. HIF-1α gene was silenced by shRNA constructed in early time; and HIF-1α-shRNA lentivirus vector was constructed in the experimental group, and then transfected into glioblastoma U87 cells with the mediation of liposome. The interference efficiency was detected by using RT-PCR and Western blotting, and cell proliferation was measured by MTT assay; cell migration in vitro was observed by migration test, and invasion and metastasis abilities were detected by Transwell booth model. Results As compared with those in cells of the control and blank groups, the mRNA and protein expressions of HIF-1α in cells of the experimental group were significantly decreased; MTT assay showed that the cell proliferation in the experimental group was significantly lower than that in the other 2 groups (P<0.05). The number of penetrating cells of the blank group, control group and experimental group in Transwell chamber invasion assay were (125.2±10.8), (118.3±8.3), (60.9±5.4), respectively, and significant differences were noted between each 2 groups (P<0.05). Conclusion The mRNA and protein levels of HIF-1α in U87 cells are efficiently depressed by HIF-1α-shRNA, and so are the proliferation, invasion and metastasis abilities of U87 cells.     

Ulinastatin suppresses endoplasmic reticulum stress and apoptosis in the hippocampus of rats with acute paraquat poisoning

Lung injury is the main manifestation of paraquat poisoning. Few studies have addressed brain damage after paraquat poisoning. Ulinastatin is a protease inhibitor that can effectively stabilize lysosomal membranes, prevent cell damage, and reduce the production of free radicals. This study assumed that ulinastatin would exert these effects on brain tissues that had been poisoned with paraquat. Rat models of paraquat poisoning were intraperitoneally injected with ulinastatin. Simultaneously, rats in the control group were administered normal saline. Hematoxylin-eosin staining showed that most hippocampal cells were contracted and nucleoli had disappeared in the paraquat group. Fewer cells in the hippocampus were concentrated and nucleoli had disappeared in the ulinastatin group. Western blot assay showed that expressions of GRP78 and cleaved-caspase-3 were significantly lower in the ulinastatin group than in the paraquat group. Immunohistochemical findings showed that CHOP immunoreactivity was significantly lower in the ulinastatin group than in the paraquat group. Terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling staining showed that the number of apoptotic cells was reduced in the paraquat and ulinastatin groups. These data confirmed that endoplasmic reticular stress can be induced by acute paraquat poisoning. Ulinastatin can effectively inhibit this stress as well as cell apoptosis, thereby exerting a neuroprotective effect.     

Effect of curcumin on nitric oxide synthase expression in Iipopolysaccharide-activated microglia cells and the anti-oxidative effect of curcumin

BACKGROUND: It has been demonstrated that curcumin can increase the activities of various anti-oxidase in blood and tissue, effectively eliminate various free radicals, reduce the production of peroxisome, and alleviate oxidative stress reaction. Whether it has the same effect on microglia? OBJECTIVE: To observe the effects of curcumin on the expressions of inducible nitric oxide synthase (iNOS), nuclear factor-κB (NF-κB), and superoxide dismutase (SOD) in microglial cell line BV stimulated by lipopolysaccharide (LPS). DESIGN: An observational comparative study. SETTING: Research Room of Biochemistry, Medical College of Nantong University. MATERIALS: Mice microglia cell line BV, iNOS and NF-κB reporter gene plasmids were presented by Dr. Bhat.NR. from the Medical University of South Carolina (USA). Curcumin was produced by the Xi'an Branch of China Chengdu Scholar Bio-Tech. Co.,Ltd.; LPS (E.Coli O26:B6), anti-mice iNOS monoclonal antibody, horseradish peroxidase labeled goat-anti-mice IgG were the products of Sigma Company (USA). METHODS: The experiments were carried out in the Research Room of Biochemistry, Medical College of Nantong University from May 2006 to April 2007. ① Detection of iNOS: The cells were seeded onto 24-well plate at the density of 1×105, After the cells had adhered to the cover glasses, the cells were grouped as negative control group (the primary antibody was replaced by phosphate buffered solution PBS); normal control group (the cells were normally cultured); LPS-treated group (the cells were treated with LPS for 24 hours); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours). The expressions of iNOS protein were detected with immunocytochemical staining. ② Determination of iNOS and NF-κB gene activities: According to the introduction of the kit for transfection, iNOS or NF-κB report gene plasmids were transiently transfected with LipofectamineTM2000 liposomes into the cells in the 24-well plate for 24 hours. The cells were divided into normal control group (the cells were normally cultured after transfected with report gene plasmids); blank plasmid group (the cells were normally cultured after transfected with blank plasmids); LPS-treated group (the cells were treated with LPS for 4 hours after transfected with report gene plasmids); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours after transfected with report gene plasmids). The content of luciferase in the cell lysis buffer was determined after cell lysis. ③ Determination of SOD activity: The cells were seeded into culture bottle at the density of 1×106, and the divided into four groups, including normal control group (the cells were normally cultured); LPS-treated group (the cells were treated with LPS for 24 hours); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours); vitamin C+LPS group (the cells were treated with vitamin C for 1 hour and LPS for 24 hours). The SOD activity was determined with xanthine oxidase and quantitative colorimetric assay. MAIN OUTCOME MEASURES: The expressions of iNOS protein, iNOS and NF-κB, and the activity of SOD were observed. RESULTS: ① Expression of iNOS protein in microglia: The expression of iNOS protein in the LPS-treated group was obviously higher than that in the negative control group (P < 0.01); Those in the curcumin+LPS group were significantly decreased as compared with that in the LPS-treated group (P < 0.01). ② Expressions of iNOS and NF-κB genes: The expressions of iNOS and NF-κB genes in the LPS-treated group were significantly higher than those in the normal control group (P < 0.01); Those in the curcumin+LPS group were significantly lower than those in the LPS-treated group (P < 0.01). ③ SOD activity: The activity of SOD in the LPS-treated group was significantly lower than those in the normal control group (P < 0.01). It in the curcumin+LPS group and vitamin C +LPS group was significantly higher than that in the LPS-treated group (P < 0.01). CONCLUSION: Curcumin could inhibit the expression of iNOS in the activated microglia, and it also has the abilities in eliminating free radicals and antagonizing lipid peroxidation.     

miR-27b影响β-catenin／Tcf-4通路调控U87胶质瘤细胞生物学行为

目的研究miR-27b在胶质瘤细胞系U87中的表达、功能及作用机制。方法在多个胶质瘤细胞系及胶质瘤标本中检测miR-27b的表达;利用反义miR-27b转染U87,下调rniR-27b的表达,应用流式细胞术和MTT法,评价细胞生长、增殖、凋亡及周期变化：采用荧光素酶法及Western印迹法检测miR-27b作用的初步机制及通路蛋白的变化。结果miR-27b在胶质瘤细胞系及胶质瘤标本中均呈高表达;转染反义miR-27b后,表达降低86％,MTT法显示转染反义miR-27b后细胞生长受到抑制且凋亡细胞增加13％;荧光素酶实验结果显示：miR-27b发挥作用可能与B—catenin／Tcf-4通路相关,Western印迹法显示通路相关蛋白STAT3、c-myc和CyclinD1在转染反义miR-27b后表达下降。结论miR-27b可促进人脑胶质瘤细胞生长、增殖,具有原癌基因的作用,其发挥作用可能与β-catenin／Tcf-4通路密切相关。     

miR-92b对神经胶质瘤细胞U251增殖及迁移侵袭的 影响

目的 探究miR-92b对神经胶质瘤细胞U251增殖及迁移侵袭的影响以及其可能机制.方法 利用miRNA芯片筛选出在神经胶质瘤细胞U251和人脑正常胶质细胞HEB中差异表达的miRNA;化学合成法制备miR-92b抑制剂,转染后用real-time PCR技术验证表达变化;CCK-8实验检测转染后细胞的增殖能力;划痕实验和Transewll实验检测转染后细胞的迁移和侵袭能力;Western blot检测细胞中胰岛素样生长因子结合蛋白3(IGFBP-3)、β-catenin和E-cadherin的表达;荧光酶素实验验证IGFBP-3是否为miR-92b的靶基因.结果 基因芯片结果显示miR-92b在神经胶质瘤细胞中表达水平高于人脑正常胶质细胞(P<0.05);CCK-8实验结果显示转染miR-92b抑制剂后,U251细胞增殖能力降低(P<0.05);划痕实验和Transwell实验结果显示转染miR-92b抑制剂后,U251细胞迁移和侵袭能力降低(P<0.05);Western blot结果显示抑制miR-92b后,IGFBP-3和E-cadherin蛋白表达增加(P<0.05),β-catenin表达减少(P<0.05);荧光素酶实验结果显示,miR-92b能直接靶向调控IGFBP-3.结论 miR-92b在神经胶质瘤中表达显著增加,其可能通过抑制IGFBP-3蛋白表达,从而促进肿瘤细胞的增殖及迁移侵袭.     

RNAi沉默Gli1基因对人胶质瘤细胞U251增殖与凋亡的影响

目的 研究RNA干扰技术(RNAi)沉默Hedgehog(Hh)信号转导途径中核心转录因子Gli1基因表达后对U251细胞增殖与凋亡以及下游因子Bcl-2、Bax、cycin D1表达的影响。 方法 合成、转染4对针对Gli1 mRNA不同位点序列的siRNA (58、59、60、61)至人胶质瘤U251细胞,RT-PCR检测细胞Gli1 mRNA的表达,筛选最有效抑制Gli1 mRNA表达的siRNA干扰片段(siRNA-Gli1);RT-PCR、Western blotting分别检测转染SiRNA-Gli后不同时间U251细胞Gli1mRNA和蛋白的表达,确定转染干扰的时间规律;实验分为3组：siRNA-Gli1组（转染筛选后siRNA-Gli1片断）、siRNA-NC组（转染阴性对照siRNA片断）、siRNA-N组（空白对照）。RT-PCR、Western blotting分别检测各组转染48 h后U251细胞cyclin D1、Bcl-2及Bax mRN和蛋白的表达,MTT、流式细胞术分别检测细胞增殖、凋亡及细胞周期的变化。 结果 干扰片断58、59、60、61、NC的转染效率均达69.2％;RT-PCR检测显示转染后48 h U251-60细胞无明显Gli1 mRNA表达,选择60作为最佳干扰片段siRNA-Gli1转染U251细胞,48h时无明显Gli1 mRNA和蛋白表达,确定48 h为转染干扰的最佳时间;转染48 h后与siRNA-N和siRNA-NC组相比,siRNA-Gli1组U251细胞Bcl-2、cyclin D1 mRNA和蛋白表达下调、Bax mRNA和蛋白上调,差异均有统计学意义(P＜0.05)。MTT检测显示24、48和72 h时siRNA-Gli1组细胞增殖抑制率均明显高于siRNA-NC和siRNA-N组,差异均有统计学意义(P＜0.05)。流式细胞术结果显示siRNA-Gli1组细胞凋亡率高于siRNA-NC和siRNA-N组,差异有统计学意义(P＜0.05),且siRNA-Gli1组G1/G0期细胞比例增加,S期细胞明显减少。 结论 针对Gli1基因设计合成的siRNA-Gli1能明显抑制人胶质瘤U251细胞生长并能诱导其凋亡,其机制可能与下调cyclin D1、Bcl-2的表达,上调Bax的表达有关。     

miR-137调控人脑胶质瘤细胞增殖侵袭能力的体内外研究

目的探讨miR-137调控人脑胶质瘤细胞的增殖侵袭性生长能力及其机制。方法采用实时PCR分析miR-137在不同品系胶质瘤细胞及不同级别胶质瘤样本中的表达;脂质体介导miR-137模拟物转染胶质瘤细胞,实时PCR检测转染后miR-137的表达;应用MTT法、流式细胞术评价细胞生长和增殖的生物学特征变化;划痕实验、transwell细胞体外迁移实验检测肿瘤细胞迁移、侵袭能力;动物实验评价体内条件下肿瘤生长能力变化;Western blot、免疫组织化学染色检测肿瘤细胞Ki-67、MMP9表达水平。结果实时PCR分析显示：miR-137在胶质瘤中低表达。miR-137模拟物转染LN229和U87细胞后,实时PCR显示：miR-137表达上调;MTT法及流式细胞术显示细胞生长受抑,出现G0/G1期阻滞;划痕实验及transwell实验证实：细胞迁移侵袭能力下降;进一步Western blot、免疫组织化学染色显示：增殖侵袭相关蛋白Ki-67、MMP9表达降低;动物实验反映：肿瘤细胞生长受抑制。结论 miR-137高表达可抑制胶质瘤细胞生长和侵袭能力,提示miR-137可作为基因治疗脑胶质瘤的候选靶点。     

miR-7沉默表皮生长因子受体对胶质瘤细胞周期的影响

目的 研究miR-7阻遏脑胶质瘤细胞增殖周期由G1期向S期转化的内在机制.方法 脂质体转染miR-7表达质粒进入人脑胶质瘤细胞株U251,原位杂交和RT-PCR方法检测外源miR-7基因的整合效果;流式细胞术检测细胞周期变化,Western blot方法检测EGFR、CyclinD1、CyclinE、CDK2、CDK4、CDK6、P16、P21和P27的蛋白表达;SPSS13.0软件进行统计学分析.结果 RT-PCR结果显示miR-7基因被成功整合入U251胶质瘤细胞,原位杂交结果示miR-7主要定位于细胞核和细胞质;转染后瘤细胞增殖速度减慢,且大部分阻滞于G1期,处于分裂期S期的细胞比例明显减少(P＜0.05);miR-7转染组EGFR、CyclinD1、CyclinE、CDK2、CDK4和CDK6蛋白表达水平均有不同程度下降(P＜0.05),其中EGFR、CyclinD1、CDK4和CDK6的降低程度更为显著(P＜0.01),P16水平显著升高(P＜0.01),P21和P27的表达没有明显变化(P＞0.05).结论 miR-7可能通过有效沉默癌基因EGFR的表达途径,抑制细胞周期G1期调节蛋白复合体CyclinD1/CDK4/6的活性,并接受P16的协同作用,共同阻遏胶质瘤由G1期向S期转化,进而抑制肿瘤增殖;miR-7有望成为一种新的胶质瘤治疗候选药物.     

MicroRNA-143-3p对缺糖缺氧的大鼠脑微血管内皮细胞的保护作用及机制

目的 探讨MicroRNA-143-3p(miR-143-3p)对缺糖缺氧的大鼠脑微血管内皮细胞的保护作用及机制。方法 建立大鼠脑微血管内皮细胞(rBMECs)缺糖缺氧(OGD)损伤模型,分为正常培养组和OGD组,qRT-PCR检测各组不同时间rBMECs中miR-143-3p的相对表达水平; 干扰rBMECs中miR-143-3p表达的实验将细胞分为正常培养组、OGD组、NC inhibitor+OGD组和miR-143-3p inhibitor+OGD组,qRT-PCR检测各组细胞中miR-143-3p相对表达水平; MTT、流式细胞术分别检测各组细胞的增殖、周期和凋亡情况。结果 与正常培养组比较,OGD组OGD处理2、4、6、8和10 h后rBMECs中miR-143-3p相对表达水平明显上调,且呈明显上升趋势; 转染miR-143-3p inhibitor能够显著降低OGD条件下rBMECs中miR-143-3p相对表达水平; 与正常培养组比较,OGD组细胞的增殖能力、周期转化能力显著降低,而细胞凋亡比例显著增加; 与NC inhibitor+OGD组比较,miR-143-3p inhibitor+OGD组细胞的增殖能力、周期转化能力显著增强,而细胞凋亡比例显著降低。结论 OGD处理显著抑制rBMECs的增殖、周期转化、促进凋亡; 干扰miR-143-3p表达对OGD条件下的rBMECs具有保护作用。     

miR-29c对脑胶质瘤U251细胞生物学活性的影响

目的 探讨miR-29c对脑胶质瘤U251细胞生物学活性的影响及可能机制.方法 体外培养胶质瘤U251细胞,实验组将miR-29c mimics转染至细胞,miR mimcs转染作为对照组.应用实时荧光定量PCR检测转染后miR-29c表达量的改变,MTT实验测定细胞增殖,流式细胞术检测细胞凋亡情况,体外侵袭实验分析miR-29c转染后细胞的体外侵袭能力,Western blot检测转染后转录因子YY1蛋白的表达.结果 荧光定量PCR结果显示:实验组miR-29c表达量为(106.65±15.51),较对照组显著上调(P＜0.01).与对照组比较,实验组U251细胞的增殖能力明显降低(P＜0.01),凋亡率增加(P＜0.05),体外侵袭能力明显减弱(P＜0.01),YY1蛋白表达下调(P＜0.01).结论 miR-29c可能通过调控YY1蛋白的表达抑制胶质瘤细胞的生物活性.     

miR-21调控人脑胶质瘤细胞侵袭能力的体外研究

目的 探讨miR-21调控人脑胶质瘤细胞侵袭能力的体外机制.方法 脂质体介导反义miR-21(AS-miR-21)转染体外常规培养的U251细胞,同时设无义寡脱氧核苷酸(ODN)组和对照组.测定荧光素酶活性验证3组细胞中miR-21的表达,Matrigel基质生长实验检测U251细胞的生长情况,Transwell细胞体外迁移实验检测U251细胞的迁移能力,Western blotting检测细胞侵袭相关蛋白局部粘着斑激酶(FAK)、基质金属蛋白酶9/2(MMP-9/2)、人基质金属蛋白酶抑制因子1(TIMP-1)、微管蛋白α(Tubulin-α)的表达,免疫荧光检测细胞Tubulin-α蛋白的形态. 结果 与对照组和无义ODN组比较,转染AS-miR-21组U251细胞miR-21表达水平降低,Matrigel基质生长实验显示转染AS-miR-21组U251细胞体外培养克隆平均直径较小,Transwell细胞体外迁移实验显示转染AS-miR-21组穿膜细胞数较少,Western blotting结果 显示转染AS-miR-21组U251细胞FAK、MMP-9/2表达较低,TIMP-1表达较高,差异均有统计学意义(P＜0.05).Tubulin-α的表达无明显变化,免疫荧光染色显示转染AS-miR-21组Tubulin-α蛋白形态扭曲. 结论 miR-21高表达可以促进U251胶质瘤细胞侵袭生长,提示miR-21可以作为基因治疗脑胶质瘤的候选靶点.     

miR-125b对胶质瘤细胞侵袭能力影响的体内研究

目的探讨体内miR-125b表达对人脑胶质瘤细胞侵袭力的影响及其可能的机制。方法培养原代胶质瘤细胞,分别转染miR-125b mimics和miR-125b inhibitor慢病毒载体,上调或下调细胞中miR-125b表达水平,种植乳鼠皮下,Transwell试验评价胶质瘤细胞侵袭能力的变化,Western Blot验证侵袭相关基质金属蛋白酶(MMP)-2、MMP-9及RECK和TIMP3蛋白的表达。结果转染miR-125b mimics能有效提高原代胶质瘤细胞中miR-125b的表达,miR-125b inhibitor有效降低miR-125b的表达;体内实验表明miR-125b mimics及inhibitor对胶质瘤侵袭能力无明显影响,不影响侵袭相关MMP-2、MMP-9及RECK、TIMP3蛋白的表达。结论体内miR-125b表达水平与原代胶质瘤细胞的侵袭能力无明显相关。