Study On The Mechanism Of Action Of PCIF1 In Vascular Endothelial Cell Senescence And The Mechanism Of Endoplasmic Reticulum Stress In The Pathogenesis Of Hypertension In Offspring Caused By Gestational Diabete

Background: The high incidence and poor prognosis of coronary heart disease(CHD)impose great burden on society,vascular aging is considered as a main risk factor for developing CHD.Cellular aging,known as senescence,mainly contributes to vascular diseases associated with inflammation and dysfunction of the endothelial cells(ECs): the senescence cells would reduce nitric oxide production and increase endothelin release,resulting in reduced vasodilation.On the other hand,senescence cells could activate a variety of pro-inflammatory cytokines and chemokines,forming a senescence-associated secretory phenotype(SASP)that promotes the development of CHD.The study of vascular endothelial cell senescence can help to understand the specific mechanisms of CHD pathogenesis.Cellular senescence is a state of cell-cycle arrest associated with aging induced by mitogenic signals,DNA damage and inflammation,which may eventually lead to organ failure and death.Senescence can be influenced by gene and environmental factors.In the progression of CHD,various risk factors such as diabetes,nutrition,smoking,stress,hypertension and circadian rhythms have effect on EC senescence through epigenetic factors.Epigenetic regulation can influence gene expression independently of the gene sequence and mainly includes DNA methylation,histone modifications and RNA modifications.Epigenetic regulation plays important roles in many physiological processes and may explain some gaps regarding the interaction between genes and diseases.Large scale analysis has revealed the abundance of RNA modifications in the human epitranscriptome.Methylation modification,the most prevalent RNA chemical modification,has been widely reported relate to cardiovascular diseases.The relationship between RNA modification and CHD is relatively poorly reported,but it has been documented that m6 A methyltransferase(MELLT3/MELLT14 and WTAP)can affect the expression of senescence-associated protein P21 and P27,impacting on cellular senescence.In the late 2018,a novel N6,2’-O-dimethyladenine(m6Am)modifying enzyme,PCIF1,was identified and characterized.Unlike the modification of m6 A,m6Am modification is mainly located in the 5’m7Gppp cap structure followed by the first base.The current experiments have demonstrated that PCIF1 is highly conserved in different species and has effects on m RNA stability and translation,suggesting that PCIF1 may play an important role in regulating life activities.We hypothesized that PCIF1 plays a role in the development of EC senescence.In this study,we first compared the expression of PCIF1 in vascular tissues and endothelial cells under different senescence states.Next,we intervened PCIF1 expression to verify the effect on cell senescence by detecting relevant senescence markers,and make a preliminary investigation on regulatory mechanisms.Methods: 1.To clarify the role of PCIF1 in vascular senescence(1)C57BL/6J mice were bred on normal diet,16-month-old mice were divided into the senescence group and 3-month-old mice were divided into the control group,aortic tissues were stripped to extract RNA,the expression of PCIF1 in tissues were compared by q RT-PCR.(2)Human primary umbilical vein endothelial cells(HUVEC)were extracted,and the cellular senescence model was constructed by repeated passaging culture.The passage 2(P2)as the young group,the passage 7(P7)as the senescence group.RNA and protein were extracted,and the changes of PCIF1 were quantified by q RT-PCR,Western blot and immunofluorescence assays respectively.(3)After specific silencing and overexpression of PCIF1 in HUVEC by si RNA and plasmid,respectively,the number of senescent endothelial cells(β-gal staining),senescence-related phenotype(scratch assay,tube formation assay),senescence-related genes changes(q RT-PCR,Western Blot)to clarify the role of PCIF1 in regulating endothelial cell senescence.2.To clarify whether PCIF1 modulate endothelial cell senescence through regulating Sirt3 expression.(1)Transcriptome sequencing was performed after specific silencing PCIF1 expression in HUVEC,and preliminary screening for downstream genes that may be regulated by PCIF1.qRT-PCR and Western blot were performed and identified Sirt3 as the key target gene for regulation.(2)After RNA transcription inhibition by actinomycin D,cells from control and PCIF1-specific silencing groups were collected at different time points.qRT-PCR was used to detect the relative expression of Sirt3 at different time.Calculating the degradation rate to the effect of PCIF1 on the m RNA stability of Sirt3.(3)Silencing PCIF1 expression while overexpressing Sirt3 in HUVEC,the senescence cells(β-gal staining)and senescence-related genes(P16,P21,P53,IL-6)changes(Western Blot)were detected separately to observe whether the effect of PCIF1 on cellular senescence could be rescued by Sirt3.Results: 1.PCIF1 was significantly decreased in senescent vascular tissues when 3-month-old mice compared with 16-month-old mice,which confirmed the association of PCIF1 with vascular senescence at the animal level.2.When compare the P2 generation endothelial cells with the P7 generation cells,PCIF1 expression was significantly decreased in senescent endothelial cells,which confirmed the association of PCIF1 with vascular endothelial cell senescence at the cellular level.3.After silencing PCIF1 expression in HUVEC,the number of senescent cells increased,cell viability decreased,senescence-related gene expression increased,and cells showed accelerated senescence;conversely,overexpression of PCIF1 delayed cell senescence.4.Sequencing results showed that 5361 genes were changed after intracellular silencing PCIF1 expression,a total of 2920 genes were down-regulated,of which 1643 genes were differentially expressed more than 1.2-fold.Functional and molecular pathway analysis(GO and KEGG)of the down-regulated genes suggested that a large number of genes were enriched in the cellular senescence pathway.5.Five candidate genes for PCIF1 regulation of endothelial cell senescence were initially screened based on gene expression changes,and Sirt3,the most up-regulated gene,was selected as the target gene after combining gene functions reported in papers and verified by q RT-PCR and Western blot.6.The rate of Sirt3 m RNA degradation was faster in the PCIF1-silenced group compared with the control group after transcriptional inhibitor treatment,PCIF1 could maintain the mRNA stability of Sirt3.7.Silencing PCIF1 expression in HUVEC accelerated cellular senescence,and the number of senescent cells and the expression of senescence-related genes was relatively reduced under Sirt3 overexpression.Conclusion: 1.PCIF1 is involved in the regulation of senescence in vascular endothelial cells 2.PCIF1 plays a protective role against cellular senescence.3 PCIF1 positively regulates Sirt3 to affect endothelial cell senescence.Background: Hypertension,a dominant risk factor for vascular diseases,imposes immense health burdens worldwide.Understanding the causes of hypertension is key to its prevention and treatment.Recent evidence suggests the effect of intrauterine environment and early life related events in the maternal generation would lead to the development of offspring hypertension,including hyperglycemia during pregnancy that can cause some effects on fetal growth and development,leading to structural and functional impairment of normal cardiovascular or renal function that continues into the onset of adult hypertension.Studies have shown that endoplasmic reticulum stress plays an important role in the development of offspring hypertension.The endoplasmic reticulum is a major sensor of cellular stress and plays an important role in protein synthesis,folding and transport and disruption of endoplasmic reticulum function leads to activation of unfolded protein responses,which ultimately disrupts cellular function and leads to the development of hypertension.In this experiment,it was hypothesized that endoplasmic reticulum stress might play an important role in the development of hypertension in the offspring due to maternal diabetes mellitus.We investigated the mechanism of endoplasmic reticulum stress in the development of hypertension in the offspring.Methods: A maternal model of diabetes mellitus was prepared by intraperitoneal injection of streptozotocin,the offspring rats showed elevated blood pressure in adulthood.After intraperitoneal injection of the endoplasmic reticulum stress inhibitor 4-phenylbutyric acid sodium salt(4-PBA)or taurodeoxycholic acid(Tudca)in offspring,the elevated blood pressure could be normalized.After execution of the offspring rats,the mesenteric vascular endothelial cells were extracted separately for primary culture,and assayed for:1.TNF-α changes in the culture medium of primary cultured endothelial cells.2.Alteration of endoplasmic reticulum stress pathway proteins in primary cultured endothelial cells.3.Altered e NOS expression in primary cultured endothelial cells.Results: 1.The diabetic mother offspring(DMO)had significantly higher levels of TNF-α in the mesenteric vascular endothelial cell culture medium compared to the control mother offspring(CMO).2.The protein expression of ATF-6 and p-IRE was elevated in the DMO compared to the CMO in mesenteric vascular endothelial cell,which could be reduced to the normal level after the intervention of endoplasmic reticulum stress inhibitors.3.The DMO showed no significant difference in total e NOS expression compared to the CMO in mesenteric vascular endothelial cell,but p-e NOS expression was decreased and could be restored to normal after endoplasmic reticulum stress inhibitor intervention.Conclusion: 1.Maternal gestational diabetes caused increased endoplasmic reticulum stress levels in mesenteric vascular endothelial cells of the offspring rats.2.Maternal gestational diabetes caused abnormal expression of p-e NOS in mesenteric vascular endothelial cell of offspring rats.