Experimental Study On MicroRNAs Increasing The Sensitivity Of Chondrosarcoma Cells To Cisplatin

BackgroundChondrosarcoma is the second most frequent primary malignant type of primary bone malignancy after osteosarcoma. Although the operation is the preferred and effective treatment to chondrosarcoma, but requires complete excision of tumor, otherwise easily relapse. Chemotherapy and radiation are not considered as active treatments since these tumors are notoriously resistant to both chemotherapy and radiation treatment, therefore a new hypothesis of the molecular mechanism of regulating chondrosarcoma resistance to chemotherapy has been widely discussed.MicroRNAs (miRNA) are small single-stranded non-coding RNAs of 19 to 25 ribonucleotides in length, that are encoded in both plant and animal genomes.Each miRNA is assigned a species prefix and an individual numerical identifier. One hundred kinds of miRNA have been found to play an important role in the proliferation, differentiation, migration, cell cycle, apoptosis and other biological processes and along with the research, miRNA playing an important role in the tumor occurrence, development, treatment and so on,which has been recognized by the public. MiRNA combined with mRNA to play regulating role, the same miRNA combined with different target in different tissues plays different role, show that the regulation mechanism of miRNA is a complex network. Under normal circumstances, the miRNA through the precise network to maintain a balance of protein levels in cells or tissues, but when its function is missing or obtained, will break the original balance. MiRNA has been reported to be involved in drug tolerance, and as a potential tumor suppressor. The expression of miRNA in many human tumor tissues is abnormal and it is provided with important significance in diagnosis, treatment and prognosis. It is found that miRNA is involved in regulating the sensitivity of tumor cells to chemotherapy, which is important for revealing the mechanism of drug resistance. Resistance to chemotherapy is one of the main reasons for the poor prognosis of tumor patients. With the development of the correlation between miRNA and tumor,more and more studies have shown that the abnormal expression of miRNA is associated with the chemotherapy tolerance. MiRNA mutation, abnormal expression and abnormal processing will affect the normal function of miRNA, which leads to abnormal expression of target protein. If the target gene is associated with tumor cells to chemotherapy sensitivity, the sensitivity of tumor cells to chemotherapy will be changed. Resistance to chemotherapy is correlation with the different position and role of miRNA in different tumor cells.Generally, it can be divided into four types: apoptosis related miRNA mediated chemotherapy resistance, drug transport related miRNA mediated chemotherapy resistance, cytothesis related miRNA mediated chemotherapy resistance, cytothesis related miRNA mediated chemotherapy resistance,drug target related miRNA mediated chemotherapy resistance. At present, the research of miRNA in tumor drug resistance is just beginning. Most of the current research on miRNA spectrum is to find out the key miRNA related to drug sensitivity by comparing the resistant cell line and parental sensitive cell line, in order to deeply study the mechanism of tumor chemotherapy tolerance and find new drug targets.Cisplatin is the first-line drug for the treatment of solid tumors, as a metal complex for platinum, its functions like a alkylating agent, interfering with DNA replication.Characterized by a broad anticancer spectrum, strong function, synergy with multiple antitumor drugs and no cross resistance, it belongs to a non-specific cycle drug and one of the most common drugs used in current combined chemotherapy. This study is divided into two parts, take cisplatin as an example to explore the mechanism of mir-100 and mir-23b recovery chemotherapy sensitivity of chondrosarcoma and provide a theoretical basis for miRNAs as a treatment of chondrosarcoma.Part I Experimental study on miR-100 increasing the sensitivity of chondrosarcoma cells to cisplatinObjective:To investigate the mechanism of miR-100 increasing the sensitivity of chondrosarcoma cells to cisplatin and provide basic theory for miR-100 as a therapeutic strategies for chondrosarcoma.Methods:1.The expression levels of miR-100 in mutiple human chondrosarcoma cell lines, normal chondrocyte lines and chondrosarcoma tissue samples from patients were determined by quantitative real-time PCR (qRT-PCR).2.Drug screening was conducted for CH-2879 chondrosarcoma cell line by concentration gradient method to obtain cisplatin-resistant CH-2879 cells(named:CH-2879/DDP). QRT-PCR was used to detect and compare with the expression level of miR-100 in the resistant cell line and its parent cell line. MTT and clone formation assay were employed to compare the proliferation abilities and clone formation abilities of these two cells under the action of cisplatin.3.The target of miR-100 was predicted based on miRNA databases such as TargetScan, Pictar and MicroRNA.4.pre-miR-100 and anti-miR-100 as well as their negative controls were transferred into CH-2879 cells with Lipofectamine 2000, then miR-100 expression in these cells after transfection was verified by qRT-PCR, and mTOR expression and the phosphorylation levels of S6K and 4EBP1 after improving or inhibiting miR-100 were examined by Western blot.5.Luciferase reporter assay was used to further verify miR-100 acting on the 3’UTR region of mTOR.6.CH-2879 cells and CH-2879/DDP cells were treated with CDDP, BEZ235 and CDDP+BEZ235 respectively. The inhibition of mTOR pathways and the sensitivity of chondrosarcoma cells to cisplatin.7.The miR-100 and its controls were transfected into CH-2879 cells and CH-2879/DDP cells respectively. High miR-100 expression and the sensitivity of chondrosarcoma cells to cisplatin were observed.8.Exogenous overexpression of S6K into mir-100 pre-transfected chondrosarcoma cells to observe the resistant changes of chondrosarcoma cells to cisplatin,after the activity recovery of the mTOR pathway.Results:1.miR-100 is down regulated in human chondrosarcoma cells and patient samples.2. CH-2879/DDP cells exhibits lower expression of miR-100, indicated that the gene is associated with chemoresistance of chondrosarcoma.3.As a hot research point,the mTOR signaling pathway has also been increasingly found implicated in tumorigenesis. As we predicted,mTOR was a direct target of miR-100 in chondrosarcoma cells. Overexpression/low of miR-100 can inhibit/recovery the expression of mTOR.4.Overexpression of miR-100 in chondrosarcoma enhances the sensitivity to cisplatin, further evidence indicated that this gene is associated with chemoresistance of chondrosarcoma.5.Restoration of the activity of mTOR pathway renders miR-100 overexpressing chondrosarcoma cells resistant to cisplatin, indicated that overexpression of miR-100 sensitizes chondrosarcoma cells to cisplatin through the inhibition of mTOR pathway.Conclusions:The downregulation of miR-100 in chondrosarcoma,compared with normal chondrocyte,indicated that miR-100 is a chondrosarcoma suppressor; The downregulation of miR-100 in CH-2879/DDP cells,compared with parental cells, not only showed that the miR-100 is a chondrosarcoma suppressor gene, but also revealed miR-100 associated with chemosensitivity of chondrosarcoma to cisplatin. mTOR is activated in a variety of malignant tumors and can become a promising therapeutic target for the treatment of cancer. In this study, we determined that mTOR was a direct target of miR-100, and first reported that over expression of miR-100 sensitized chondrosarcoma cells to cisplatin through the inhibition of mTOR pathway, provides basic theory for miR-100 as a therapeutic strategies for chondrosarcoma.PartⅡ Experimental study on miR-23b increasing the sensitivity of chondrosarcoma cells to cisplatinObjective:To investigate the mechanism of miR-23b increasing the sensitivity of chondrosarcoma cells to cisplatin and provide basic theory for miR-100 as a therapeutic strategies for chondrosarcoma.Methods:1.The expression levels of miR-23b in mutiple human chondrosarcoma cell lines, normal chondrocyte lines and chondrosarcoma tissue samples from patients were determined by qRT-PCR.2.pre-miRs and contral-miRs were transferred into SW1353 cells with Lipofectamine 2000, and detect the proliferation of cells in different time points after transfection.3. Under normal cell culture conditions, CH-2879 cells and CH-2879/DDP cells were treated with cisplatin for 48 hours, and the cell morphology was observed by microscope and the expression of miR-23b was detected by qRT-PCR.4. According to previous reports, we predict and verify that Src is a direct target of miR-23b.5. Using different concentration of cisplatin to treat CH-2879 cells, after 48 hours, the cells were collected for Western blot to verificate the effect of miR-23b overexpression on Src-Akt pathway.6.pre-miR-negative and pre-miR-23b were transferred into SW-1353 and CH-2879 cells with Lipofectamine 2000, then miR-23b expression in these cells after transfection was verified by qRT-PCR, Src and p-Src after improving or inhibiting miR-23b were examined by Western blot.7. By using Lipofectamine 2000, containing wild type and mutant Src3’UTR transfected cells,Luciferase reporter gene method was used to further verify miR-23b acting on the 3’UTR of Src.8.contral-miR and pre-miR-23b were transferred into CH-2879 cells, SW1353 cells and CH-2879/DDP cells, using different concentration of cisplatin to treat these cells and using MTT to determine cell activity.9.The miR-23b was transfected into CH-2879 cells and CH-2879/DDP cells.to observe miR-23b overexpression increasing the sensitivity of chondrosarcoma cells to cisplatin.10.Exogenous overexpression of Src into mir-23b pre-transfected chondrosarcoma cells to observe the resistant changes of chondrosarcoma cells to cisplatin,after the activity recovery of the Src-Akt pathway.Results:1.miR-23b is down regulated in chondrosarcoma patient samples.2.Cisplatin resistant chondrosarcoma cells exhibits lower expression of miR-23b,indicated that the gene is associated with chemoresistance of chondrosarcoma.3.Src kinase is involved in the proliferation, migration and invasion of tumor. CH-2879/DDP cells exhibit upregulated activities of Src-Akt pathway. 4.Src is a direct target of miR-23b in chondrosarcoma cells.5.Overexpression of miR-23b sensitizes chondrosarcoma cells to cisplatin through direct suppression of Src-Akt pathway.6.Restoration of Src-Akt pathway in miR-23b overexpressing cells renders chondrosarcoma cells resistant to cisplatin.Conclusions:Same as miR-100,the down-regulation of miR-23b expression in chondrosarcoma cells,indicated that miR-23b is a chondrosarcoma suppressor and associated with chemosensitivity of chondrosarcoma to cisplatin. We confirmed by experiment, Src was a direct target of miR-23b in chondrosarcoma cells, Akt is a cytoplasmic serine and threonine kinasethat positively regulates metabolism, cell cycle progression and cell survival.MiR-23b can negative regulate the Src-Akt pathway to improve the sensitivity of chondrosarcoma cells, our research provides basic theory for miR-23b as a therapeutic strategies for chondrosarcoma.