The Role And Molecular Mechanisms Of Micro RNAs In Drug Resistance Of Osteosarcoma

【Background】Osteosarcoma is a rare highly malignant bone tumor that occurs predominantly in adolescents and young adults. A defining feature of osteosarcoma is the high rate of metastasis and the most common site of metastasis is the lungs. In the era before the use of chemotherapy, over 85% of post-surgery patients continued to develop metastases. Chemotherapy routinely plays an important role in the treatment of advanced osteosarcoma. Now, although the percentage of patients cured has increased between 60% and 70% by neoadjuvant chemotherapy coupled with limb-sparing surgery, the prognosis remains poor for most patients with metastatic or recurrent osteosarcoma.This poor prognosis is mostly due to the development of drug resistance by osteosarcoma cells after chemotherapy. Currently, neoadjuvant chemotherapy drugs for osteosarcoma include cisplatin(C DDP), adriamycin(ADM), methotrexate(MTX) and ifosfamide(IFO).C isplatin or cis-diamminedichloroplatinum(II) is one of the most active anticanceragents, being widely used against various tumors including osteosarcoma. Because osteosarcoma cells often acquire resistance to drugs and even develop multiple drug resistance, which results in treatment failure. In this regard, CDDP-resistance has become one of major clinical problem to overcome. Though extensive study has been done on drug resistance, there is no mechanism completely to explain the clinical response to drug resistance. In recent years, a large number of studies have shown that micro RNAs(mi RNAs) are a class of small non-coding RNAs, negatively regulate gene expression at post-transcriptional level and are encoded by the genomes of a wide range of multicellular organisms. mi RNAs participate in a variety of cell signaling pathways and regulate more than 30 percent human genes. mi RN As have been shown to play crucial roles in cancer development, progression and many physiological and pathological processes, including cancer drug resistance. Indeed, some of the protein associated with drug resistance mechanisms are also regulated by mi RNAs. So we postulated that mi RN A could be such class of modulators located in the upstream of drug resistance mechanisms of osteosarcoma. These mi RN A molecules can be as osteosarcoma biomarkers and treatment targets.【Aims】To establish a new cisplatin- resistant human osteosarcoma cell line and investigate its biological characteristics. To investigate the expression of mi RN A in cisplatin-resistant human osteosarcoma cell line and parental cell line. To discuss the roles of mi RNA in the underlying mechanisms of osteosarcoma drug resistance.To provide a useful tool in the gene treatment of osteosarcoma drug resistance.【Methods】1. The human osteosarcoma cell line SOSP-9607 was exposed to cisplatin by stepwisely increasing the concentrations in medium to select for the drug-resistant subline, SOSP-9607/CDDP cells.2. The morphological features were observed using inverted microscopy. The growth curves of SOSP-9607 and SOSP-9607/CDDP cells were drawn to calculate the doubling time. FCM was also used to determine the distribution of the cell cycle. The MTT assay was performed to test the drug resistance of SOSP-9607 and SOSP-9607/CDDP cells. Transwell assay was used to examine invasion capability of SOSP-9607/CDDP and SOSP-9607 cells. RT-PCR was performed to determine the m RNA expression levels of drug-resistance-related and apoptosis-related genes, MDR1, MRP1, MRP2, LRP, ABC G2, GST-π, Bcl-2 and Bax in both cell lines. Immunocytochemistry and western blot were performed to determine the expression levels of MRP1.3. The differentially expressed mi RNAs between SOSP-9607 and SOSP-9607/CDDP cells were identified by mi RN A profiling of these two cell lines using mi RN A microarray.4. Real-time RT-PCR analysis was used to validate the results obtained by microarray profiling. Five micro RNAs were selected for real-time RT-PCR verification.5. Bioinformatics anaylsis was used for mi RNA target prediction. BAK1 was the potential target of mi R-25.6. The pre-mi R-25 was transfected into SOSP-9607 cells to up-regulate mi R-25 expression and the anti- mi R-25 was transfected into SOSP-9607/CDDP cells to down-regulate mi R-25 expression.7. MTT assay was used to determine the effect of mi R-25 on drug sensitivity of osteosarcoma cells(SOSP-9607 and SOSP-9607/CDDP) in vitro. Flow cytometry was used to determine the effect of mi R-25 on drug-induced apoptosis in osteosarcoma cells. The putative target gene of mi R-25 was validated by Western blot and RT-PCR.8. Luciferase reporter assay was used to validate the putative target gene(BAK1)of mi R-25.【Results】1. The cisplatin-resistant human osteosarcoma cell line(SOSP-9607/CDDP) was established by stepwise exposure methods after 12 months.2. SOSP-9607/CDDP cells exhibited changes in morphology, proliferation rate, doubling time, cell cycle distribution and invasion capability were detected as compared with SOSP-9607 cells. SOSP-9607/CDDP cells were 6.24-fold resistant to cisplatin in comparison with SOSP-9607 cells, and also exhibited cross-resistance to CBP, MTX and ADM. There were more SOSP-9607/CDDP cells in G0/G1 phase and less in S phase as compared to SOSP-9607 cells. There was no significant difference between the two cells in G2/M phase.SOSP-9607/CDDP cells overexpressed MRP1, MRP2, and GST-π. Immunocytochemistry and western blot assay show that MRP1 protein levels were increased significantly in SOSP-9607/CDDP cells compared with SOSP-9607 cells.3. Eighteen microRNAs were differentially expressed in SOSP-9607/CDDP cells compared with that of SOSP-9607 cells. Of these, 11 were over expressed and 7 were under expressed in SOSP-9607/CDDP cells.4. The results of real-time RT-PCR assay( mi R-25,-29 c,-1290,-194) consistented with that of mi RN A microarray.5. The results of bioinformatics prediction method show that there are binding sites for seed sequence of mi R-25 in 3 ’-UTR of BAK1.6. The pre- mi R-25 and anti- mi R-25 were respectively transfected into SOSP-9607 and SOSP-9607/CDDP cells. RT-PCR analysis showed that the transfection was effective, while no significant changes were observed in the m RN A level of BAK1. However, the increased BAK1 protein level in SOSP-9607 cells and the decreased BAK1 protein level in SOSP-9607/CDDP cells were observed using western blot analysis.7. The pre- mi R-25 and anti- mi R-25 were respectively transfected into SOSP-9607 and SOSP-9607/CDDP cells. Flow cytometry analysis showed that SOSP-9607 cells exhibited lower sensitivity to CDDP- induced apoptosis compared tocontrol-transfected ones and SOSP-9607/CDDP cells exhibited higher sensitivity to CDDP- induced apoptosis compared to control-transfected ones.8. The pre- miR-25 and anti- miR-25 were respectively transfected into SOSP-9607 and SOSP-9607/CDDP cells. MTT assay showed that overexpression of mi R-25 sensitized SOSP-9607/CDDP cells to different anticancer drugs whereas inhibition of them conferred SOSP-9607 cells drug resistance.9. The pmir Glo-vector luciferase reporter assay showed that a significant decrease of luciferase activity in the presence of pre- mi R-25 in SOSP-9607 cells was observed. These results experimentally confirm that BAK1 is a direct target for mi R-25 in SOSP-9607 cells.【Conclusions】We established a new cisplatin-resistant human osteosarcoma cell line, SOSP-9607/CDDP. This cell line is an invaluable tool to study the resistance of anticancer drugs. There were 18 differentially expressed micro RNAs in SOSP-9607/CDDP cells compared with that of SOSP-9607 cells, suggesting the involvement of these micro RN As in the pathogenesis of osteosarcoma drug resistance. BAK1 is a direct target for mi R-25 in SOSP-9607 cells. mi R-25 may play an important role in the development of osteosarcoma drug resistance.