Role And Mechanism Of MiR-23a-5p In Renal Medulla During Hyperosmotic Stress Response

Background: In normal physiological conditions,renal meddullary cells are exposed to extremely harmful hyperosmotic conditions,which can reach up to 3200 mOsmol/kg.Studies have shown that microRNA(miRNA)play important protective role in the response of hyperosmotic stress in renal medullary cells.However,how many miRNA function in the response of hyperosmotic stress,and their mechanisms by which they regulate are not fully elucidated.Objective: to reveal the miRNAs that play regulatory roles under hyperosmotic stress,and elucidate their functions and regulatory mechanisms by which they function under hyperosmotic stress.Methods and Results: To systematically identify miRNAs that play regulatory role under hyperosmotic stimulation,the models of mIMCD3 cells under hyperosmotic stress was developed,and were subjected to a high-throughput transcriptome sequencing.High-throughput sequencing results were verified by qPCR.The results showed that after 6 hours of hyperosmotic stress,12 miRNAs were significantly down-regulated and 3 miRNAs were significantly up-regulated.In these changed miRNAs,miR-23a-5p was the most downregulated miRNA,so that was chosen for further study.By qPCR detection,the expression of miR-23a-5p gradually decreased with the increase of osmotic pressure.At the same time,we also detected other kidney cells including 293 A and HK2 in the same treatment of hypertonicity.under hypertonic stress,miR-23a-5p was slightly upregulated in 293 A.By contrast,miR-23a-5p showed a sharp downregulation at early stage and recover to normal quickly,indicating that the downregulation of miR-23a-5p is cell type specific.To investigate the function of miR-23a-5p during the response of hypertonic stress,the miR-23a-5p analogue or inhibitor was transfected into mIMCD3 cells.We detected the cellular activity by MTS assay.The results showed that overexpression of miR-23a-5p could reduces cellular viability.In contrast,inhibition of miR-23a-5p significantly could enhance cellular viability.However,the effect of miR-23a-5p overexpression or inhibition on cellular activity appears to be more pronounced under hypertonic conditions.Afterwards,it was found through the EDU cell proliferation assay that miR-23a-5p did not affect cell proliferation significantly under isotonic conditions.However,under hyperosmotic stress,overexpression of miR-23a-5p significantly inhibited cell proliferation while inhibition of miR-23a-5p significantly promoted cell proliferation,suggesting that the function of miR-23a-5p in proliferatio was hypertonicity-dependent.To study the mechanism of miR-23a-5p function under osmotic stress,we performed high-throughput transcriptome sequencing of m IMCD3 cells by hypertonic treatment and inhibition of miR-23a-5p expression.We comprehensively compared the deep-sequencing results,and bioinformatic prediction by Targetscan we identified 14 candidate targets at downstream of miR-23a-5p.We verified these fourteen candidate target genes by qPCR.Four candidate target genes were selected for 3?-UTR assay and further functional studies.In addition,we have also verified on animalstreated with water deprivation.Real-time fluorescence quantitative PCR was used to detect the expression of miR-23a-5p from mouse kidneies.CONCLUSION: miR-23a-5p was significantly down-regulated in renal medullary cells under hypertonic stress,which resulted in meddullary cell survive and proliferation under hypertonic stress.