Molecular Epidemiology Study Of SIRT1-MRPS5 Axis In Metabolic Reprogramming Of Human Liver Cancer Stem Cells

Liver cancer is an aggressive disease with a poor outcome.Approximately 75%of all liver cancer arises in Asia,with China accounting for over 50%of the world's burden.Previous studies have identified some stemness markers,which are used to isolate cancer stem cells?CSCs?.These cells are widely accepted as the root cause of tumor relapse,metastasis,and chemoresistance.Although the CSC hypothesis has recently been experimentally validated,there is few effective strategy to limit their behavior and control tumor progression.Energy metabolism is the base of biological activities,and cancer metabolism is one of the hot filed of research in cancer biology.Recent studies have demonstrated that tumors reprogram pathways of nutrient acquisition and metabolism to meet the bioenergetic,biosynthetic,and redox demands of malignant cells.These reprogrammed activities are now recognized as hallmarks of cancer.Therefore,it logically flows that if these activities provide benefits to the malignant cell,then some of them might be suitable therapeutic targets.There is a growing body of evidence to suggest that tumor cells with stem-like properties preferentially use mitochondrial respiration to generate energy.In contrast to CSCs,most differentiated cancer cells primarily rely on aerobic glycolysis to generate energy,known as the Warburg effect.Therefore,the differentiation of CSCs should couple with switch of energy metabolism.Metabolic reprogramming endows cancer cells with the ability to adjust metabolic pathways to support heterogeneously biological processes.However,it is not known how the reprogrammed activities are implemented during differentiation of liver cancer stem cells?CSCs?.Based on the above understanding of liver cancer research,we decided to draw support from molecular epidemiology to find the key molecule in metabolic reprogramming of liver cancer stem cells,and validate its role in large sample of clinical data and TCGA database.Our previous study has shown that SIRT1 is highly expressed in liver CSCs.Given SIRT1 is a deacetylase which engages in various metabolic pathway,but its role in metabolism of liver cancer remains unclear.Mitochondrial ribosome was only regarded as an organelle responsible for protein synthesis in mitochondrion,the past decade has revealed new roles of mitochondrial ribosomal proteins?MRPs?in apoptotic signaling,anti-aging and the regulation of cell proliferation.Given that the mitochondrion is the hub of cellular energy metabolism,whether MRPs independently participate in metabolic pathway of cancer cells is currently not known.Studying above question contributes to thoroughly understanding of the metabolism of liver cancer cells and provides potential targets for clinical work.In according to the experimental results and the screening of TCGA and KEGG database,we found mitochondrial ribosomal protein S5?MRPS5?might play key role in metabolism of liver cancer cells.In order to identify the role of MRPS5 in metabolic reprogramming of liver cancer stem cells,we divided this study into 5 sections.Firstly,we measured the oxygen consumption rate?OCR?of cells by the Seahorse XFp analyzer,detected intracellular reactive oxygen species?ROS?levels and lactate levels,and detected mitochondrial kinetics.It was found that liver cancer stem cells mainly rely on oxidative phosphorylation for energy supply rather than stem cells on glycolysis.Then we applied mitochondrial complex I specific inhibitor metformin treatment in cell and mouse xenograft models and found that mitochondrial complex I function is closely related to stemness of liver cancer stem cell.Further,we used bioinformatics methods to analyze the correlation between all mitochondrial ribosomal coding genes and Nduf gene expression in the TCGA database and screen for mitochondrial ribosomal proteins highly correlated with mitochondrial complex I function.Finally,TCGA and KEGG databases were used to conduct oxidative phosphorylation,pyruvate metabolism and tricarboxylic acid cycle?TCA?gene set variation analysis?GSVA?of different mitochondrial ribosomal proteins?MRPs?to determine the correlation between MRPs and different metabolic processes.We found that MRPS5 is highly correlated with mitochondrial complex I function and oxidative phosphorylation of cells,and is relatively less involved in other metabolic processes than oxidative respiration.Secondly,We first studied the relationship between intracellular NAD⁺levels and MRPS5 by knocking down MRPS5 and exogenously supplementing NAD⁺precursors?NAM?,and found that knockdown of MRPS5 can significantly reduce intracellular NAD⁺levels.Then Western blot analysis of protein expression and mitochondrial morphology analysis revealed that MRPS5 plays an important role in mitochondrial biogenesis.Meanwhile,we used the treatment of hydrogen peroxide to simulate the tumor microenvironment of active oxygen accumulation and judged the mitochondrial unfolded protein reaction by detecting the expression of HSP60.It was found that MRPS5 activates mitochondria unfolded protein reaction and enhances the anti-oxidative stress of liver cancer stem cells.Through the above experiments,we found that MRPS5 is essential for enhanced hepatocyte stem cell mitochondrial function and clarifies the specific mechanism.After that,we knocked down MRPS5 to detect the tumor sphere and clone formation ability of the liver cancer stem cells and the tumorigenic ability of the transplanted tumor,and compared the tumor initiation ability of knockdown MRPS5 and normal liver cancer stem cells by means of limiting dilution and orthotopic transplantation.We found that MRPS5 plays an important role in the maintenance of stemness of liver cancer cells.Further,we performed immunohistochemical analysis of clinical tumor tissue samples and performed survival analysis on patient information,and found that high expression of MRPS5 was associated with poor prognosis in clinical patients.In the third part,we first predicted the acetylation modification site on MRPS5 protein by bioinformatics,and then used point mutation and co-immunoprecipitation experiments to confirm that amino acid 365 is the acetylation modification site of MRPS5.Then we applied Sirtuin protein overexpression and specific inhibitor treatment to find that SIRT1 can significantly reduce the acetylation level of MRPS5 and demonstrate that SIRT1 can interact with MRPS5 through both endogenous and exogenous co-immunoprecipitation experiments.Through the above experiments,MRPS5 can be acetylated at amino acid 365 and SIRT1 is its specific deacetylase.Further,we identified different subcellular localizations of MRPS5 by Western-Blot and immunohistochemistry.Using bioinformatics methods,we found that nuclear sequence and mitochondrial localization sequences are present in the protein sequence of MRPS5.After that,we confirmed the acetylation status of MRPS5 determine its subcellular localization by Western-Blot,immunoprecipitation and immunofluorescence.Deacetylated MRPS5 localizes to mitochondria and acetylated MRPS5 aggregates into the nucleus.Finally,we demonstrated that mitochondrial function can be enhanced only when MRPS5 is localized on mitochondria by comparing the mutant and wild-type functions of MRPS5.In the fourth part,we first tested the oxygen consumption rate and glycolysis level of the cells by Seahorse XFp analyzer.It was found that the cell oxygen consumption rate of liver cancer stem cells decreased significantly and the level of glycolysis increased after SIRT1was knocked down.Then we used the characteristics of deacetylated MRPS5 that could not enter the nucleus to construct MRPS5 mutants to compare the effects of nuclear proteins.The expression of glycolytic-related proteins was detected by Western-Blot and RT-PCR,and energy metabolism was detected by Seahorse.We found that MRPS5 enters the nucleus and promotes the expression of glycolytic-related proteins and enhances the glycolysis function of the cells.Finally,we used metformin to interfere mitochondrial function and compare the conversion of energy metabolism in different cells and found that MRPS5 enhanced the metabolic plasticity of liver cancer cells.Finally,in the fifth part,we analyzed the clinical significance of the SIRT1/MRPS5 axis in clinical samples and verified it in the TCGA database.First,immunohistochemical analysis of liver cancer tissue samples from clinical patients was performed,and then the samples were grouped according to the level of SIRT1 expression and the plasmon localization of MRPS5.The samples were then subjected to SOX2 and EpCAM analysis.From the results,we found that the expression of stemness markers was higher in the SIRT1^High/Cytoplasmic-MRPS5^High group,indicating a higher proportion of liver cancer stem cells in this group.Then we performed a logistic regression analysis of clinicopathological information under R language conditions and found that patients in SIRT1^High/Cytoplasmic-MRPS5^High group had higher risk of large tumor volume,high pathological stage,metastasis,recurrence and capsule infiltration than SIRT1^Low/Nuclear-MRPS5^High group.We then performed a survival analysis of the two groups of patients and found that the overall survival time of the SIRT1^High/Cytoplasmic-MRPS5^High group was shorter.Further,we combined survival and pathological indicators for COX regression analysis to screen out valuable risk factors,and judged the importance of different risk factors in prognosis through decision tree analysis.From the results,we found that recurrence is the most important risk factor affecting the overall survival time of patients,and the expression of SIRT1-MRPS5 axis has a high weight in the prognosis of non-recurrent liver cancer patients,so it can be used as a new prognosis index in clinical work.To validate the results,we repeated all of the above analysis using the clinical information in the TCGA database.In the analysis of the decision tree model,we found that the subcellular localization data of MRPS5 defined the critical value of SIRT1expression from a new dimension,thus confirming the relationship between SIRT1-MRPS5axis and prognosis of HCC patients and realized Two-way verification of the conclusions of the study by actual clinical data and bioinformatic database data.A list of different sections and main conclusions are shown as following:1.Liver cancer stem cells rely mainly on oxidative phosphorylation for energy supply and use biological information database to statistically screen research targets.?1?liver CSCs mainly rely on OXPHOS to generate ATP;?2?liver CSCs maintain more enhanced mitochondrial function than non-CSCs;?3?Interfering the mitochondrial respiration in liver CSCs lead to reduced self-renewal capacity;?4?Interfering the function of mitochondrial complex-I in liver CSCs lead to reduced self-renewal capacity;?5?MRPS5 gene correlated with the coding genes of complex-I core subunits and supernumerary subunits;?6?MRPS5 is one of the MRPs proteins that has the strongest correlation with OXPHOS;?7?MRPS5 less involved in other metabolism rather than OXPHOS.2.MRPS5 enhances mitochondrial function and participates in maintaining stemness properties of liver CSCs?1?MRPS5 enhances mitochondrial respiration of liver CSCs by promoting the production of NAD⁺;?2?MRPS5 is necessary for mitochondrial biogenesis;?3?MRPS5 promote the generation of NAD⁺,which activate the UPR^mt to against oxidative stress;?4?MRPS5 both enhances and maintains the health of mitochondrial function;?5?MRPS5 enhances the self-renewal capacity of liver CSCs;?6?MRPS5 participates in tumor initiation and progression.3.The acetylation and subcellular location of MRPS5?1?MRPS5 could be acetylated at K365 site;?2?SIRT1 is the specific deacetylase of MRPS5;?3?Analysis of clinical liver cancer samples and hepatoma cell lines revealed both a cytoplasmic and nuclear distribution of MRPS5;?4?The subcellular location of MRPS5 is decided by its acetylation status,acetylaed MRPS5 gather in nuclei while deacetylated MRPS5 guided by mitochondrial targeting domain and translocate to mitochondrion;?5?Only mitochondria locating MRPS5 promote the mitochondrial function.4.MRPS5 enhanced metabolic plasticity of liver cancer cells?1?Acetylated MRPS5 translocation to the nucleus is necessary to enhance glycolysis of liver cancer cells;?2?MRPS5 enhances the flexibility of energy metabolism of liver cancer cells;?3?MRPS5 participates in the metabolic reprogramming during the differentiation of liver CSCs.6.Clinical epidemiological study of SIRT1-MRPS5 axis?1?Tumor recurrence is the major risk of poor prognosis of liver cancer patients;?2?Patients in the SIRT1^High/Cytoplasmic-MRPS5^High group are more prone to tumor recurrence,have faster tumor progression,and have a worse prognosis;?3?Subcellular localization information of MRPS5 is critical for determining the critical value of SIRT1 expression;?4?SIRT1/MRPS5 axis participates in metabolic reprogramming to facilitate tumor progression and may serve as a promising therapeutic target of liver cancer.In conclusion,the above data suggest that liver CSCs relied on the enhanced mitochondrial function to maintain stemness properties,which is different from aerobic glycolysis playing main roles in the differentiated non-CSCs.We found that liver CSCs exhibit increased mitochondrial respiratory capacity and complex-I of mitochondrial was necessary for stemness properties of liver CSCs via regulation of mitochondrial respiration.Bioinformatics analysis reveals that mitochondrial ribosomal protein S5?MRPS5?is closely related with the function of complex-I.Further experiments confirmed that MRPS5 promoted the production of NAD⁺,which is necessary for enhanced mitochondrial function in liver CSCs.MRPS5 played a critical role for liver CSCs to maintain stemness properties and to participate in tumor progression.Mechanistically,the acetylation status of MRPS5 is directly regulated by NAD⁺dependent deacetylase sirtuin-1?SIRT1?,which is abundant in liver CSCs and decreased during differentiation.Deacetylated MRPS5 locates in mitochondria to promote the function complex-I and the generation of NAD⁺to enhance mitochondrial respiration.Conversely,the acetylated MRPS5 gathered in nuclei leads to increased expression of glycolytic proteins and promotion of the Warburg Effect.Therefore,liver CSCs transform mitochondrial-dependent energy supply to a Warburg phenotype by the dual function of MRPS5.Clinical analysis of SIRT1 and MRPS5 expression in tumor tissues showed the SIRT1^High/Cytoplasmic-MRPS5^High profile was associated with poor prognosis hepatocellular carcinoma?HCC?patients,and the profile of SIRT1/MRPS5 axis had a higher weight in estimation of prognosis of HCC patients without tumor recurrence.