FAT1 knockdown in immortalized astrocyte and glioma cell lines
ABSTRACT: The FAT1 gene was knocked down using 2 independent siRNAs, in immortalized human astrocytes and U87 and U251 glioma cell lines. A non-targeted scramble siRNA was used as a control. Scramble, FAT1 siRNA#1 and FAT1 siRNA#2 were transfected into each cell line, in duplicate, and RNA analyzed using the Affymetrix U133A 2.0 platform.
Project description:Human four-and-a-half LIM domains protein 1 (FHL1) is a member of the FHL protein family, which serves an important role in multiple cellular events by interacting with transcription factors using its cysteine-rich zinc finger motifs. A previous study indicated that FHL1 was downregulated in several types of human cancer and served a role as a tumor suppressive gene. The overexpression of FHL1 inhibited tumor cell proliferation. However, to the best of our knowledge, there is no evidence to confirm whether FHL1 affected glioma growth, and the molecular mechanisms through which FHL1 represses tumor development remain unclear. In the present study, the expression level of FHL1 was determined using immunohistochemical staining in 114 tumor specimens from patients with glioma. The results indicated that FHL1 expression was negatively associated with the pathological grade of gliomas. Furthermore, Kaplan-Meier survival curves demonstrated that the patients with an increased FHL1 expression exhibited a significantly longer survival time, suggesting that FHL1 may be a prognostic marker for glioma. The protein level of FHL1 was relatively increased in the U251 glioma cell line compared with that in the U87 cell line. Therefore, FHL1 was knocked down in U251 by siRNA and overexpressed in U87, and it was identified that FHL1 significantly decreased the activation of PI3K/AKT signaling by interacting with AKT. Further experiments verified that FHL1 inhibited the growth of gliomas in vivo by modulating PI3K/AKT signaling. In conclusion, the results of the present study demonstrated that FHL1 suppressed glioma development through PI3K/AKT signaling.
Project description:We investigated the effects of aquaporin 5 (AQP5) gene silencing on the proliferation, migration and apoptosis of human glioma cells through regulating the EGFR/ERK/p38MAPK signaling pathway. qRT-PCR was applied to examine the mRNA expressions of AQP5 in five human glioma cell lines. U87-MG, U251 and LN229 cells were selected and assigned into blank, vector, AQP5 siRNA and FlagAQP5 groups. MTT assay was used to measure cell proliferation. Flow cytometry (FCM) with AnnexinV-FITC/PI double staining and PI staining were employed to analyze cell apoptosis and cell cycle respectively. Scratch test was used to detect cell migration. Western blotting was performed to determine the EGFR/ERK/p38 MAPK signaling pathway-related proteins. Results showed that the positive expression of AQP5 in primary glioblastoma was associated with the tumor size and whether complete excision was performed. The mRNA expressions of AQP5 in cell lines of U87-MG, U251 and LN229 were significantly higher than in U373 and T98G. The proliferation rates of U87-MG, U251 and LN229 cells in the AQP5 siRNA group were lower than in the vector and blank groups. The apoptosis rate increased in the AQP5 siRNA group compared with the vector group. Scratch test demonstrated that AQP5 gene silencing could suppress cell migration. Compared with the vector and blank groups, the AQP5 siRNA group showed decreased expressions of the ERK1/2, p38 MAPK, p-ERK1/2 and p-p38 MAPK proteins. AQP5 gene silencing could inhibit the cell proliferation, reduce cell migration and promote the cell apoptosis of U87-MG, U251 and LN229 by suppressing EGFR/ERK/p38 MAPK signaling pathway.
Project description:Glioma is the most frequent primary brain tumor. Recently, the upregulation of microRNA (miR)-23a was found to be associated with glioma, but the molecular mechanism by which miR-23a promotes glioma growth remains to be unveiled. In the present study, we found that miR-23a was significantly upregulated in glioma tissues compared to their matched adjacent tissues. miR-23a was also highly expressed in glioma cell lines SHG44, U251, and U87 cells. Moreover, we identified homeobox D10 (HOXD10) as a novel target for miR-23a. The expression of HOXD10 was significantly reduced in glioma tissues and cell lines, and miR-23a negatively regulates the protein expression of HOXD10 in U251 and U87 cells. We further showed that miRNA-23a promoted U251 and U87 cell invasion, at least partially, by directly targeting HOXD10 and further modulating MMP-14. These findings suggest that miR-23a may serve as a promising therapeutic target for glioma.
Project description:Purpose:To investigate the specific function of long noncoding RNA FGD5 antisense RNA 1 (lncRNA FGD5-AS1) in glioma. Materials and Methods:The level of FGD5-AS1 was detected in clinical samples and cell lines by qRT-PCR. Small interfering RNA (siRNA) of FGD5-AS1 or scramble siRNA was transfected into U87 cell lines to examine the role of FGD5-AS1 on glioma development. The proliferation of glioma cells was tested by Cell Counting Kit-8 (CCK-8), the migration and invasion of glioma cells were tested by transwell assay without matrigel or with matrigel. Western blot was used to detect the protein expression, and XAV-939 was used to inhibit wnt/?-catenin pathway. The effect of FGD5-AS1 on tumorigenesis of glioma was confirmed by xenograft nude mice model. Results:FGD5-AS1 was significantly increased in glioma tissues and cells. Loss of FGD5-AS1 inhibited the proliferation, migration and invasion of U87 cells. Furthermore, overexpression of FGD5-AS1 increased the mRNA and protein levels of ?-catenin and cyclin D1. Blocking of wnt/?-catenin using XAV-939 reversed the promotion role of FGD3-AS1 on glioma cells' migration and invasion. The in vivo tumor growth assay showed that FGD3-AS1 accelerated glioma tumorigenesis with activating wnt/?-catenin pathway. Conclusion:Our research emphasized FGD5-AS1 acting as an oncogene by regulating wnt/?-catenin signaling pathway, thus providing some novel experimental basis for clinical treatment of glioma.
Project description:Poor prognosis of glioblastoma multiforme is strongly associated with the ability of tumor cells to invade the brain parenchyma, which is believed to be the major factor responsible for glioblastoma recurrence. Therefore, identifying the molecular mechanisms driving invasion may lead to the development of improved therapies for glioblastoma patients. Here, we investigated the role of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2), an enzyme catalyzing collagen cross-linking, in the biology of glioblastoma invasion. PLOD2 mRNA was significantly overexpressed in glioblastoma compared to low-grade tumors based on the Oncomine datasets and REMBRANDT database for human gliomas. Kaplan-Meier estimates based on the TCGA dataset demonstrated that high PLOD2 expression was associated with poor prognosis. In vitro, hypoxia upregulated PLOD2 protein in U87 and U251 human glioma cell lines. siRNA knockdown of endogenous HIF-1? or treatment of cells with the HIF-1? inhibitor PX-478 largely abolished the hypoxia-mediated PLOD2 upregulation. Knockdown of PLOD2 in glioma cell lines led to decreases in migration and invasion under normoxia and hypoxia. In addition, levels of phosphorylated FAK (Tyr 397), an important kinase mediating cell adhesion, were reduced in U87-shPLOD2 and U251-shPLOD2 cells, particularly under hypoxic conditions. Finally, orthotopic U251-shPLOD2 xenografts were circumscribed rather than locally invasive. In conclusion, the results indicated that PLOD2 was a gene of clinical relevance with implications in glioblastoma invasion and treatment strategies.
Project description:The brain microenvironment has emerged as an important component in malignant progression of human glioma. However, astrocytes, the most abundant glial cells in the glioma microenvironment, have as yet a poorly defined role in the development of this disease, particularly with regard to invasion. Here, we co-cultured human astrocytes with human glioma cell lines, U251 and A172, in an in vitro transwell system in order to ascertain their influence on migration and invasion of gliomas. mRNA and protein expression assays were subsequently used to identify candidate proteins mediating this activity. Astrocytes significantly increased migration and invasion of both U251 and A172 cells in migration and invasion (plus matrigel) assays. Membrane type 1 matrix metalloproteinase (MMP14) originating from glioma cells was identified in qRT-PCR as the most highly up-regulated member of the MMP family of genes (~ 3 fold, p < 0.05) in this system. A cytokine array and ELISA were used to identify interleukin-6 (IL-6) as a highly increased factor in media collected from astrocytes, especially under co-culture conditions. IL-6 was also the key cytokine inducing cytomembrane MMP14 expression, the active form of MMP14, in glioma cells. Knockdown of MMP14 with siRNA led to decreased migration and invasion. Taken together, our results indicated that cytomembrane MMP14 was induced by IL-6 secreted from astrocytes, thereby enhancing the migration and invasion of glioma cells through activation of MMP2. Therefore, this IL-6 and MMP14 axis between astrocytes and glioma cells may become a potential target for treatment of glioma patients.
Project description:miRNA-184 is an oncogene in human hepatocellular carcinoma but acts as a tumor suppressor in tongue squamous cell carcinoma. Studies have shown that miR-184 was down-regulated in glioma and TNFα-induced protein 2 (TNFAIP2) was closely related to tumorigenesis. This study aimed to determine the functions of miR-184 in glioma and the mechanisms of miRNA-184-TNFAIP2 mediated glioma progression.Real-time reverse-transcription PCR detected expression of miR-184 and TNFAIP2. U87 and U251 cells were transfected with miR-184 mimic, inhibitor, or negative control miRNA, and their invasion abilities were assayed. Cellular proliferation was measured by the cell counting kit-8 assay. miR-184 effects on glioma cell apoptosis and cell cycle were assessed by flow cytometer. Biological information software have predicted that miR-184 could target TNFα-induced protein 2 (TNFAIP2), Which was further validated by Western blot and qRT-PCR in glioma cells. In vivo, U87 cells transduced with either lentiviral over-expressed miR-184 or control lentivirus were injected into nude mice subcutaneously and intracranial respectively.Expression of miR-184 was significantly lower in glioma tissues and cell-lines compared to normal brain tissues. Protein and mRNA expression of TNFAIP2 were inversely correlated with miR-184 in glioma. In vitro, proliferation and invasion abilities were also decreased in U87 and U251 cells after transfection with miR-184 mimic. In vivo, the xenografted tumor size in the miR-184 overexpressing group were smaller than the miR-NC group. Concordantly, U87 and U251 cells transfected with miR-184 mimic had a higher apoptosis rate, triggering an accumulation of cells at the G0/G1 phase and decreased cells in S-phase.miR-184 could regulate TNFAIP2 expression and affected its translation in glioma. miR-184 could also inhibit glioma progression and might serve as a novel therapeutic target in glioma.
Project description:Background: CAPON has two isoforms in human brain: long form of CAPON (CAPON-L) and short form of CAPON (CAPON-S). Recent studies have indicated the involvement of CAPON in tumor cell growth. We aimed to reveal the role of the two CAPON isoforms in the proliferation of glioma cells in this study. Materials and Methods: Lentivirus-mediated stable cell lines with CAPON-L or CAPON-S overexpression were established in U87 and U251 glioma cells. Cell counting kit-8 and colony formation assays were used to evaluate cell proliferation. Western blot analysis of cell cycle-related proteins and flow cytometry were performed to analyze cell cycle progression. Some important molecules of the AKT/mTOR pathway and P53 were also measured by Western blot analysis. Results: Overexpression of CAPON-L showed a significantly inhibitory role in U251 cells, while it exhibited a promoting role in U87 cells. Consistently, overexpressing CAPON-L impeded the cell cycle progression and down-regulated the expression levels of Cyclin D1, CDK4 and CDK6 in U251 cells, whereas it up-regulated the CDK6 level in U87 cells. The overexpression of CAPON-L significantly decreased the phosphorylation and/or total levels of AKT, mTOR and S6 in U251 cells, while it did not affect these signaling molecules in U87 cells, except for a significant increase in the phosphorylation of AKT at Thr-308 site. Transfecting constitutively active AKT (myr-AKT) partially reversed the decreased phosphorylation of AKT and S6 in the CAPON-L-overexpressing U251 cells. In addition, we found a significant decrease in the wild-type P53 level in the CAPON-L-overexpressing U87 cells. The overexpression of CAPON-S also inhibited cell proliferation, blocked cell cycle progression, and decreased the AKT/mTOR pathway activity in U251 cells. Conclusion: The effects of CAPON-L overexpression on glioma cell proliferation are dependent on the AKT/mTOR/P53 activity. The overexpression of CAPON inhibits U251 cell proliferation through the AKT/mTOR signaling pathway, while overexpressing CAPON-L promoted U87 cell proliferation, possibly through down-regulating the P53 level.
Project description:Effective suicide gene delivery and expression are crucial to achieving successful effects in gene therapy. An ideal tumor-specific promoter expresses therapeutic genes in tumor cells with minimal normal tissue expression. We compared the activity of the FOS (FBJ murine osteosarcoma viral oncogene homolog) promoter with five alternative tumor-specific promoters in glioma cells and non-malignant astrocytes. The FOS promoter caused significantly higher transcriptional activity in glioma cell lines than all alternative promoters with the exception of CMV. The FOS promoter showed 13.9%, 32.4%, and 70.8% of the transcriptional activity of CMV in three glioma cell lines (U87, U251, and U373). Importantly, however, the FOS promoter showed only 1.6% of the transcriptional activity of CMV in normal astrocytes. We also tested the biologic activity of recombinant adenovirus containing the suicide gene herpes simplex virus thymidine kinase (HSV-tk) driven by the FOS promoter, including selective killing efficacy in vitro and tumor inhibition rate in vivo. Adenoviral-mediated delivery of the HSV-tk gene controlled by the FOS promoter conferred a cytotoxic effect on human glioma cells in vitro and in vivo. This study suggests that use of the FOS-tk adenovirus system is a promising strategy for glioma-specific gene therapy but still much left for improvement.
Project description:Proteolipid protein 2 (PLP2) is an integral ion channel membrane protein of the endoplasmic reticulum. The protein has been shown to be highly expressed in many cancer types, but its importance in glioma progression is poorly understood. Using publicly available datasets (Rembrandt, TCGA and CGGA), we found that the expression of PLP2 was significantly higher in high-grade gliomas than in low-grade gliomas. We confirmed these results at the protein level through IHC staining of high-grade (n = 56) and low-grade glioma biopsies (n = 16). Kaplan-Meier analysis demonstrated that increased PLP2 expression was associated with poorer patient survival. In functional experiments, siRNA and shRNA PLP2 knockdown induced ER stress and increased apoptosis and autophagy in U87 and U251 glioma cell lines. Inhibition of autophagy with chloroquine augmented apoptotic cell death in U87- and U251-siPLP2 cells. Finally, intracranial xenografts derived from U87- and U251-shPLP2 cells revealed that loss of PLP2 reduced glioma growth in vivo. Our results therefore indicate that increased PLP2 expression promotes GBM growth and that PLP2 represents a potential future therapeutic target.