Cisplatin in combination with temozolomide has been in clinical trial in malignant glioma patients [18–20]. The combination of temozolomide and cisplatin is safe and effective in the treatment of chemotherapy-naïve GBM patients, and also in pre-treated patients with high-grade glioma refractory to single-agent temozolomide [21, 22]. However, cancer cells can develop a resistant phenotype

to cisplatin in many patient cases with very poor clinical outcomes [23]. Mechanisms associated with chemoresistance Selleck BB-94 to cisplatin have been investigated, such as Selleck Necrostatin-1 up-regulation of drug transporter proteins, aberrancies in DNA damage repair, and apoptosis induction [24]. However, mechanisms of how tumors become resistant to cisplatin have still not been clearly established [25]. To study chemoresistance in glioma, we established a cisplatin-resistant glioblastoma cell line U251R, which

is 3.1 fold resistant to cisplatin compared to its parental cell U251. MiRNA expression signature analyzed by microarray identified 16 miRNAs as down-regulated in U251R. Let-7b is one of the most significantly suppressed miRNA. Furthermore, over-expression of Let-7b significantly re-sensitized U251R cells to cisplatin through inhibition of cyclin D1 expression. Cyclin D1 knockdown dramatically increased cisplatin-induced apoptosis and G1 arrest. Taken together, our results suggested that cisplatin treatment leads to Let-7b suppression, which in turn up-regulates cyclin D1 expression, resulting in resistance to cisplatin. Therefore, Let-7b may be considered as a marker for early find more diagnosis of cisplatin resistance, and restoration of Let-7

in glioblastoma could be a new strategy for cisplatin-resistant cancer treatment in the future. Materials and methods Reagents, antibodies, and vectors Fetal bovine serum for cell culture and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). Anti-β-actin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Bcl-2, Bax, and ppRb antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-cyclin D1 antibody was from Abcam (Cambridge, MA, USA). Let-7b mimics expression vector was purchased Florfenicol from Wuhan Genesil Biotechnology (Wuhan, Hubei, China). Cell culture Human neuronal glioblastoma cell line U251 was a gift from Dr. Zhongping Chen (Sun Yat-Sen University, Guangzhou, Guangdong, China). U251 cell line was maintained in Dulbecco’s Modified Eagle’s Medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Invitrogen), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen), in a 5% CO2 humidified atmosphere at 37°C. Generation of cisplatin-resistant U251 cells in vitro To generate a cisplatin-resistant cell line, U251 cells were exposed to increasing concentrations of cisplatin. Cisplatin concentrations were increased stepwise from 0.1 μg/mL to 0.