| Part I Expressions of miR-17-5p and 130b-3p in glioma stem cells and relationship between the both miR and HIF-laBackground and objective:The radiotherapeutic effectiveness of malignant glioma is not ideal,and the potential reason may be the increment of hypoxia inducible factor-1 alpha(HIF-la)expression,which is induced by radiation-mediated microRNA(miR)in glioma stem cells after radiotherapy.In the study,we examined the differences of miR-17-5p,miR-130b-3p,and HIF-1 a expressions in SU3 and SU3-5R glioma stem cells and the changes of the three indices in SU3cells after irradiation,and our aim was to investigate the relationship between the both miR and HIF-la expression,which might be associated with the radioresistance.Methods:The SU3 and SU3-5R glioma stem cells were respectively seeded in 6-well culture plates and cultured in a DMEM/F12 high glucose medium containing 10%fetal bovine serum for 24 h,the cultured cells were then collected and the total RN A and protein were extracted to measure the levels of intracellular miR-17-5p,miR-130b-3p,and HIF-la mRNA or/and protein expressions.Additionally,the SU3 glioma stem cells were divided into the control and radiation groups,and cultured for 24 h,the radiation-treated cells were then given irradiation 8 Gray.After6,12,and 24 hof irradiation,the cells were collected and the total RNA and protein were extracted to determine the levels of intracellular miR-17-5p,miR-130b-3p,and HIF-1α mRNA or/and protein expressions.Results:Compared with the SU3 cells,the levels of intracellular miR-17-5p,miR-130b-3p,and HIF-1a mRNA or/and protein expressions in the SU3-5R cells were higher(P<0.01).At 12 and 24 h after treatment of SU3 cells with irradiation 8 Gray,the three indices in the radiation group were also higher as compared with the control group(P<0.05or P<0.01).Conclusion:The intracellular miR-17-5p,miR-130b-3p,and HIF-1 a expressions in the SU3-5R cells were much higher than those in the SU3 cells,and the treatment of SU3 cells with irradiation could simultaneously increase the three indices.These results suggested that the both miR might be related to the radioresistance generation of SU3 cells.Part II MiR-17-5p-and 130b-3p-mediated radioresistance of glioma stem cells and signaling pathwayBackground and objective:Our aforementioned results showed that the irradiation could induce the miR-17-5p,miR-130b-3p,and radioresistance-related HIF-1 a expressions.In the present study,we used the miR-17-5p or miR-130b-3p mimic-or inhibitor-transfected SU3 cells,and further confirmed the relationship between the both miR andradioresistance of SU3 cells and investigated the possible signaling pathway.Methods:The SU3 cells were transfected with miR-17-5p or miR-130b-3p mimic or inhibitor and given irradiation 10 Gray after 24 h of transfection,the cell viability was then determined according to the MTT method after 0,12,and 24 h of irradiation.Additionally,following transfection of SU3 cells with miR-17-5p or miR-130b-3p mimic or inhibitor for 36 h,the cultured supernatant glucose,intracellular glucose 6-phosphate dehydrogenase(G6PDH),6-phosphogluconate dehydrogenase(6PGDH),reduced nicotinamide adenine dinucleotide phosphate(NADPH),reduced glutathion(GSH),and glutathione peroxidase(GSH-Px)levels,intracellular HIF-1α and phosphatase and tensin homolog(PTEN)mRNA and protein expressions,and intracellular glucose transporter(GLUT)-1,GLUT-3,protein kinase B(AKT),and p-AKT protein expressions were detected.The bioinformatic analysis was used to predict the possible target genes of miR-17-5p and 130b-3p,and the differences of the target gene mRNA and protein expressions in SU3 and SU3-5R cells were compared.The interaction of the target gene and the both miR was examined by luciferase reporter gene assay.Results:After 12 and 24 h of irradiation 10 Gray,the viability of miR-17-5p or miR-130b-3p mimic-transfected SU3 cells was obviously increased(P<0.05),whereas that of miR-17-5p or miR-130b-3p inhibitor-transfected SU3 cells was obviously decreased(P<0.05).In the miR-17-5p or miR-130b-3p mimic-transfected SU3 cells,the expressions of intracellular HIF-1α,GLUT-1,GLUT-3,AKT,andp-AKT proteins were higher(P<0.05),the levels of intracellular G6PDH,6PGDH,NADPH,GSH-Px,and GSH were also higher(P<0.05 or P<0.01),while the intracellular PTEN expression and cultured supernatant glucose were lower(P<0.05).The reverse effects could be observed in the miR-17-5p or miR-130b-3p inhibitor-transfected SU3 cells.The PTEN mRNA and protein expressions in the SU3-5R cells were decreased as compared with the SU3 cells(P<0.05 or P<0.01).The bioinformatic analysis predicted that the PTEN might be a potential target gene of miR-17-5p or miR-130b-3p,and the activity of the luciferase reporter gene containing PTEN wild type 3’-UTR was obviously lower(P<0.05).Conclusion:The miR-17-5p and 130b-3p might cause the radioresistance of SU3 cells,and the mechanisms were associated with the enhancement of antioxidant production,which was from the increments of PTEN/HIF-1α signaling pathway-controlled glucose transmembrance transport and phosphopentose pathway metabolism.Part Ⅲ Vitexin,an inhibitor of HIF-1α,enhances the radiotherapy sensitizations of SU3 cells and xenograft tumorBackground and objective:Our aforementioned results showed that the miR-17-5p and 130b-3p could enhance the phosphopentose pathway metabolism and induce the radioresistance by affecting PTEN/HIF-la signaling pathway.In the present study,we examined whether vitexin,an inhibitor of HIF-1α,had the radio-sensitizations on SU3 cells and xenograft tumor,and further investigated the possible mechanisms.Methods:In vitro,the miR-17-5p or miR-130b-3p mimic-transfected SU3 cells were treated with 25-100 μM vitexin for 24 h,and then given irradiation 10 Gray,the cell viability was determined after 12 h of irradiation.Also,after treatment of SU3-5R cells with 50-200μM vitexin for 24 h,the cell viability was determined after 12 h of irradiation 10 Gray.The miR-17-5p and 130b-3p expressions were determined by real-time polymerase chain reaction method after treatment of SU3 or SU3-5R cells with 25-100 μM vitexin for 24 h.In vivo,the nude mice were subcutaneously injected once with 50 μ1 suspension containing 2×107/ml SU3 cells into left paw to induce xenograft tumor.28 days later,these mice were assigned to 3 groups in terms of the tumor volume,namely control,radiation,and vitexin+radiation groups.The mice in the last group were daily given vitexin 75 mg/kg by intraperitoneal injection,while the mice in the former two groups were treated with an equivalent volume of 5%DMSO solution according to the same manner.The radiation-treated mice were given local tumor irradiation with high-energy X-ray(linear accelerator)on the 3rd,10th,and 17th days during the vitexin treatment,and the dose of irradiation was 10 Gy/time.Four days after irradiation of the third time,these animals were fasted for 12 h and killed.Blood and tumor tissues were collected for related parameter measurements.The experimental treatment lasted for 21 days.Results:After treatment of miR-17-5p or miR-130b-3p mimic-transfected SU3 cells with vitexin for 24 h and subsequent irradiation,the cell viability was decreased,especially in the 50 and 100 μM vitexin-treated cells(P<0.05 or P<0.01).After treatment of SU3-5R cells with 50 and 100 μM vitexin for 24 h and subsequent irradiation,the cell viability was also reduced(P<0.05 or P<0.01).After treatment of SU3 or SU3-5R cells with 50 and 100μM vitexin for 24 h,the levels of intracellular miR-17-5p and 130b-3p expressions were significantly decreased(P<0.05 or P<0.01).After treatment of xenograft tumor mice with vitexin for 21 days and radiotherapy,the tumor volume and weight,tumor tissue GSH and GSH-Px contents,miR-17-5p and 130b-3p expressions,and HIF-1α,GLUT-1,GLUT-3,and VEGF protein expressions were lower,whereas the tumor tissue PTEN protein expression was higher compared with those of the irradiation group(P<0.05 or P<0.01).Conclusion:Vitexin could enhance the radiotherapy sensitizations of SU3 cells and xenograft tumor,and its mechanisms might be correlated to the reduction of miR-17-5p and 130b-3p expressions,which might subsequently decrease the interaction with PTEN and attenuate the HIF-1α-controlled glucose transmembrance transport,phosphopentose pathway metabolism,and antioxidant production. |
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