| Purpose: Currently the inefficiency of chemotherapy for malignant glioma is obvious,and the mechanism of chemotherapy resistance remains unclear.1.At present study,the common used chemotherapeutic drug cisplatin,was used to the induction of human glioblastoma cell line,and microarray analysis and information biology methods were used in order to find the long noncoding RNA(LncRNA)-AC023115.3,whose change is the most obvious.Thus we seek to confirm the AC023115.3 could regulate cell apoptosis induced by cisplatin through autophagy pathway.2.The precise mechanisms regarding the effect of AC023115.3 on cell apoptosis induced by cisplatin through regulating autophagy were clearly elucidated,thus the signaling pathways that could affect the efficacy of chemotherapy for malignant glioma were found.All these could provide new ideas for promoting the therapeutic research regarding the chemosensitivity of glioma.Methods:1.Following the use of cisplatin,the apoptosis-related protein expressions were tested in human glioblastoma cell line U87 MG by Western blotting.Then we established different glioma cell models that could be tolerant of different doses of cisplatin;microarray analysis and bioinformatics tools were used to analyse the LncRNAs expression profile.During this process,the most obviously up-regulated LncRNA could be found(AC023115.3),then the qPCR were further exerted to confirm the up-regulated LncRNA(AC023115.3).Besides,U87 MG was treated with different doses of cisplatin,and the time-dependent expression profile of LncRNA-AC023115.3was confirmed.Furthermore,same methods were used and the results in U251 MG cell line were found to be in conformity with those in U87 MG.2.In U87 MG cells,AC023115.3 was knocked down using SiRNA,and then afterthe use of cisplatin,the methods of quantitative PCR and RT-PCR were used for detection the expression of Lnc RNA AC023115.3,and Western blotting for the detection of apoptosis-related protein expression changes,flow cytometric analysis for cell apoptosis ratio changes.Conversely,overexpression of AC023115.3 in U87 MG were induced and then exerted cisplatin induction,change of each index were analysed.Same detection methods were further used to elucidate the effect of AC023115.3 on apoptosis of U251 MG cell line.3.In U251 MG cells,cisplatin were used after the induction of overexpression of AC023115.3,while in U87 MG cells,cisplatin were used after the knockdown of AC023115.3,then the analysis of LC3 formation in cells were exerted via punctate fluorescence images.Besides,protein expression of LC3-type II were detected compared to type I,and level of p62 protein were detected through Western blotting.Then,ultrastructural analysis by transmission electron microscopy(TEM)was used to count the number of autophagic vacuoles per cell,thus the regulatory effect of AC02115.3 on autophagy could be determined.4.Targeted shRNA of autophagy-related gene 5(ATG5)were transfected into U87 MG and U251 cells.After the induction by cisplatin,the Western blotting was used to detect the autophagy and changes of apoptosis-related proteins,and MTT was used to detect cell viability.Then selective autophagy inhibitor 3-methyladenine(3-MA)were used followed by the induction of cisplatin,changes of autophagy and apoptosis-related proteins were detected in U87 MG and U251 MG cells,as well as cell viability.Then the double transfection were exerted using both the AC023115.3 si RNA and ATG5 shRNA to detect the changes of apoptosis induced by cisplatin in U87 MG cells,thus to determine the relationship between apoptosis and autophagy caused by AC023115.3induced by cisplatin.The method of 3-MA were used to further confirm the results.5.In U87 MG and U251 MG cells induced by cisplatin,protein expression changes of BCL2 family were detected by Western blotting.Then the U87 MG cells with AC023115.3 knockdown were induced by cisplatin to detect the expression of MCL1 protein through Western blotting.In U87 MG cells,quantitative PCR and RT-PCR wereused to exert detection of the effect of AC023115.3 on MCL1 in the mRNA level.The effect AC023115.3 on the protein level of MCL1 were detected using the protein half-life experiments and ubiquitination assay.The effect of AC023115.3 on MCL1phosphorylation(Ser 159)were also determined by Western blotting.In U87 MG and U251 MG cells,changes of GSK3 beta protein expression were detected after the induction of cisplatin,after that,GSK3 beta protein expression were detected again in with AC023115.3 knockdown in U87 MG and AC023115.3 overexpression in U251 MG cells.Finally,we exert the overexpression of GSK3 beta in AC023115.3 knockdown U87 MG cells,and exert the GSK3 beta knockdown in U251 MG cells of AC023115 overexpression using shRNA,to detect changes of MCL1 protein expressions induced by cisplatin.Thus the relationship between AC023115.3,GSK3 and MCL1 could be actually determined by the studies mentioned above.6.U87 MG cells were treated with cisplatin followed by detection of the level of GSK3 beta mRNA by qRT-PCR.U87 MG cells with AC023115.3 knockdown were induced by cisplatin,then detection of GSK3 beta mRNA were exerted by qPCR.In the RIP experiment,the combining situation of Ago2 antibody and AC023115.3 were detected to test whether they could combine with microRNA.Then the potential microRNA may be predicted that could combine with AC023115.3 via LNCipedia website.After that,the most likely one,the miR-26 a,was further verified by report gene experiments.Then the AC023115.3 sequences,GSK3 beta 3'UTR(non encoding region)sequence containing miR-26 a binding sites,and AC023115.3 mutated sequence were inserted into the carrier of the reporter gene,in order to detect whether miR-26 was combined to AC023115.3.Then transfected GSK3 beta 3'UTR sequence into U87 MG and U251 MG cells,followed by induction of cisplastin,and luciferase activity was then detected.In U87 MG cells transfected with GSK3 beta 3'UTR sequence,knockdown AC023115.3 or overexpress the miR-26 a followed by induction of cisplatin,and luciferase activity was detected.And GSK3 beta protein expression level was determined by Western blot.Finally,in U87 MG and U251 MG cells,the GSK3 beta3'UTR,miR-26 a and AC023115.3 were cotransfected followed by luciferase activity test,thus to clarify the relationships between AC023115.3,miR-26 a and GSK3 beta.7.Exogenous AC023115.3 was transfected into U87 MG cells,and then a Flag-labeled MCL1 was transfected into U87 MG cells again,followed by induction of cisplatin.changes of apoptosis-and autophagy-related proteins were detected by Western blotting.Then in U251 MG cells with AC023115.3 overexpression,GSK3 beta was knocked down,apoptosis-and autophagy-related protein were then detected.The analogue inhibitors of miR-26 a or miR-26 a were transfecteed into U87 MG cells followed by induction of cisplatin,apoptosis-and autophagy-related protein changes were then detected.In turn are iR-26 a inhibitors or miR-26 a analogues in U87 MG cells overexpressing GSK3 beta after cisplatin induced apoptosis and the changes of Western,blot detection of autophagy related protein.AC023115.3 was knocked down through siRNA in U87 MG cells,and then miR-26 a inhibitors were transfected into cells,apoptosis-and autophagy-related protein were detected followed by induction of cisplatin.Finally in U87 MG cells with overexpressed AC023115.3,miR-26 simulation was transfected into cells,then detect apoptosis-and autophagy-related proteins by Western blot.Through the studies of various aspects mentioned above,the relationship between the important roles of AC023115.3 and miR-26a-GSK3 beta-MCL1 signaling pathway could be clarified.Results:1.Cisplatin could promote the apoptosis of glioma cells,microarray analysis showed that the expression of AC023115.3 was up-regulated in many lncRNAs triggered by cisplatin,and this up-regulated expression could increase with the increase of drug dose or time increased.Up-regulated AC023115.3 expression could combat cisplatin-resistance in glioma cells.2.Western blotting and flow cytometry analysis revealed that in glioma cells,the down-regulated expression of AC023115.3 could down-regulate the apoptosis-related proteins,reduce the number of apoptotic cells;On the contrary,the up-regulated expression of AC023115.3 could cause increase of apoptosis-related proteins andincrease the number of apoptotic cells.AC023115.3 could promote cell apoptosis induced by cisplatin.3.Cellular fluorescence experiment,Western blotting analysis and TEM ultrastructural results all showed that: increased expression of AC023115.3 could decrease autophagy-related indicators in glioma cells,and conversely,down-regulated expression of AC023115.3 could enhance the autophagy-related indexes.AC023115.3thus can inhibit autophagy under the induction of cisplatin.4.With the treatment of shRNA of the autophagy-related gene 5(ATG5)or the inhibitor 3-methyladenine or autophagy(3-MA),apoptosis induced by cisplatin was enhanced in glioma cells,and cell viability was decreased.Decreased apoptosis-induction of cisplatin caused by down-regulatin of AC023115.3 could be attenuated by knocking down ATG5 or 3-MA.AC023115.3 could enhance cisplatin-induced apoptosis through inhibition of autophagy.5.The results of Western blotting suggested that after treatment with cisplatin,MCL1 protein expression levels in glioma cells was decreased.Knockdown of AC023115.3 could suppress the decreased expression of MCL1 and improve the stability of MCL1 protein,thus inhibit the increased ubiquitination of MCL1 induced by cisplatin.Cisplatin can cause changes of the phosphated level of MCL1 protein in glioma cells and increase the expression of GSK3 beta,while the GSK3 expression was positively regulated by AC023115.3.Cotransfection could determine that AC023115.3in glioma cells coud down-regulate the expression of MCL1 through GSK3 beta.6.AC023115.3 could be of no effect on the level of GSK3 beta mRNA in glioma cells with treatment of cisplatin.RNA immunoprecipitation test revealed that AC023115.3 may occur in conjunction with the microRNA,and LNCipedia prediction and reporter gene test were used to screened out the most possible miRNA(mi R-26a).Then reporter gene test and cotransfected were used to determine the relationship between GSK3 beta,miR-26 a and AC023115.3,AC02115.3 plays important part in sponge-adsorption role of mi R-26 a and promotes the expression of GSK3 beta.7.Up-regulated AC023115.3 expression can promote apoptosis of glioma cells induced by cisplatin and decrease the autophagy,however this effect can be inhibited by MCL1.Down-regulated GSK3 beta could inhibit the apoptosis-enhancement of AC023115.3,and promote the autophagy.The use of MiR-26 a inhibitors can enhance cisplatin-induced apoptosis and decrease the autophagy,exogenous miR-26 a expression could decrease cell apoptosis and promote autophagy,and after the overexpression of GSK3 beta,these effects generated by miR-26 a would then disappear.The role of MiR-26 a on the apoptosis and autophagy induced by cisplatin could depend on GSK3 beta.8.Knockdown of AC023115.3 could inhibit cisplatin-induced apoptosis in glioma cells and promote the autophagy;however these effect could be reversed by inhibitors of miR-26 a.On the contrary,the increased expression of miR-26 a could attenuate apoptosis of glioma cells induced by overexpression of AC023115.3.Above all,AC023115.3 could enhance cisplatin-induced apoptosis and inhibit autophagy through regulating miR-26a-GSK3 beta-MCL1 pathway.Conclusions:1.AC023115.3 can be induced by cisplatin and inhibit the chemoresistance of glioma.2.AC023115.3 could promote cisplatin-induced apoptosis of glioma cells through reducing MCL1-mediated autophagy.3.AC023115.3 plays a role in miR-26 a sponge and inhibits its activity.4.AC023115.3 could enhance cisplatin-induced apoptosis by regulating the miR-26a-GSK3 beta-MCL1 signaling pathway and optimizes the chemosensitivity of glioma cells. |
PDF Full Text Request