MiR-181b modulates chemosensitivity of glioblastoma multiforme cells to temozolomide by targeting the epidermal growth factor receptor

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1 DOI /s LABORATORY INVESTIGATION MiR-181b modulates chemosensitivity of glioblastoma multiforme cells to temozolomide by targeting the epidermal growth factor receptor Yunxiang Chen 1 Rui Li 1 Minhong Pan 2 Zhumei Shi 1 Wei Yan 1 Ning Liu 1 Yongping You 1 Junxia Zhang 1 Xiefeng Wang 1 Received: 17 September 2016 / Accepted: 6 May 2017 Springer Science+Business Media New York 2017 Abstract Temozolomide (TMZ) is a promising chemotherapeutic agent to treat Glioblastoma multiforme (GBM). However, resistance to TMZ develops quickly with a high frequency. The mechanisms underlying GBM cells resistance to TMZ are not fully understood. MicroRNAs (mir- NAs) are small, non-coding RNA molecules that regulate protein expression by cleaving or repressing the translation of target mrnas. Recently, mirnas have been discovered to play important roles in drug resistance. A previous study showed that mir-181b in involved in glioma tumorigenesis. Thus, it would be valuable to explore the functions and mechanisms of mir-181b in regulating GMB cells sensitivity to TMZ. In this study, quantitative real-time reverse transcription PCR (qrt-pcr) data indicated that mir- 181b was significantly downregulated in recurrent GBM tissues compared with initial GBM tissues. We also found that mir-181b overexpression increased the chemo-sensitivity of GBM cells to TMZ and potentiated TMZ-induced apoptosis in vitro and in vivo. Moreover, we demonstrated that the epidermal growth factor receptor (EGFR) was a direct target of mir-181b: restoration of EGFR rescued the Yunxiang Chen, Rui Li and Minhong Pan have contributed equally to this work. * Junxia Zhang * Xiefeng Wang 1 Department of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University, 300, Guangzhou road, Nanjing, People s Republic of China 2 Department of Pathology, The First Affiliated Hospital of Nanjing Medical University, 300, Guangzhou road, Nanjing, People s Republic of China inhibitory effects of mir-181b and TMZ treatment. Taken together, our data support strongly an important role for mir-181b in conferring TMZ resistance by targeting EGFR expression. Keywords MiR-181b Glioblastoma multiforme Temozolomide Epidermal growth factor receptor Introduction Glioblastoma multiforme (GBM), a Grade IV brain tumor according to the World Health Organization (WHO) classification, is one of the most aggressive human cancers [1, 2]. Current treatments include surgical resection and radio/chemotherapy; however, the median survival remains months from time of diagnosis and a nearly 100% local recurrence rate following maximal therapy has been reported [3, 4]. One of the most common reasons for GBM recurrence is resistance to therapeutic drugs [5, 6]. Furthermore, treatment of drug resistant-gbm is more difficult because of its limited repair mechanisms and anatomical complexities [7 9]. Therefore, it is urgent to explore the molecular mechanisms involved in GBM to develop effective therapeutic interventions. The epidermal growth factor receptor (EGFR, also referred to as ERBB1 or HER1) is a member of the HER superfamily of receptor tyrosine kinases, together with ERBB2, ERBB3, and ERBB4 [10, 11]. Binding of growth factors to EGFR leads to autophosphorylation of receptor tyrosine kinase and activation of the downstream signal transduction pathways, which are involved in regulating cellular proliferation, differentiation, and survival [12, 13]. EGFR is commonly overexpressed and amplified in gliomas, and contributes to uncontrolled Vol.:( )

2 proliferation and survival of GBM cells [14, 15]. Multiple EGFR inhibitors have been developed that can inhibit tumor growth, such as erlotinib and gefitinib; however, many patients do not respond well to EGFR inhibitors, including those with non-small-cell lung cancer or GBM [16 18]. Meanwhile, EGFR inhibition can cooperate in a schedule-dependent manner with low doses of temozolomide (TMZ) to reduce GBM cell growth [19, 20]. Unfortunately, the mechanisms related to resistance to EGFR inhibitors remain unclear; however, evidence of the roles for micrornas (mirnas) in determining drug-sensitivity/resistance is emerging. MiRNAs are small regulatory RNA molecules that have been identified recently in the progression of various cancers and are proposed as novel targets for anticancer therapies [21, 22]. MiRNAs target the 3 untranslated regions (UTRs) of oncogenes and tumor suppressor genes, thus contributing to the tumorigenesis of various human cancers [23, 24]. Previous studies demonstrated that mirnas could mediate EGFR signaling to influence tumor growth and drug resistance [25 27]. For example, mirna-556 regulates EGFR directly and enhances drug-resistance, correlating significantly with the clinical response and survival following anti-egfr therapy [28]. Our previous studies demonstrated that mir-181b functions as a tumor suppressor gene by inhibiting tumor cell growth, migration, and invasion [29]. However, the detailed role and underlying mechanism of mir-181b s regulation of GMB cells sensitivity to TMZ is unclear. In the present study, overexpression of mir-181b downregulated EGFR and enhanced the apoptosis induced by TMZ. We confirmed that mir-181b influences the EGFR pathway and enhances the sensitivity of GBM cells to TMZ in vitro and in vivo. Therefore, mir-181b may serve as a potential target for overcoming TMZ resistance in human GBM treatment. Materials and methods Tissue collection Twenty Human GBM tissue samples, eight normal brain tissues, and ten paired initial and recurrent GBM tissues with particular clinical data were obtained from the Department of Neurosurgery at the first affiliated hospital of Nanjing medical university. All the tissues were immediately snap-frozen and stored in liquid nitrogen after surgical removal. This study was approved by the hospital Ethics Committee, and written informed consent was obtained from all patients. Cell culture and treatment Human U87 and U251 GBM cells were purchased from ATCC (the American Type Culture Collection, USA) and maintained in Dulbecco s modified Eagle medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 2 mm glutamine (Sigma, USA), 100 µg/ml penicillin (Sigma, USA), and 100 µg/ml streptomycin (Sigma, USA), and incubated at 37 C with 5% CO 2. For cell transfection, Lipofectamine 2000 (Invitrogen, USA) was used according to the manufacturer s instruction. Cell transfection Cells at 50 70% confluence were transfected using Lipofectamine 2000 (Invitrogen). A vector expressing EGFR or the empty vector (both 2 μg) were transfected into U87 and U251 cells according to the manufacturer s instructions. Chemo sensitivity assay The cells were seeded into 96-well plates at per well. After 24 h, the cells were treated with different concentrations of TMZ. The absorption of the cells were measured after 48h using a Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan). Lentivirus packaging and stable cell lines The lentiviral packaging kit was purchased from Open Biosystems (Huntsville, AL, USA). Lentiviruses carrying hsa-mir-181b or hsa-mir-negative control (mir-nc) were packaged following the manufacturer s manual. Lentiviruses were packaged in HEK293T cells and collected from the medium supernatant. Stable cell lines were established by infecting lentiviruses into U87 and U251 cells and selecting them using puromycin. Cell apoptosis assay To measure cell apoptosis, cells in the log phase of growth were harvested by centrifugation for 5 min at 500 g and subjected to annexin V/propidium iodide (PI) staining using an annexin V-FITC Apoptosis Detection Kit (Bio- Vision, CA), according to the manufacturer s protocol. Annexin V and PI cells were used as controls. Annexin V + and PI cells were designated as apoptotic, and Annexin V + and PI + cells displayed necrotic features. The

3 results were evaluated by fluorescence activated cell sorting (FACS) flow cytometry. Western blotting analysis The total proteins of cells were extracted using protein lysis buffer (20 mm Tris HCL, ph 8.0, 120 mm NaCl, 1% Nonidet P-40, 10% glycerol, 1 mm CaCl 2, 1 mm MgCl 2, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium fluoride, 1 mm sodium orthovanadate, and a protease inhibitor mixture). Lysates containing 20 μg of total protein were separated on 8 or 10% SDS-PAGE gels. Proteins were then transferred onto a polyvinylidenedifluoride (PVDF) membrane (Bio-Rad) using a semidry transfer unit (Bio-Rad). Membranes were blocked with 5% nonfat dried milk for 2 h and then incubated with primary antibodies followed by mouse anti-rabbit secondary antibodies. The following primary antibodies were used: EGFR (1:1000, Cell Signaling Technology, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:3000, Abcam, UK). An ECL Western Blotting Kit (Pierce) and X-ray film were used, and signal intensities were determined using Image J software (NIH). Luciferase assay A luciferase assay was performed as described in our previous study [29]. The 3 UTR of EGFR was amplified by PCR from human cdna using the following primers: EGFR forward primer: ccgag CTC AAT AAT AAC TCG GAT TCC AGCCC, EGFR reverse primer: ccctc GAG TTC TGA ACC ATT TCT TCC TTGAT. The complementary sequence in the 3 UTR of EGFR (GAA TGT ) was replaced by (GCA CGC ). The PCR products were digested using SacI and HindIII and inserted into pmirreporter. These constructs were then validated by sequencing. HEK-293 cells were seeded into 24-well plates and co-transfected with the wild-type or mutated EGFR 3 -UTR reporter plasmids and prl-tk, or transfected with mir-181b and mir-nc. Luciferase assays were performed 24 h after transfection using the Dual Luciferase Reporter Assay System (Promega). Tumorigenesis in nude mice Nude mice (male BALB/cA-nu, 6 weeks old) were purchased from the Shanghai Experimental Animal Center (Chinese Academy of Sciences, Shanghai, China) and maintained in specific pathogen-free conditions. Sixteen mice were randomly divided into two groups. In one group, U87 cells stably expressing mir-181b were injected subcutaneously into both flanks of each mouse ( cells in 100 μl), and in the other group, U87 cells stably expressing mir-nc (as a negative control) were similarly injected. When tumors became visible, half of the mice of each group were treated with TMZ by intraperitoneal injection and the other half were not; the tumor sizes were measured using vernier calipers every 2 days. The tumor volume was calculated according to the formula: volume = 0.5 length width 2. The mice were euthanized after 18 days, and tumors were photographed and weighed. Statistical analysis Results showed in figures are expressed as mean ± SD. Data was analyzed using Student s t test in GraphPad Prism (GraphPad Software Inc. La Jolla, CA). P values less than 0.05 were considered to be statistically significant. Results MiR 181b levels are downregulated in human GBM tissues and are especially low in recurrent tissues In our previous research, we showed that mir-181b had a markedly low expression in human glioma tissues. To further investigate the expression of mir-181b in human GBM tissues, we tested new samples and performed a qrt-pcr analysis. The results showed that mir-181b had very low expression in GBM compared with normal brain tissues (Fig. 1a). To evaluate the roles of mir-181b in neoplasm recurrence, ten paired initial and recurrent GBM tissues were collected and analyzed for their mir-181b expression levels. As shown in Fig. 1b, mir-181b expression was significantly downregulated in the recurrent GBM tissues, which suggested that mir-181b has important biological functions in tumor recurrence. Overexpression of mir 181b sensitizes GBM cells to TMZ and induced apoptosis To explore the potential role of mir-181b in chemotherapy, we treated U87 and U251 cells stably expressing mir- 181b or mir-nc with different concentrations of TMZ. As shown in Fig. 2a, b, overexpression of mir-181b in GBM cells significantly increased their chemo-sensitivity to TMZ treatment, and cell viability was reduced significantly with increasing concentrations of TMZ compared with the mir-nc group. To further investigate the effect of mir-181b on apoptosis in the presence of TMZ treatment, FACS analysis was performed to detect cell apoptosis rates. The combination of mir-181b and TMZ treatment induced significant apoptosis (Fig. 2c, d). These findings implied that mir-181b overexpression increases the chemo-sensitivity of GBM cells to TMZ and potentiates TMZ-induced apoptosis.

4 A B Relative mir-181b expression (Normalized by U6) Initial (n=10) ** Recurrent (n=10) Fig. 1 MiR-181b levels are downregulated in human glioblastoma multiforme (GBM) tissues and are especially low in recurrent tissues. a Relative mir-181b expression levels were analyzed by quantitative real-time reverse transcription PCR (qrt-pcr) in normal (n = 8) and GBM tissues (n = 20). U6 RNA levels were used as an internal control. **p < 0.01 indicates a significant difference. b Relative expression levels of mir-181b in initial and recurrent cancer tissues; **p < 0.01 Fig. 2 Overexpression of mir-181b sensitizes glioblastoma multiforme (GBM) cells to temozolomide (TMZ) and induces apoptosis. The GBM cell lines U87 and U251 were infected with mir-181b or mir-nc lentivirus to establish stable cell lines. a and b TMZ sensitivity in U87/miR-NC, U87/miR-181b, U251/miR-NC, and U251/ mir-181b cell lines were tested using a Cell Counting Kit-8 (CCK-8) assay. Data are presented as the mean ± SD from three independent experiments with triple replicates per experiment. **p < c and d Cells were treated with 200 μm TMZ or not, and cell apoptosis was analyzed by flow cytometry after 48 h; **p < 0.01

5 MiR 181b directly targets and inhibits EGFR expression EGFR was reported to be overexpressed and amplified in GBM, in which it promotes GBM cell proliferation, apoptosis and invasion. Using bioinformatics analysis (microrna.org), we found that EGFR is a potential target of mir-181b (Fig. 3a). To confirm this, reporter constructs were made containing the putative mir-181b binding sites from the EGFR 3 -UTR regions (WT), or a mutated version of the same binding site (Mut), as indicated in Fig. 3b. Overexpression of mir-181b inhibited EGFR WT, but not Mut reporter activities (Fig. 3b), demonstrating that mir-181b can specifically target the EGFR 3 -UTR via the binding site sequences. Next, we used western blotting to show that the EGFR protein expression was decreased in U87 cells overexpressing mir-181b compared with cells expressing mir-nc (Fig. 3c). In addition, we found that the expression levels of EGFR were significantly upregulated in human GBM tissues compared with those in normal brain tissues using qrt-pcr (Fig. 3d). Furthermore, there was a significant inverse correlation between EGFR and mir- 181b in the GBM tissues (Fig. 3e, Spearman s correlation r = ). This inverse correlation indicated a possible relationship between mir-181b and EGFR. Fig. 3 mir-181b directly targets and inhibits epidermal growth factor receptor (EGFR) expression. a Putative seed-matching sites or mutant sites (red) between mir-181b and the 3 -untranslated region (UTR) of EGFR. b A luciferase reporter assay was performed on U87 cells to detect the relative luciferase activities of the wild-type (WT) and mutant (Mut) EGFR reporters. A renilla luciferase vector was used as an internal control; **p < c Total proteins of mir- 181b and mir-nc-expressing cells were subjected to western blotting and detected for EGFR expression levels. d The expression levels of EGFR in normal and human GBM tissues were determined by quantitative real-time reverse transcription PCR (qrt-pcr) analysis, and the fold changes were obtained from the ratio of EGFR to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels; **p < e Spearman s correlation analysis was used to determine the correlation between the expression levels of EGFR and mir-181b in human glioblastoma multiforme (GBM) specimens

6 Overexpression of EGFR reverses the inhibitory effects of mir 181b and its apoptotic induction effect in the presence of TMZ To further investigate whether the effects of mir-181b and TMZ in chemotherapy are mediated via EGFR, a plasmid expressing the EGFR cdna was transfected into mir- 181b- or mir-nc-expressing cells. Cell viability under different concentrations of TMZ was assayed using the CCK-8 proliferation assay. The results showed that overexpression of EGFR reversed the mir-181b-induced chemo-sensitivity of GBM cells to TMZ (Fig. 4a, b). We then examined whether the restoration of EGFR could rescue the anti-apoptotic effects of mir-181b. As shown in Fig. 4c, d, the cell apoptosis rate of mir-181b-overexpression plus TMZ treatment was rescued by overexpression of EGFR compared with control cells. Based on these findings, our results indicated that mir-181b sensitizes GBM cells to TMZ, and induces an apoptotic effect combined with TMZ by targeting EGFR. MiR 181b enhances the chemo sensitivity of TMZ in vivo To assess the functions of mir-181b in GBM chemo-sensitivity in vivo, a subcutaneous xenotransplanted GBM model was employed. U87 cells overexpressing mir-nc or mir-181b were collected and injected subcutaneously into both posterior flanks of male BALB/c nude mice, respectively. After 8 days, the mir-nc group was randomly divided into two groups: mir-nc and mir-nc+tmz. TMZ was delivered to the mir-nc+tmz group by peritoneal injection. The same procedure was also applied to the mir-181b group and the tumor volumes were monitored every 2 days during the tumor progression (Fig. 5a). The mice were sacrificed on day 18 and the tumors were excised, after which the tumors were photographed and weighed (Fig. 5b, c). The mir-181b-treated xenografts group had a significant decrease in tumor volume and weight when treated with TMZ compared with the mir-nc group. To reveal the molecular mechanisms of mir-181 in tumor growth, total protein was extracted to perform western blotting analysis to analyze the EGFR expression levels. The results demonstrated the EGFR levels were significantly downregulated in mir-181b overexpressing tumors (Fig. 5d). Thus, mir-181b not only inhibits tumor growth, but also enhances the sensitivity of TMZ chemotherapy via its target EGFR in vivo. Discussion TMZ is the most widely used alkylating agent to treat GBM; however, drug resistance often leads to tumor Fig. 4 Overexpression of EGFR reversed the mir- 181b-induced chemo-sensitivity of GBM cells to TMZ. mir- 181b- or mir-nc-overexpressing cells were transfected with vector or epidermal growth factor receptor (EGFR). a and b TMZ sensitivity were tested using a Counting Kit-8 (CCK- 8) assay; **p < 0.01 indicates significant difference compared with the mir-nc+vector group # p < 0.05, ## p < 0.01 indicate significant differences compared with the mir-181b+ vector group. c and d Cells were treated with 200 μm TMZ or not, and cell apoptosis was analyzed by flow cytometry after 48 h; **p < 0.01

7 Fig. 5 MiR-181b enhances the chemo-sensitivity of temozolomide (TMZ) in vivo. mir-181b- or mir-nc-overexpression cells were dispersed in 100 μl of serum-free DMEM medium and were subcutaneously injected into each side of posterior flank of nude mice (n = 4), then half the mice in each group were treated with TMZ by peritoneal injection. a, b and c Tumors were measured every 2 days after they were observed by the naked eye and their volumes were calculated using the following formula: volume = 0.5 Length Width2. The tumor was excised and weighed after 18 days with representative pictures of tumors shown (Bar 10 mm). d Whole proteins were extracted from xenografts and subjected to western blotting assay for epidermal growth factor receptor (EGFR) expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression served as an internal control recurrence and poor outcome [30, 31]. Therefore, it is pivotal to improve current chemotherapy regimens by identifying new predictive biomarkers based on the existing treatment. Accumulating evidence indicates that the aberrant expression of mirnas is associated with human GBM chemotherapy resistance and sensitivity. Wang et al. confirmed that inhibition of mir-381 enhanced the sensitivity of neurofilament, light polypeptide (NEFL)-mediated stemness factors to TMZ in GBM. MiR-125b has been reported to control apoptosis and TMZ resistance by targeting TNFAIP3 and NKIRAS2 in GBM [32]. Our previous studies focused on the roles of mir-181b in glioma. Meanwhile, mir-181b has also been reported to affect tumor chemo-sensitivity. For example, the suppression of mir181b enhanced the sensitivity of hepatocellular carcinoma cells to doxorubicin. In addition, Wang et al. showed that increased mir-181b modulated GBM cells sensitivity to TMZ by targeting MEK1 [33]. In the present study, we found that mir-181b was decreased between paired initial and recurrent GBMs that were insensitive to TMZ treatment. Moreover, overexpression of mir-181b enhanced the chemo-sensitivity of GBM cells to TMZ by inducing cell apoptosis. These data were further supported by the finding that mir-181b-treated xenografts group had a significant decrease in tumor volume and weight when treated with TMZ compared with the mir-nc group. Therefore, overexpression of mir-181b might not only limit cancer growth, but also sensitize cells to TMZ in vitro and in vivo. Most mirnas function by inhibiting effective mrna translation of target genes. To date, several targets of 13

8 mir-181b have been identified, such as NOVA1, MDM2, LATS2, HMGB1, and MCL-1 [34 36]. Our previous research showed that mir-181b inhibited GBM cell growth by targeting IGF1R [29]. In the present study, we uncovered a novel role of mir-181b for TMZ resistance in GBM and identified EGFR as a direct target of mir-181b. Aberrant expression of EGFR occurs in the majority of gliomas, and activation of EGFR signaling plays a central role in GBM and other human cancers, including lung, breast, and colorectal [37 40]. Accumulating evidence suggests that mirnas can regulate EGFR signaling, correlate with EGFR expression, and influence chemotherapy resistance. For example, mir-21 is responsible for modulating EGFR signaling and is involved in the responses of GBM cells to anti-egfr treatment [41]. Furthermore, mir-146b-5p could target EGFR and reduce the in vitro migration and invasion of glioma [42]. Here, we found that the relative luciferase level for EGFR was significantly decreased in the mir-181b-overexpression group compared with that in the mir-nc group, and western blotting analysis further confirmed these findings. Additionally, overexpression of EGFR partially abolished the effect induced by mir- 181b plus TMZ treatment. Meanwhile, the xenograft data showed that the expression of EGFR in mir-181b-overexpressing cells treated with TMZ was lower that that in the non-treated cells, suggesting that mir-181b is a potential strategy for EGFR-targeted therapy. In conclusion, this study demonstrated that EGFR, as a direct target of mir-181b, might be involved in the mir- 181b-mediated chemo-resistance of GBM cells to TMZ. Further studies are required to verify this association. Acknowledgements This work was supported in part by the National Natural Science Foundation of China [Grant Nos , , , , ], and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical approval This study was approved by the Hospital Ethics Committee. Informed consent For the glioma tissues, written informed consent was obtained from all patients. References 1. Cloughesy TF, Cavenee WK, Mischel PS (2014) Glioblastoma: from molecular pathology to targeted treatment. Ann Rev Pathol 9:1 25. doi: /annurev-pathol Aldape K, Zadeh G, Mansouri S, Reifenberger G, von Deimling A (2015) Glioblastoma: pathology, molecular mechanisms and markers. Acta Neuropathol 129: doi: / s Wen PY, Reardon DA (2016) Neuro-oncology in 2015: progress in glioma diagnosis, classification and treatment. 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