Lingchao Chen

VHL regulates the effects of miR-23b on glioma survival and invasion via suppression of HIF-1α/VEGF and β-catenin/Tcf-4 signaling

Aberrant microRNA expression has been implicated in the development of human cancers. Here, we investigated the oncogenic significance and function of miR-23b in glioma. We identified that the expression of miR-23b was elevated in both glioma samples and glioma cells, indicated by real-time polymerase chain reaction analyses. Down-regulation of miR-23b triggered growth inhibition, induced apoptosis, and suppressed invasion of glioma in vitro. Luciferase assay and Western blot analysis revealed that VHL is a direct target of miR-23b. Restoring expression of VHL inhibited glioma proliferation and invasion. Mechanistic investigation revealed that miR-23b deletion decreased HIF-1α/VEGF expression and suppressed β-catenin/Tcf-4 transcription activity by targeting VHL. Furthermore, expression of VHL was inversely correlated with miR-23b in glioma samples and was predictive of patient survival in a retrospective analysis. Therefore, we demonstrated that downregulation of miR-23b suppressed tumor survival through targeting VHL, leading to the inhibition of β-catenin/Tcf-4 and HIF-1α/VEGF signaling pathways.

miR-137 is frequently down-regulated in glioblastoma and is a negative regulator of Cox-2

MicroRNAs are strongly implicated in cancer but their specific roles and functions in the major cancers have yet to be fully elucidated. In this study, we defined the expression and function of miR-137, which we found to be downregulated in glioma samples and glioma cells by qRT-PCR. Ectopic expression of miR-137 in glioma cell lines inhibited proliferation and invasion. Using computational and expression analysis, Cox-2 was identified as a candidate target of miR-137. Reporter assay with 3UTR of Cox-2 cloned downstream of the luciferase gene showed reduced luciferase activity in the presence of miR-137, providing strong evidence that miR-137 was a direct regulator of Cox-2. Expression analysis further revealed that Cox-2 was elevated in glioma and associated with survival of patients. Furthermore, we observed that Cox-2 knockdown resulted in effects similar to those with miR-137 transfection in glioma cells. In conclusion, our study demonstrates that miR-137 deregulation is common in glioma, and restoration of its function inhibits cell proliferation and invasion, suggesting that miR-137 may act as a tumour suppressor.

MiR-218 reverses high invasiveness of glioblastoma cells by targeting the oncogenic transcription factor LEF1

The invasive behavior of glioblastoma multiforme (GBM) cells is one of the most important reasons for the poor prognosis of this cancer. For invasion, tumor cells must acquire an ability to digest the extracellular matrix and infiltrate the normal tissue bordering the tumor. Preventing this by altering effector molecules can significantly improve a patients prognosis. Accumulating evidence suggests that miRNAs are involved in multiple biological functions, including cell invasion, by altering the expression of multiple target genes. The expression levels of miR-218 correlate with the invasive potential of GBM cells. In this study, we found that miR-218 expression was low in glioma tissues, especially in GBM. The data showed an inverse correlation in 60 GBM tissues between the levels of miR-218 and MMP mRNAs (MMP-2, -7 and -9). Additionally, ectopic expression of miR-218 suppressed the invasion of GBM cells whereas inhibition of miR-218 expression enhanced the invasive ability. Numerous members of the MMP family are downstream effectors of the Wnt/LEF1 pathway. Target prediction databases and luciferase data showed that LEF1 is a new direct target of miR-218. Importantly, western blot assays demonstrated that miR-218 can reduce protein levels of LEF1 and MMP-9. We, therefore, hypothesize that miR-218 directly targets LEF1, resulting in reduced synthesis of MMP-9. Results suggest that miR-218 is involved in the invasive behavior of GBM cells and by targeting LEF1 and blocking the invasive axis, miR-218-LEF1-MMPs, it may be useful for developing potential clinical strategies.

Glioblastoma with an oligodendroglioma component: distinct clinical behavior, genetic alterations, and outcome

Glioblastomas (GBMs) containing foci that resemble oligodendroglioma are defined as GBM with oligodendroglioma component (GBMO). However, whether GBMO is a distinct clinicopathological variant of GBM or merely represents a divergent pattern of differentiation remains controversial. We investigated 219 consecutive primary GBMs, of which 40 (18.3%) were confirmed as GBMOs. The clinical features and genetic profiles of the GBMOs were analyzed and compared with the conventional GBMs. The GBMO group showed more frequent tumor-related seizures (P= .027), higher frequency of IDH1 mutation (31% vs. <5%, P= .015), lower MGMT expression (P= .016), and longer survival (19.0 vs. 13.2 months; P= .022). In multivariate Cox regression analyses, presence of an oligodendroglioma component was predictive of longer survival (P= .001), but the extent of the oligodendroglial component appeared not to be linked to prognosis (P= .664). The codeletions of 1p/19q, somewhat surprisingly, were infrequent (<5%) in both GBMO and conventional GBM. In addition, the response to aggressive therapy differed: the GBMO group had no survival advantage associated with aggressive treatment protocols, whereas a clear treatment effect was observed in the conventional GBM group. Collectively, the clinical behavior and genetic alterations of GBMO thus differs from those of conventional GBM. Presence of an oligodendroglial component may therefore be a useful classification and stratification variable in therapeutic trials of GBMs.

MiR-139 Inhibits Mcl-1 Expression and Potentiates TMZ-Induced Apoptosis in Glioma

Mcl‐1, an antiapoptotic member of the Bcl‐2 family, is overexpressed in human glioblastoma, conferring a survival advantage to tumor cells. The mechanisms underlying its dysregulation have not been clarified. In this study, we explored the involvement of micro‐RNAs that acted as endogenous sequence‐specific suppressors of gene expression.

Downregulation of miR-221/222 sensitizes glioma cells to temozolomide by regulating apoptosis independently of p53 status

A previous study showed that miR-221/222 can regulate cell apoptosis. p53 is a well known tumor suppressor which can influence the chemosensitivity of glioma cells. However, the effect of miR-221/222 in gliomas with different p53 status is unknown. Here, we demostrate that knockdown of miR-221/222 increases apoptosis in human gliomas of different p53 types (U251 cells, p53 mutant-type; LN308 cells, p53 null-type; and U87 cells, p53 wild-type). Furthermore, the effect of miR-221/22 caused no change of p53 expression in the glioma cells studied. In addition, when a specific siRNA against p53 was employed in U87 cells, no attenuation of apoptosis was found after knockdown of miR-221/222. Importantly, we found that As-miR-221/222-treated cells increased expression of Bax, cytochrome c, Apaf-1 and cleaved-caspase-3. Our results showed that low expression of miR-221/222 sensitized glioma cells to temozolomide (TMZ); in addition, ectopic expression of PUMA by pcDNA-PUMA had a similar effect. Taken together, our study indicates that downregulated miR-221/222 can sensitize glioma cells to TMZ by regulating apoptosis independently of p53 status.

MiR-410 regulates MET to influence the proliferation and invasion of glioma.

MET, the receptor for hepatocyte growth factor receptor (HGF), has been reported to trigger multiple and sometimes opposing cellular responses in various types of tumor cells. It has been implicated in the regulation of tumor-cell survival, proliferation, angiogenesis, invasion and metastasis. However, the MET regulatory mechanism in glioma is not well known. MicroRNAs are a class of small noncoding RNAs that play important roles in a variety of biological processes including human cancers. In this study, we used computational and expressional analysis to identify that the seed sequence of miR-410 matched the 3 UTR of the MET mRNA. Besides, the expression of miR-410 was inversely associated with MET in human glioma tissues. Using luciferase and western blot assay, we certified that miR-410 directly targeted MET in glioma cells. While restoring expression of miR-410 led to proliferation inhibition and reduced invasive capability in glioma cells. Furthermore, we showed that miR-410 played an important role in regulating MET-induced AKT signal transduction. While downregulation of MET by RNAi, we observed that MET knockdown resulted in effects similar to that with miR-410 transfection in glioma cells. Our findings suggest that miR-410, a direct regulator of MET, may function as a tumor suppressor in human gliomas.

MiR-24 regulates the proliferation and invasion of glioma by ST7L via β-catenin/Tcf-4 signaling

MicroRNAs are strongly implicated as affecting glioma, but their specific roles and functions have yet to be fully elucidated. In this study, we defined the expression and function of miR-24, which we found to be upregulated in glioma samples and glioma cells by qRT-PCR. Downregulation of miR-24 in glioma cell lines inhibited proliferation and invasion and induced apoptosis. Using computational and expression analysis, ST7L was identified as a candidate target of miR-24. A reporter assay with the 3UTR of ST7L cloned downstream of a luciferase gene showed increased luciferase activity in the absence of miR-24, providing strong evidence that miR-24 is a direct regulator of ST7L. Furthermore, we observed that restoration of ST7L activity resulted in effects that were similar to those from transfecting a miR-24 inhibitor into glioma cells. Mechanistic investigation revealed that the deletion of miR-24 suppressed β-catenin/Tcf-4 transcription activity by targeting ST7L. In conclusion, our study demonstrates that miR-24 upregulation is common in glioma and that suppression of miR-24 expression inhibits cell proliferation and invasion, suggesting that miR-24 may act as an oncogene in glioma.

Blockage of a miR-21/EGFR regulatory feedback loop augments anti-EGFR therapy in glioblastomas

Epidermal growth factor receptors (EGFR) expression is frequently amplified in human glioblastoma cells. Nimotuzumab, a monoclonal antibody (mAb) against EGFR, has been used globally in clinics as an anti-cancer agent. It is largely unknown whether the blockade of miR-21, a microRNA that is upregulated in glioma cells, could amplify the effects of nimotuzumab. Herein, we have demonstrated that miR-21 directly targets von Hippel-Lindau (VHL) and peroxisome-proliferator-activated receptor α (PPARα) and that miR-21 regulates EGFR/AKT signaling through VHL/β-catenin and the PPARα/AP-1 axis. Further, the expression of miR-21 is regulated by EGFR via the activation of β-catenin and AP-1. These data indicate that a feedback loop exists between miR-21 and EGFR. We also show that the combination of nimotuzumab and an inhibitor of miR-21 is superior to single-agent therapy. These results clarify a novel association between miR-21 and EGFR in the regulation of cancer cell progression.

Expression and function of miR-27b in human glioma.

Our previous miRNAs profiling study showed that miR-27b was up-regulated in glioma cells compared with H4 low grade astrocytoma cells. However, the main function of miR-27b in glioma in not known yet. The aim of this study was to investigate the expression and function of miR-27b in the pathogenesis of glioma. Real-time PCR showed that miR-27b was up-regulated in glioma samples and glioma cells. Down-regulation of miR-27b triggered growth inhibition, induced apoptosis and inhibited invasion in glioma cells. Furthermore, TOPflash luciferase activity was decreased significantly, while FOPflash luciferase did not change significantly. In addition, Western blot assay showed that STAT3, c-myc and cyclin D1 were knocked down after treatment with miR-27b inhibitor. These findings suggest that aberrantly up-regulated miR-27b may be one of the critical factors that contribute to malignancy in human gliomas.