The Epigenetic Regulatory Mechanisms Of Mitochondria During H₂O₂-induced Cellular Premature Senescenc

Cellular senescence is a process of progressive dysfunction,loss of disease resistance and homeostasis disorder.Cell senescence is one of the endings for cell and a sign of maturation.It has been reported that environmental exogenous oxidative stress can accelerate the process of aging,which leading to the appearance of premature aging phenotypes in cells,tissues and individuals.Mitochondria are semi-autonomous organelles and play a key role in aging,which can interact with epigenetic mechanisms in a variety of ways and are likely to participate in the aging process of the human body.And this process has been proven to be complex and finely regulated by epigenetics as well as gene expression map changes are closely related.But the epigenetic variation of premature senescence is the trigger or the accompanying consequences remain unclear.However,epigenetic regulation serves as a bridge between environmental factors and genomic modifications.The epigenetic modified genome contains adaptive or harmful bidirectional biological effects that may be persistent or heritable,but may change.The mitochondrial epigenetics（mitoepigenetics）refers to the epigenetic modification of genes encoded by mitochondria itself,such as mtDNA methylation and hydroxy methylation,and the epigenetic regulation of mitochondria by the metabolites of mitochondria and nucleus,including the regulation of non-coding RNA or nuclear coding for mitochondrial-related functional gene promoter methylation modification.Therefore,systematically studying the epigenetic regulation mechanism during premature senescence can help to reveal the underlying causes of aging and its related diseases,which can provide the possibility for early mitochondrial epigenetic targeted interventions,and it has important scientific significance.1.Objective:It aims to study the alterations of mitochondrial biological characteristics,mitochondrial DNA methylation microenvironment and the epigenetic modification of its core transcriptional regulation factors during cellular replicative and premature senescence induced by hydrogen peroxide in human embryonic lung fibroblasts（HEFs）.The mtDNA epigenetic modification code during premature senescence was searched in human embryonic lung fibroblasts,which it can provide the scientific theoretical basis for mitochondrial toxicity assessment of oxidative stress and possible epigenetic interventions target for other age-related disorders.2.Materials and Methods:2.1 Test substance and method of treatmentThe cell test substance was hydrogen peroxide at a concentration of 400μmol/L.Firstly,the 30%hydrogen peroxide was prepared in an intermediate concentration of10 mmol/L with PBS,and then serum-free L-DMEM was used to adjusted to a concentration of 400μmol/L according to the addition of the cell culture flask.The 400μmol/L toxic solution should be freshly prepared.Hydrogen peroxide was stored at 4°C and protected from light.2.2 Cell line and groupingThe cell number of normal human embryonic lung fibroblasts was CCC-HCF-1,obtained from a 15-week-old embryonic tissue,and established by the Cell Resource Center for Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences,XY=46,PDL=10（population doubling levels,PDL）.2.3 Cell culture and H₂O₂ treatmentHEFs were maintained at 37°C in culture flasks containing L-DMEM supplemented with 10%newborn calf serum and 1%penicillin/streptomycin solution under 5%CO2,95%relative humidity and aseptic operation.Cell passage was performed at 1:4 when the cell confluence reached 90%and cell counting.Based on the cell model which has been successfully established by our research team,the young cell group of normal embryonic lung fibroblasts was 22PDL,the middle-aged cell group was 35PDL,and the replicative senescence cell group was49PDL.The cellular premature senescence model was induced by 400μM H₂O₂ once a day at the same time and with 2 hours each time,after four consecutive days the premature senescence models were classified into the premature senescence initiation group（PSi）and the premature senescence persistence group（PSp）.To sum up,there were five senescent cell groups in this study:22PDL（young）,35PDL（mid-age）,49PDL（replicative senescence）,PSi and PSp.2.4 Research methods2.4.1 Detection of general biological characteristics alterations in mitochondria during premature senescence induced by H₂O₂Based on the normal human embryonic lung fibroblast replicative senescence and H₂O₂-induced premature senescence model,the alterations of mitochondria general biological characteristics were aim to observed.The cell senescence situation was identified by Senescence-associated specificβ-galactosidase staining.The mitochondrial distribution and the content were observed with confocal laser scanning microscope during cellular senescence.The ATP contents in mitochondria were detected by luminometer.The enzyme content of 8-hydroxydeoxyguanosine（8-OHdG）in mitochondria was investigated by ELISA assay.The Q-PCR assay was used to measure the mtDNA copy number（mtCN）.The protein expression for TFAM,TFB2M,POLRMT and NRF1 was measured by Western blot.2.4.2 Epigenetic microenvironmental changes and regulation of mitochondrial DNA methylation during premature senescenceThe ELISA colorimetric assay was performed to explore the total methylation level of mtDNA.The total enzyme activities of mitochondrial DNMTs（mtDNMTs）were measured by the Elisa-like methods.The Q-PCR assay was used to detect the mRNA expression levels of mtDNMT1.The mtDNA methylation related protein expression of mitochondrial DNA methyltransferases1（mtDNMT1）,DNA methyltransferases 3A and DNA-binding domain proteins were detected by Western blot.2.4.3 The expression of mitochondrial core transcriptional regulators and methylation level of promoter region during premature senescenceThe Q-PCR assay was used for the mRNA levels of TFAM,TFB2M,POLRMT,NRF1.The protein expression levels of mitochondrial transcription factors such as NRF1,POLRMT,TFAM and TFB2M both in cell and its mitochondria were detected by Western blot.And the total DNA of 22PDL,49PDL and PSp groups were used to analyzed the methylation of CpG sites with bisulfite treatment sequencing（BSP）for TFAM and TFB2M.2.4.4 Mitochondrial Methylated DNA Immunoprecipitation（mt-Me DIP）was performed to analyze mtDNA methylated fragments during cellular prematuremtMeDIP-sequencing was used to detect the overall status of mtDNA methylation.And bioinformatics was preformed to analyze the epigenetic regulatory networks and key molecular events involved in mtDNA methylation during premature senescence and identify the mitochondrial DNA senescence-related differences basic gene.2.4.5 Statistical analysis.All experiments were repeated for three or more times.Statistics were assessed using SPSS 13.0 Statistical data were expressed as mean土SEM,the analysis among all groups of datas by one-way ANOVA.In all cases,t<0.05 was considered statistically significant.3.Results3.1 Mitochondrial toxicity during premature senescence induced by H₂O₂During cellular senescence,the mitochondria were tightly distributed around their nuclei and showed dispersive distribution around the nuclei.35PDL had the largest number of mitochondria but PSi had the least.There was no significant difference between 22PDL and 35PDL,the ATP content of 49PDL and PSi were close and decreased rapidly compared with 22PDL;and the ATP content of PSp was higher than22PDL.The levels of mt-8-OHdG in 35PDL and PSp were no significant difference but much lower than 22PDL;49PDL and PSi had no differences but much higher than22PDL.The relative mtDNA copy number（mtCN）of 35PDL,49PDL and PSp were higher than 22PDL except PSi.All of the differences were statistically significant（P<0.05）3.2 Variational characteristic in mitochondrial DNA epigenetic microenvironment and its regulatory mechanism during cellular premature senescenceThe results of ELISA showed that the mtDNA methylation level increased gradually in 35PDL while decreased in 49PDL during replicative senescence.But the difference of mtDNA methylation level between PSi and PSp was not statistically significant（P>0.05）and were slight down-regelation than 22PDL.The mRNA level of mtDNMT1 and mitochondrial total DNMTs activity showed a gradual increased level during replicative senescence,while decreased dramatically in PSi.But in PSp the mRNA level of mtDNMT1 and mtDNMTs activity were raised to equivalent to that of22PDL（P<0.05）.The mitochondrial protein expression of mtDNMT1 in 35PDL and49PDL were higher than 22PDL while PSi and PSp were decreased.The expression of DNMT3A in 35PDL in mitochondria was still significantly higher than 22PDL,while the expression level of 49PDL and PSi was lower than 22PDL,and the difference between PSp and 22PDL was not statistically significant.The expression of MBD2 in35PDL was higher than 22PDL,while other groups were lower than 22PDL.3.3 Expression of mitochondrial core transcriptional regulators and methylation analysis of its promoter region during premature senescenceThe results of qPCR shown that the mRNA levels of TFAM,TFB2M and POLRMT in 35PDL were higher than 22PDL,but NRF1 was decreased.In 49 PDL,the mRNA levels of TFAM,TFB2M and POLRMT were lower than 22PDL,but NRF1was increased.During premature senescence,all of the mRNA levels were lower than22PDL.The mRNA levels of TFAM,TFB2M in PSp were higher than 22PDL while POLRMT and NRF1 were decreased.The alterations at cellular protein level of TFAM,TFB2M,POLRMT and NRF1 were similar to those at the mRNA level.Western blot showed that the TFAM expression level of 22PDL was the highest in the mitochondrial protein,and 49PDL,PSi and PSp were lower than 22PDL and there was no significant difference among them（P<0.05）.The protein expression of TFB2M showed that 35PDL was higher than 22PDL,but there was no significant difference between 49PDL and22PDL while the same to PSi and PSp,which were lower than 22PDL.The expression level of POLRMT in 35PDL was significantly higher than that in 22PDL;49PDL and PSi were higher than 22PDL,but lower than 35PDL;there was no difference between49PDL and PSi,and there was no significant difference between PSp and22PDL（P>0.05）.The protein expression of NRF1 in 35PDL and 49PDL were higher than 22PDL,but the expressions of PSi and PSp were lower than 22PDL.The difference between the groups was statistically significant（P<0.05）.The results of BSP experiment showed that there were no statistically significant difference in the proportion of methylation at CpG sites of TFAM promoters among the cell groups,and all of the methylation degree are less than 1%.The proportion of CpG methylation sites of TFB2M transcriptional regulatory region of 49PDL was higher than that of 22PDL and PSp,and the difference between 22PDL and PSp was not statistically significant.3.4 The sequencing analysis of mtDNA methylated fragmentmtDNA methylation 5-mC immunoprecipitation combined sequencing showed that there were differences in mtDNA methylation abundance of 22PDL,49PDL and PSp.The specific mtDNA methylated fragments of 22PDL and PSp were basically consistent,and the specific mtDNA methylated fragments of 49PDL were ND5 and ND6.The common mtDNA methylation fragments were ND1,2,COX2,3,ATP6,8,ND4,ND4L,ND5,ND6 and CYB.According to the analysis of KEGG pathway in bioinformatics,the occurrence of specific mtDNA methylation fragments involves oxidative phosphorylation and some age-related diseases such as Parkinson’s disease,Alzheimer’s disease,Huntington’s disease and non-alcoholic fatty liver disease（NAFLD）.In the Gene Ontology project（GO）analysis,the biological processes mainly involve oxidative phosphorylation and ATP metabolism.The cell components were mainly involved in mitochondrial endometrial protein complex and respiratory chain.The major molecular functions involved are NADH dehydrogenase activity and oxidoreductase/REDOX enzyme activity.4.Conclusion4.1 The premature senescence of human embryonic lung fibroblasts induced by H₂O₂has a certain degree of mitochondrial toxicity.4.2 The cellular replicative and premature senescence induced by H₂O₂exhibited different mitochondrial epigenetic microenvironmental characteristics.4.3 The mitochondrial core transcriptional were decreased during both senescence.Methylation of TFB2M transcriptional regulatory regions is involved in the regulation of its mRNA expression.4.4 The replicative and premature senescence have the same reduction fragments of mtDNA methylation（ND1,ND2,ND4,ND4L,ND5,ND6,COX1～3,ATP6,ATP8,CYB and d-loop）.In addition to the above mtDNA fragments,the methylation level of COX1 fragment during premature senescence was elevated.The alterations of mtDNA methylation that lead to cellular senescence existed in the NADH dehydrogenase family associated with oxidative phosphorylation and ATP metabolism.