Increased DKC1 expression in glioma and its significance in tumor cell proliferation, migration and invasion.

The dyskeratosis congenita 1 (DKC1) gene is located on the X chromosome at Xq28. Dyskerin encoded by the DKC1 gene is associated with the formation of certain small RNAs and the telomerase activity. Inherited mutations in DKC1 inactivate the dyskerin and causes dyskeratosis congenital, which is characterized by skin defects, hematopoiesis failure, and increased susceptibility to cancer. DKC1 reportedly up-regulates in several human cancers, including renal cell carcinoma and prostate cancer. Dyskerin is deregulated in B-chronic lymphocytic leukemia and breast carcinomas, but its expression and function in glioma have hardly been investigated. Hence, we were prompted to collect tissue samples and implement cell experiments. Our study reveals that DKC1 expression is significantly increased in the pathological tissues of glioma compared with that in normal tissues. The increased staining of DKC1 is related to the World Health Organization stages of tumors. DKC1 knockdown also significantly inhibits glioma cell growth by altering the expression of cell cycle-relative molecules to arrest at the G1 phase. In the transwell chamber, DKC1 knockdown glioma cells exhibit low motility. Consistent with classic oncogenic pathways, N-cadherin, HIF-1α, and MMP2 expression levels are lower compared with those of the control group. Therefore, DKC1 up-regulation in gliomas is common and necessary for extensive tumor growth. The phenotype of glioma cell lines after DKC1 down-regulation suggests its use as a valuable clinical treatment strategy.

RNA-seq of 272 gliomas revealed a novel, recurrent PTPRZ1-MET fusion transcript in secondary glioblastomas.

Studies of gene rearrangements and the consequent oncogenic fusion proteins have laid the foundation for targeted cancer therapy. To identify oncogenic fusions associated with glioma progression, we catalogued fusion transcripts by RNA-seq of 272 gliomas. Fusion transcripts were more frequently found in high-grade gliomas, in the classical subtype of gliomas, and in gliomas treated with radiation/temozolomide. Sixty-seven in-frame fusion transcripts were identified, including three recurrent fusion transcripts: FGFR3-TACC3, RNF213-SLC26A11, and PTPRZ1-MET (ZM). Interestingly, the ZM fusion was found only in grade III astrocytomas (1/13; 7.7%) or secondary GBMs (sGBMs, 3/20; 15.0%). In an independent cohort of sGBMs, the ZM fusion was found in three of 20 (15%) specimens. Genomic analysis revealed that the fusion arose from translocation events involving introns 3 or 8 of PTPRZ and intron 1 of MET. ZM fusion transcripts were found in GBMs irrespective of isocitrate dehydrogenase 1 (IDH1) mutation status. sGBMs harboring ZM fusion showed higher expression of genes required for PIK3CA signaling and lowered expression of genes that suppressed RB1 or TP53 function. Expression of the ZM fusion was mutually exclusive with EGFR overexpression in sGBMs. Exogenous expression of the ZM fusion in the U87MG glioblastoma line enhanced cell migration and invasion. Clinically, patients afflicted with ZM fusion harboring glioblastomas survived poorly relative to those afflicted with non-ZM-harboring sGBMs (P < 0.001). Our study profiles the shifting RNA landscape of gliomas during progression and reveled ZM as a novel, recurrent fusion transcript in sGBMs.

MiRNA-181b suppresses IGF-1R and functions as a tumor suppressor gene in gliomas.

MicroRNAs (miRNAs) are single-stranded, 18- to 23-nt RNA molecules that function as regulators of gene expression. Previous studies have shown that microRNAs play important roles in human cancers, including gliomas. Here, we found that expression levels of miR-181b were decreased in gliomas, and we identified IGF-1R as a novel direct target of miR-181b. MiR-181b overexpression inhibited cell proliferation, migration, invasion, and tumorigenesis by targeting IGF-1R and its downstream signaling pathways, PI3K/AKT and MAPK/ERK1/2. Overexpression of IGF-1R rescued the inhibitory effects of miR-181b. In clinical specimens, IGF-1R was overexpressed, and its protein levels were inversely correlated with miR-181b expression. Taken together, our results indicate that miR-181b functions in gliomas to suppress growth by targeting the IGF-1R oncogene and that miR-181b may serve as a novel therapeutic target for gliomas.

Fenofibrate induces G0/G1 phase arrest by modulating the PPARα/FoxO1/p27 kip pathway in human glioblastoma cells.

Fenofibrate, a fibric acid derivative, is known to possess lipid-lowering effects. Although fenofibrate-induced peroxisome proliferator-activated receptor alpha (PPARα) transcriptional activity has been reported to exhibit anticancer effects, the underlying mechanisms are poorly understood. In this study, we investigated the mechanisms behind the antiproliferative effects of fenofibrate in U87MG cells (human glioma cell line) using the WST-8 Cell Proliferation Assay Kit. Furthermore, we examined genome-wide gene expression profiles and molecular networks using the DAVID online software. Fenofibrate reduced the expression of 405 genes and increased the expression of 2280 genes. DAVID analysis suggested that fenofibrate significantly affected cell cycle progression and pathways involved in cancer, including the mTOR signaling pathway and insulin signaling pathway. Results of flow cytometry analysis indicated that fenofibrate induced cell cycle G0/G1 arrest in U87MG cells. Furthermore, we identified the FoxO1-p27(kip) signaling axis to be involved in fenofibrate-induced cell cycle arrest. Our findings suggest that in addition to its known lipid-lowering effects, fenofibrate may be used as an antitumor agent in glioma therapy.

MiR-1224-5p acts as a tumor suppressor by targeting CREB1 in malignant gliomas.

The dysregulation of miR-1224-5p has been reported in several human cancers. However, the expression and function of miR-1224-5p in glioma remains unknown. The aim of our study was to investigate the effect of miR-1224-5p on glioma cells and to determine its functional signaling mediators. Using 198 glioma samples within the Chinese Glioma Genome Atlas expression dataset, we demonstrated that miR-1224-5p expression is decreased in high-grade gliomas when compared with low-grade gliomas. Differential miR-1224-5p expression in 50 randomly selected samples was verified by in situ hybridization. The expression of miR-1224-5p was shown to positively correlate with overall survival in 82 glioblastoma patients. Exogenous expression of miR-1224-5p in glioma cells suppressed proliferation and invasion and promoted apoptosis. Target prediction algorithms identified a consensus miR-1224-5p recognition site in the 3'UTR of the cAMP response element-binding protein (CREB1) gene, and this sequence was shown to directly confer miR-1224-5p repression in luciferase reporter assays. Furthermore, exogenous miR-1224-5p expression was shown to down-regulate CREB1, as well as its downstream target genes matrix metalloproteinase-9 and B-cell lymphoma-2. Conversely, over-expression of CREB1 reversed the effect of miR-1224-5p on the proliferation, invasion, and apoptosis of glioma cells. These data indicate that miR-1224-5p may inhibit tumor-associated activity in malignant gliomas by targeting CREB1.

Vitamin D3 suppresses the cholesterol homeostasis pathway in patient-derived glioma cell lines.

Glioblastoma is one of the most common malignant brain tumors. Vitamin D, primarily its hormonally active form calcitriol, has been reported to have anti-cancer activity. In the present study, we used patient-derived glioma cell lines to examine the effect of vitamin D3 and calcitriol on glioblastoma. Surprisingly, vitamin D3 showed a more significant inhibitory effect than calcitriol on cell viability and proliferation. Vitamin D receptor (VDR) mediates most of the cellular effects of vitamin D, and thus we examined the expression level and function of VDR via gene silencing and gene knockout experiments. We observed that VDR does not affect the sensitivity of patient-derived glioma cell lines to vitamin D3, and the gene encoding VDR is not essential for growth of patient-derived glioma cell lines. RNA sequencing data analysis and sterolomics analysis revealed that vitamin D3 inhibits cholesterol synthesis and cholesterol homeostasis by inhibiting the expression level of 7-dehydrocholesterol reductase, which leads to the accumulation of 7-dehydrocholesterol and other sterol intermediates. In conclusion, our results suggest that vitamin D3, rather than calcitriol, inhibits growth of patient-derived glioma cell lines via inhibition of the cholesterol homeostasis pathway.

Primary Skull Base Chordomas: A Clinicopathological Analysis of 94 Patients.

OBJECTIVE: To investigate prognostic factors in patients with primary skull base chordoma (PSBC) to guide future therapeutic advances. METHODS: This retrospective cohort study of 94 PSBC patients was conducted in 2 institutions from January 2006 to December 2013. Independent predictors for progression-free survival (PFS) and overall survival were established with multivariate Cox regression analysis. RESULTS: Age (P = 0.006), extent of resection (P = 0.037), and radiotherapy (RT) (P = 0.027) were established as independent predictors for PFS in PSBC patients. Similarly, age (P = 0.002), extent of resection (P = 0.048), and RT (P = 0.015) were established as independent predictors for overall survival. Meta-analysis manifested that lower MIB-1 correlated with longer PFS in skull base chordoma patients (P < 0.001). RT doubled the 5-year PFS rate from 28.6 ± 12.1% to 61.6 ± 10.7% (P = 0.031) and increased the 5-year overall survival rate from 54.5 ± 13.8% to 84.2 ± 8.4% (P = 0.020) in the subtotal resection/partial resection and MIB-1 labeling index (STR/PR+MIB-1 LI) <2% subgroup. In contrast, in the STR/PR+MIB-1 LI ≥2% subgroup, the survival benefit of RT remained uncertain. Further analysis revealed no survival difference between different RT modalities in STR/PR PSBC patients. CONCLUSIONS: In PSBC patients, age, extent of resection, and adjuvant RT all are independent predictors for PFS. Lower MIB-1 LI is associated with longer PFS in PSBC patients. Adjuvant RT is necessary for PSBC patients who undergo STR/PR with MIB-1 LI <2%. Patients who undergo GTR or STR/PR with MIB-1 LI ≥2% seem nonresponsive to RT.

Gossypin induces G2/M arrest in human malignant glioma U251 cells by the activation of Chk1/Cdc25C pathway.

Gossypin is a flavone that was originally isolated from Hibiscus vitifolius and has traditionally been used for the treatment of diabetes, jaundice, and inflammation. Recently, gossypin was found to have potent anticancer properties; however, its effect on human gliomas still remain unknown. To investigate the potential anticancer effects of gossypin on malignant gliomas and analyze the associated molecular mechanisms, we treated human glioma U251 cells with gossypin. Our study showed that the treatment of U251 cells with gossypin inhibited cell proliferation in a dose- and time-dependent manner and was observed to be minimally toxic to normal human astrocytes. Gossypin's effect on cell cycle distribution was observed, and we found that it induced G2/M-phase arrest in U251 cells. An analysis of cell-cycle regulatory proteins indicated that the arresting effect of gossypin on the cell cycle at G2/M phase was involved in the phosphorylation of cell division cycle 25C (Cdc25C) tyrosine phosphatase via the activation of checkpoint kinase 1 (Chk1). These findings indicate that gossypin is a potential treatment of gliomas because of gossypin's potential to regulate the proliferation of U251 cells via the cell-cycle regulatory proteins Chk1 and Cdc25C.

[Expression of RAS protein in glioma and its effect on the growth of human glioma cells].

OBJECTIVE: To study the expression of RAS protein in human glioma tissues and its influence on tumor growth. METHODS: RAS protein expression in glioma tissues was determined by immunohistochemical (IHC) staining. Subsequently, MTT cell proliferation assay, flow cytometry and Western blotting were used to assay U251 cells with reduced RAS expression. RESULTS: The expression of RAS in glioma was increased and strongly correlated with pathological grade. Downregulation of RAS resulted in glioma cells growth suppression and increased apoptosis. CONCLUSION: The expression level of RAS protein in human glioma was increased. Downregulation of RAS can inhibit glioblastoma cell growth through the RAS signal pathway.

MicroRNA roles in beta-catenin pathway.

ß-catenin, a key factor in the Wnt signaling pathway, has essential functions in the regulation of cell growth and differentiation. Aberrant ß-catenin signaling has been linked to various disease pathologies, including an important role in tumorigenesis. Here, we review the regulation of the Wnt signaling pathway as it relates to ß-catenin signaling in tumorigenesis, with particular focus on the role of microRNAs. Finally, we discuss the potential of ß-catenin targeted therapeutics for cancer treatment.

MiR-221 and miR-222 target PUMA to induce cell survival in glioblastoma.

BACKGROUND: MiR-221 and miR-222 (miR-221/222) are frequently up-regulated in various types of human malignancy including glioblastoma. Recent studies have reported that miR-221/222 regulate cell growth and cell cycle progression by targeting p27 and p57. However the underlying mechanism involved in cell survival modulation of miR-221/222 remains elusive. RESULTS: Here we showed that miR-221/222 inhibited cell apoptosis by targeting pro-apoptotic gene PUMA in human glioma cells. Enforced expression of miR-22/222 induced cell survival whereas knockdown of miR-221/222 rendered cells to apoptosis. Further, miR-221/222 reduced PUMA protein levels by targeting PUMA-3'UTR. Introducing PUMA cDNA without 3'UTR abrogated miR-221/222-induced cell survival. Notably, knockdown of miR-221/222 induces PUMA expression and cell apoptosis and considerably decreases tumor growth in xenograft model. Finally, there was an inverse relationship between PUMA and miR-221/222 expression in glioma tissues. CONCLUSION: To our knowledge, these data indicate for the first time that miR-221/222 directly regulate apoptosis by targeting PUMA in glioblastoma and that miR-221/222 could be potential therapeutic targets for glioblastoma intervention.

Use of thymidine kinase gene-modified endothelial progenitor cells as a vector targeting angiogenesis in glioma gene therapy.

OBJECTIVE: The poor prognosis of patients with glioma is due to infiltrative growth of glioma cells, which correlates with their ability to induce angiogenesis. Tumor angiogenesis is supported by the mobilization and functional incorporation of endothelial progenitor cells (EPCs). The aim of this study was to propose the use of gene-modified EPCs as a vector system that allowed systemic gene delivery into multiple areas of tumor angiogenesis for glioma therapy. METHODS: Thymidine kinase (TK) gene-modified EPCs were mixed with glioma cells or human umbilical vein endothelial cells (HUVECs) at varying ratios for ganciclovir in vitro. Cell proliferation was evaluated by MTT assay, and apoptosis was examined by annexin-V and phosphatidylserine (propidium iodide) staining. EPCs were injected via tail vein into nude mice bearing glioma, and EPC incorporation into the tumor was determined immunohistochemically. The antitumor effects of TK gene-modified EPCs in vivo were evaluated by apoptosis assay and microvessel density analysis. RESULTS: TK gene-modified EPCs exerted a potent bystander effect on glioma cells and HUVECs by induction of apoptosis via caspase activation in vitro. EPCs incorporated preferentially into glioma vasculatures. Furthermore, TK gene-modified EPCs clearly augmented the antitumor effect by inhibition of angiogenesis following repeated intravenous injection in vivo. CONCLUSION: The results indicate the feasibility of EPC-based gene delivery into disseminated areas of tumor angiogenesis as a rational strategy for glioma gene therapy.

[Effect of bone morphogenetic protein 4 on glioma stem cell proliferation and apoptosis in vitro].

OBJECTIVE: To investigate the role of bone morphogenetic protein 4 (BMP4) on the proliferation and apoptosis in glioma stem cells. METHODS: Stem cells were isolated from a human glioma cell line U87 by using vincristine and characterized by immunofluorescence assay. Proliferation and apoptosis were determined by soft agar colony assay and flow cytometry; Cyclin D1, Bcl-2 and Bax were detected by Western blot analysis. RESULTS: BMP4 inhibited cell proliferation and promoted apoptosis in U87 glioma stem cells. Moreover, Bcl-2 and Cyclin D1 expression were decreased by BMP4, while Bax level was elevated. CONCLUSION: BMP4 can inhibit U87 glioma stem cells proliferation through downregulating Cyclin D1 level, and promote apoptosis through induction of Bax expression and inhibition of Bcl-2 level. It suggests that BMP4 plays an important role in human glioma stem cell biology.

[Effect of osteopontin silencing by lentivirus-mediated delivery of siRNA on glioma cell invasion and apoptosis].

OBJECTIVE: To investigate the effect of osteopontin silencing on the invasion and apoptosis of U251 cells. METHODS: The invasion, apoptosis and levels of uPA, MMP-2 and MMP-9 were determined by invasion assay, flow cytometry, Western blot and real-time fluorescence quantitative PCR respectively. RESULTS: Osteopontin small interference RNA (siRNA) inhibited osteopontin expression and cell invasion, promoted apoptosis in U251 cells. In addition, the expression of Bcl-2, uPA, MMP-2 and MMP-9 was decreased, while Bax level was elevated. CONCLUSION: Osteopontin siRNA can inhibit U251 cells invasion via the down-regulation of uPA, MMP-2 and MMP-9 levels, and promote apoptosis through induction of Bax expression and inhibition of Bcl 2 level. It suggests that osteopontin plays an important role in human glioma progression.

[Antiglioma activity of endothelial progenitor cells transduced with HSV-TK via inhibiting angiogenesis in vitro and in vivo].

OBJECTIVE: To investigate the potentiality of herpes simplex virus thymidine kinase transduced endothelial progenitor cells (EPC-TK) as angiogenesis-targeting vector in the glioma treatment in vitro and in vivo. METHODS: EPC-TK were mixed with human umbilical vein endothelial cells (HUVECs), U87 or U251 cells at various ratios for ganciclovir (GCV) treatment. The bystander effect was observed by counting the survival cells using MTT assay, and the apoptotic cells were determined by annexin-V and propidium iodide (PI) staining. EPC-TK, EPCs, or phosphate buffered saline (PBS) were injected into the nude mice model of glioma xenograft by tail vein, for the EPC-TK group, EPC group, and PBS group, respectively. And then the changes of tumor volume and tumor vasculature were observed. RESULTS: GCV killed most EPC-TK and reduced the number of other viable cells in a cell:cell ratio-dependent and time-dependent manner. EPC-TK obviously inhibited tumor growth. The tumor volumes on day 21 were 1741.20+/- 576.10 mm(3), 3275.52 +/- 710.86 mm(3) and 3033.09+/-1134.86 mm(3) in the EPC-TK, EPC and PBS group, respectively. EPC-TK also displayed a significant effect on the inhibition of tumor angiogenesis. CONCLUSION: EPC-TK can exert a potent antiglioma effect via inhibiting angiogenesis.

[Construction and screening an effective anti-miR-221 RNAi vector in vitro].

OBJECTIVE: To construct and screen an effective anti-miR-221 vector of siRNA. METHODS: Four hairpin structure of siRNA transcript templates targeting miR-221 and a negative control were synthesized, then ligated with pGCSIL-GFP vector and a pEGFP-miR-221 which express pre-miR-221 was also constructed. All the recombinants were sequenced. The confirmed pGCSIL-GFP recombinants by combining with pEGFP-miR-221 were transfected into 293T cells seperately. The expressed Flag protein was detected by Western blot to evaluate the inhibition effect of targeting sequences. Then a recombinant with the highest anti-miR-221 effect was screened and transfected into U87 glioma cell, and its anti-tumor effect was evaluated by MTT and FCM. RESULTS: The resulting recombinants were confirmed by sequencing which demonstrated that the recombinant plasmids contained the correct sequences of designed transcript templates. The results of Western blot indicated the expression of Flag of No 1 recombinant plasmid group was inhibited heavily with a 34.3% expression level by compared with control group. The proliferation inhibition and induced apoptosis by this recombinant with an apoptosis ratio of 21.89% in U87 cell were also observed. CONCLUSION: The anti-miR-221 expression siRNA espression recombinants were constructed successfully, and one sequence with the highest inhibition efficiency was screened out, which could inhibit U87 cell proliferation and induce cell apoptosis, and could be used to suppress target gene for further study in tumor biology.

[The construction of lentivirus-mediated RNAi vector containing hTERT].

OBJECTIVE: To construct a recombinant lentivirus RNA interference (RNAi) vector carrying hTERT gene, and to obtain the titer of the lentiviral stock for investigating the expression in the eukaryotic cells and the effect on the hTERT gene silencing in the eukaryotic cells. METHODS: Two complimentary oligos of small interference RNA (siRNA) with hairpin structures targeting the hTERT gene and a negative control were synthesized, then ligated with pLVTHM vector and sequenced. The recombinant vectors were then transfected with viral packaging mix into T293 cells, viral supernatant was harvested to determine the titer. U87 cells infected by virus were harvested and the expression of hTERT, telomerase activity and apoptosis were detected by reverse transcription-PCR(RT-PCR), TRAP assay and flow cytometry separately. RESULTS: Sequencing data showed that the constructed plasmids contained the correct sequences of hTERT siRNA transcript templates. A vector producing cell line T293 was established, and the titer for transfection was obtained. RT-PCR and TRAP flow cytometry analyses demonstrated that hTERT shRNA expression construct could suppress the expression of hTERT and telomerase activity and induce apoptosis. CONCLUSION: A lentivirus RNAi vector targeting hTERT gene was successfully constructed, which decreased the expression of hTERT and telomerase activity effectively and induced apoptosis. It has set up a research platform for the gene therapy of tumors which take hTERT as the target.

[Study on combined gene therapy for malignant gliomas transfected with antisense hTERT/PTEN in vitro and in vivo].

OBJECTIVE: To study inhibitory efficacy of combined gene therapy for malignant gliomas transfected with antisense human telomerase reverse transcriptase (hTERT)/PTEN in vitro and in vivo. METHODS: To construct two adenovirus recons which contained antisense hTERT and wild-type PTEN respectively with high performance homologous recombination system in bacteria. The two adenovirus recons were transfected into U251 human malignant glioma cells combinedly or respectively in vitro and in vivo. U251 cell proliferation in vitro was determined by MTT assay and flow cytometry, tumor growth in vivo was measured by the volume of glioma in nude mice. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP) assay. Expression of hTERT and PTEN protein was detected by Western blotting methods. RESULTS: After transfection in vitro, the growth of U251 cells was inhibited significantly. The inhibitory effect was time-dependent. The strongest inhibition was observed in combined transfection group, at the 6th day, the survival rate was 37.6%, telomerase activity (only 28.8TPG) was inhibited significantly, hTERT protein expression was inhibited significantly too, which was 0.2106, but PTEN protein expression was increased significantly, which was 0.9630. In vivo, the growth of tumors was also effectively inhibited. CONCLUSION: Growth of malignant glioma cells is effectively inhibited after transfection with combined antisense hTERT and PTEN in vitro and in vivo.