Sequence-dependent Synergistic Inhibition Of Human Glioma Cell Lines By Chemotherapy In Combination With MiR-21 Gene Therapy

Drug insensitivity or resistance has always been a major problem in the treatment of glioma. The purpose of present study is to investigate the chemosensitivity of miR-21 inhibitor to glioma cells and the sequence-dependent cytotoxic effects of combination of miR-21 inhibitor with chemotherapeutic drugs, based on mechanisms of miR-21 in regulation of resistance-related signaling pathways. By using PAMAM dendrimer as vector, miR-21 inhibitor was combined with temozolomide (TMZ), paclitaxel (Taxol) and doxorubicin (DOX) respectively to treat glioma cells. The results of In-situ hybridization showed that PAMAM could effectivly mediate the transfection of the miR-21 inhibitor, leading to the obviously decrease of miR-21 expression in tumor cells, and meanwhile, resulting in 2- to 3-fold decrease in the concentrations caused 50% growth inhibition (IC50) for DOX, TMZ and Taxol respectively when used together to treat the tumor cells. The Q value analysis was employed to evaluate the combination effect of different sequence treatment for two glioma cell lines. For LN229 cell line, the concomitant treatments exhibited strongest synergistic inhibition effects on the cell growth. While for U251 cell lines, simultaneous treatment only yelided weak additive effect. The synergetic inhibition effect appeared when treated the cells with miR-21 inhibitor followed by chemotherapeutic drugs 4h later or the reverse sequence.In order to comprehensively study the combination mechanism of sequence-dependent cytotoxic effects for miR-21 and chemotherapeutic drugs, TMZ and three different genetic background glioma cell lines LN229 (PTEN wild), U251 (PTEN mutant) and U87 (PTEN lost) were chosen to analyze the changes in drug resistance-related signal pathways and their relationships with the antitumor effect of the combine treatment. Both the results of Real time PCR and In-situ hybridization demonstrated that the cell proliferation exhibited a negative correlation with miR-21 expression level in any cases of combining treatments. And the flow cytometry analysis and the Western blot detection indicated that the apoptosis introduced by the combining treatment relied to the STAT3 status in tumor cells. Whatever the cell lines, the best suppression effect for the glioma cells always be acquired at the lowest expression level of STAT3. Taken together, this study provides evidence that the combination of miR-21 inhibitor with chemotheraputants could be an effective therapeutic strategy for the inhibition of glioma tumor, but the inhibition effect was influenced by the treatment sequence of gene therapy and chemotherapy. Our researches provide an opportunity to make use of oncogenic pathway signatures to guide the use of targeted therapeutics.