The Dexamethasone-induced Inhibition of Proliferation, Migration, and Invasion in Glioma Cell Lines Is Antagonized by Macrophage Migration Inhibitory Factor (MIF) and Can Be Enhanced by Specific MIF Inhibitors

Glioblastomas (GBMs) are the most frequent and malignant brain tumors in adults. Glucocorticoids (GCs) are routinely used in the treatment of GBMs for their capacity to reduce the tumor-associated edema. Few in vitro studies have suggested that GCs inhibit the migration and invasion of GBM cells through the induction of MAPK phosphatase 1 (MKP-1). Macrophage migration inhibitory factor (MIF), an endogenous GC antagonist is up-regulated in GBMs. Recently, MIF has been involved in tumor growth and migration/invasion and specific MIF inhibitors have been developed on their capacity to block its enzymatic tautomerase activity site. In this study, we characterized several glioma cell lines for their MIF production. U373 MG cells were selected for their very low endogenous levels of MIF. We showed that dexamethasone inhibits the migration and invasion of U373 MG cells, through a glucocorticoid receptor (GR)- dependent inhibition of the ERK1/2 MAPK pathway. Oppositely, we found that exogenous MIF increases U373 MG migration and invasion through the stimulation of the ERK1/2 MAP kinase pathway and that this activation is CD74 independent. Finally, we used the Hs 683 glioma cells that are resistant to GCs and produce high levels of endogenous MIF, and showed that the specific MIF inhibitor ISO-1 could restore dexamethasone sensitivity in these cells. Collectively, our results indicate an intricate pathway between MIF expression and GC resistance. They suggest that MIF inhibitors could increase the response of GBMs to corticotherapy.     

LncRNA-ATB promotes TGF-β-induced glioma cells invasion through NF-κB and P38/MAPK pathway

Glioma constitutes the most aggressive primary intracranial malignancy in adults. We previously showed that long noncoding RNA activated by TGF-β (lncRNA-ATB) promoted the glioma cells invasion. However, whether lncRNA-ATB is involved in TGF-β-mediated invasion of glioma cells remains unknown. In this study, quantitative real-time polymerase chain reaction and western blot analysis were used for detecting the mRNA and protein expression of related genes, respectively. Transwell assay was performed to assess the impact of lncRNA-ATB on TGF-β-induced glioma cells migration and invasion. Immunofluorescence staining was utilized to characterize related protein distribution. Results showed that TGF-β upregulated lncRNA-ATB expression in glioma LN-18 and U251 cells. Overexpression of lncRNA-ATB activated nuclear factor-κB (NF-κB) pathway and promoted P65 translocation into the nucleus, thus facilitated glioma cells invasion stimulated by TGF-β. Similarly, lncRNA-ATB markedly enhanced TGF-β-mediated invasion of glioma cells through activation P38 mitogen-activated protein kinase (P38/MAPK) pathway. Moreover, both the NF-κB selected inhibitor pyrrolidinedithiocarbamate ammonium and P38/MAPK specific inhibitor SB203580 partly reversed lncRNA-ATB induced glioma cells invasion mediated by TGF-β. Collectively, this study revealed that lncRNA-ATB promotes TGF-β-induced glioma cell invasion through NF-κB and P38/MAPK pathway and established a detailed framework for understanding the way how lncRNA-ATB performs its function in TGF-β-mediated glioma invasion.     

Curcumin inhibits adverse psychological stress-induced proliferation and invasion of glioma cells via down-regulating the ERK/MAPK pathway

Curcumin is a natural polyphenol extracted from the rhizome of Curcuma that has an important antitumour effect, but its effect on adverse psychological stress-induced tumour proliferation and invasion has not been reported to date. Here, we found that curcumin not only inhibited the growth of xenografts in chronically stressed nude mice, but also decreased the expression of matrix metalloproteinase (MMP)-2/9 and CD147 in tumour tissues. Exogenous norepinephrine (NE) was used to stimulate glioma cells to simulate the stress environment in vitro, and it was found that curcumin inhibited the NE-induced proliferation and invasion of glioma cells in a dose-dependent manner. Further research found that the effects of NE on glioma cells could lead to the activation of the mitogen-activated protein kinase (MAPK) signalling pathway through β-adrenergic receptor, while curcumin suppressed the level of extracellular signal–regulated kinase (ERK)1/2 phosphorylation. In addition, blocking ERK1/2 expression with U0126 resulted in the down-regulated expression of CD147, which further led to the decreased expression of MMP-2 and MMP-9. Curcumin could also inhibit the expression of cyclin D1/CDK4/6 and anti-apoptotic protein Bcl-2/Bcl-XL induced by NE, and induced cell cycle changes and increased apoptosis. Therefore, curcumin may be a potential candidate drug for preventing and treating the progression of glioma induced by adverse psychological stress.     

Claudin-7 inhibits human lung cancer cell migration and invasion through ERK/MAPK signaling pathway

Tight junctions are the most apical component of the junctional complex critical for epithelial cell barrier and polarity functions. Although its disruption is well documented during cancer progression such as epithelial–mesenchymal transition, molecular mechanisms by which tight junction integral membrane protein claudins affect this process remain largely unknown. In this report, we found that claudin-7 was normally expressed in bronchial epithelial cells of human lungs but was either downregulated or disrupted in its distribution pattern in lung cancer. To investigate the function of claudin-7 in lung cancer cells, we transfected claudin-7 cDNA into NCI-H1299, a human lung carcinoma cell line that has no detectable claudin-7 expression. We found that claudin-7 expressing cells showed a reduced response to hepatocyte growth factor (HGF) treatment, were less motile, and formed fewer foot processes than the control cells did. In addition, cells transfected with claudin-7 dramatically decreased their invasive ability after HGF treatment. These effects were mediated through the MAPK signaling pathway since the phosphorylation level of ERK1/2 was significantly lower in claudin-7 transfected cells than in control cells. PD98059, a selective inhibitor of ERK/MAPK pathway, was able to block the motile effect. Claudin-7 formed stable complexes with claudin-1 and -3 and was able to recruit them to the cell-cell junction area in claudin-7 transfected cells. When control and claudin-7 transfected cells were inoculated into nude mice, claudin-7 expressing cells produced smaller tumors than the control cells. Taken together, our study demonstrates that claudin-7 inhibits cell migration and invasion through ERK/MAPK signaling pathway in response to growth factor stimulation in human lung cancer cells.     

MEK/ERK-dependent uPAR expression is required for motility via phosphorylation of P70S6K in human hepatocarcinoma cells

Motility and invasiveness events require specific intracellular signaling cascade activations. In cancer liver cells, one of these mechanisms could involve the MAPK MEK/ERK cascade activation which has been shown over expressed and activated in hepatocellular carcinoma. To study whether the MEK/ERK cascade is involved in the motility of HCC, we examined the effect of MEK inhibitor and ERK2 silencing using monolayer wound-healing assays and fluoroblock invasion systems. Evidence was provided that the MAPK cascade is a key transduction pathway which controls HCC cells motility and invasiveness. We could disconnect proliferation to motility using mitomycin C and we established that RNAi-mediated inhibition of ERK2 led to strongly reduced cell motility. To improve our understanding, we analysed the regulation and the role of urokinase receptor (uPAR) in this process. We provided evidence that uPAR was under a MEK/ERK dependent mechanism and blocking uPAR activity using specific antagonist or inhibiting its expression by RNA interference which resulted in complete inhibition of motility. Moreover, we found in MAPK inhibited cultures and in uPAR silencing cells that p70S6K phosphorylation on residue Thr-389 was significantly reduced, whereas Ser-421/Thr-424 phosphorylation did not change. We highlighted that the FRAP/mTOR pathway did not affect motility and Thr-389 phosphorylation. Furthermore, we demonstrated that p70S6K inhibition by RNA interference completely inhibited hepatocarcinoma cell motility. Therefore, targeting uPAR and/or MEK/ERK/S6K by RNA interference could be a major therapeutic strategy for the future treatment of invasive hepatocarcinoma cells.     

miR-491-5p靶向调控SHOC2对食管鳞癌细胞增殖、迁移及侵袭的影响

目的探讨miR-491-5p对食管鳞癌细胞增殖、迁移及侵袭的影响及作用机制。 方法培养永生化食管上皮细胞株HET-1A和人食管癌细胞株EC109,EC9706,KYSE510,qRT-PCR检测细胞中miR-491-5p和富含亮氨酸重复蛋白SHOC2 （SHOC2） mRNA水平。EC109细胞分为空白对照组、miR- 491-5p组、miR-NC组、miR-491-5p+pcDNA-SHOC2组和miR-491-5p+pcDNA组,MTT检测细胞增殖,Transwell检测细胞迁移和侵袭,Western Blot法检测CyclinD1、Vimentin、E-cadherin以及MAPK/ERK信号通路相关蛋白水平。双荧光素酶报告基因实验验证miR- 491- 5p与SHOC2之间调控关系。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,两两比较采用SNK-q检验。 结果食管鳞癌细胞EC109、EC9706和KYSE510中miR-491-5p表达水平低于HET-1A细胞（0.32±0.06、0.62±0.10、0.61±0.08比1.00±0.08）,差异具有统计学意义（F = 106.340,P < 0.001）;SHOC2 mRNA表达水平高于HET- 1A细胞（2.85±0.16、1.73±0.10、1.45±0.06比1.02±0.09）,差异具有统计学意义（F = 464.949,P < 0.001）。miR-491-5p组EC109细胞培养72 h后的OD值、细胞迁移数、侵袭数及CyclinD1、Vimentin、p-MEK和p-ERK蛋白水平均低于miR-NC组（0.70±0.06比1.42±0.08,65.01±10.36比150.01±12.48,70.03±10.26比140.02±11.85,0.30±0.03比0.93±0.16,0.41±0.05比0.86±0.08,0.32±0.06比0.95±0.11,0.40±0.06比0.92±0.13）,差异具有统计学意义（F = 236.565、159.440、120.706、101.071、98.619、130.766、77.046,P均< 0.001）,E-cadherin蛋白水平高于miR-NC组（0.89±0.13比0.48±0.08）,差异具有统计学意义（F = 816.432,P < 0.001）。miR-491-5p在EC109细胞中负调控SHOC2表达,SHOC2过表达逆转了miR-491-5p过表达对EC109细胞增殖、迁移和侵袭及MAPK/ERK信号通路的影响。 结论miR-491-5p可抑制食管鳞癌细胞的增殖、迁移和侵袭,其作用机制可能与下调SHOC2表达抑制MAPK/ERK信号通路活性有关。     

Fisetin Inhibits Migration and Invasion of Human Cervical Cancer Cells by Down-Regulating Urokinase Plasminogen Activator Expression through Suppressing the p38 MAPK-Dependent NF-κB Signaling Pathway

Fisetin (3,3’,4’,7-tetrahydroxyflavone), a naturally occurring flavonoid, has been reported to inhibit proliferation and induce apoptosis in several cancer types. However, its effect on the anti-metastatic potential of cervical cancer cells remains unclear. In the present study, we found that fisetin inhibits the invasion and migration of cervical cancer cells. The expression and activity of urokinase plasminogen activator (uPA) was significantly suppressed by fisetin in a dose-dependent manner. We also demonstrated that fisetin reduces the phosphorylation of p38 MAPK, but not that of ERK1/2, JNK1/2, or AKT. Addition of a p38 MAPK inhibitor, SB203580, further enhanced the inhibitory effect of fisetin on the expression and activity of uPA and the invasion and motility in cervical cancer cells. Fisetin suppressed the TPA (tetradecanoylphorbol-13-acetate)-induced activation of p38 MAPK and uPA, and inhibited the TPA-enhanced migratory and invasive abilities. Furthermore, the promoter activity of the uPA gene was dramatically repressed by fisetin, which disrupted the nuclear translocation of NF-κB and its binding amount on the promoter of the uPA gene, and these suppressive effects could be further enhanced by SB203580. This study provides strong evidence for the molecular mechanism of fisetin in inhibiting the aggressive phenotypes by repression of uPA via interruption of p38 MAPK-dependent NF-κB signaling pathway in cervical cancer cells and thus contributes insight to the potential of using fisetin as a therapeutic strategy against cervical cancer by inhibiting migration and invasion.     

Erlotinib inhibits colon cancer metastasis through inactivation of TrkB-dependent ERK signaling pathway

The distal metastasis is the main cause of death in patients with colon cancer. Tyrosine receptor kinase B (TrkB) and ERK signals may be the potential targets for the treatment of colon cancer metastasis. This study aims to investigate whether erlotinib inhibits distant metastasis of colon cancer by regulating TrkB and ERK signaling pathway. Human colon adenocarcinoma cell lines (SW480 and Caco-2) pretreated with exogenous C-X-C motif chemokine ligand 8 (CXCL8) were used to assess the suppressive effect of erlotinib on tumor metastasis, including anoikis, epithelial-mesenchymal transformation (EMT), migration, and invasion. Through TrkB overexpression, Akt suppression, and ERK suppression, the roles of TrkB, Akt, and ERK in erlotinib-induced metastasis inhibition of colon cancer cells were explored. The results showed that erlotinib alleviated CXCL8-induced metastasis of the colon cancer cells. Overexpression of TrkB in colon cancer cells eliminated the effect of erlotinib on anoikis, inhibition of EMT, migration, and invasion, and downregulation of p-ERK and p-Akt. Furthermore, the inhibition of ERK activation instead of Akt activation was found to participate in erlotinib-mediated metastasis resistance, including anoikis, inhibition of EMT, migration, and invasion. In conclusion, erlotinib inhibits colon cancer cell anoikis resistance, EMT, migration, and invasion by inactivating TrkB-dependent ERK signaling pathway.     

Suppression of FAM83D Inhibits Glioma Proliferation,Invasion and Migration by Regulating the AKT/mTOR Signaling Pathway

ObjectiveTo explore the mechanism by which the family with sequence similarity 83, member D (FAM83D)-mediated AKT/mTOR signaling pathway activation affects the proliferation and metastasis of glioma cells.MethodsFAM83D protein expression in glioma cells and tissues was detected by western blotting. Glioma U87 and U251 cells were selected and divided into the Mock, siNC, siFAM83D, FAM83D, MK2206 and FAM83D + MK2206 groups. Cell proliferation was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and clone formation assays, while invasion and migration were evaluated by Transwell assays and wound healing tests. The protein expression of members of the AKT/mTOR pathway was determined via western blotting. Xenograft models were also established in nude mice to observe the in vivo effect of FAM83D on the growth of glioma.ResultsFAM83D was upregulated in glioma patients, especially in those with Stage III-IV. In addition, cells treated with siFAM83D had significant downregulation of p-AKT/AKT and p-mTOR/mTOR, with decreased proliferation and colony numbers, as well as decreased invasion and migration compared to the Mock group. However, FAM83D overexpression could activate the Akt/mTOR pathway and promote the proliferation, invasion and migration of glioma cells. Moreover, treatment with MK2206, an inhibitor of AKT, reversed the promoting effect of FAM83D on the growth of glioma cells. The in vivo experiments demonstrated that silencing FAM83D could inhibit the in vivo growth of glioma cellsConclusionFAM83D was upregulated in glioma and silencing FAM83D suppressed the proliferation, invasion and migration of glioma cells via inhibition of the AKT/mTOR pathway.     

Advanced glycation end products promote triple negative breast cancer cells via ERK and NF-κB pathway

Advanced glycation end products (AGEs) are harmful compounds generated by nonspecific glycation of proteins and lipids. The accumulation of AGEs is associated with various diseases, including breast cancer. AGEs have been shown to promote a breast cancer cell line by enhancing proliferation, invasion and migration. In this study, we investigated the effect and associated mechanism of AGEs on triple negative breast cancer cells. AGEs enhanced the proliferation, tumorigenicity, invasion and migration of primary breast cancer cells. AGEs also enhanced the RNA and protein expression of matrix metalloproteinase (MMP)-9 and its gelatinase activity. Enhanced MMP-9 expression was mediated by extracellular-signal regulated kinase (ERK) and nuclear factor kappa B (NF-κB) pathways. Moreover, inhibitors of ERK and NF-κB signaling attenuated the effect of AGEs on tumorigenicity, invasion and migration of primary breast cancer cells. Taken together, we suggest that AGEs directly promote primary breast cancer cells via the ERK and NF-κB pathway, which may lead to advanced therapeutic modalities of breast cancer.     

Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI-A down regulates c-myc gene expression and endothelial cell proliferation.

Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation.     

Epstein-Barr virus latent membrane protein 2A regulates c-Jun protein through extracellular signal-regulated kinase

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in both EBV-infected cells and EBV-associated malignancies. However, the function of LMP2A is still veiled. In this study, LMP2A was found to induce the kinase activities of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase JNK/SAPK. Furthermore, the downstream effector c-Jun showed hyperphosphorylation under LMP2A expression. The phosphorylation could be inhibited by the ERK pathway inhibitor PD98059, indicating that ERK may contribute to the phosphorylation of c-Jun in LMP2A-expressing cells. The impact on c-Jun phosphorylation by mitogen-activated protein kinase (MAPK) is suggested to increase c-Jun protein stability, and this was also observed in LMP2A-expressing cells by a protein synthesis inhibition assay. Moreover, LMP2A-induced cell invasion was inhibited in the presence of the ERK pathway inhibitor. Taken together, we suggest that LMP2A may exploit MAPK kinases and affect both the phosphorylation and stability of c-Jun protein. Additionally, LMP2A may thereby promote the mobility of the cells. In doing so, it may enhance the mobility of EBV-infected cells and contribute to the metastatic process of malignant cells. Here we demonstrated the first evidence of LMP2A-induced migration and the underlying pathways accounting for it.     

Anti-metastatic effects of ginsenoside Rd via inactivation of MAPK signaling and induction of focal adhesion formation

Ginsenoside Rd is a protopanaxadiol-type ginsenoside found in ginseng and is the active ingredient in several Oriental herbal medicines. We investigated the effects of ginsenoside Rd on tumor invasion and metastasis in the human hepatocellular carcinoma HepG2 and its possible mechanism of action. HepG2 cells were treated with ginsenoside Rd at different concentrations. Scratch wound and Boyden chamber assays were used to determine the effects of ginsenoside Rd on the migration and invasiveness of HepG2 cells, respectively. The molecular mechanisms by which ginsenoside Rd inhibited the invasion and migration of HepG2 cells were investigated by RT-PCR, Western blotting, gelatin zymography, promoter assay, and treatment with inhibitors of MAPK signaling. Immunofluorescence analysis was conducted to evaluate the effect of ginsenoside Rd on focal adhesion formation in HepG2 cells. Treatment with ginsenoside Rd dose- and time-dependently inhibited the migration and invasion of HepG2 cells. It achieved this by reducing the expression of MMP-1, MMP-2, and MMP-7, by blocking MAPK signaling by inhibiting the phosphorylation of ERK and p38 MAPK, by inhibition of AP-1 activation, and by inducing focal adhesion formation and modulating vinculin localization and expression. Treatment of HepG2 cells with ginsenoside Rd significantly inhibited metastasis, most likely by blocking MMP activation and MAPK signaling pathways involved in cancer cell migration. These findings may be useful for the development of novel chemotherapeutic agents for the treatment of malignant cancers.     

ERK5 and its role in tumour development

The MEK5 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 5]/ERK5 pathway is the least well studied MAPK signalling module. It has been proposed to play a role in the pathology of cancer. In the present paper, we review the role of the MEK5/ERK5 pathway using the 'hallmarks of cancer' as a framework and consider how this pathway is deregulated. As well as playing a key role in endothelial cell survival and tubular morphogenesis during tumour neovascularization, ERK5 is also emerging as a regulator of tumour cell invasion and migration. Several oncogenes can stimulate ERK5 activity, and protein levels are increased by a novel amplification at chromosome locus 17p11 and by down-regulation of the microRNAs miR-143 and miR-145. Together, these finding underscore the case for further investigation into understanding the role of ERK5 in cancer.     

LRRC4 inhibits human glioblastoma cells proliferation, invasion, and proMMP-2 activation by reducing SDF-1 alpha/CXCR4-mediated ERK1/2 and Akt signaling pathways

Gliomas take a number of different genetic routes in the progression to glioblastoma multiforme, a highly invasive variant that is mostly unresponsive to current therapies. The alpha-chemokine stromal cell-derived factor (SDF)-1 alpha binds to the seven transmembrane G-protein-coupled CXCR-4 receptor and acts to modulate cell migration and proliferation by activating multiple signal transduction pathways. Leucine-rich repeats containing 4 (LRRC4), a putative glioma suppressive gene, inhibits glioblastoma cells tumorigenesis in vivo and cell proliferation and invasion in vitro. We also previously demonstrated that LRRC4 controlled glioblastoma cells proliferation by ERK/AKT/NF-kappa B signaling pathway. In the present study, we demonstrate that CXC chemokine receptor 4 (CXCR4) is expressed in human glioblastoma U251 cell line, and that SDF-1 alpha increases the proliferation, chemotaxis, and invasion in CXCR4+ glioblastoma U251 cells through the activation of ERK1/2 and Akt. The reintroduction of LRRC4 in U251 cells inhibits the expression of CXCR4 and SDF-1 alpha/CXCR4 axis-mediated downstream intracellular pathways such as ERK1/2 and Akt leading to proliferate, chemotactic and invasive effects. Furthermore, we provide evidence for proMMP-2 activation involvement in the SDF-1 alpha/CXCR4 axis-mediated signaling pathway. LRRC4 significantly inhibits proMMP-2 activation by SDF-1 alpha/CXCR4 axis-mediated ERK1/2 and Akt signaling pathway. Collectively, these results suggest a possible important "cross-talk" between LRRC4 and SDF-1 alpha/CXCR4 axis-mediated intracellular pathways that can link signals of cell proliferation, chemotaxis and invasion in glioblastoma, and may represent a new target for development of new therapeutic strategies in glioma.     

Inhibiting of circ-TLK1 inhibits the progression of glioma through down-regulating PANX1 via targeting miR-17-5p

Glioma remains the most common malignant tumors in the central nervous system and often has poor prognosis. In recent years, it has been gradually revealed that non-coding RNA effects glioma progression. In this study, we aimed to investigate the significance of circular RNA TLK1 (Circ-TLK1) in predicting the survival of glioma patients as well as its role in glioma development via both in-vitro and in-vivo experiments. We found that Circ-TLK1 was conspicuously up-regulated in glioma tissues compared with adjacent normal tissues, and the up-regulated Circ-TLK1 was significantly correlated with glioma patients’ larger tumor volume and higher grades. Functionally, Circ-TLK1 over-expression facilitated glioma growth, migration and invasion, inhibited cell apoptosis, and accelerated PANX1/MAPK/ERK expression, while Circ-TLK1 low expression had the opposite effects. In addition, bioinformatics analysis showed that miR-17-5p was a potential target of Circ-TLK1 and targeted at PANX1. Furthermore, through dual luciferase viability assay, Circ-TLK1 acted as a competing endogenous RNA by sponging miR-17-5p, which targeted and inhibited PANX1/MAPK/ERK expression. MiR-17-5p overexpression mitigated glioma progression, which was significantly inhibited with Circ-TLK1 upregulation. In conclusion, this study confirmed a novel axis of Circ-TLK1-miR-17-5p-PANX1 in modulating glioma development, providing more references for glioma diagnosis and targeted therapy.

     

Gartanin induces cell cycle arrest and autophagy and suppresses migration involving PI3K/Akt/mTOR and MAPK signalling pathway in human glioma cells

In central nervous system, glioma is the most common primary brain tumour. The diffuse migration and rapid proliferation are main obstacles for successful treatment. Gartanin, a natural xanthone of mangosteen, suppressed proliferation, migration and colony formation in a time‐ and concentration‐dependent manner in T98G glioma cells but not in mouse normal neuronal HT22 cells. Gartanin, at low micromole, led to cell cycle arrest in G1 phase accompanied by inhibited expression level of G1 cell cycle regulatory proteins cyclin D1, while increased expression level of cyclin‐dependent kinase inhibitor p27Kip1. In addition, the secretion and activity of matrix metalloproteinases 2/9 (MMP‐2/‐9) were significantly suppressed in T98G cells treated with gartanin, and it might result from modulating mitogen‐activated protein kinases (MAPK) signalling pathway in T98G glioma cells. Moreover, gartanin significantly induced autophagy in T98G cells and increased GFP‐LC3 punctate fluorescence accompanied by the increased expression level of Beclin 1 and LC3‐II, while suppressed expression level of p62. Gartanin treatment resulted in obvious inhibition of PI3K/Akt/mTOR signalling pathway, which is important in modulating autophagy. Notably, gartanin‐mediated anti‐viability was significantly abrogated by autophagy inhibitors including 3‐methyladenine (3‐MA) and chloroquine (CQ). These results indicate that anti‐proliferation effect of gartanin in T98G cells is most likely via cell cycle arrest modulated by autophagy, which is regulated by PI3K/Akt/mTOR signalling pathway, while anti‐migration effect is most likely via suppression of MMP‐2/‐9 activity which is involved in MAPK signalling pathway.     

MicroRNA-145 Is Downregulated in Glial Tumors and Regulates Glioma Cell Migration by Targeting Connective Tissue Growth Factor

Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3′-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3′-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.