| Objection:Malignant glioma is one of the most malignant tumors in the human brain. It is easy to recur and difficult to cure. The existence of glioma stem cells is considered to be one of the main reasons. In recent years, more and more studies have indicated that miRNAs and its related signal transduction pathways are related to the occurrence and development of human cancer stem cells. Among them, the expression level of miR-125b is closely related to the glioma stem cells, which can regulate the proliferation, invasion and apoptosis of glioma stem cells. However, the specific mechanism of the action is still not very clear. Temozolomide (TMZ) is a first-line anti-glioma drug; however, the study found that glioma stem cells are generally resistant to TMZ. However, the sensitivity of glioma stem cells to TMZ could be improved after miR-125b was inhibited. Recent studies have indicated that glioma stem cells may be associated with PI3K/Akt, Bcl-2/caspase, and high expression of ABCG2 in glioma stem cells, but not related to the expression of 06-methyl guanine-DNA-methyl transferase (MGMT). The effect of PI3K/Akt pathway inhibition on the biological characteristics of glioma stem cells (GSCs) was higher than that of non glioma stem cells (GSCs). And direct inhibition of Bcl-2 expression or Bcl-2/caspase signaling pathway could induce apoptosis of glioma stem cells. ABCG2 transporter is a new drug delivery pump related to tumor MDR, which can lead to the drug efflux of chemotherapeutic drugs with different chemical structures and targets at different locations in the cells. Demethoxycurcumin (DMC) was found to be a natural inhibitor of the ABCG2 gene, which could effectively inhibit the expression of ABCG2 in cells. This study aimed to improve the TMZ concentrations in glioma stem cells by inhibition of ABCG2 induced by DMC, and further inhibit the miR-125b expression level to increase glioma stem cell sensitivity to TMZ for achieving better effects of anti-glioma stem cells, and explore the combined functional mechanisms. And enhance the theory and practice basis of glioma stem cells to chemotherapy.Methods:Clinical different grades of glioma tissues and normal brain tissue from brain injury and brain swelling were collected. And the expression of miR-125b in different grades of glioma tissue and normal brain tissue were detected using real-time fluorescence quantitative PCR. The primary glioma cells were cultured from the pathological diagnosis of WHO-IV grade clinical glioma cell tumor tissue. Then the CD 133 positive glioma stem cells were separated using immunomagnetic beads separation method, and identified by Nestin and CD 133 immunofluorescence staining. Establish the models of primary glioma stem cells in vitro culture and in vivo xenografts. Then the effects of different concentrations of DMC on the growth of glioma stem cells were evaluated, and the mechanisms of anti-glioma stem cells were studied. Further, the function and mechanisms of DMC and TMZ combined treatment were analyzed. After that, glioma stem cells were transfected with up-regulated and down regulated expression vectors of miR-125b, and the transfection efficiency was measured by real-time fluorescence quantitative PCR. Then, the effects of DMC or TMZ on the proliferation and apoptosis of glioma stem cells were analyzed after changing the expression level of miR-125b in glioma stem cells. And we analyzed the changes of the related signal pathway, and found out the regulation of the target signal pathway. Finally, the combined effects of miR-125b inhibitor, TMZ and DMC on anti-glioma stem cells were evaluated.Result:(1) miR-125b was low expression in normal brain tissue. It was slightly elevated in WHO-II, grade III gliomas tissue compared with normal brain tissues. However, it showed significantly higher expression of more than ten times in WHO-IV grade of glioma tissues compare to normal brain tissues. It was also over-expressed in glioma stem cells. (2) We have successfully isolated and cultured primary glioma stem cells in vitro and identified as CD 133 and Nestin double positive by immunohistochemistry. (3) DMC can effectively inhibit the proliferation of glioma stem cells, and induce its apoptosis, andits effect is better than TMZ. In vitro experiment showed that the combined use of the two has a synthetic effect; however, in vivo results showed no synthetic effect of the combined use of DMC and TMZ. But in vivo anti-tumor effect is still significantly better than TMZ. Its effect is related to the inhibition of JAK/STAT3 activation. (4) miR-125b inhibitor and TMZ alone did not induce apoptosis of glioma stem cells. Combined miR-125b inhibitor and TMZ intervention showed that the inhibition of miR-125b expression could promote the proliferation and apoptosis of glioma stem cells induced by TMZ. The mechanism was related to the miR-125b targeting BAK1 gene, the PI3K/AKT signaling pathway inhibiton, and so on. (5) miR-125b could regulate the sensitivity of DMC to glioma stem cells. Oer expression of miR-125b could resist DMC induced apoptosis of glioma stem cells, and its mechanism might be related to the miR-125b targeted BAK1 expression. (6) The effect of miR-125b inhibitor combined with DMC and TMZ on the anti-glioma stem cells was significantly better than that in other two combined groups.Conclusion:The study confirms inhibition of miR-125b expression in glioma stem cells can effectively increase the chemotherapy sensitivity of glioma stem cells to TMZ and DMC, and found that DMC is significantly better than TMZ on anti-glioma stem cells both in vitro and in vivo. Thus, we propose that the targeted miRNA regulation may be a new way to increase the sensitivity of glioma stem cells to chemotherapy drugs, which provides a new direction for the treatment of glioma stem cells in the future.
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