Effects Of Mitochondrial Pyruvate Carrier On Glycolysis Metabolism And Stemness Regulation

Mitochondria are semiautomatic organelles with bilayer membrane structure in eukaryotic cells.They are involved in bioenergy metabolism,biosynthesis,signal transduction and cell adaptive regulation in stress response.At the same time,mitochondria take part in cell differentiation,metabolite transport,apoptosis and other processes.Most of the metabolism-related enzymes are located in the mitochondrial matrix,but the protein located in mitochondrial inner membrane plays an important role in the catalytic response of the metabolites in the mitochondria and the matrix.It is known that some small molecules are free to diffuse the outer membrane of the mitochondria,but require a specific carrier to transport to the mitochondrial inner membrane.Mitochondrial transport of pyruvate through mitochondrial intima is the basis for glucose,fatty acid and amino acid metabolism.In 2012,the studies revealed that the carrier of the transport of pyruvate on the mitochondrial inner membrane consists of two proteins,MPC1 and MPC2,which form a protein complex to act as a transporter.In yeast,there is MPC3 gene that is homologous to the MPC2 gene.The reports of MPC1 mutations in clinical patients indicated that the incidence of MPC1 mutations in the population is higher.Mitochondrial membrane carriers,such as mitochondrial ADP/ATP vector and amino acid carrier,have correlation with the occurrence of inflammation or cancer.The treatment method of pyruvate metabolism has been used for Parkinson and Aermeicihaimo patients.In view of these,we established the MPC1 gene knockout mice and studied the effects of MPC1 gene deficient on fertility,body weight and glucose tolerance.Subsequently,we used immunocytochemistry to detect the protein expression in different prostate and esophagea cancer cell lines.And the expression of MPC1 and MPC2 in prostate and esophageal cancer tissues was detected by immunohistochemistry.The MPC1 gene knockout cell line of prostate cancer RM-1 and esophageal cancer KYSE450 were made by CRISPR/Cas9 gene editing system.Moreover,the energy metabolism and stemness influence of MPC1 gene knockout on these cells were analyzed.Finally,we explored the relationship between the MPC1 gene expression and EMT with different culture.Part Ⅰ:Establishment of mitochondrial pyruvate carrier 1 gene knockout mice and preliminary gene function analysisMethods1.The MPC1 gene knockout mice were obtained by the effective CRISPR/Cas9 system for embryo gene edition.2.The living habits,body weight and reproductive capacity of MPC1 gene knockout(KO)mice were studied.3.The possible off-target sites by CRISPR/Cas9 gene editing were examined.4.After MPC1 gene knockout,the glucose and lipid metabolism relative genes expression in mice tissues were detected.Moreover,the glucose tolerance and insulin sensitivity were tested.Results1.The MPC1 heterozygous KO mice were established successfully by CRISPR/Cas9 system.The MPC1 protein expression was verified by Western blotting.2.MPC1 heterozygous KO mice were healthy and had no significant developmental disorder sign.There were only 5 homozygous MPC1 mutant offspring mice,and the overall birth rate of MPC1 hetrozygous KO mice was much lower than the WT mice.In addition,all of these homozygous KO mice died within 6 months.The weight of MPC1 hetrozygous KO female mice was significantly higher than WT mice after 10 weeks.3.The possible off-target sites were detected.For the possible off-target loci chr12:+78920516 and chr12:+78920523,there were 9 mice with DNA mutations.For the off-target sites chr12:-112097058 and chr12:-112097051,4 mice had multiple base mutations.For off-target chrX:+140020779,there was no mutation in mice.No off-target mutations were found in the NO.1 and NO.17 mice maintained after long time mating.4.Compared to the WT mice,the mice with MPC1 heterozygous mutations have different glucose and fat metabolism genes expression.MPC1 heterozygous KO mice had reduced glucose tolerance.In the insulin tolerance test,the glucose level was the same between MPC1 heterozygous KO mice and WT mice.However,the glucose utilization of MPC1 hetrozygous KO mice decreased during fasting.Part Ⅱ:MPC1 and MPC2 expressions are associated with favorable clinical outcomes in prostate cancer and esophageal cancerMethods1.The expression of MPC1 and MPC2 in cell lines was verified by Western blotting and immunocytochemistry(ICC).2.Immunohistochemical(IHC)method was used to detect the expression of MPC1 and MPC2 in 88 cases of prostate cancer.The correlation between MPC1/2 expression and clinicopathological features and survival were statistically analysed.3.The expression of MPC1 and MPC2 in 138 cases of esophageal cancer were detected by IHC.The correlation between MPC1/2 expression and clinicopathological features and survival were detected.4.SPSS 17.0 was used for statistic analysis.The correlation between MPC1 and MPC2 protein expression and clinical parameters was analyzed by Chi-square analysis or Fisher’s accurate test.The correlation of MPC1 and MPC2 protein expression was analysed by linear regression.Survival analysis was performed using the Kaplan-Meier curve by the log-rank test.COX regression was used to analyze the effect of each parameter on prognosis.Results1.The ICC detection showed that the expression of MPC1 and MPC2 in DU145 cell line was very low,but the expression of MPC1 and MPC2 in LNCaP cell line was high.The expression of MPC1 and MPC2 in HetlA was higher than that in KYSE70 and KYSE450.The MPC expression in KYSE70 was significantly lower than that of KYSE450 cell line.2.The expression of MPC1 or MPC2 in most of the prostate cancer tissues was low or negative.Of the 88 cases,only 29 cases(32.95%)had positive expression of MPC1 protein,and 23 cases(26.14%)had positive expression of MPC2 protein.The MPC1 expression was negatively correlated with UICC staging(p=0.031),and the MPC2 expression was negatively correlated with UICC staging(p=0.000)and lymph node metastasis(p=0.002).Linear regression analysis showed positive correlation between MPC1 and MPC2 protein expression in prostate cancer tissue samples(r=0.375,p=0.006).The overall survival was lower in patients with MPC negative expression(p=0.007 and p=0.02,respectively).3.The expression of MPC1 protein was positive in 36 cases(26.09%).33 cases(23.91%)showed positive expression of MPC2 protein in 138 cases of esophageal squamous cancer tissues.The expression of MPC 1 protein was negatively correlated with clinical stage(p=0.038)and lymph node metastasis(p=0.024).The expression of MPC2 protein was correlated with tumor depth(p=0.046),clinical stage(p=0.016)and distant metastasis(p=0.034)were negatively correlated.Linear regression analysis showed positive correlation between MPCl and MPC2 protein expression in esophageal cancer samples(r=0.569,p=0.000).The overall survival was significantly lower in patients with weak expression of MPC in 138 patients(p=0.002 and p=0.01,respectively).4.Multivariate analysis showed that MPC1 and MPC2 could be used as predictors of prognosis in prostate cancer patients(MPC1:RR=0.654,95%CI:0.621-0690,p<0.001;MPC2:RR=0.696,95%CI:0.660-0.734,p<0.001).In patients with esophageal cancer,MPC2(RR=0.655,95%CI:0.439-0.978,p=0.039)could be used as a prognosis factor.Part Ⅲ:Mitochondrial pyruvate carrier function determines cell stemness and metabolic reprogramming in cancer cellsMethods1.Using the CRISPR-mediated gene editing system,the MPC1 gene knockout was performed on prostate cancer cell line RM-1 and esophageal cancer cell line KYSE450,and monoclonal was selected to establish a stable MPC1 gene knockout cell line.2.The expression of MPC1 protein was verified by ICC,immunofluroescnce and Western blotting.The changes of pyruvate,lactate,glucose and ATP concentration were detected,and the changes of oxygen consumption and extracellular acidification rate were analyzed by Seahorse XFe96.Based on the above experiments,changes of pyruvate transportation,glycolytic rate and mitochondrial oxidative phosphorylation in MPC1 gene knockout cell could be calculated.3.After MPC1 gene KO,the cell proliferation ability and cell cycle was detected,transwell assay and wound healing were used to detect the ability of migration,flow cytometry was used to detect the ROS,clone formation experiment was used for chemo-or radiotherapy sensitivity detection,and western blotting and flow cytometry were used for stem cell markers Notchl,Hifla,Nanog,ALDH and CD44 expression detection.Results1.Two stable MPC1 knockout cell lines were successfully established in the RM-1 cell line and KYSE450 cell line.2.After MPC1 gene KO,the cells showed pyruvate metabolism dysfunction,the glucose consumption and lactate production increased,oxidative respiratory capacity and ATP production decreased.The cells showed metabolic reprogramming with oxidative phosphorylation inhibition and enhanced glycolysis metabolism.3.The proliferation and cell cycle of the MPC1 KO cells were inhibited,the cellular ROS content was increased and the ROS elimination ability was decreased,but the cell migration ability was enhanced,in addition to significantly higher chemotherapy and radiotherapy resistance.Stem cell makers like Notch1,Hif1α,Nanog,ALDH and CD44 were significantly upregulated in the MPC1 KO cells,suggesting that MPC1 gene knockout cells were tumor stem cell-like cells.Part Ⅳ:Glutamine deprivation culture induced EMT in mitochondrial pyruvate carrier dysfunction tumor cellsMethods1.With or without glutamine culture,MPC1 KO cell morphology,proliferation and cell cycle were detected.2.The activation of cell anaplerotic pathway was verified by cell glutamine consumption,metabolomics changes and anaplerotic pathway inhibition detection.3.With glutamine deprivation culture,the epithelial-mesenchymal morphology was verified by F-actin.EMT related protein expression was verified by Western blotting,and the migration ability of cells was verified by transwell expreiments.4.By detection of glucose consumption and oxidative respiratory capacity in MPC1 knockout cells,the cells’ Warburg features could be studied when the cells MPC1 gene KO or when the cells were facing glutamine deprivation.Gene expression difference were analyzed by GO and KEGG pathway with transcriptome detection.Results1.Cells with MPC1 knockout were more dependent on glutamine for growth.Without glutamine,cell proliferation slowed down and cell cycle was inhibited.Different cells responded to glutamine deprivation differently.The KYSE450 and KYSE450 MPC1KO cells showed cell growth slowing down,while the RM-1 cells grew slowly and the RM-1 MPC1-/-cells showed different morphology.2.The glutamine consumption rate of RM-1 MPC1-/-cells was significantly higher than that of RM-1 cells,and the metabolites were significantly changed.With treatment of L-alanine aminotransferase competitive inhibitor P-chloro-L-alanine,the RM-1 MPC1 KO cells grew more slowly and the apoptosis rate was significantly increased.3.RM-1 MPC1-/－ cells showed epithelial-mesenchymal transition,and the EMT related proteins expressions were significant changed.The cells showed significantly higher ability of invasion and metastasis in vitro.4.Glucose was used as the main source of energy with glutamine deprivation in MPC1 gene knockout cells,which forced cells to be prone to glycolysis and generate ATP.Cell transcriptional studies revealed alterations in sevral pathways in these cells.Conclusions1.We established the MPC1 KO mice by CRISPR/Cas9 system,which confirmed that this system has high efficiency for gene editing in mice.Study on MPC1 gene heterozygous KO mice showed that the fertility rate was decreased significantly,and the mice body weight,glucose tolerance and insulin resistance changed significantly.2.The expression rate of MPC1/2 protein in prostate and esophageal cancer tissues was low.It was found that MPC1/2 negative expression was associated with poor prognosis,suggesting that glycolysis relative gene expression plays an important role in the development of cancer.3.MPC1 gene knockout cells showed pyruvate transport dysfunction,mitochondrial oxidative phosphorylation inhibition and enhanced glycolysis rate,indicated these cells were prone to Warburg effect.The MPC1 gene knockout cells showed cell cycle inhibition,enhanced invasive ability and significantly higher stemness,which indicated the molecular basis of poor prognosis in patients with decreased MPC1 expression.The MPC1 expression can be used as a maker of tumor malignancy.4.MPC1 gene knockout cells showed tricarboxylic acid cycle blocked and anaplerotic metabolic pathway activated.With glutamine deprivation,the cells were turned into EMT feature,increased glycolysis and corresponding transcriptome changes,indicating the significance of cancer microenviroment based therapeutic studies.