CircAFF2 Promotes Neuronal Cell Injury in Intracerebral Hemorrhage by Regulating the miR-488/CLSTN3 Axis.

BACKGROUND: Intracerebral hemorrhage (ICH), a subtype of devastating stroke, carries high morbidity and mortality worldwide. CircRNA AFF2 (circAFF2) was significantly increased in ICH patients, but the underlying mechanism of circAFF2 is unknown. METHODS: Hemin was employed to treat neuronal cells to mimic ICH in vitro. Mice were injected with collagenase VII-S to establish in vivo ICH models. Genes and protein expressions were detected using qRT-PCR and Western blotting. The interaction among circAFF2, miR-488, and CLSTN3 was validated by dual-luciferase reporter assay and RNA-RIP. Cell viability, MDA, iron, GSH, and lipid ROS were examined using the MTT, the commercial kits, and flow cytometry, respectively. ICH injury in mice was evaluated using neurological deficit scores and brain water measurements. RESULTS: CircAFF2 was significantly increased in ICH in vivo and in vitro models. CircAFF2 bound to miR-488 and knockdown of circAFF2 or overexpression of miR-488 inhibited hemin-induced injury of neuronal cells as indicated by increased cell viability and reduced markers of oxidative stress and lipid peroxidation. CLSTN3 was the downstream target of miR-488. Silencing of circAFF2 or miR-488 overexpression reduced CLSTN3 expression and protected against the injury of neuronal cells. In vivo experiments finally confirmed that circAFF2 knockdown attenuated mice ICH injury via the miR-488/CLSTN3 axis. CONCLUSION: CircAFF2 promotes the injury of neuronal cells and exacerbates ICH via increasing CLSTN3 by sponging miR-488, suggesting that circAFF2 may be a potential therapeutic target for ICH treatment.

Integrated profiling identifies CACNG3 as a prognostic biomarker for patients with glioma.

Gliomas are the most common malignant primary brain tumors in adults with poor prognoses. The purpose of this study is to explore CACNG3 as a prognostic factor that is closely related to the progression and survival outcome of gliomas and to provide a potential new molecular target for the diagnosis and treatment of glioma patients. CACNG3 expression and related clinical data were collected from three major databases of The Chinese Glioma Genome Atlas (CGGA), The Cancer Genome Atlas (TCGA), and Gene Expression Omnibus (GEO). The CGGA dataset was used as a training set, and TCGA and GEO datasets obtained from the GEO database were used for validation. CACNG3 was expressed at low levels in the tumor group, and the overall survival (OS) in patients with low CACNG3 expression is shorter. Furthermore, CACNG3 expression was negatively associated with glioma grades, which was confirmed in the IHC results of clinical samples. The expression level of CACNG3 in the IDH1 wide-type group, 1p/19q non-codel group, and mesenchymal subtype group was significantly reduced, and the results showed that CACNG3 could serve as a biomarker for the mesenchymal molecular subtype. In addition, the univariate and multivariate analysis verified the prognostic value of CACNG3 in predicting the OS of gliomas of all grades. The results of functional annotation and pathway enrichment analysis of differently expressed genes(DEGs), showed that CACNG3 might affect the development of glioma by interfering with synaptic transmission. Moreover, temozolomide (TMZ), commonly used in the treatment of glioma, increased CACNG3 expression in a dose and time-dependent manner. Therefore, CACNG3 plays a vital role in the occurrence and development of gliomas and can serve as a potential biomarker for targeted therapy and further investigation in the future.

IRAK4 exacerbates traumatic brain injury via activation of TAK1 signaling pathway.

Although multiple signaling pathways contributing to the pathophysiological process have been investigated, treatments for traumatic brain injury (TBI) against present targets have not acquired significant clinical progress. Interleukin-1 receptor-associated kinase 4 (IRAK4) is an important factor involved in regulating immunity and inflammation. However, the role of IRAK4 in TBI still remains largely unknown. Therefore, using a controlled cortical impact model (CCI), we investigated the function and molecular mechanism of IRAK4 in the context of TBI. IRAK4 was found to be activated in a time-dependent manner after TBI and mainly expressed in neurons. Inhibition of IRAK4 by siRNAs could significantly alleviates neuroinflammation, neuron apoptosis, brain edema, brain-blood barrier (BBB) dysfunction and improves neurological deficit in the context of CCI. Mechanistically, IRAK4 exacerbates CCI via activation of TAK1 signaling pathway. Interestingly, PF-0665083, an IRAK4 inhibitor, inhibits phosphorylation of IRAK4 and attenuates CCI-induced secondary injury. It could be conclude that IRAK4 plays a critical role in TBI-induced secondary injury and the underlining mechanism may be related to activation of TAK1 signaling pathway. PF-0665083 may serve as a potential treatment strategy to relieve TBI.

miR-720 is a key regulator of glioma migration and invasion by controlling TARSL2 expression.

Glioblastoma (GBM) is the most lethal type of primary brain tumor and is characterized by diffuse infiltrative growth. However, the mechanisms that control this phenotype remain largely unknown. Emerging evidence has demonstrated that the abnormal expression of microRNAs and their target genes are involved in the migration and invasion of glioma cells. In this study, we demonstrated that microRNA-720 (miR-720) was significantly upregulated in glioma tissues and cells. Functional experiments showed that overexpression of miR-720 promotes glioma migration and invasion, while downregulation of miR-720 inhibits glioma migration and invasion. Meanwhile, we found that threonyl-tRNA synthetase like-2 (TARSL2) was a direct and functional target of miR-720 in glioma. Reintroduction of TARSL2 into glioma cells repressed the invasion promoting function of miR-720, whereas downregulation of TARSL2 reversed the anti-invasion function of anti-miR-720. Furthermore, quantitative real-time polymerase chain reaction results showed that miR-720 was inversely correlated with TARSL2 expression in 40 GBM tissues. Finally, in vivo experiments showed that miR-720 promotes glioma growth and upregulates invasion-related genes in nude mice. Overall, our findings suggest increasing miR-720 enhances glioma migration and invasion through downregulation of TARSL2, which may provide novel insight into the treatment of glioma.

TGF-ß1 modulates temozolomide resistance in glioblastoma via altered microRNA processing and elevated MGMT.

BACKGROUND: Our previous studies have indicated that miR-198 reduces cellular methylguanine DNA methyltransferase (MGMT) levels to enhance temozolomide sensitivity. Transforming growth factor beta 1 (TGF-ß1) switches off miR-198 expression by repressing K-homology splicing regulatory protein (KSRP) expression in epidermal keratinocytes. However, the underlying role of TGF-ß1 in temozolomide resistance has remained unknown. METHODS: The distribution of KSRP was detected by western blotting and immunofluorescence. Microarray analysis was used to compare the levels of long noncoding RNAs (lncRNAs) between TGF-ß1-treated and untreated cells. RNA immunoprecipitation was performed to verify the relationship between RNAs and KSRP. Flow cytometry and orthotopic and subcutaneous xenograft tumor models were used to determine the function of TGF-ß1 in temozolomide resistance. RESULTS: Overexpression of TGF-ß1 contributed to temozolomide resistance in MGMT promoter hypomethylated glioblastoma cells in vitro and in vivo. TGF-ß1 treatment reduced cellular MGMT levels through suppressing the expression of miR-198. However, TGF-ß1 upregulation did not affect KSRP expression in glioma cells. We identified and characterized 2 lncRNAs (H19 and HOXD-AS2) that were upregulated by TGF-ß1 through Smad signaling. H19 and HOXD-AS2 exhibited competitive binding to KSRP and prevented KSRP from binding to primary miR-198, thus decreasing miR-198 expression. HOXD-AS2 or H19 upregulation strongly promoted temozolomide resistance and MGMT expression. Moreover, KSRP depletion abrogated the effects of TGF-ß1 and lncRNAs on miR-198 and MGMT. Finally, we found that patients with low levels of TGF-ß1 or lncRNA expression benefited from temozolomide therapy. CONCLUSIONS: Our results reveal an underlying mechanism by which TGF-ß1 confers temozolomide resistance. Furthermore, our findings suggest that a novel combination of temozolomide with a TGF-ß inhibitor may serve as an effective therapy for glioblastomas.

MicroRNA-206 attenuates glioma cell proliferation, migration, and invasion by blocking the WNT/ß-catenin pathway via direct targeting of Frizzled 7 mRNA.

Glioma is one of the most prevalent primary malignant brain tumours among adults, and accumulating evidence has shown that dysregulation of microRNAs (miRNAs) is associated with various types of cancers, including glioma. It is necessary to gain a better understanding of the roles and mechanisms of action of miRNAs in WNT-driven glioblastoma multiforme (GBM). Here, we report that miR-206 inhibits the WNT/ß-catenin pathway by directly targeting Frizzled 7 (FZD7) mRNA and functions as a tumour suppressor in glioma. The expression of miR-206 in human glioma samples and glioma cells was assessed by reverse-transcription quantitative PCR, fluorescence in situ hybridisation, and histological analysis. Cell Counting Kit-8, colony formation, 5-ethynyl-2'-deoxyuridine incorporation, flow-cytometric, wound healing, Transwell invasion, and three-dimensional migration assays were performed to examine glioma cell proliferation, migration, and invasion in vitro. The effects of miR-206 in vivo were investigated in a xenograft nude-mouse model. MiR-206 expression was significantly lower in glioma specimens than in normal control samples. FZD7 was confirmed as a direct target gene of miR-206. GBM cell proliferation, migration, and invasion were blocked after restoration of miR-206 expression. Moreover, intracranial glioma models revealed an inhibitory effect of miR-206 on intracranial glioma tumour growth. Our results suggest that miR-206 plays a key role in the blockade of the WNT/ß-catenin signalling pathway by down-regulating FZD7 and may be a promising therapeutic agent against malignant glioma and other WNT-driven tumours.

FRK suppresses human glioma growth by inhibiting ITGB1/FAK signaling.

Fyn-related kinase (FRK), a member of the Src-related tyrosine kinase family, functions as a tumor suppressor in several malignancies. We previously showed that FRK overexpression inhibited the growth of glioma cells. However, it is unknown whether FRK is equally effective against intracranial glioma in vivo, and the mechanism by which FRK influences glioma cell growth remains unclear. In this study, we found that tumor volume was reduced by about one-third in mice with FRK overexpression, which showed improved survival relative to controls. Immunofluorescence analysis revealed that FRK overexpression inhibited glioma cell proliferation and induced their apoptosis. Importantly, in vitro we further found that FRK decreased the expression of integrin subunit ß1 (ITGB1) at both the mRNA and protein levels. FRK also inhibited transactivation by ITGB1, resulting in the suppression of its target proteins AKT and focal adhesion kinase (FAK). ITGB1 overexpression promoted glioma cell growth and partially reduced FRK-induced growth suppression. These results indicate that FRK inhibits human glioma growth via regulating ITGB1/FAK signaling and provide a potential therapeutic target for the treatment of glioma.

ECT2/PSMD14/PTTG1 axis promotes the proliferation of glioma through stabilizing E2F1.

BACKGROUND: Epithelial cell transformation sequence 2 (ECT2) is upregulated in glioma and promotes glioma cell proliferation. A preliminary experiment showed a positive correlation between ECT2 and pituitary tumor-transforming gene 1 (PTTG1). The aim of this study was to explore how ECT2 affects PTTG1 to influence the proliferation of glioma cells. METHODS: The expression of ECT2 in glioma was detected by western blot and reverse transcription PCR. The effect of ECT2 on glioma proliferation was examined using cell proliferation-related assays and in vivo experiments. The effect of ECT2 on the stability of E2F transcription factor 1 (E2F1) and the expression of PTTG1 were examined by western blot, co-immunoprecipitation, and in vivo ubiquitination assays. RESULTS: ECT2 was upregulated in gliomas and was negatively correlated with prognosis; its downregulation inhibited glioma cell proliferation. Furthermore, ECT2 regulated PTTG1 expression by affecting the stability of E2F1, thereby affecting the glioma cell proliferation. In addition, the deubiquitinating enzyme proteasome 26S subunit, non-ATPase 14 (PSMD14) affected the degradation of E2F1 and regulated the stability of E2F1. Interestingly, ECT2 regulated the expression of PSMD14. CONCLUSION: In this study, we clarify a new mechanism by which ECT2 regulates the expression of PTTG1 and thus affects the proliferation of glioma cells: ECT2 influences the stability of E2F1 by regulating the expression of the deubiquitinating enzyme PSMD14, thereby affecting the expression of PTTG1. Intensive and extensive understanding of the mechanism of ECT2 in glioma proliferation may provide an opportunity for the development of new molecular therapeutic targets for glioma treatment.

Fstl1/DIP2A/MGMT signaling pathway plays important roles in temozolomide resistance in glioblastoma.

Temozolomide was recognized as the first-line therapy for glioblastoma to prolong the survival of patients noticeably, while recent clinical studies found that some patients were not sensitive to temozolomide treatment. The possible mechanisms seemed to be methylguanine-DNA-methyltransferase (MGMT), mismatch repair, PARP, etc. And the abnormal expression of MGMT might be the most direct factor. In this study, we provide evidence that Fstl1 plays a vital role in temozolomide resistance by sequentially regulating DIP2A protein distribution, H3K9 acetylation (H3K9Ac), and MGMT transcription. As a multifunctional protein widely distributed in cells, DIP2A cooperates with the HDAC2-DMAP1 complex to enhance H3K9Ac deacetylation, prevent MGMT transcription, and increase temozolomide sensitivity. Fstl1, a glycoprotein highly expressed in glioblastoma, competitively binds DIP2A to block DIP2A nuclear translocation, so as to hinder DIP2A from binding the HDAC2-DMAP1 complex. The overexpression of Fstl1 promoted the expression of MGMT in association with increased promoter H3K9Ac. Upregulation of Fstl1 enhanced temozolomide resistance, whereas Fstl1 silencing obviously sensitized GBM cells to temozolomide both in vivo and in vitro. Moreover, DIP2A depletion abolished the effects of Fstl1 on MGMT expression and temozolomide resistance. These findings highlight an important role of Fstl1 in the regulation of temozolomide resistance by modulation of DIP2A/MGMT signaling.

PBX3/MEK/ERK1/2/LIN28/let-7b positive feedback loop enhances mesenchymal phenotype to promote glioblastoma migration and invasion.

BACKGROUND: Brain invasion by glioblastoma (GBM) determines recurrence and prognosis in patients, which is, in part, attributed to increased mesenchymal transition. Here, we report evidence favoring such a role for the Pre-B-cell leukemia homebox (PBX) family member PBX3. METHODS: Western blot, immunohistochemistry, qRT-PCR and datasets mining were used to determined proteins or genes expression levels. Wound-healing and transwell assays were used to examine the invasive abilities of GBM cells. Dual-luciferase reporter assays were used to determine how let-7b regulates PBX3. Chromatin-immunoprecipitation (ChIP) and rescue experiments were performed to investigate the involved molecular mechanisms. Orthotopic mouse models were used to assess the role of PBX3 in vivo. RESULTS: We found that PBX3 expression levels positively correlated with glioma mesenchymal markers. Ectopic expression of PBX3 promoted invasive phenotypes and triggered the expression of mesenchymal markers, whereas depletion of PBX3 reduced GBM cell invasive abilities and decreased the expression of mesenchymal markers. In addition, inhibition of PBX3 attenuated transforming growth factor-ß (TGFß)-induced GBM mesenchymal transition. Mechanistic studies revealed that PBX3 mediated GBM mesenchymal transition through activation of MEK/ERK1/2, leading to increased expression of LIN28 by c-myc. Increased LIN28 inhibited let-7b biogenesis, which then promoted the pro-invasive genes, such as HMGA2 and IL-6. Furthermore, let-7b suppressed PBX3 by directly targeting 3'-UTR of PBX3. Thus, repressed let-7b by PBX3 amplifies PBX3 signaling and forms a positive feedback loop to promote GBM mesenchymal transition. CONCLUSIONS: These data highlight the importance of PBX3 as a key driver of mesenchymal transition and potential therapeutic target.

Delivery of MGMT mRNA to glioma cells by reactive astrocyte-derived exosomes confers a temozolomide resistance phenotype.

The glioma-astrocyte interaction plays an important role in tumor microenvironment remodeling; however, the underlying mechanism has not been completely clarified. In this study, we show that glioma cells stimulate normal human astrocyte (NHA) into reactive astrocyte (RAS) in a non-contact manner. Additionally, the amount of O6-alkylguanine DNA alkyltransferase (MGMT) mRNA in exosomes (EXOs) released by RAS was significantly increased compared with that in non-reactive NHA. Importantly, MGMT-negative glioma cells can take up RAS-EXOs and acquire a temozolomide (TMZ)-resistant phenotype via the translation of exogenous exosomal MGMT mRNA both in vitro and in vivo. Our findings illuminate a novel phenomenon that may be a potent mechanism underlying glioma recurrence in which glioma-associated NHAs protect MGMT-negative glioma cells from TMZ-induced apoptosis by the functional intercellular transfer of exosomal MGMT mRNA.

ERK1/2/mTOR/Stat3 pathway-mediated autophagy alleviates traumatic brain injury-induced acute lung injury.

Acute lung injury (ALI) is one of several complications in patients with traumatic brain injury (TBI). Autophagy is a primary homeostatic process that promotes cell survival under stress. Accumulating evidence implicates autophagy in the pathogenesis of ALI under various conditions. However, the role of autophagy in TBI-induced ALI remains unknown. The aim of this study was to adjust autophagy with pharmacological agents to determine its functional significance in TBI-induced ALI. Rats were preconditioned with autophagy promoter rapamycin or inhibitor 3-methyladenine before they were challenged with TBI. Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126, mechanistic target of rapamycin (mTOR) inhibitor rapamycin, and signal transducer and activator of transcription 3 (Stat3) inhibitor S31-201 were used to test the role of ERK1/2/mTOR/Stat3 signaling pathway in regulating autophagy. Autophagy is activated in lung tissues after TBI. Enhancement of autophagy suppressed apoptosis, inflammation and oxidative stress in lung tissues, which were activated after TBI, whereas inhibition of autophagy aggravated these critical pathological changes. Autophagy also improved TBI-induced impairment in pulmonary barrier function, oxygenation function and static compliance. Furthermore, TBI-induced autophagy was mediated by ERK1/2/mTOR/Stat3 pathway, which may serve to reduce ALI and improve pulmonary barrier function, oxygenation function and static compliance. These findings are important for the prevention and treatment of TBI-induced ALI.

MicroRNA-520d-5p inhibits human glioma cell proliferation and induces cell cycle arrest by directly targeting PTTG1.

Glioma accounts for the majority of primary malignant brain tumors in adults and is highly aggressive. Although various therapeutic approaches have been applied, outcomes of glioma treatment remain poor. Acquiring a better understanding of the pathogenic mechanisms is essential to the design of effective therapeutic strategies. Previous studies have found that miR-520d-5p was negatively correlated with glioma grade, but its role and mechanism in glioma progression remain largely unknown. In the present study, we reported that miR-520d-5p directly targeted the Pituitary Tumor Transforming Gene 1 (PTTG1) and functioned as a tumor-suppressor in glioma. The expression of miR-520d-5p in glioma cells and specimens were detected by Quantitative reverse transcription-PCR and Fluorescence in situ hybridization (FISH). The effects of miR-520d-5p on glioma progression was examined by cell-counting kit 8, colony formation, 5-ethynyl-2-deoxyuridine (EDU) and flow cytometry assays. Using bioinformatics and luciferase reporter assays, we identified PTTG1 as a novel and direct target of miR-520d-3p. A xenograft model was used to study the effect of miR-520d-5p on tumor growth and angiogenesis. We found that miR-520d-5p expression was significantly decreased in glioma cell lines and tissues. Overexpression of miR-520d-5p showed a significant inhibitory effect on cell proliferation and accompanied cell cycle G0/G1 arrest in U87-MG and LN229 glioma cells. PTTG1 was a novel and direct target of miR-520d-5p, and the protein expression of PTTG1 was markedly reduced after overexpression of miR-520d-5p in U87-MG and LN229 cells. Overexpression of PTTG1 reversed the inhibitory effect of miR-520d-5p on glioma cell proliferation. In vivo studies confirmed that miR-520d-5p overexpression retarded the growth of U87 xenograft tumors, which was accompanied by reduced expression of PTTG1. In conclusion, these results provide compelling evidence that miR-520d-5p functions as an anti-onco-miRNA, which is important in inhibiting cell proliferation in GBM, and its anti-oncogenic effects are mediated chiefly through direct suppression of PTTG1 expression. Therefore, we suggest that miR-520d-5p is a potential candidate for the prevention of glioblastoma.

MicroRNA-936 induces cell cycle arrest and inhibits glioma cell proliferation by targeting CKS1.

Accumulating evidence shows that microRNAs play important roles in cancers, including glioma. MiRNAs have been shown to participate in a variety of cellular functions including cell apoptosis, cell proliferation, neural development, and stem cell differentiation. Previous studies reported that miR-936 levels were downregulated in glioma specimens. Here, we further investigate the potential role of miR-936 in glioma. Quantitative reverse transcription-PCR was applied to detect the expression of mir-936 in glioma specimens. The direct targets of miR-936 were identified by bioinformatics analysis and were further validated by immunoblotting and luciferase reporter assay. The effects of miR-936 on glioma cell proliferation and cell cycle of glioma cells were analyzed by Cell-Counting Kit 8 assay, colony formation, 5-ethynyl-2-deoxyuridine (EDU) and flow cytometry assays. A xenograft model was used to study the effect of miR-936 on tumor growth and angiogenesis. Expression levels of miR-936 were greatly downregulated in glioma specimens, CKS1 was confirmed as a direct target of miR-936. The glioma cell cycle was blocked to G1 by negatively regulating CKS1 and its downstream signaling pathway, Akt-ERK1/2. Furthermore, overexpression of CKS1 rescued the inhibitory effects of miR-936. In vivo studies revealed that increased levels of miR-936 delayed the growth of tumors. Taken together, mir-936 may act as a glioma suppressor by targeting CKS1.

Fstl1 Promotes Glioma Growth Through the BMP4/Smad1/5/8 Signaling Pathway.

BACKGROUND: Gliomas result in the highest morbidity and mortality rates of intracranial primary central nervous system tumors because of their aggressive growth characteristics and high postoperative recurrence. They are characterized by genetic instability, intratumoral histopathological variability and unpredictable clinical behavior in patients. Proliferation is a key aspect of the clinical progression of malignant gliomas, complicating complete surgical resection and enabling tumor regrowth and further proliferation of the surviving tumor cells. METHODS: The expression of Fstl1 was detected by western blotting and qRT-PCR. We used cell proliferation and colony formation assays to measure proliferation. Then, flow cytometry was used to analyze cell cycle progression. The expression of Fstl1, p-Smad1/5/8 and p21 in GBM tissue sections was evaluated using immunohistochemical staining. Furthermore, we used coimmunoprecipitation (Co-IP) and immunoprecipitation to validate the relationship between Fstl1, BMP4 and BMPR2. Finally, we used orthotopic xenograft studies to measure the growth of tumors in vivo. RESULTS: We found that follistatin-like 1 (Fstl1) was upregulated in high-grade glioma specimens and that its levels correlated with poor prognosis. Fstl1 upregulation increased cell proliferation, colony formation and cell cycle progression, while its knockdown inhibited these processes. Moreover, Fstl1 interacted with bone morphogenetic protein (BMP) 4, but not BMP receptor (BMPR) 2, and competitively inhibited their association. Furthermore, Fstl1 overexpression suppressed the activation of the BMP4/Smad1/5/8 signaling pathway, while BMP4 overexpression reversed this effect. CONCLUSION: Our study demonstrated that Fstl1 promoted glioma growth through the BMP4/Smad1/5/8 signaling pathway, and these findings suggest potential new glioblastoma treatment strategies.

EZH2 alteration driven by microRNA-524-5p and microRNA-324-5p promotes cell proliferation and temozolomide resistance in glioma.

Recent data have been shown that EZH2 is a critical oncogene via the repression of tumor suppressor genes in human cancers. In our study, we performed a genome-wide miRNA screen with a bioinformatics analysis to identify EZH2 specific miRNAs. Of these miRNAs, miR-524-5p and miR-324-5p were decreased in glioma tissues, and confered poor prognosis for glioma patients. Upregulation of miR-524-5p and miR-324-5p reduced glioma cell proliferation and increased temozolomide (TMZ) chemosensitivity by targeting EZH2. Importantly, the effection of miR-524-5p and miR-324-5p on cell proliferation and TMZ chemosensitivity in glioma were reversed by expression of EZH2 cDNA. Further, miR-524-5p and miR-324-5p overexpression suppressed glioma growth and prolonged survival in an intracranial xenograft model. Multivariate Cox regression analysis revealed that miR-524-5p was an independent prognostic factor in gliobalstoma patients. Taken together, these data indicate that miRNA-driven EZH2 repression may provide evidence of the molecular mechanism for gliomagenesis and the novel therapeutic targets for glioma.

MicroRNA-141-3p promotes glioma cell growth and temozolomide resistance by directly targeting p53.

Glioblastoma multiforme is the most common primary malignancy in the brain and confers a uniformly poor prognosis. MicroRNAs have been shown to activate or inhibit tumorigenesis. Abnormalities in the p53 signaling pathway are found in various cancers and correlate with tumor formation. We examined the expression of microRNA-141-3p (miR-141-3p) in glioma of different grades by analysis of expression profiling databases and clinical specimens. Cell proliferation and flow cytometry assays were performed to evaluate the promotion of miR-141-3p in proliferation, cell cycle, apoptosis, and temozolomide resistance of glioblastoma cells in vitro. Bioinformatics analyses, luciferase reporter assays, and immunoblotting showed that p53 is a target gene of miR-141-3p. A significant inverse correlation was observed between expression of miR-141-3p and p53 in glioma and normal brain tissues (R2=0.506, P<0.0001). Rescue experiments indicated that overexpression of p53 significantly reversed the alterations in proliferation, cell cycle distribution, and temozolomide resistance measured by cell apoptosis induced by miR-141-3p overexpression. In an orthotopic mouse model of human glioma, inhibition of miRNA-141-3p reduced the proliferation and growth of glioma cells in the brain and significantly prolonged the survival of glioma-bearing mice. We suggest that miR-141-3p promotes glioblastoma progression and temozolomide resistance by altering p53 expression and therefore may serve as a new diagnostic marker and therapeutic target for glioma in the future.

MicroRNA-1179 inhibits glioblastoma cell proliferation and cell cycle progression via directly targeting E2F transcription factor 5.

Glioblastoma multiforme (GBM) is an extraordinary aggressive disease that requires more effective therapeutic options. In the past few years, many microRNAs (miRNAs) have been demonstrated to have important roles in promoting GBM progression. However, little is known about the role of miR-1179 in GBM. In the present study, we found that miR-1179 was significantly downregulated in glioma tissues and cell lines. Functional experiments showed that introduction of miR-1179 dramatically suppressed GBM cell proliferation and cell cycle progression. Importantly, treatment of miR-1179 strongly inhibited tumor growth in a subcutaneous GBM model. Further studies showed that E2F transcription factor 5 (E2F5), a key transcription factor that controls cell cycle transition, was a direct target of miR-1179. Silencing of E2F5 inhibited the proliferative ability of GBM cells and induces cell cycle arrest, which were consistent with the effects of miR-1179 overexpression. More importantly, reintroduction of E2F5 into GBM cells reversed the tumor-suppressive function of miR-1179. Finally, we demonstrated that miR-1179 expression was negatively correlated with E2F5 messenger RNA (mRNA) levels in high-grade gliomas. Our findings provide new insights into the role of miR-1179 in the progression of GBM, and implicate the potential application of miR-1179 in GBM therapy.

MicroRNA-1468-5p inhibits glioma cell proliferation and induces cell cycle arrest by targeting RRM1.

MicroRNAs are associated with different types of cancers. In this study, we found that miR-1468-5p could inhibit growth and cell cycle progression in glioma by targeting ribonucleotide reductase large subunit M1 (RRM1). First, we analyzed miR-1468-5p expression in different glioma grades and the prognostic significance of its expression in glioblastoma multiform patients from the Chinese Glioma Genome Atlas. Then, we expressed miR-1468-5p in U87 and U251 cells and assessed the effects on proliferation and cell cycle progression using cell counting kit-8, colony formation, EdU and flow cytometry assays. Western blotting and luciferase reporter assays identified RRM1 as a novel direct target of miR-1468-5p. Experiments to determine the role of RRM1 in glioma showed that RRM1 expression was significantly higher in glioma than in normal brain tissues, and silencing RRM1 with small-interfering RNAs decreased proliferation and suppressed cell cycle progression, which indicated that RRM1 had pro-tumor functions. miR-1468-5p overexpression suppressed RRM1 expression, reduced glioma cell proliferation and induced cell cycle arrest, which was partially rescued by forced RRM1 expression. In summary, our study revealed that the regulatory mechanism of miR-1468-5p in glioma cell cycle progression involved direct regulation of RRM1 expression, suggesting that RRM1 may be a potential therapeutic target for glioma.