Current anti-cancer chemotherapeutic agents for glioblastoma have not significantly improved the survival of glioblastoma patients during the past ten years . Those patients succumb to their disease mostly for the reason of chemoresistance. Chemoresistance may be either inherent (intrinsic resistance), or induced by chemotherapeutic drugs (acquired resistance) . Intrinsic resistance to anti-cancer drugs results from various factors, including somatic cell genetic diversification in tumors and individual variations of patients. Acquired drug resistance occurs when a tumor that initially sensitive to an anti-cancer drug becomes resistant to that treatment. One prevalent reason for acquisition of chemoresistance is induction of energy-dependent transporter proteins that pump anti-cancer drugs out of cells, and other mechanisms of chemoresistance including resistance to drug-induced apoptosis may also play an important role in acquired drug resistance. Furthermore, recent study indicates that intrinsic and acquired resistances have some similar profiles . So far, there is no effective strategy to overcome chemoresistance. Moreover, drug resistance can only be identified after long-time treatment until now. Therefore, early diagnosis to indicate drug resistance is essential for optimizing therapeutic strategy, avoiding unnecessary treatment and drug-induced side effects. In view of this fact, the research on mechanisms of chemoresistance regulation, the early diagnosis of drug resistance, and the development of novel and effective anti-cancer therapies against glioblastoma are urgently required. In this study, Let-7b down-regulation is associated with acquired cisplatin resistance in U251R cells. Let-7b mimics re-sensitized U251R cells to cisplatin through suppression of cyclin D1 protein expression. Based on these findings, Let-7b might be considered as an early diagnostic marker of cisplatin resistance, and restoration of Let-7b could overcome cisplatin resistance in glioblastoma cells.
Recently, miRNA has been proved as one of the critical regulators during glioma progression. Both up-regulation and down-regulation of miRNAs are involved in the development of glioblastomas and chemoresistance. Shi et al. showed that over-expression of miR-21 could attenuate TMZ-induced apoptosis in U87MG cells through up-regulation of Bax, reduction of Bax/Bcl-2 ratio and caspase-3 activity, demonstrated that miR-21 over-expression is associated with resistance to chemotherapeutic drug TMZ . Furthermore, Li et al. demonstrated that miRNA-21 targets LRRFIP1 which inhibits NF-κB activation. NF-κB pathway is activated upon miR-21 over-expression, exhibits significant anti-apoptotic efficacy, and contributes to VM-26 resistance in glioblastoma . Based on these findings, miR-21 could be a potential target to increase the chemotherapeutic efficacy during glioblastoma treatment. Another study indicated that using an established U251 cell line resistant to temozolomide, Ujifuku et al. performed an analysis of miRNA expression in this cell line and its parental cell line. Three miRNAs miR-195, miR-455-3P, and miR-10a were identified as the most up-regulated miRNAs in the U251 cell line resistant to temozolomide. Knockdown of miR-195 inhibited tumor cell growth, suggesting that it could be a potential target for treatment of glioblastoma with acquired TMZ resistance . In our study, Let-7b was down-regulated in acquired cisplatin-resistant U251R cells. Furthermore, ectopic Let-7b can increase the sensitivity of U251R cells to cisplatin through inhibition of cyclin D1 expression. In this regard, Let-7b could overcome cisplatin resistance in glioblastoma cells, indicating that it could be applied to treat glioblastoma patients with cisplatin resistance.
It is known that Let-7 modulates chemosensitivity in various types of cancer. Let-7 inhibited gemcitabine chemoresistance in pancreatic cancer , and could also negatively modulate the chemoresistance in Head and neck cancer . Sugimura et al. showed that Let-7b and Let-7c expression were down-regulated in cisplatin-resistant esophageal cancer cell lines compared with their parent cell lines . Transfection of Let-7 into esophageal cancer cell lines restored their sensitivity to cisplatin. Furthermore, low expression of Let-7b and Let-7c in before-treatment patients is correlated with poor response to cisplatin-based chemotherapy, so Let-7 can also be used as a marker to predict the sensitivity to cisplatin treatment . Moreover, Let-7b down-regulated cyclin D1 expression through targeting 3’-UTR of cyclin D1 mRNA, and inhibited cell cycle progression in melanoma cells . Let-7 also regulates cyclin D1 in other types of tumors. It is reported that Let-7 miRNA inhibited cell growth partially by decreasing mRNA expression of cell cycle stimulators MYC and cyclin D1 in thyroid cancer . Zhao et al. demonstrated that Let-7b regulates neural stem cell proliferation and differentiation by targeting cyclin D1 . Our results also indicated that down-regulation of Let-7b was correlated with cisplatin resistance in glioblastoma cells, and Let-7b could attenuate cyclin D1 expression then dampen chemoresistance of U251R cells to cisplatin. Overall, restoration of Let-7 in glioblastoma may offer a new approach for cancer treatment in the future.
Cyclin D1 belongs to a family of protein kinases that involved in cell cycle regulation. Cyclin D1 has been proved to be associated with chemoresistance to cisplatin-based therapy. Noel et al. demonstrated that cyclin D1 expression was significantly higher in chemoresistant testicular germ tumor cell lines comparing with the parental cells. Furthermore, cyclin D1 knockdown in combination with cisplatin treatment inhibited tumor cell growth more effectively than single treatments . In pancreatic tumor cells, over-expression of cyclin D1 also dramatically reduced chemosensitivity and prolonged survival time upon cisplatin treatment, and knockdown of cyclin D1 resulted in impaired resistance to cisplatin-induced apoptosis [41, 42]. Moreover, inhibition of cyclin D1 expression in human pancreatic cancer cells enhances their responsiveness to multiple chemotherapeutic agents other than cisplatin, including 5-fluorouracil, 5-fluoro-2'-deoxyuridine, and mitoxantrone These findings demonstrate that up-regulation of cyclin D1 may be a major reason of cisplatin resistance in multiple tumors. In this regard, cyclin D1 could be a potential marker for treatment evaluation and a candidate target to improve the treatment of cisplatin-resistant tumors. Our study indicated that Let-7b might down-regulate cyclin D1 protein expression through targeting its 3’-UTR. Therefore, cyclin D1 down-regulation induced by restoration of Let-7 in tumors might be a novel therapeutic strategy for cisplatin-resistant glioblastoma treatment.
To sum up, we generated a cisplatin-resistant glioblastoma cell line U251R, and analyzed miRNA expression profiles in U251R compared with its parental cell line U251. Microarray data indicated that Let-7b was dramatically down-regulated in U251R cells compared with U251 cells. Furthermore, ectopic expression of Let-7b remarkably inhibited U251R cell chemoresistance to cisplatin through cyclin D1 expression blockade. Cyclin D1 knockdown significantly promoted cisplatin-induced apoptosis and G1 arrest. In conclusion, Let-7b could be considered as a novel marker of cisplatin resistance during early diagnosis, and more importantly, restoration of Let-7 in tumor cells could offer a novel therapeutic approach for cisplatin-resistant glioblastoma treatment.