Comparative Transcriptome Analysis And Mitochondrial Protein Changes In Tumor Cells Under Energy Stress

ObjectivePrevious studies have shown that cells use glucose for production of ATP,and that glucose starvation activates cellular energy sensor,AMPK.Its full complex,consists of α,β,and γ.Recently,some studies have found that the activated AMPK is located on the lysosomal membrane.In light of the important role of mitochondria in ATP production and metabolism,the question is whether the activated AMPK distributes on the mitochondrial membrane? Is there any information exchange and communication occurred between the nucleus and mitochondria of tumor cells under glucose starvation? What proteins are changed in transcription levels and on mitochondria under these two different conditions? These problems are not clear at present.Therefore,the purpose of this study is to explore the changes of transcriptome and mitochondrial proteome under glucose starvation,and to find out potential new responsive genes in this process.Methods1.He La cells were normally cultured or treated with glucose starvation.Then the total RNA was extracted and transcriptome sequencing was performed.Database comparisons and software analyses were performed to find genes with significant differences in expression.2.Lentivirus was used to infect He La cells and construct cell lines that overexpressed mitochondrial proteins.Then the cell line was immunoprecipitated to obtain mitochondrial proteins,and a new method for purification of mitochondria was established.3.The above tumor cells were treated with glucose starvation,and their mitochondrial proteins were extracted.Western blots were used to verify the changes of mitochondrial proteins before and after glucose starvation and the localization of AMPK on mitochondria.Results1.By analyzing the results of RNA sequencing,we found that 107 genes were significantly up-regulated and 48 genes were significantly down-regulated in He La cells under glucose starvation.2.Two important subclusters were found through GO enrichment analysis.The genes of one subcluster was significantly up-regulated under glucose starvation,and is mainly concentrated in nucleosome,ER stress response and autophagy pathway.The genes in the other subcluster were significantly down-regulated under glucose starvation and were mainly concentrated in mitochondria and other membrane organelles.3.Through KEGG enrichment analysis,we found that the differentially expressed genes in response to glucose starvation were mainly concentrated in alcoholism,systemic lupus erythematosus,endoplasmic reticulum protein processing,cancer,AMPK signaling pathway.In the signaling pathway of alcoholism and systemic lupus erythematosus,the expression of histones H2 B,H3 and H4 are up-regulated.The expression of CHOP,GADD34,BIP and HERP were up-regulated in the signal pathway of protein processing in ER,activating the endoplasmic reticulum unfolded protein response.In the cancer signaling pathway,the expressions of p21,c-Jun,c-Fos and GPCR were up-regulated,and the expression of Cyclin D1 was down-regulated.In addition,genes for mitochondrial quality control like AMPK,ULK1 and PGC-1αare up-regulated.However,no significant differentially expressed genes were found in the signaling pathways of glucose metabolism,tricarboxylic acid cycle and oxidative phosphorylation.4.After lentivirus infection,He La cells showed specific bands of HA in western blot experiments,which proved that we successfully constructed stable cell lines with overexpression of mitochondrial marker protein OMP25.Then the mitochondria were isolated by mitochondrial immunoprecipitation(mito-IP)and the purity of mitochondria was confirmed by western blot by using the endoplasmic reticulum,mitochondrial,golgi,nucleus,lysosome and peroxidosome markers.5.Mitochondria were purified from cells cultured in normal or glucose starvation conditions respectively,and the changes of AMPK in mitochondria before and after starvation were verified by western blotting.AMPK was activated under glucose starvation condition as evidenced by the phosphorylation of T172 at AMPK.AMPKαand AMPKβ were both found in mitochondrial lysates under the glucose starvation or normal cultured condition,while AMPKγ was not found in either condition.Conclusion1.Under glucose starvation,tumor cells accumulated a large amount of unfolded proteins,activated the unfolded protein response of endoplasmic reticulum and mitochondria.And the nuclear and mitochondrial genes of He La cells were significantly differentially expressed,which involved the related functions of chromatin remodeling and mitochondrial regulation.Glucose starvation regulates a variety of biological processes through transcription factors,such as cell growth,cell apoptosis,AMPK-related mitophagy,and mitochondrial biogenesis.2.AMPK was located on mitochondria in both normal condition and glucose starvation,but there were only AMPKα and AMPKβ subunits and no AMPKγsubunits,indicating that AMPK location on mitochondria was not related to the presence or absence of glucose.AMPK can be localized on lysosomes as well as mitochondria.