DZNep raises miR-200c expression to delay the invasion and migration of MGC-803 gastric carcinoma cells / 生理学报

The aim of the present study was to investigate the regulatory effects of histone methylation modifications on the expression of miR-200c, as well as invasion and migration of gastric carcinoma cells. Gastric carcinoma cell line, MGC-803, were treated by 2.5 μmol/L histone methyltransferase inhibitor, DZNep. The expression of miR-200c was detected by real-time quantitative PCR (qRT-PCR). The epithelial-mesenchymal transition (EMT) indicators (ZEB1/2 and E/N-cadherin), EZH2, EED, SUZ12 and H3K27me3 expressions were detected by Western blot. Cell migration and invasion abilities were detected by Transwell and scratch tests. The result showed that, compared with DMSO (control) group, DZNep significantly increased the expression of miR-200c to about 2.1 times, inhibited ZEB1, ZEB2, and N-cadherin expressions, and activated E-cadherin expression; Also, DZNep decreased the protein expressions of EZH2, EED, SUZ12 and H3K27me3; Moreover, DZNep could inhibit MGC-803 cell invasive and migrative abilities, as well as MMP9 expression. These results suggest DZNep raises miR-200c expression to delay the invasion and migration of gastric carcinoma cells, and the underlying mechanisms involve the regulations of EMT-related proteins and polycomb repressive complex 2.

Knock-down of IKKε inhibits proliferation and invasion of U251 glioma cells in vitro / 中华神经医学杂志

Objective To investigate the effect of IKKε knock-down on the biological characteristics of U251 glioblastoma cells. Methods IKKε small interfering RNA (IKKε siRNA) mediated by lipofectamine were transfected into U251 cells while cells transfected with scrambled siRNA and control cells were prepared.RT-PCR was employed to detect expressions of IKKε in the transfected cells.The cell proliferation was determined by MTT assay.Flow cytometry was used to monitor changes in cell cycle.Cell invasion was evaluated by Transwell assay.Moreover,the proliferating cell nuclear antigen (PCNA),cyclin D 1 and matrix metalloproteinase-9 (MMP9) that regulated proliferation,invasion and cycle progression of the transfected cells and the changes of NF-κB after transfection were examined by Western blotting. Results RT-PCR revealed that the proliferation of U251 cells was inhibited,the cell cycle was arrested in G0/G1 phase and the invasive activity was attenuated in cells transfected with IKKe siRNA,with significant differences as compared with the cells transfected with scrambled siRNA and control cells (P＜0.05).The expressions ofPCNA,MMP9 and cyclin D1 were down-regulated in the IKKε knock-down cells, as compared with the other 2 groups. In addtion, transposition of NF-κB reduced from the cytoplasm to the nucleus after transfection. Conclusion As IKKε plays a vital role in proliferation and invasion of glioma cells,it may serve as a potential target ofgene therapy for glioma.

Preliminary study on aberrant expression of miRNAs related to the formation of the distinction between pediatric and adult types of brainstem gliomas / 中华外科杂志

<p><b>OBJECTIVES</b>To study the different expression of miRNA between pediatric and adult types of brainstem gliomas, and to provide the target miRNAs for explore the mechanism and miRNA interference of the malignant progression of pediatric BSG.</p><p><b>METHODS</b>miRNA expression profiles in orthotopic models which could simulate the BSG heterogeneity were examined by microarray and analyzed to obtain the aberrantly expressed miRNAs. The two types of human BSG tissue were utilized to verify the microarray data by qRT-PCR and in situ hybridization for the putative causative miRNAs.</p><p><b>RESULTS</b>There were 216 miRNAs detected in both the pediatric BSG group and the adult BSG group, 39 miRNAs to be differential expressed in the pediatric BSG group versus adult group, including 10 up-regulated and 29 down-regulated. qRT-PCR and in situ hybridization indicated good consistency with that of the microarray method.</p><p><b>CONCLUSIONS</b>Aberrantly expressed miRNA may serve as putative causative involvement of malignant progression of pediatric BSG, thereby might be potentially novel targets for therapy.</p>

Inhibitory effects of knocking down microRNA-19a and microRNA-19b on glioma cell growth in vitro / 中华神经医学杂志

objective To investigate the effects of knocking down of miR-19a and miR-19b on the biological characteristics of SNB19 glioblastoma cells. Methods Oligonucleotides inhibitor of miR-19a and miR-19b (miR-19a inhibitor or miR-19b inhibitor) mediated by lipofectamine2000 were transfected to SNB19 cells to knock down miR-19a and miR-19b; control group (without transfection),group D (performing transfection with nonsense sequence) and group E (performing transfection with both miR-19a inhibitor and miR-19b inhibitor) were established. Real time PCR was conducted to detect the expressions ofmiR-19a and miR-19b in these groups after the transfection. The cell proliferation rate and cell cycle kinetics were detected by 3-(4, 5-Dime- -thylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively; the cell invasive ability was evaluated by Transwell assay.Results As compared with those in control group and group D, the expressions of miR-19a and miR-19b, proliferation activity and invasive ability of cells in the miR-19a/19b inhibitor transfected cells (group A/B) were significantly reduced (P＜0.05). The expressions of miR-19a and miR-19b and the proliferation activity and invasive ability of cells 2, 3, 4 and 5 d after the transfection in group E were significantly reduced as compared with those in group A/B (P＜0.05). Delayed cell cycle in group A/B and group E was noted as compared with that in control group and group D; and group E enjoyed more obviously delayed eell cycle than group A/B (P＜0.05). Conclusion MiR-19a and miR-19b might be oncomiRs, and may be candidate target miRNAs for gene therapy of glioma.

Suppressing effects of RNA interference targeting AKT1 and phosphatidylinositol 3-kinase p85 on the invasion ability of malignant glioma U251 cells in vitro / 中华神经医学杂志

Objective To investigate the suppressing effects of RNA interference (RNAi)targeting AKT1 and phosphatidylinositol 3-kinase p85 (PI3KP85) on the invasion ability of malignant glioma U251 cells in vitro.Methods Normal control group,negative control group (U251 cells being transfected by nonsense sequence of adenovirus) and gene treatment group were chosen in the experiment.A recombinant rctrovims expressing short interference RNA (siRNA) sequence targeting AKTi and PI3KP85 genes was established and transfected into the U251 cells in the gene treatment group.The silencing effect of RNAi on AKTI and PI3KP85 expressions was identified by real time PCR and Westem blotting,respectively.Western blotting was also employed to analyze the expression of some functional proteins.Enzyme-linked immuno sorbent assay (ELISA) was used to detect the changes of concentration of ectocytic matrix metalloproteinases 2 (MMP2) and MMPg.The invasion ability of U251 cells in the 3 groups was evaluated by scarification and Transwell assay.Results The expressions of AKT1 and PI3KP85 in U251 cells in the gene treatment group were dramatically down-regulated.As compared with the normal control and negative control groups,the gene treatment group showed significantly lower expression level of MMP2 and MMP9(P<0.05);meanwhile tissue inhibitor of matrix metalloproteinase-2(TIMP2)in the gene treatment group was significantly up-regulated.ELISA also indicated obvious changes of concentration of ectocytic MMP2 and MMP9.The scarification and Transwell assay showed that the invasion ability in the gene treatment group was significantly decreased as compared with that in the other 2 groups (P<0.05).Conclusion RNAi targeting AKT1 and PI3KP85 can significantly down-regulate the expressions of AKT1 and PI3KP85 and decrease the invasion ability of U251 cells in vitro.

The effect of silencing Dicer by small interference RNA on the biological characteristics of human glioma cells / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the effect of silencing Dicer by small interference RNA (siRNA) to suppress the global microRNA (miRNAs) expression on the biological characteristics of TJ905 glioblastoma cells.</p><p><b>METHODS</b>The silencing effect of RNA interference on Dicer expression was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunofluorescence staining. The cell proliferation rate and cell cycle kinetics were detected by MTT assay and flow cytometry respectively, and the cell invasive ability was evaluated by transwell assay.</p><p><b>RESULTS</b>The siRNA targeting Dicer suppressed the expression of Dicer in TJ905 cells. Meanwhile, the proliferation activity and invasive ability were significantly enhanced in cells transfected with Dicer siRNA compared to those cells transfected with scrambled siRNA and the control cells.</p><p><b>CONCLUSION</b>Suppression of Dicer expression renders the glioma cells harboring more aggressive phenotype. This preliminary finding suggests that global lower expression of miRNAs may play an oncogenic role.</p>

Inhibitory effect of knocking down microRNA-221 and microRNA-222 on glioma cell growth in vitro and in vivo / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the inhibitory effect of knocking down microRNA(miR)-221 and miR-222 on human glioma cell growth and its possible mechanism.</p><p><b>METHODS</b>miRNA-221/222 antisense oligonucleotides (antisense miR221/222) were transfected into human glioma U251 cells by lipofectamine. Northern blot analysis was conducted to detect the mRNA expression of miR-221/222 in the control and transfected cell groups. The proliferation activity of cells was determined by MTT assay. Cell invasion ability was examined by transwell assay, and cell cycle kinetics and apoptosis were detected with flow cytometry. The expression of relevant proteins was analyzed by Western blotting. The therapeutic efficacy of antisense miR221/222 on the growth of xenograft tumors in nude mice were also observed.</p><p><b>RESULTS</b>In the antisense miR-221/222-transfected cells, the expression of miR-221/222 was significantly reduced; the cell invasion ability was suppressed, cell cycle was blocked at G(0)/G(1) phase, and apoptotic cells were increased. The growth of xenograft tumors treated with antisense miR-221/222 was also inhibited. In antisense miR-221/222 treated tumor cells, the expression of bcl-2 was down-regulated while connexin43, p27, PUMA, caspase-3, PTEN, TIMP3 and Bax up-regulated, and p53 expression not changed.</p><p><b>CONCLUSION</b>There is a significant inhibitory effect of antisense miR-221/222 on the growth of human glioma U251 cells. miR-221/222 may be considered as a candidate target for gene therapy of human gliomas.</p>

Suppressive effect of knockdown of miR-21 expression on U87 human glionto growth in vivo / 中华神经医学杂志

Objective To study the suppressive effect of knockdown of miR-21 on the U87 human giioma xenograft growth and the possible mechanism. Methods Nude mice bearing U87 human glioblastoma subcutaneously were treated with miRNA-21 anfisense oligonucleotides(AS-miR-21)intratumomlly every 3 d until the observation peded ended.The tumor volume of the mice treated withAS-miR-21 was measured regularly as compared with that in the control untreated mice and in the mice treated with scramble oligonucelotides(ODN).Finally,the tumors were removed from nude mice for the examination.In-sire hybridization and real-time PCR were conducted to detect the miRNA expression of miR-21.The biological charaetedsties of the tumors were evaluated by HE and immunohistochemieal staining, and the cell apoptosis was detected by TUNEL method. Resulls During the observation period,the tumor growth was delayed and the final tumor volume of AS-miR-21 heated group was smaller than that in the control and scramble ODN treatedg roup(F=6-056,P=0.007).The expression of miRNA precursor was knocked down in As-miRNA treated tunlors compared with that in untreated or scramble ODN treated tumors.Histopathological examination exhibited the appearance of degraded malignancy.The expressions of PCNA and MMP-9 were down-regulated while Septin-7 and P21 were up-regulated and apoptotic index was increased significantly (F=141.021,P=000) as well.Conclusion The suppressive effect of anti-miR-21 ODNs on the growth of U87 human glioma xenogratts is significant and miR-21 Call be taken as a candidate for gene therapy ofhuman glioma.

Study on the anti-invasion effect of SEPT7 gene for U251MG glioma cell in vitro / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the anti-invasion effect of SEPT7 gene on U251MG glioma cells and its possible molecular mechanism.</p><p><b>METHODS</b>Recombinant adenovirus vector carrying SEPT7 gene (rAd5-SEPT7) was transduced to human glioma cell line U251MG, and empty adenovirus vector was used as control. Tumor invasion was examined by Transwell method and 3 D-Matrigel assay, and tumor cell migration by wound-healing method and 2 D-Matrigel assay. Three major molecular events associated with cell motility and migration, including changes of expression in MMP2, MMP9, MT1-MMP, TIMP1 and TIMP2, the alteration of integrin alpha(v)beta(3) expression, and the structural change of cytoskeleton protein, tubulin-alpha, in U251 cells transduced with rAd5-SEPT7 were studied by Western blotting, immunofluorescence and laser scanning confocal microscope, respectively.</p><p><b>RESULTS</b>The invasive and migratory capabilities of cells transduced with rAd5-SEPT7 were inhibited. The expression of extracellular matrix metalloproteinases MMP-2, MMP-9, MT1-MMP and integrin alpha(v)beta(3) was significantly decreased, while the expression of matrix metalloproteinase inhibitor TIMP1, TIMP2 was upregulated. Intracellular cytoskeleton protein-tubulin-alpha in U251 cells exhibited prominent morphological changes which including the appearance of distortion and aggregation resulting from redistribution of tubulin-alpha, and this feature of alteration was similar to the tubulin-alpha structure in normal non-tumor cells.</p><p><b>CONCLUSION</b>SEPT7 gene can inhibit the invasion and migration ability of U251 glioma cells. Its molecular mechanism may include that SEPT7 gene reverses the imbalanced state of MMPs/TIMPs, downregulates the expression of integrin alpha(v)beta(3) and alters the structure of tubulin-alpha of U251MG glioma cells. It is suggested that SEPT7 gene could be a good candidate for gene therapy of gliomas.</p>

A study on the chemical constituents from Polyporus ellissi / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents of Polyporus ellissi.</p><p><b>METHOD</b>Silica gel column chromatography was applied for the isolation and purification of the constituents. The structures were established by means of spectroscopic and chemical data.</p><p><b>RESULT</b>Six compounds were obtained and identified as cerebroside B (I), cerebroside D (II), ergosterol peroxide (III), 9(11)-dehydroergosterol peroxide (IV), mannitol (V) and palmitate-1-glycerol (VI).</p><p><b>CONCLUSION</b>Compounds (I) and (II) were isolated from the genus Polyporus for the first time.</p>