The Mechanisms Of MiR-129 Regulating Fibroblast Cellular Senescence And Aging Through Intracellular Calcium Signaling

Background:Cellular senescence is mainly characterized by stable cell cycle arrest,accompanied by senescence-associated secretory phenotype(SASP)secreted by senescent cells,which plays a critical role in pathophysiological processes such as embryonic development,tumor and body aging.More and more studies have shown that intracellular calcium signaling are involved in the regulation of cellular senescence.The previous study of our group found that inositol 1,4,5-trisphosphate receptor 2(ITPR2),the endoplasmic reticulum calcium release channel,is a new regulator of senescence.However,the transcriptional regulation mechanism of ITPR2 in the process of cellular senescence is not fully understood.Objective:Screen whether there are endogenous small molecules that regulate ITPR2 expression at the transcriptional level and modulate cellular senescence.Methods:(1)The candidate small miRNA molecules that inhibit ITPR2 transcription were preliminarily screened from databases.Furthermore,the mimics and inhibitors of candidate miRNA were transfected into MRC5 cells,and the candidate miRNA regulated expression of ITPR2 was analyzed by RT-qPCR.Then verify whether the candidate miRNA regulates the expression of ITPR2 in the similar cell lines IMR90 cells and WI38 cells.Based on the possible binding sites of candidate miRNA and ITPR2 3’-UTR predicted by miRbase website,the corresponding ITPR2 mutants were designed,and the luciferase assay was used to verify the direct binding sites of miRNA to ITPR2 3’-UTR.(2)Mimics and inhibitors of miRNA were transfected into MRC5 cells,and the levels of cytoplasmic calcium,mitochondrial calcium,mitochondrial membrane potential,mitochondrial reactive oxygen species(ROS)and cytoplasmic ROS were detected by flow cytometry(FCM).DNA damage responses(DDR)were detected by immunofluorescence(IF)and Western Blot.(3)MRC5 cells were transfected with miRNA mimics and then treated with ITPR agonists,and miRNA inhibitors were treated with calcium chelator or antioxidants.FCM was used to analyze whether miRNA regulates the levels of ROS in mitochondria and cytoplasm via calcium signaling.IF and Western Blot were applied to analyze whether miRNA regulates DDR through calcium signaling.(4)Mimics and inhibitors of miRNA were transfected into MRC5 cells.RT-qPCR,Western Blot,SA-β-gal and crystal violet staining were used to analyze whether miRNA regulates cellular senescence through calcium signaling.Then,the expression level of miRNA was detected in replicative senescence model and therapy-induced senescence model,as well as in PBMCs of healthy old and young individuals.(5)In the mouse lung aging model induced by intratracheal instillations of bleomycin and natural aging mouse model,miR-NC agomir or miR-129 agomir was injected via intraperitoneally.The Immunosenescent phenotype of mice was analyzed by FCM,the proportion of senescent cells in lung tissue was analyzed by SA-β-gal staining,and the expression of lung senescence-associated genes was analyzed by RT-qPCR and Western Blot.Results:(1)Based on the Venny diagram analysis of 4 miRNA databases,we preliminarily screened out 8 candidate miRNA that repressed ITPR2 transcription.Cell experiments showed that miR-129 inhibited the expression of ITPR2 most significantly among the 8 candidates.After co-transfection of psiCHECK2-ITPR2 3’-UTR wt and miR-129 mimics,the ratio of Renilla/Firefly Luciferase in 293T cells was significantly lower than that in the control group,which indicating that miR-129 bound to ITPR2 3’-UTR and inhibited its expression.(2)FCM showed that miR-129 mimics significantly decreased the levels of calcium in cytoplasm and mitochondria,reduced the decrease of mitochondrial membrane potential,and decreased the levels of mitoROS and total ROS,while miR-129 inhibitors had the opposite effect.In addition,knockdown of ITPR2 or MCU partially reversed the upregulation of cytoplasmic and mitochondrial calcium levels,the decrease of mitochondrial membrane potential,and the upregulation of mitoROS and total ROS levels induced by miR-129 inhibitors.IF and Western Blot showed that miR-129 mimics significantly reduced the production of 53BP1 foci and the expression of p-H2AX protein,whereas miR-129 inhibitors had the opposite effect.In addition,knocking down of ITPR2 or MCU can partially reverse the effect of miR-129 inhibitors.(3)The results of FCM,IF and Western Blot showed that ITPR agonists upregulated the phenotype of decreased mitoROS,ROS and DNA damage induced by miR-129 mimics,while calcium chelators and antioxidants reversed the phenotype of increased mitoROS,ROS and DNA damage induced by miR-129 inhibitors.(4)The results of RT-qPCR,Western Blot,SA-β-gal and crystal violet staining showed that miR-129 mimics up-regulated the expression of Ki67 and down-regulated the expression of ITPR2,p16 and p21,reduced the proportion of senescent cells and promoted cellular proliferation,while miR-129 inhibitors had the opposite effect.In addition,knocking down of ITPR2 or MCU partially reversed cellular senescence induced by miR-129 inhibitors.In replicative senescence model,therapy-induced senescence model and elderly PBMCs,the expression of miR-129 was significantly down-regulated,and there was a significant negative correlation between ITPR2 and miR-129 in human PBMCs.miR-129 mimics partially reversed the upregulation of cytoplasmic and mitochondrial calcium levels,the decrease of mitochondrial membrane potential,the upregulation of mitoROS and total ROS levels,the increase of DNA damage,proliferation inhibition,the increase of SA-β-gal activity and the down-regulation of Ki67 expression,as well as the upregulation of ITPR2,p21 and p16 induced by bleomycin.(5)In the therapy-induced lung aging mouse model,miR-129 agomir partially reversed the phenotype of bleomycin-induced up-regulation of SA-β-gal activity,CDKN1A and p21 expression in lung tissue,and further,miR-129 agomir partially reversed the decrease of naive T cell frequency and the increase of memory T cell frequency induced by bleomycin in lung tissue.In the natural aging mouse model,miR-129 agomir partially reversed the up-regulation of SA-β-gal activity and the expression of CDKN2A,p16 and IL-6 in the lung tissue of aged mice.miR-129 agomir partially reversed the decrease of naive T cell frequency and the increase of memory T cell frequency in the lung tissue of aged mice.Conclusion:(1)miR-129 directly binds to the 3’-UTR of ITPR2 and inhibits its expression;(2)miR-129 controls a cascade of mitochondrial membrane potential,mitochondrial and cytoplasmic ROS levels,and DNA damage via intracellular calcium signaling;(3)miR-129 regulates fibroblast cellular senescence through intracellular calcium signaling;(4)miR-129 partially reverses therapy-induced aging and natural aging phenotypes.