MiR-184and MiR-150Promote Renal Mesangial Cell Senescence By Directly Targeting Autophagy Gene Rabl And Rab31

Kidney experiences a more rapid aging process of all the organs, which ischaracterized by renal function decreasing gradually. Glomerular hypertrophy,mesangial matrix hyperplasia, glomerulosclerosis and tubulointerstitial fibrosis alwayswere found in the renal organs of old animals and humans. However, the mechanism ofrenal aging is not clear. Therefore, elucidating the mechanisms of kidney aging willprovide important implications for the treatment of kidney disease. One of the mostimportant innate cells in kidney tissue is glomerular mesangial cell (GMC), which altersthe aging process of kidnyey via its cellular senescence, however, the mechanisms arestill unclear so far. Thus, having a better understanding on the cellular senescence ofGMC will be beneficial for preventing aging-related renal disease.MicroRNA (miRNA), widely existed in eucaryotic organisms and highlyconserved in sequences and functions, is a non-coding single strand RNA which reducesthe expression of targeted genes by degradating mRNA or inhibiting its translation.Current studies have verified that miRNA plays significant role in many physiologicaland pathological process, including development, immunity and cancer. Autophagefacilitates the elimination of cellular damaged moleculs and organelles, which is closlyassociated with cell aging. Our study is to determine the role of miRNA differentialexpression in kidney aging process and its influence on cell autophage even on theregulation of GMC senescence, both in vivo and in vitro.Part1. Effect of caloric restriction on miRNA expression in old rat kidny tissuesand the determination of differentially expressed miRNACaloric restriction (CR) was administrated to interfere the aging process of ratkidney and the pathological change of the tissues was manifested by PAS staning.Moreover, chip scan was employed to determine the alternation of miRNA expression profile between normal diet and calory restricted diet feeding rats. Furthermore, thedifferentially expressed miRNA was performed by RT-PCR. The results suggested that24-month rats exhibited obvious characteristics of aged kidney tissues. Compared withnormal diet group, rats in the CR group showed less GMC hyperplasia. Chip scan andcluster analysis indicated that miR-132, miR-184and miR-150were statisticallydownregulated in24-month-old rat renal tissues fed with calory restricted diets(p<0.05-0.01). RT-PCR showed that compared with normal diet group, expression ofmiR-150, miR-132and miR-184in CR group were reduced by66%，66.9%and96.7%(p<0.05-0.01). The outcomes showed that caloric restriction inhibited kidney aging andmiR-132, miR-184and miR-150may involved in the regulation of kidney aging.Part2: miR-184and miR-150modulated autophage-mediated GMC cell damageand agingFirstly, AL rats were sacrificed and3kinds of kidney innate cellls (Glomerularepithelial cell (GEC); Glomerular mesangial cell (GMC); Renal tubular epithelial cell(RTEC)) were isolated to analyze the expression of miR-184or miR-150using RT-PCR.The results showed that miR-184and miR-150were dominantly expressed in GMCcells（p <0.01-0.05）, therefore, the GMC which is isolated from3-month and24-monthrats, was selected for further studies in vitro to evaluate the influence of oxidative stresson cell aging. Exogenous miRNA mimics or inhibitor were constructed andadministrated to GMCs to intervene the expression of miR-184or miR-150, and cellularoxidative stress and cell aging were measured. We found that the GMC came from24-month rats showed clear aging characteristcs suggested by presence of95%SA-β-gal and SAHF positive cells. Compared with cells from3-month rats, cellularbiomolecules oxidative products (MDA,8-OhdG and oxidative carbonyl protein)accumulated (p<0.01) while miR-184and miR-150reduced in aged cells. Moreover,LC3, biomarker of autophage activity, reduced and p62, ubiquitin-proteasome systembiomarker, increased in old cells. Additionally, miR-184or miR-150mimics inducedoverexpression of miR-184or miR-150reduced the LC3levels and blocked theautophage activity, while, the adminstration of miR-184or miR-150inhibitor showed the opposite effects. Moreover, oxidative stress augmented in aging cells and miR-184or miR-150regulated cell aging via inhibiting autophage. After raising miR-184andmiR-150expression, oxidative products accumalated and SA-β-gal and SAHF positivecells elevated（p<0.05）.. Above all, miR-184and miR-150inhibited regulated cell agingby inhibiting autophage activities.Part3: miR-184or miR-150regulated autophage by interating with3′-UTR ofRab1和Rab31mRNAThe potential miR-184or miR-150targeted genes were predicted usingBioinformatics software (miRanda、Targetseans and PicTar) and239-649genes formiR-184were screeed and209-2825for miR-150corresponding to different programs.Among all the genes, autophage-related genes were selected and many nembers of Rabsubfamily, targets for both miR-184and miR-150, were considered to be associatedwith autophagy bodies’ formation and maturity. Thus, Rab1and Rab31were selectedfor further studies. MiR-150and miR184inhibitor didn’t change the presence of Rab1and Rab31mRNA, but they significantly downregulated the translation of Rab1andRab31proteins (p<0.05-0.01). Dual luciferase report system were employed to detectthe interaction between miR-150/miR184and target mRNA3′-UTR and the resultsconfirmed that their interaction downregulated luciferase activity (p<0.05-0.01).MiR-150/miR184played important roles in regulating cell aging through directlyinhibiting the expression of autophage-related Rab in post-transcription level. Overall,miR184and miR-150interacted with Rab mRNA3′-UTR to degradate mRNA orinhibit their expression,which inhibited their regulation of autophage furtherly.In summary, we found that calory restriction and aging affected the expression ofmiR-150and miR184in kidney tissues, which were mostly expressed in GMC cells. Invitro studies verifyed that miR-150and miR-184interacted with their common targetsRab1and Rab31mRNA to inhibite GC autophage activities, which regulated cellularoxidative stress and promoted the aging in post-transcription level.