Study On The Changes Of Circular RNA Expression Profiles In HK-2 Cells Cultured With High Glucose And Caspase-1 Inhibitor Intervention

Objective:This experiment aimed to detect and verify the circ RNAs expression profiles and changes of HK-2 cells?human proximal tubular epithelial cells?cultured in vitro with normal glucose,high glucose,high glucose and VX-765?caspase-1 inhibitor?,to investigate which circ RNAs may be involved in the inflammation and pyroptosis mechanisms in renal tubulointerstitial damage of diabetic nephropathy.Methods:HK-2 cells cultured in vitro were divided into 3 groups: normal glucose group,high glucose group,high glucose with VX-765 group,Trizol extraction of total RNA after simultaneous culture for 24 hours.The circ RNA expression levels of three groups of cells were detected using an Arraystar Human circ RNA Array V2 chip.The collected array images were analyzed by Agilent Feature Extraction software?version11.0.1.1?,and data was normalized using the lim software package.Differentially expressed circ RNAs between two groups were determined by fold change,and the circ RNAs expression patterns were analyzed by stratified clustering.Then targetscan and mi Randa-based mi RNA target prediction software were used to predict the possible micro RNAs interacting with circ RNAs.Ten circ RNAs were screened by strict screening conditions for further q RT-PCR validation and bioinformatics analysis.Combining with literatures to preliminarily investigate the circ RNAs that may be involved in the pathogenesis of diabetic tubulointerstitial lesions,may be related to inflammation and pyroptosis.Results:1.Compared with normal glucose group,there were 92 circRNAs that were up-regulated in high glucose group and 25 circirc RNAs that were down-regulated in high glucose group,The most significant up-regulation was hsa_circ RNA₄02294?7.0299804-fold?,and the most significant down-regulation was hsa_circ RNA₀01906?-22.9722809-fold?.In high glucose + VX-765 group,compared with high glucose group,there were 54 circirc RNAs that were up-regulated and 103 circrc RNAs were down-regulated,The most significant up-regulation was hsa_circ RNA₄04975?100.4332781 times?,and the most significant down-regulation was hsa_circ RNA₁03142?-21.0658222 times?.2.The caspase-1 inhibitor?VX-765?inhibited the high expression of 54 circ RNAs under high glucose status and the low expression of 14 circ RNAs under high glucose status.These circ RNAs may be involved in the inflammatory and pyroptotic lesion of the diabetic tubulointerstitial injury.3.hsa_circ RNA₁02747,hsa_circ RNA₄05650,hsa_circ RNA₁04854 verified by q RT-PCR showed statistical significance among the three groups of cells.4.Each circ RNA has detailed annotation information,and there are five micro RNAs sorted according to the strength of their competition binding order,which can be used for subsequent ce RNA mechanism analysis or other functional mechanisms.Conclusions:In this experiment,abnormal expression of circRNAs was observed in high glucose group compared with normal glucose group in HK-2 cells cultured in vitro.After the addition of caspase-1 inhibitor?VX-765?to the high glucose group,the circ RNAs expression profile significantly changed compared with the high glucose group,suggesting that circ RNAs may be involved in diabetic nephropathy with tubulointerstitial lesions,possibly related to inflammation and pyroptosis...