Chunzhi Zhang

High level of miR-221/222 confers increased cell invasion and poor prognosis in glioma

BackgroundMiR-221 and miR-222 (miR-221/222), upregulated in gliomas, can regulate glioma cell cycle progression and apoptosis, respectively. However, the association of miR-221/222 with glioma cell invasion and survival remains unknown.MethodsInvasion capability of miR-221/222 was detected by mutiple analyses, including diffusion tensor imaging (DTI), transwell, wound healing and nude mouse tumor xenograft model assay. Further, the target of miR-221/222 was determined by luciferase reporter, western blot and gene rescue assay. The association of miR-221/222 with outcome was examined in fifty glioma patients.ResultsMiR-221/222 expression was significantly increased in high-grade gliomas compared with low-grade gliomas, and positively correlated with the degree of glioma infiltration. Over-expression of miR-221/222 increased cell invasion, whereas knockdown of miR-221/222 decreased cell invasion via modulating the levels of the target, TIMP3. Introduction of a TIMP3 cDNA lacking 3’ UTR abrogated miR-221/222-induced cell invasion. In addition, knockdown of miR-221/222 increased TIMP3 expression and considerably inhibited tumor growth in a xenograft model. Finally, the increased level of miR-221/222 expression in high-grade gliomas confers poorer overall survival.ConclusionsThe present data indicate that miR-221 and miR-222 directly regulate cell invasion by targeting TIMP3 and act as prognostic factors for glioma patients.

MicroRNA-21 Expression is regulated by β-catenin/STAT3 Pathway and Promotes Glioma Cell Invasion by Direct Targeting RECK

MicroRNA‐21 (miR‐21) expression is increased in many types of human malignancy, including glioma. Recent studies report that miR‐21 regulates cell invasion by targeting RECK, however, the underlying transcriptional regulation of miR‐21 in glioma cells remains elusive.

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-221/222 is the regulator of Cx43 expression in human glioblastoma cells

The miR-221/222 cluster is significantly upregulated in malignant glioma cells and regulates the expression of multiple genes associated with glioma cell proliferation, invasion and apoptosis, which was shown in our previous studies. Cx43 has been identified as a tumor suppressor and major component for the establishment of gap junction intercellular communication (GJIC) in glial cells, which is frequently reduced or deleted in high-grade gliomas. According to bioinformatic analysis, connexin 43 (Cx43) may be one of the target genes of miR-221/222. The aim of the present study was to validate Cx43 as a target gene of miR-221/222 and to determine whether overexpression of miR-221/222 is one of the molecular mechanisms for the reduced expression of Cx43 in malignant gliomas. We transfected miR-221/222 antisense oligonucleotides (AS-miR-221/222) into U251 human glioblastoma cells using a lipofectamine method. Northern blot analysis was conducted to detect the expression of the miR-221/222 cluster. Luciferase reporter assays were exploited to confirm Cx43 as a target gene of miR-221/222. Cx43 expression was assessed by western blotting and immunofluorescence staining. Scrape loading and dye transfer (SLDT) assays were used for examination of GJIC. Proliferation and invasion of U251 cells were evaluated by MTT and transwell assays, respectively. Cell cycle kinetics and apoptosis were determined with flow cytometry. We found that expression of the miR-221/222 cluster was significantly reduced while Cx43 expression was upregulated in U251 cells transfected with AS-miR-221/222, and the GJIC deficiency in parental U251 cells was re-established. Moreover, the luciferase activity determined by the luciferase reporter assay was enhanced in AS-miR-221/222-treated cells, and cell proliferation and invasion were suppressed while apoptosis was induced. We conclude that miR-221/222 function as oncogenic microRNAs in human gliomas, at least in part, by targeting Cx43.

MicroRNA-221 and -222 Regulate Radiation Sensitivity by Targeting the PTEN Pathway

PURPOSE MicroRNAs (miRNAs) are noncoding RNAs inhibiting expression of numerous target genes by posttranscriptional regulation. miRNA-221 and miRNA-222 (miRNA-221/-222) expression is elevated in radioresistant tumor cell lines; however, it is not known whether and how miRNAs control cellular responses to ionizing irradiation. METHODS AND MATERIALS We used bioinformatic analyses, luciferase reporter assay, and genetic knockdown and biochemical assays to characterize the regulation pathways of miRNA-221/-222 in response to radiation treatment. RESULTS We identified the PTEN gene as a target of miRNA-221/-222. Furthermore, we found that knocking down miRNA-221/-222 by antisense oligonucleotides upregulated PTEN expression. Upregulated PTEN expression suppressed AKT activity and increased radiation-induced apoptosis, resulting in enhancement of radiosensitivity in tumor cells. CONCLUSIONS miRNA-221/-222 control radiation sensitivity by regulating the PTEN/AKT pathway and can be explored as novel targets for radiosensitization.

Abstract 776: Sensitization of glioblastoma cells to temozolomide induced antitumor effect by PLGA-aspirin microsphere through modulation of β-catenin transactivation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Malignant gliomas are the most prevalent and deadliest brain neoplasms. Although temozolomide (TMZ) was currently used for treating glioblastoma patients, the recurrence was commonly existed due to the acquired therapeutic resistance. Thus the combination chemotherapy with TMZ is considered a promising therapeutic strategy in overcoming therapeutic resistance and improving treatment efficacy. In this study, we performed PLGA microspheres of aspirin and TMZ by spray drying technique and detected their cytotoxicity to glioblastoma cells. In vitro assay, PLGA-aspirin (PLGA-A) microsphere treatment revealed a slight inhibition of proliferation, invasion, as well as slightly induced apoptosis of A172 and U87 cells through inhibition of β-catenin transactivation in comparison with PLGA microsphere and Control group. In addition, the limited antitumor effect was also demonstrated in vivo. However, PLGA-aspirin-temozolomide (PLGA-A-T) microsphere treatment group displayed enhancing antitumor efficacy compared with PLGA-temozolomide (PLGA-T) microsphere group. IC50 values were dramatically decreased in cells treated with PLGA-A-T microsphere, to a greater extent than those treated with PLGA-A microsphere. Meanwhile, the PLGA-A-T microsphere significantly enhanced apoptosis in both A172 cells and U87 cells, and cell proliferation and invasiveness were obviously weakened. In response to the inhibition of β-catenin signaling, β-catenin/TCF4 transcriptional activity and STAT3 luciferase activity was strongly inhibited, as well as a greater decrease of mRNA and protein expression levels of β-catenin, TCF4, pAKT and pSTAT3.Similar results were also observed in vivo, intratumoral injection of PLGA-A-T microsphere significantly downregulated expression of β-catenin, TCF4, pAKT, pSTAT3 and PCNA, and also delayed tumor growth in nude mice harboring subcutaneous U87 xenografts. These results indicated that the synergistic cytotoxic effect of aspirin and temozolomide was achieved by aspirin- temozolomide -loaded PLGA nanoparticles through inhibition of β-catenin transactivation, offering the potential for improved treatment of glioblastomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 776. doi:1538-7445.AM2012-776