Study on the Molecular Mechanism of Let-7b Regulates Glioma Cell Chemoresistance
|School||Central South University|
|Keywords||glioma cell drug resistance microarray let-7b cisplatin|
Background:Gliomas account for approximately40%of all central nervous system tomors, and these leisions are locally invasive and recur even after aggressive resection, radiation, and chemotherapy are performed. The prognosis for patients with high-grade gliomas, which include anaplastic astrocytoma(WHO Ⅲ) and glioblastoma multiforme (GBM, WHO IV), remains dismal.Hence, there is a great hope for novel therapeutic approaches. MicroRNA(miRNA) are a family of approximately22-nuleotide-long, highiy conserved, non-coding, single-stranded small RNAs, which induce target gene silence by inhibiting translation or by targeting messenger RNA(mRNA) for degradation in a post-transcriptional fashion. MiRNAs involver in a series of biological events, including development, hematopoiesis, organ generation, apoptosis, development and progress of cancers. Currently, accumulation evidence has suggested that microRNAs may play an important role in the mechanism of drug resistance.Objective:To analyze the difference in microRNAs expression between U251and U251/CDDP cells and explore the mechanism and association between microRNA and drug resistance of glioma cell.Methods:1、malignant glioma cell line U251was used to induce cisplatin resistance subline by gradual elevation of cisplatin concentration in culture. MTT assay was performed to assess the cytotoxicity of cisplatin in U251and U251/CDDP cell lines. MicroRNA microarray was applied to detect the microRNA expression profile of human glioma cell line U251and its drug resistant model U251/CDDP. The results of the microarray were confirmed by real-time PCR. Special microRNA was synthesized in vitro and transfected to U251and U251/CDDP to reverse the drug resistance of the model.2、U251and U251/CDDP cell lines were transfected with let-7b micmics to improve its expression. Apoptosis was analyzed by Annexin V-FITC staining. Western blot analysis was used to determine expression of Bcl-2、Bax and active caspase-3proteins after cisplatin treatment. Flow cytometry(FCM) was used to determine the cell cycles. A computational search revealed a conserved target site of let-7b within the3’-untranslated region of CCND1, and protein expression of CCND1、ppRb after let-7b transfected were determined by Western blot. CCND1and ppRb probein expressions were detected by WB after transfection of siRNA-CCND1. Then, apoptosis and cell cycles in the two glioma cells were analyzed by flow cytometry. Result:1、A cisplatin resistant subline U251/CDDP has been successfully established, which could be stably cultured in the presence of0.5ug/ml cisplatin. The IC50value of U251/CDDP was3.1times as high as that of U251cells.(1.4±0.1ug/ml and4.4±0.9ug/ml respectively). The drug resistance index of U251/CDDP cells relative to the patental U251cells was3.1. Microarray analysis of U251to U251/CDDP cells identified57differentially expressed genes, including23up-regulated and34down-regulated ones in U251/CDDP. Of these differentially expressed microRNAs, miR-182、miR-224showed significantly increased expression,and Let-7b、miR-125b、miR-107、miR-203showed significantly lowered expression in U251/CDDP cells as indicated by the results of microarray analysis and RT-PCR. IC50of combination of let-7b、miR-125b、miR-107、miR-203、miR-182、miR-224and CDDP in U251glioma cells were0.63±0.09、1.47±0.06、1.33±0.07、1.1±0.02、1.17±0.10、0.77±0.07ug/ml; while IC50of combination of let-7b、miR-125b、miR-107、miR-203、miR-182、miR-224and CDDP in U251/CDDP glioma cells were1.62±0.03、5.52±0.47、5.87±1.56、3.72±0.96、2.58±0.22、2.74±0.59ug/ml。Let-7b restoration in U251and U251/CDDP cells rendered the cells2-3-fold more sensitive to the other microRNAs, based on IC50data. In a word, we found that there were differential expression of microRNAs between U251and U251/CDDP cells, and let-7b may paly an important role in the mechanism of drug resistance.2、Over-expression of let-7b in U251and U251/CDDP cells have no effect on the expression of protein such as Bcl-2、Bax、caspase-3. However, Combination of let-7b and cisplatin inhibited Bcl-2protein, increased Bax protein and the activity of caspase-3more significantly when compared with negative group, let-7b group, and cisplatin group. Over-expression of let-7b in U251and U251/CDDP cells resulted to inhibition of cell growth and arrest in G0/G1phase, and induced apoptosis. Reporter assays indiacted that CCND1was a direct target of let-7b. Both of let-7b and si-CCNDl inhibited the expression of CCND1and ppRb protein.Conclusion:1、U251/CDDP cells show a differnet microRNA expression profile from its parental U251cells, suggesting the involvement of microRNAs in tumor cell drug resistance. This finding provides a expremental basis for further study of mechanism.2、Transfected with let-7b micmics, let-7b can enhance the chemo-sensitivity of U251and U251/CDDP.3、Let-7b modulated chemosensitivity of U251and U251/CDDP glioma cells to chemotherapeutic agents by regulating the expression of CCND1protein.4、The anti-let-7b expression siRNA expression recombinants were constructed successfully. It could inhibit the expression of CCND1and ppRb protein, arrest in G0/G1phase and induced apoptosis.