الملخص
Objective To establish U251 cells with inhibited expression of interferon-γ inducible protein 30 (IFI30), and to investigate the effect of IFI30 on cell biological function as well as its underlying mechanism. Methods Three knockdown sequences which target IFI30 were designed online and 3 small interfering RNAs (siRNA) were synthesized. After transfection, the inhibition efficiency was detected by real-time quantitative PCR. The siRNA sequence with the highest inhibition efficiency was selected to create short hairpin RNA (shRNA) plasmids. The recombinant plasmids and packaging plasmids were co-transfected into HEK293T cells to prepare lentivirus. The glioma U251 cells were transfected with lentivirus, and the positive cells were screened by puromycin. CCK-8 assay, 5-ethyl-2'-deoxyuridine (EdU) and colony formation assays were used to analyze cell proliferation; the flow cytometry was used to analyze cell cycle and apoptosis; the TranswellTM assay was used to detect cell invasion; the wound-healing assay was employed to detect cell migration, and western blot analysis to detect the protein expresison of cyclin D1, B-cell lymphoma factor 2 (Bcl2), epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), signal transducer and activator of transcription 1 (STAT1). Results The sequence which effectively target IFI30 was screened and U251 cell line capable of inhibiting the IFI30 expression was successfully established. When IFI30 expression was knocked down, the proliferation of U251 cells was inhibited, along with increased ratio of cells in the phase G0/G1, the decreased phase S, the increased rate of cell apoptosis. The cell invasion and migration capabilities was also reduced. The decreased expression of cyclin D1, Bcl2 and N-cadherin were observed in U251 cells, and the expression of E-cadherin and the phosphorylation of STAT1 were found increased. Conclusion Knockdown of IFI30 inhibits the proliferation, invasion and migration of human glioma cell U251 and promotes its apoptosis by activating STAT1.
الموضوعات
Humans , Cyclin D1/genetics , HEK293 Cells , Interferon-gamma , RNA, Small Interfering , Apoptosis/genetics , Cadherins , Cell Proliferation/genetics , Glioma/genetics , Proto-Oncogene Proteins c-bcl-2 , Oxidoreductases Acting on Sulfur Group Donors , STAT1 Transcription Factor/geneticsالملخص
OBJECTIVE@#To investigate the growth-inhibitory and pro-apoptotic effects of piroctone olamine (PO) on glioma cells and explore the underlying mechanism.@*METHODS@#Human glioma cell lines U251 and U373 were treated with PO and the changes in cell proliferation were detected using CCK-8 assay and EdU assay. Clone formation assay and flow cytometry were used to examine the changes in clone formation ability and apoptosis of the treated cells. Mitochondrial membrane potential of the cells and morphological changes of the mitochondria were detected using JC-1 staining and a fluorescence probe, respectively. The expressions of mitochondrial fission protein DRP1 and the fusion protein OPA1 were determined with Western blotting. Transcriptome sequencing and differential gene enrichment analysis was performed, and the expression levels of PI3K, AKT and p-AKT in the treated cells were verified using Western blotting.@*RESULTS@#CCK-8 assay showed that PO significantly inhibited the proliferation of U251 and U373 cells in a time- and dose-dependent manner (P < 0.001). EdU test showed that the proliferative activity of PO-treated cells was significantly decreased, and the number of cell colonies also decreased significantly (P < 0.01). PO treatment significantly increased apoptotic rates (P < 0.01), decreased mitochondrial membrane potential and caused obvious changes in mitochondrial morphology of the cells. Pathway enrichment analysis showed that the down-regulated genes were significantly enriched in the PI3K/AKT pathway, which was verified by Western blotting showing significantly down-regulated expression levels of PI3K, AKT and p-AKT in PO-treated cells (P < 0.05).@*CONCLUSION@#PO interferes with mitochondrial fusion and fission function through the PI3K/AKT pathway, thereby inhibiting the proliferation and increasing apoptosis of glioma cells.
الموضوعات
Humans , Glioma , Mitochondrial Dynamics , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-aktالملخص
OBJECTIVE@#To investigate the effect of lactic acid-induced upregulation of PLEKHA4 expression on biological behaviors of glioma cells and the possible molecular mechanism.@*METHODS@#GEO database and GEPIA2 website were used to analyze the relationship between PLEKHA4 expression level and the pathological grade of glioma. A specific PLEKHA4 siRNA was transfected in glioma U251 and T98G cells, and the changes in cell proliferation ability were assessed by real-time cell analysis technology and Edu experiment. The colony-forming ability of the cells was evaluated using plate cloning assay, and cell cycle changes and cell apoptosis were analyzed with flow cytometry. The mRNA expression of PLEKHA4 was detected by PCR in glioma samples and controls and in glioma cells treated with lactic acid and glucose. Xenograft mice in vivo was used to detect tumor formation in nude mice; Western blotting was used to detect the expressions of cyclinD1, CDK2, Bcl2, β-catenin and phosphorylation of the key proteins in the MAPK signaling pathway.@*RESULTS@#The results of GEO database and online website analysis showed that PLEKHA4 was highly expressed in glioma tissues and was associated with poor prognosis; PLEKHA4 knockdown obviously inhibited the proliferation and attenuated the clone-forming ability of the glioma cells (P < 0.05). Flow cytometry showed that PLEKHA4 knockdown caused cell cycle arrest in G1 phase and promoted apoptosis of the cells (P < 0.01). PLEKHA4 gene mRNA expression was increased in glioma samples and glioma cells after lactate and glucose treatment (P < 0.01). PLEKHA4 knockdown, tumor formation ability of nude mice decreased; PLEKHA4 knockdown obviously lowered the expression of cyclinD1, CDK2, Bcl2 and other functional proteins, inhibited the phosphorylation of ERK and p38 and reduced the expression of β-catenin protein (P < 0.01).@*CONCLUSION@#PLEKHA4 knockdown inhibited the proliferation of glioma cells and promoted apoptosis by inhibiting the activation of the MAPK signaling pathway and expression of β-catenin. Lactic acid produced by glycolysis upregulates the expression of PLEKHA4 in glioma cells.
الموضوعات
Humans , Animals , Mice , Up-Regulation , beta Catenin/metabolism , Mice, Nude , Brain Neoplasms/pathology , Lactic Acid , Cell Line, Tumor , Glioma/pathology , Cell Proliferation , Apoptosis , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , Gene Expression Regulation, Neoplasticالملخص
Toll-like receptors (TLRs) play crucial roles in the innate immune system by recognizing pathogen-associated molecular patterns derived from various microbes and self-derived molecules derived from damaged cells.TLRs can express in multiple cells in the retina, such as glia cells,retinal pigment epithelium (RPE) as well as photoreceptor ceils.When combined with special ligands,TLRs can initiate intracellular signal transduction pathways, induce specific gene expression, and upregulate the expression of costimulatory molecules as well as stimulate the secretion of inflammatory factors which take part in resisting to infection.Furthermore, these inflammatory factors may have an effect on retinal neovascular diseases via regulating angiogenic growth factors level.Microglia cells are the most important part in retinal innate immune system as they can participate in immune surveillance and tissue repair.Among the recognition receptors of microglia cells,TLRs play a significant role in regulating microglia cells which involved in retinal infection and inflammation as well as ischemic retinal diseases.In this review,we focused on the role of TLRs in microglia cells.