Caveolin-1 is a negative regulator of tumor growth in glioblastoma and modulates chemosensitivity to temozolomide

Caveolin-1 (Cav-1) is a critical regulator of tumor progression in a variety of cancers where it has been shown to act as either a tumor suppressor or tumor promoter. In glioblastoma multiforme, it has been previously demonstrated to function as a putative tumor suppressor. Our studies here, using the human glioblastoma-derived cell line U-87MG, further support the role of Cav-1 as a negative regulator of tumor growth. Using a lentiviral transduction approach, we were able to stably overexpress Cav-1 in U-87MG cells. Gene expression microarray analyses demonstrated significant enrichment in gene signatures corresponding to downregulation of MAPK, PI3K/AKT and mTOR signaling, as well as activation of apoptotic pathways in Cav-1-overexpressing U-87MG cells. These same gene signatures were later confirmed at the protein level in vitro. To explore the ability of Cav-1 to regulate tumor growth in vivo, we further show that Cav-1-overexpressing U-87MG cells display reduced tumorigenicity in an ectopic xenograft mouse model, with marked hypoactivation of MAPK and PI3K/mTOR pathways. Finally, we demonstrate that Cav-1 overexpression confers sensitivity to the most commonly used chemotherapy for glioblastoma, temozolomide. In conclusion, Cav-1 negatively regulates key cell growth and survival pathways and may be an effective biomarker for predicting response to chemotherapy in glioblastoma.     

IL‐17A promotes cell migration and invasion of glioblastoma cells via activation of PI3K/AKT signalling pathway

Glioblastomas (GBMs) are the most common of both benign and malignant primary brain tumours, in which the inflammatory and immunologic abnormalities are involved. Interleukin‐17A (IL‐17A) plays an important role in various inflammatory diseases and cancers. Several recent studies revealed that the expression of IL‐17A was overexpressed in human GBMs tissue. However, the accurate role of IL‐17A in GBMs remains unclear. In this study, we aimed to explore the effect of IL‐17A on cell migration and invasion of GBMs and the mechanism by which the effects occurred. We found that exogenous IL‐17A promoted significantly cell migration and invasion abilities in two GBMs cell lines (U87MG and U251) in a time‐dependent manner. In addition, the protein expressions of PI3K, Akt and MMP‐2/9 were increased in the GBMs cells challenged by IL‐17A. Furthermore, a tight junction protein ZO‐1 was down‐regulated but Twist and Bmi1 were up‐regulated. Treatment with a PI3K inhibitor (LY294002) significantly reduced the abilities of both migration and invasion in U87MG and U251 cells. LY294002 treatment also attenuated the IL‐17A causing increases of protein levels of PI3K, AKT, MMP‐2/9, Twist and the decreases of protein level of ZO‐1 in the U87MG and U251 cells. Taken together, we concluded that IL‐17A promotes the GBM cells migration and invasion via PI3K/AKT signalling pathway. IL‐17A and its related signalling pathways may be potential therapeutic targets for GBM.     

The role of A-kinase interacting protein 1 in regulating progression and stemness as well as indicating the prognosis in glioblastoma

BackgroundA-kinase interacting protein 1 (AKIP1) is recently implicated in the pathogenesis of several solid tumors, while its role in glioblastoma multiforme (GBM) is largely unknown. Therefore, the current study aimed to investigate the effect of AKIP1 on GBM cell malignant behaviors, stemness, and its underlying molecular mechanisms.MethodsU-87 MG and A172 cells were transfected with control or AKIP1 overexpression plasmid; control or AKIP1 siRNA plasmid. Then cell proliferation, apoptosis, invasion, CD133⁺ cell proportion, and sphere formation assays were performed. Furthermore, RNA-Seq was performed in U-87 MG cells. Besides, AKIP1 expression was detected in 25 GBM and 25 low-grade glioma (LGG) tumor samples.ResultsAKIP1 was increased in several GBM cell lines compared to the control cell line. After transfections, it was found that AKIP1 overexpression increased cell invasion, CD133⁺ cell proportion, and sphere formation ability while less affecting cell proliferation or cell apoptosis in U-87 MG and A172 cells. Moreover, AKIP1 siRNA achieved the opposite effect in these cells, except that it inhibited cell proliferation but induced cell apoptosis to some extent. Subsequent RNA-Seq assay showed several critical carcinogenetic pathways, such as PI3K/AKT, Notch, EGFR tyrosine kinase inhibitor resistance, Ras, ErbB, mTOR pathways, etc. were potentially related to the function of AKIP1 in U-87 MG cells. Clinically, AKIP1 expression was higher in GBM tissues than in LGG tissues, which was also correlated with the poor prognosis of GBM to some degree.ConclusionsAKIP1 regulates the malignant behaviors and stemness of GBM via regulating multiple carcinogenetic pathways.     

Cloning, expression, and characterization of TNFSF14 (LIGHT) gene in mefugu, Takifugu obscurus

Tumor necrosis factor receptor-associated factor 6 (TRAF6), which plays an important role in inflammation and immune response, is an essential adaptor protein for the NF-κB (nuclear factor κB) signaling pathway. Recent studies have shown that TRAF6 played an important role in tumorigenesis and invasion by suppressing NF-κB activation. However, up to now, the biologic role of TRAF6 in glioma has still remained unknown. To address the expression of TRAF6 in glioma cells, four glioma cell lines (U251, U-87MG, LN-18, and U373) and a non-cancerous human glial cell line SVG p12 were used to explore the protein expression of TRAF6 by Western blot. Our results indicated that TRAF6 expression was upregulated in human glioma cell lines, especially in metastatic cell lines. To investigate the role of TRAF6 in cell proliferation, apoptosis, invasion, and migration of glioma, we generated human glioma U-87MG cell lines in which TRAF6 was either overexpressed or depleted. Subsequently, the effects of TRAF6 on cell viability, cell cycle distribution, apoptosis, invasion, and migration in U-87MG cells were determined with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry analysis, transwell invasion assay, and wound-healing assay. The results showed that knockdown of TRAF6 could decrease cell viability, suppress cell proliferation, invasion and migration, and promote cell apoptosis, whereas overexpression of TRAF6 displayed the opposite effects. In addition, the effects of TRAF6 on the expression of phosphor-NF-κB (p-p65), cyclin D1, caspase 3, and MMP-9 were also probed. Knockdown of TRAF6 could lower the expression of p-p65, cyclin D1, and MMP-9, and raise the expression of caspase 3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, invasion, and migration of U-87MG cell, as well as inhibition of apoptosis of U-87MG cell by abrogating activation of NF-κB.     

受体酪氨酸激酶Axl对胶质母细胞瘤细胞增殖、凋亡和侵袭的影响

摘要 目的：研究受体酪氨酸激酶Axl在胶质母细胞瘤组织和细胞系U-118MG细胞中的表达情况及其对U-118MG细胞增殖、凋亡、侵袭的影响。方法：收集2015年3月至2018年5月在本院进行手术切除并经病理分型证实的胶质母细胞瘤组织标本（n=30）,另取脑外伤手术中因作内减压而切除的正常脑组织作为对照（n=28）。采用荧光实时定量 (qRT-PCR)检测正常脑组织和胶质母细胞瘤肿瘤组织中Axl mRNA表达水平;采用Western blot检测人小神经胶质HM细胞、U-118MG细胞以及Axl-shRNA转染后U-118MG细胞中Axl蛋白表达水平;采用CCK-8检测Axl-shRNA转染后U-118MG细胞增殖能力;采用流式细胞术检测Axl-shRNA转染后U-118MG细胞凋亡水平;采用Transwell小室实验检测Axl-shRNA转染后U-118MG细胞的侵袭能力。结果：在胶质母细胞瘤组织中Axl mRNA表达水平显著高于正常脑组织（P<0.05）;U-118MG细胞Axl蛋白表达水平显著高于人小神经胶质细胞系HM细胞,差异有统计学意义（P<0.05）;转染Axl-shRNA后,U-118MG细胞中Axl蛋白表达水平显著降低（P<0.05）。与U-118MG细胞和转染control-shRNA细胞相比, 转染Axl-shRNA的U-118MG细胞增殖能力降低（P<0.05）,凋亡水平升高（P<0.05）,侵袭能力降低（P<0.05）。结论：在胶质母细胞瘤组织和U-118MG细胞中,Axl表达水平显著增高,并且Axl表达水平与U-118MG细胞增殖、凋亡及侵袭密切关联。     

Ganoderic acid A holds promising cytotoxicity on human glioblastoma mediated by incurring apoptosis and autophagy and inactivating PI3K/AKT signaling pathway

Ganoderic acid A (GA‐A), recognized as a lanostanetriterpene isolated from Ganoderma lucidum, demonstrates an efficient antitumor activity in multiple cancers. To date, it is unclear whether and how GA‐A functions on human glioblastoma (GBM). To unravel the functional significance of GA‐A on human glioblastoma (GBM), the cell‐counting kit‐8 and transwell assays were used to detect proliferation, migration, and invasion of human GBM cell after GA‐A treatment. Then, we utilized the flow cytometry and western blot to further evaluate the effect of GA‐A on GBM cell. Further, activities of autophagy and PI3K/AKT signaling were assessed by Western blot assay. We found that GA‐A significantly inhibited proliferation, migration, and invasion of GBM cell. Additionally, GA‐A markedly triggered cell apoptosis, which incarnated an elevation trend in apoptotic percentage, simultaneously, an increased level of proapoptosis protein (Bax and active caspase‐3) and a decreased level of antiapoptosis protein (Bcl‐2), induced by GA‐A treatment. Meanwhile, levels of two well‐known autophagy markers (beclin 1 and LC3 II) increased while another autophagic substrate (P‐62) was reduced. Moreover, the expressions levels of phosphorylated AKT, mTOR, p‐P70S6K, and cyclin D1 in the PI3K/AKT pathway were significantly reduced, which revealed GA‐A repressed the activation of PI3K/AKT signaling pathway. Collectively, these results indicate that GA‐A may encourage U251 cell growth and invasion/migration inhibition, apoptosis, and autophagy through the inactivation of PI3K/AKT signaling pathway in human GBM. Hence, GA‐A may be a potent antitumorigenic agent for human GBM in future clinical practice.     

Inhibition of fatty acid synthase suppresses U-2 OS cell invasion and migration via downregulating the activity of HER2/PI3K/AKT signaling pathway in vitro

FASN plays an important role in the malignant phenotype of various tumors. Our previous studies show that inhibition FASN could induce apoptosis and inhibit proliferation in human osteosarcoma (OS) cell in vivo and vitro. The aim in this study was to investigate the effect of inhibition FASN on the activity of HER2/PI3K/AKT axis and invasion and migration of OS cell. The expression of FASN, HER2 and p-HER2(Y1248) proteins was detected by immunohistochemistry in OS tissues from 24 patients with pulmonary metastatic disease, and the relationship between FASN and p-HER2 as well as HER2 was investigated. The results showed that there was a positive correlation between FASN and HER2 as well as p-HER2 protein expression. The U-2 OS cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid. FASN mRNA was measured by RT-PCR. Western blot assays was performed to examine the protein expression of FASN, HER2, p-HER2(Y1248), PI3K, Akt and p-Akt (Ser473). Migration and invasion of cells were investigated by wound healing and transwell invasion assays. The results showed that the activity of HER2/PI3K/AKT signaling pathway was suppressed by inhibiting FASN. Meanwhile, the U-2OS cells migration and invasion were also impaired by inhibiting the activity of FASN/HER2/PI3K/AKT. Our results indicated that inhibition of FASN suppresses OS cell invasion and migration via down-regulation of the “HER2/PI3K/AKT” axis in vitro. FASN blocker may be a new therapeutic strategy in OS management.     

Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway

Ovarian cancer is the leading cause of death from gynecological malignancy for women. The amplification of the PI3K catalytic subunit (p110) and the lost function of PTEN are frequently detected in ovarian cancer cells. PI3K plays an important role in tumorigenesis. To specifically inhibit PI3K activity in ovarian cancer cells, we constructed small interfering RNA (siRNA) against p110. The expression of p110 siRNA significantly decreased cell migration, invasion, and proliferation compared to the siSCR control cells. The expression of p110 siRNA induced CDK inhibitor p27^KIP1 levels, and decreased levels of cyclin D1, CDK4, and phosphorylated retinoblastoma protein. PI3K transmits the mytogenic signal through AKT. AKT has three isoforms in the cells: AKT1, AKT2 and AKT3. We found that inhibition of AKT1 is sufficient to affect cell migration, invasion, and proliferation. Expression of AKT1 siRNA had a similar effect as p110 siRNA in the cells. We showed the roles of specific PI3K and AKT isoforms in the cells, which are important to understanding the mechanism of PI3K/AKT signaling in ovarian cancer cells. Both p110 and AKT1 siRNA-expressing cells decreased the activation of p70S6K1. Inhibition of p70S6K1 activity by its siRNA also decreased cell migration, invasion, and proliferation associated with the induction of p27^KIP1 levels, and with the inhibition of cell cycle-associated proteins including cyclin D1, CDK2, and phosphorylated retinoblastoma protein. This study demonstrates the important role of the PI3K/AKT/mTOR/p70S6K1 pathway in cell proliferation, migration, and invasion in ovarian cancer cells by using siRNA-mediated gene silencing as a reverse genetic method.     

Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, functions by inducing apoptosis in human glioblastoma U87MG cells

Malignant glioblastoma is one of the most common malignant tumors in the neurological system. Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, appears to exhibit various biological activities, including antitumor effect, but the function and mechanism of this new agent on glioblastoma cells has not previously been determined. In the present study, we investigated the proliferation change of human glioblastoma U87MG cells exposured to different concentrations (0.9-14.8 microM) of Tubeimoside V (1) for a certain time. The results showed that Tubeimoside V (1) significantly suppressed U87MG cell proliferation in a time- and dose-dependent manner (IC(50) = 3.6 microM). Flow cytometric analysis of DNA in U87MG cells showed that Tubeimoside V (1) induces the prominent appearance of a sub-G1 peak in the cell cycle suggestive of apoptosis. Furthermore, U87MG cells' treatment with Tubeimoside V (1) resulted in nuclear condensation with apoptotic bodies observed by both fluorescence and electron microscopy. The result of annexin V/PI assay showed that phosphatidylserine externalization began after treatment, and then increased in the following 24h. Molecular changes explored through Western-blot staining showed Tubeimoside V (1) decreased the expression levels of Bcl-2 protein and increased the expression levels of Bax protein. The novel findings suggest that the cytotoxic actions of Tubeimoside V (1) toward U87MG cells result from the induction of cell apoptosis. Overall, our data demonstrate that Tubeimoside V (1) is an efficient apoptotic killing agent of glioblastoma cells and suggest that this mechanism may play a critical role in anti-tumor chemotherapy.     

Cdc42 and the guanine nucleotide exchange factors Ect2 and trio mediate Fn14-induced migration and invasion of glioblastoma cells

Malignant glioblastomas are characterized by their ability to infiltrate into normal brain. We previously reported that binding of the multifunctional cytokine TNF-like weak inducer of apoptosis (TWEAK) to its receptor fibroblast growth factor-inducible 14 (Fn14) induces glioblastoma cell invasion via Rac1 activation. Here, we show that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated glioma cells display an increased activation of Cdc42, and depletion of Cdc42 using siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14, showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2 abrogates both TWEAK-induced Cdc42 and Rac1 activation, as well as subsequent TWEAK-Fn14-directed glioma cell migration and invasion. In contrast, Trio depletion inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces astrocyte migration within the brain, corroborating the in vitro importance of the TWEAK-Fn14 signaling cascade in glioblastoma invasion. Our results suggest that the TWEAK-Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-Rho GTPase signaling pathway present innovative drug targets for glioma therapy.     

Clozapine, a neuroleptic agent, inhibits Akt by counteracting Ca2+/calmodulin in PTEN-negative U-87MG human glioblastoma cells

Clozapine (CZP), a dibenzodiazepine derivative with a piperazinyl side chain, is in clinical use as an antipsychotic drug. This study investigated the effect of CZP on the modulation of the PI3K/Akt/GSK-3beta pathway in PTEN-negative U-87MG glioblastoma cells. Treatment with CZP rapidly inhibited the basal and EGF-induced phosphorylation of Akt. The inhibition of Akt resulted in the dephosphorylation of GSK-3beta and increased GSK-3beta kinase activity. A voltage-sensitive Ca(2+) channel blocker and calmodulin (CaM) antagonists inhibited Akt phosphorylation, whereas elevation of the intracellular Ca(2+) concentration prevented CZP-induced dephosphorylation of Akt and GSK-3beta, suggesting that Ca(2+)/CaM participates in the inhibition of Akt by CZP in U-87MG cells. In addition, similar to LY294002, CZP arrested cell cycle progression at G0/G1 phase, which was accompanied by decreased expression of cyclin D1. The reduction in the cyclin D1 level induced by CZP was abrogated by the inhibition of GSK-3beta, the inhibition of proteasome-dependent proteolysis, or an increase in the intracellular Ca(2+) concentration. These results suggest that the antipsychotic drug CZP modulates the PI3K/Akt/GSK-3beta pathway by counteracting Ca(2+)/CaM in PTEN-negative U-87MG glioblastoma cells.     

NETRIN-4 Protects Glioblastoma Cells FROM Temozolomide Induced Senescence

Glioblastoma multiforme is the most common primary tumor of the central nervous system. The drug temozolomide (TMZ) prolongs lifespan in many glioblastoma patients. The sensitivity of glioblastoma cells to TMZ is interfered by many factors, such as the expression of O-6-methylguanine-DNA methyltransferase (MGMT) and activation of AKT signaling. We have recently identified the interaction between netrin-4 (NTN4) and integrin beta-4 (ITGB4), which promotes glioblastoma cell proliferation via activating AKT-mTOR signaling pathway. In the current work we have explored the effect of NTN4/ITGB4 interaction on TMZ induced glioblastoma cell senescence. We report here that the suppression of either ITGB4 or NTN4 in glioblastoma cell lines significantly enhances cellular senescence. The sensitivity of GBM cells to TMZ was primarily determined by the expression of MGMT. To omit the effect of MGMT, we concentrated on the cell lines devoid of expression of MGMT. NTN4 partially inhibited TMZ induced cell senescence and rescued AKT from dephosphorylation in U251MG cells, a cell line bearing decent levels of ITGB4. However, addition of exogenous NTN4 displayed no significant effect on TMZ induced senescence rescue or AKT activation in U87MG cells, which expressed ITGB4 at low levels. Furthermore, overexpression of ITGB4 combined with exogenous NTN4 significantly attenuated U87MG cell senescence induced by TMZ. These data suggest that NTN4 protects glioblastoma cells from TMZ induced senescence, probably via rescuing TMZ triggered ITGB4 dependent AKT dephosphorylation. This suggests that interfering the interaction between NTN4 and ITGB4 or concomitant use of the inhibitors of the AKT pathway may improve the therapeutic efficiency of TMZ.     

Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cells

The small GTPase Cdc42 has been implicated as an important regulator of cell migration. However, whether Cdc42 plays similar role in all cancer cells irrespective of metastatic potential remains poorly defined. Here, we show by using three different breast cancer cell lines with different metastatic potential, the role of Cdc42 in cell migration/invasion and its relationship with a number of downstream signaling pathways controlling cell migration. Small interfering RNA (siRNA)-mediated knockdown of Cdc42 in two highly metastatic breast cancer cell lines (MDA-MB-231 and C3L5) resulted in enhancement, whereas the same in moderately metastatic (Hs578T) cell line resulted in inhibition of intrinsic cellular migration/invasion. Furthermore, Cdc42 silencing in MDA-MB-231 and C3L5 but not Hs578T cells was shown to be accompanied by increased RhoA activity and phosphorylation of protein kinase C (PKC)-δ, extracellular signal regulated kinase1/2 (Erk1/2), and protein kinase A (PKA). Pharmacological inhibition of PKCδ, MEK-Erk1/2, or PKA was shown to inhibit migration of both control and Cdc42-silenced MDA-MB-231 cells. Furthermore, introduction of constitutively active Cdc42 was shown to decrease migration/invasion of MDA-MB-231 and C3L5 but increase migration/invasion of Hs578T cells. This decreased migration/invasion of MDA-MB-231 and C3L5 cells was also shown to be accompanied by the decrease in the phosphorylations of PKCδ, Erk1/2, and PKA. These results suggested that endogenous Cdc42 could exert a negative regulatory influence on intrinsic migration/invasion and some potentially relevant changes in phosphorylation of PKCδ, Erk1/2, and PKA of some aggressive breast cancer cells.     

Retracted: MicroRNA-99b suppresses human cervical cancer cell activity by inhibiting the PI3K/AKT/mTOR signaling pathway

Cervical cancer is common cancer among women with high morbidity. MicroRNAs (miRs) are involved in the progression and development of cervical cancer. This study aimed to explore the effect of miR-99b-5p (miR-99b) on invasion and migration in cervical cancer through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. The microarray-based analysis was used to screen out differentially expressed miRNAs. Expression of miR-99b, PI3K, AKT, mTOR, and ribosomal protein S6 kinase (p70S6K) was determined in both cervical cancer tissues and paracancerous tissues. Next, alteration of miR-99b expression in cervical cancer was conducted to evaluate levels of PI3K, AKT, mTOR, p70S6K matrix metallopeptidase 2, epithelial cell adhesion molecule, and intercellular adhesion molecule 1, as well as the effect of miR-99b on cell proliferation, invasion, migration, cell cycle distribution, and apoptosis. The results demonstrated that miR-99b expression was decreased and levels of PI3K, AKT, mTOR, and p70S6K were elevated in cervical cancer tissues. More important, overexpressed miR-99b repressed the PI3K/AKT/mTOR signaling pathway, inhibited cell proliferation, invasion, and migration, blocked cell cycle entry, and promoted apoptosis in cervical cancer. These results indicate that miR-99b attenuates the migration and invasion of human cervical cancer cells through downregulation of the PI3K/AKT/mTOR signaling pathway, which provides a therapeutic approach for cervical cancer treatment.     

Nobiletin, a citrus flavonoid, suppresses invasion and migration involving FAK/PI3K/Akt and small GTPase signals in human gastric adenocarcinoma AGS cells

Nobiletin, a compound isolated from citrus fruits, is a polymethoxylated flavone derivative shown to have anti-inflammatory, antitumor, and neuroprotective properties. This study has investigated that nobiletin exerted inhibitory effects on the cell adhesion, invasion, and migration abilities of a highly metastatic AGS cells under non-cytotoxic concentrations. Data also showed nobiletin could inhibit the activation of focal adhesion kinase (FAK) and phosphoinositide-3-kinase/Akt (PI3K/Akt) involved in the downregulation of the enzyme activities, protein expressions, messenger RNA levels of matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-2 (MMP-9). Also, our data revealed that nobiletin inhibited FAK/PI3K/Akt with concurrent reduction in the protein expressions of Ras, c-Raf, Rac-1, Cdc42, and RhoA by western blotting, whereas the protein level of RhoB increased progressively. Otherwise, nobiletin-treated AGS cells showed tremendously decreased in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, nobiletin significantly decreased the levels of phospho-Akt and MMP-2/9 in Akt1-cDNA-transfected cells concomitantly with a marked reduction in cell invasion and migration. These results suggest that nobiletin can reduce invasion and migration of AGS cells, and such a characteristic may be of great value in the development of a potential cancer therapy.     

MiR-330-Mediated Regulation of SH3GL2 Expression Enhances Malignant Behaviors of Glioblastoma Stem Cells by Activating ERK and PI3K/AKT Signaling Pathways

MicroRNAs are currently considered as an active and rapidly evolving area for the treatment of tumors. In this study, we elucidated the biological significance of miR-330 in glioblastoma stem cells (GSCs) as well as the possible molecular mechanisms. SH3GL2 is mainly distributed in the central nervous system and considered to be a tumor suppressor in many tumors. In the present study, we identified miR-330 as a potential regulator of SH3GL2 and we found that it was to be inversely correlated with SH3GL2 expression in GSCs which were isolated from U87 cell lines. The expression of miR-330 enhanced cellular proliferation, promoted cell migration and invasion, and dampened cell apoptosis. When the GSCs were co-transfected with the plasmid containing short hairpin RNA directed against human SH3GL2 gene and miR-330 mimic, we found that miR-330 promoted the malignant behavior of GSCs by down-regulating the expression of SH3GL2. Meanwhile, the ERK and PI3K/AKT signaling pathways were significantly activated, leading to the decreased expression of apoptotic protein and increased expression of anti-apoptotic protein. Furthermore, in orthotopic mouse xenografts, the mice given stable over-expressed SH3GL2 cells co-transfected with miR-330 knockdown plasmid had the smallest tumor sizes and longest survival. In conclusion, these results suggested that miR-330 negatively regulated the expression of SH3GL2 in GSCs, which promoted the oncogenic progression of GSCs through activating ERK and PI3K/AKT signaling pathways. The elucidation of these mechanisms will provide potential therapeutic approaches for human glioblastoma.     

LncRNA MYU对胶质瘤细胞周期分布、增殖、转移和凋亡以及PI3K/Akt信号通路的影响

摘要 目的：探讨长链非编码RNA（LncRNA）MYU对胶质瘤细胞周期分布、细胞增殖、迁移、侵袭和凋亡的影响,并初步探讨其作用机制。方法：实时荧光定量PCR（RT-qPCR）检测人脑正常胶质细胞HEB和胶质瘤细胞（U-251MG、A172、SHG139）中LncRNA MYU的表达情况。选取SHG139细胞,分为正常对照（NC）组、si-con组、si-LncRNA MYU组进行转染实验,行RT-qPCR检测转染效果。分别采用流式细胞术、细胞计数试剂盒（CCK-8）、Transwell实验检测沉默LncRNA MYU对SHG139细胞周期分布和凋亡、细胞增殖、细胞迁移和侵袭的影响。蛋白免疫印迹（Western blot）法检测基质金属蛋白酶2（MMP-2）、MMP-9、裂解的半胱氨酸天冬氨酸蛋白酶3（Cleaved caspase-3）、Cleaved caspase-9以及磷脂酰肌醇-3-羟激酶/蛋白激酶B（PI3K/Akt）信号通路相关蛋白表达情况。结果：LncRNA MYU在胶质瘤细胞株中比人脑正常胶质细胞中的表达水平显著升高（P＜0.05）,因此选择表达量最高的SHG139细胞进行转染实验。沉默LncRNA MYU能够显著诱导SHG139细胞G0-G1期阻滞、抑制细胞增殖、迁移和侵袭并诱导细胞凋亡（P＜0.05）。沉默LncRNA MYU可显著抑制MMP-2、MMP-9、p-PI3K和p-AKT表达并促进Cleaved caspase-3、Cleaved caspase-9表达（P＜0.05）。结论：沉默LncRNA MYU可诱导胶质瘤细胞G0-G1期阻滞,抑制细胞增殖、迁移和侵袭,促进细胞凋亡,其机制可能与抑制PI3K/AKT信号通路有关。