| Part one The function of CD133 expression on the cell proliferation of glioblastoma stem cells treated by cisplatinThe tumor progenitor cells in Glioblastoma(GB)were CD133~+(CD133~+)cells.The expression level of CD133 is closely related to the disease severity and prognosis of GB patients,and a higher expression of CD133 indicates a worse prognosis.In addition,clinical data shows that patients with high expression of CD133 have low sensitivity to cisplatin drug therapy,but how CD133 is involved in the regulation of cisplatin resistance is still unclear.Therefore,targeted inhibition of CD133 expression may be an effective strategy for GB treatment.In this part of the study,we compared some biological characteristics of CD133~+and CD133~-GB cells,and further discussed the role of CD133 in cisplatin resistance,in order to discover the molecular mechanism of CD133 involved in cisplatin resistance in GB,and to provide a theoretical basis for the development of the medicine for targeted therapy.We separated CD133~-and CD133~+cells from GB cells(T89G and U87MG)by FACS flow cytometry,and detected the apoptosis of the two cells treated with cisplatin.Then we measured mRNA levels of CD133 in tumor cells before and after cisplatin treatment.Finally,we analyzed the proliferation ability of CD133~+cells by testing cell proliferation.The results showed that cisplatin treatment could significantly increase apoptosis of CD133~-T89G and U87MG cells.Compared with CD133~-cells,the apoptosis of CD133~+T89G and U87MG cells treated with cisplatin was significantly reduced.These results suggest that CD133 expression in GB cells may contribute to cisplatin resistance.The result also demonstrated that cisplatin treatment significantly increased the expression of CD133 in CD133~+T98G and U87MG cells.The cell proliferation experiments showed that the proliferation capacity of CD133~+T98G cells was significantly higher than that of CD133~-T98G cells.Part two The regulatory mechanism of miR-29a in cisplatin resistance of CD133~+glioblastomaStudies have shown that the miR-29 family can be used as biomarkers for a variety of malignant tumors,and the occurrence,development and prognosis of tumors can be evaluated by detecting the expression level of miR-29 in serum or tumor tissues.More and more studies on the members of the miR-29family have shown that they play an important role in pathophysiological processes in tumor biology.Recent studies have demonstrated that,whether used alone or in combination with other proven disease-specific microRNAs,the miR-29 family has a potential application as a predictive biomarker for early diagnosis of malignant tumors.This part of the study intends to explore the correlation between miRNA expression level and drug resistance of tumor cells by detecting the level of miR-29a combined with the intervention treatment of cisplatin.We used an online tool(http://www.microrna.org)to search for microRNAs targeted by CD133~-and regulated by cisplatin,and tested the expression level of all 21 candidate microRNAs.We then identified the binding sites in the CD133 promoter region of miR-29a,overexpressed miR-29a in CD133~+GB stem cells,and also combined it with cisplatin treatment,and then detected cell apoptosis.Finally,we explored the role of miR-29a in cd133-induced cisplatin resistance in vivo.The results showed that the expression of miR-29a(has-miR-29a)in CD133~+GB cells treated with cisplatin was significantly down-regulated.The identification of binding sites showed that cisplatin could regulate the expression of CD133 by inhibiting the expression of miR-29a.After overexpressing miR-29a in CD133~+GB cells and detecting cell apoptosis after cisplatin treatment,we discovered that the expression of CD133 decreased after overexpression of miR-29a.MiR-29a significantly increased the apoptosis of CD133~+GB cells after cisplatin treatment,suggesting that miR-29a overexpression reverses cisplatin resistance in CD133~+GB stem cells.In vivo experiments,the result also showed that the combined treatment of cisplatin and miR-29a overexpression inhibited tumor growth significantly better than that of cisplatin alone.Conclusion:1.CD133 expression may lead to cisplatin resistance in GB cells and the cisplatin treatment significantly increased CD133 expression in tumor cells.2.CD133~+GB cells showed stronger resistance to cisplatin treatment.The overexpression of miR-29a was sufficient to reverse the resistance of CD133~+GB cells to cisplatin.Overexpression of miR-29a increased the sensitivity of CD133~+cells to cisplatin and significantly inhibited tumor growth in CD133~+tumor-bearing mice. |
