Yunhui Liu

Knockdown of long non-coding RNA XIST exerts tumor-suppressive functions in human glioblastoma stem cells by up-regulating miR-152

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Great interest persists in useful therapeutic targets in GBM. Aberrant expression of long non-coding RNAs (lncRNAs) has been functionally associated with many cancers. Here, we elucidated the function and the possible molecular mechanisms of lncRNA XIST in human glioblastoma stem cells (GSCs). Our results proved that XIST expression was up-regulated in glioma tissues and GSCs. Functionally, knockdown of XIST exerted tumor-suppressive functions by reducing cell proliferation, migration and invasion as well as inducing apoptosis. The in vivo studies also showed that knockdown of XIST suppressed tumor growth and produced high survival in nude mice. Further, there was reciprocal repression between XIST and miR-152. Mechanistic investigations defined the direct binding ability of the predicted miR-152 binding site on the XIST. In addition, XIST and miR-152 are probably in the same RNA induced silencing complex (RISC). Finally, miR-152 mediated the tumor-suppressive effects that knockdown of XIST exerted. Taken together, these results provided a comprehensive analysis of XIST in GSCs and important clues for understanding the key roles of lncRNA-miRNA functional network in human glioma.

Long non-coding RNA CASC2 suppresses malignancy in human gliomas by miR-21.

Long non-coding RNAs (lncRNAs) are aberrantly expressed in many diseases including cancer. LncRNA CASC2 (cancer susceptibility candidate 2) has been characterized as a tumor suppressor in endometrial cancer and colorectal cancer. However, the role and function of CASC2 in human gliomas remain unknown. In this study, we confirmed that CASC2 was lowly expressed in glioma tissues as well as in U251 and U87 glioma cell lines. Overexpression of CASC2 inhibited the malignancy of glioma cells, including proliferation, migration, and invasion, and promoted cell apoptosis. MicroRNA-21 (miR-21) has been reported to be overexpressed in human glioma tissues and cell lines, which is responsible for the malignant progression of glioma. We found that up-regulated CASC2 decreased the expression of miR-21 significantly and there is a reciprocal repression between CASC2 and miR-21 in an Argonaute2-dependent manner. Furthermore, bioinformatics, luciferase reporter assays and pull-down assay confirmed that miR-21 binds to CASC2 in a sequence-specific manner. Introduction of miR-21 largely abrogated CASC2-mediated inhibition of glioma cell proliferation, migration, and invasion, and promotion of cell apoptosis. This study demonstrated that CASC2 plays a tumor suppressive role in glioma via negative regulation of miR-21, which may be a novel therapeutic target for treating gliomas.

Gas5 Exerts Tumor-suppressive Functions in Human Glioma Cells by Targeting miR-222.

Aberrant expression of noncoding RNAs in glioma cells, including long noncoding RNAs (lncRNAs) and microRNAs, may participate in the progression of glioma. Encoded by Growth Arrest-Specific 5 (GAS5) gene, lncRNA Gas5 was reported to be a negative regulator for survival and proliferation of several cancers. Here, Gas5 is found to be downregulated in glioma specimens and U87 and U251 glioma cell lines. We showed that the introduction of Gas5 by plasmid transfection increased the expression of tumor suppressor Bcl-2-modifying factor (bmf) and Plexin C1 via directly targeting and reducing the expression of miR-222. Downregulated expression of miR-222 inhibited U87 and U251 cell proliferation and promoted the apoptosis by upregulating bmf. As downstream signaling molecules of bmf, Bcl-2 and Bax were involved in the process. Meanwhile, knockdown of miR-222 attenuated U87 and U251 cell migration and invasion by upregulating Plexin C1, and cofilin was a crucial regulator targeted by Plexin C1. Gas5 combined with the knockdown of miR-222 resulted in the smallest tumor volumes and the longest survivals of nude mice in vivo. In summary, we show that Gas5 suppresses tumor malignancy by downregulating miR-222, which may serve as a promising therapy for glioma.

TTBK2 circular RNA promotes glioma malignancy by regulating miR-217/HNF1β/Derlin-1 pathway

BackgroundCircular RNAs are a subgroup of non-coding RNAs and generated by a mammalian genome. Herein, the expression and function of circular RNA circ-TTBK2 were investigated in human glioma cells.MethodsFluorescence in situ hybridization and quantitative real-time PCR were conducted to profile the cell distribution and expression of circ-TTBK2 and microRNA-217 (miR-217) in glioma tissues and cells. Immunohistochemical and western blot were used to determine the expression of HNF1β and Derlin-1 in glioma tissues and cells. Stable knockdown of circ-TTBK2 or overexpression of miR-217 glioma cell lines (U87 and U251) were established to explore the function of circ-TTBK2 and miR-217 in glioma cells. Further, luciferase reports and RNA immunoprecipitation were used to investigate the correlation between circ-TTBK2 and miR-217. Cell Counting Kit-8, transwell assays, and flow cytometry were used to investigate circ-TTBK2 and miR-217 function including cell proliferation, migration and invasion, and apoptosis, respectively. ChIP assays were used to ascertain the correlations between HNF1β and Derlin-1.ResultsWe found that circ-TTBK2 was upregulated in glioma tissues and cell lines, while linear TTBK2 was not dysregulated in glioma tissues and cells. Enhanced expression of circ-TTBK2 promoted cell proliferation, migration, and invasion, while inhibited apoptosis. MiR-217 was downregulated in glioma tissues and cell lines. We also found that circ-TTBK2, but not linear TTBK2, acted as miR-217 sponge in a sequence-specific manner. In addition, upregulated circ-TTBK2 decreased miR-217 expression and there was a reciprocal negative feedback between them in an Argonaute2-dependent manner. Moreover, reintroduction of miR-217 significantly reversed circ-TTBK2-mediated promotion of glioma progression. HNF1β was a direct target of miR-217, and played oncogenic role in glioma cells. Remarkably, circ-TTBK2 knockdown combined with miR-217 overexpression led to tumor regression in vivo.ConclusionsThese results demonstrated a novel role circ-TTBK2 in the glioma progression.

CRNDE affects the malignant biological characteristics of human glioma stem cells by negatively regulating miR-186

The long non-coding RNA Colorectal neoplasia differentially expressed (CRNDE) is a novel gene that activated early in colorectal neoplasia, but it is also up-regulated in many other solid tumors. Herein, the function and underlying mechanism of CRNDE in regulating glioma stem cells (GSCs) were investigated. We found that CRNDE expression was up-regulated while miR-186 expression was down-regulated in GSCs. Overexpression of CRNDE could promote the cellular proliferation, migration, invasion and inhibit the apoptosis in GSCs. Overexpression of miR-186 exerted functions of inhibiting the proliferation, migration and invasion of GSCs and promoting apoptosis. And CRNDE decreased the expression levels of XIAP and PAK7 by binding to miR-186 and negatively regulating it. In addition, miR-186 binded to XIAP and PAK7 3′UTR region, and decrease the expression of them, thus regulating the expression levels of downstream target proteins such as caspase 3, BAD, cyclin D1 and MARK2. The in vivo effect of CRNDE and miR-186 showed that the tumor formation rate was minimum in tumor-bearing nude mice with the knockdown of CRNDE and the overexpression of miR-186. In conclusion, CRNDE played an oncogenic role of GSCs through the negative regulation of miR-186. Both CRNDE and miR-186 could be regarded as potential targets in the glioma therapy.

CRNDE Promotes Malignant Progression of Glioma by Attenuating miR-384/PIWIL4/STAT3 Axis

Colorectal neoplasia differentially expressed (CRNDE) is the most upregulated long noncoding RNA (lncRNA) in glioma. Herein, the function and potential molecular mechanisms of CRNDE and miR-384 were illustrated in glioma cells. CRNDE overexpression facilitated cell proliferation, migration, and invasion, while inhibited glioma cells apoptosis. Quantitative real-time polymerase chain reaction (PCR) demonstrated that miR-384 was downregulated in human glioma tissues and glioma cell lines. Moreover, restoration of miR-384 exerted tumor-suppressive functions. In addition, the expression of miR-384 was negatively correlated with CRNDE expression. A binding region between CRNDE and miR-384 was confirmed using luciferase assays. Moreover, CRNDE promoted cell malignant behavior by decreasing miR-384 expression. At the molecular level, treatment by CRNDE knockdown or miR-384 overexpression resulted in a decrease of piwi-like RNA-mediated gene silencing 4 (PIWIL4) protein. Besides, PIWIL4 was identified as a target of miR-384 and plays an oncogenic role in glioma. Similarly, downstream proteins of PIWIL4 such as STAT3, cyclin D1, VEGFA, SLUG, MMP-9, caspase 3, Bcl-2, and bcl-xL were modulated when treated with miR-384 and PIWIL4. Remarkably, CRNDE knockdown combined with miR-384 overexpression led to tumor regression in vivo. Overall, these results depicted a novel pathway mediated by CRNDE in glioma, which may be a potential application for glioma therapy.

MiR-152 functions as a tumor suppressor in glioblastoma stem cells by targeting Krüppel-like factor 4

Glioblastoma (GBM) is the most common central nervous system tumor and the molecular mechanism driving its development is still largely unknown, limiting the treatment of this disease. In the present study, we explored the potential role of miR-152 in glioblastoma stem cells (GSCs) as well as the possible molecular mechanisms. Our results proved that miR-152 was down-regulated in human GSCs. Restoring the expression of miR-152 dramatically reduced the cell proliferation, cell migration and invasion as well as inducing apoptosis. Mechanistic investigations defined Krüppel-like factor 4 (KLF4) as a direct and functional downstream target of miR-152, which was involved in the miR-152-mediated tumor-suppressive effects in GSCs. Meanwhile, this process was coincided with the down-regulated LGALS3 that could be bound and promoted by KLF4, leading to attenuate the activation of MEK1/2 and PI3K signal pathways. Moreover, the in vivo study showed that miR-152 over-expression and KLF4 knockdown produced the smallest tumor volume and the longest survival in nude mice. Taken together, these results elucidated the function of miR-152 in GSCs progression and suggested a promising application of it in glioma treatment.

Krüppel-like factor 4 regulates blood-tumor barrier permeability via ZO-1, occludin and claudin-5.

Blood‐tumor barrier (BTB) constitutes an efficient organization of tight junctions which significantly reduce permeability for chemotherapy drugs. Krüppel‐like factor 4 (KLF4), a member of the Krüppel‐like family, has been documented in endothelial cells and may serve as an essential regulator of endothelial barrier function. However, our knowledge about the expression and function of KLF4 in the endothelial cells of BTB still remains unclear. In this study, we sought to investigate the role of KLF4 in regulation of BTB function as well as the potential molecular mechanisms. Quantitative RT‐PCR, Western blot, and immunofluorescence assays demonstrated that KLF4 was down‐regulated in the glioma endothelial cells (GECs) which were obtained through endothelial cells co‐cultured with glioma cells. Short hairpin RNA targeting KLF4 impaired the integrity of BTB detected by trans‐endothelial electric resistance assay, and meanwhile reduced the expression of ZO‐1, occludin and claudin‐5, demonstrated by quantitative RT‐PCR, Western blot, and immunofluorescence assays. Depletion of KLF4 increased BTB permeability to small molecules detected by permeability assays. Furthermore, luciferase assays and chromatin immunoprecipitation assays showed that KLF4 up‐regulated the promoter activities and interacted with “CACCC” DNA sequence presented in the promoters of ZO‐1, occludin, and claudin‐5. GATA‐1, GATA‐6, Sp1, and Sp3 factors participated in KLF4 regulation of promoter activities through binding to the promoters of tight junctions related proteins. Collectively, our results indicated that KLF4 is a key transcriptional regulator of BTB function by regulating expressions of tight junction related proteins, which would draw growing attention to KLF4 as a potential target for glioma therapy. J. Cell. Physiol. 229: 916–926, 2014.

Role of RhoA/ROCK Signaling in Endothelial-Monocyte-Activating Polypeptide II Opening of the Blood–Tumor Barrier

The purpose of the present study was to determine the potential for RhoA/ROCK signaling to play a role in endothelial-monocyte-activating polypeptide (EMAP) II-induced increase in blood–tumor barrier (BTB) permeability in rat brain microvascular endothelial cells (RBMECs). In the present study, we used an in vitro BTB model, a RhoA inhibitor (C3 exoenzyme) and a ROCK inhibitor (Y27632) to determine whether RhoA/ROCK pathway play a role in the process of TJ disassembly, stress fiber formation, MLC and cofilin phosphorylation, as well as increase of BTB permeability induced by EMAP II. The results revealed that BTB permeability was increased by EMAP II induction, and C3 exoenzyme or Y27632 could partially inhibit the EMAP II-induced increase of BTB permeability. The significant down-regulations in tight junction (TJ)-associated proteins occludin, claudin-5 and ZO-1 and stress fiber formation by EMAP II administration were observed, which were partly prevented by C3 exoenzyme or Y27632 pretreatment. Moreover, the significant increases in RhoA activity, myosin light chain (MLC) and cofilin phosphorylation by EMAP II administration were observed, MLC and cofilin phosphorylation were partly inhibited by C3 exoenzyme or Y27632 pretreatment. The present study demonstrates that the activation of RhoA/ROCK signaling in RBMECs was required for the increase of BTB permeability and these effects are related with the ability for RhoA/ROCK to mediate TJ disassembly and stress fiber formation by phosphorylating cofilin and MLC.

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.