Effects Of MiR-200b-3p Inhibition On TRPC6 And BK_Ca Channels In Podocytes

Podocytes,also known as glomerular epithelial cells,are highly differentiated cells which are attached to the glomerular basement membrane?GBM?.Podocytes consists of three parts including the cell body,the primary processes and foot processes.Owing to its special structure,podocytes together with glomerular basement membrane constitute the glomerular blood filtration,which plays an important role as a barrier to prevent protein loss.Recently,more and more researches suggest that the development and process of many kidney diseases are closely related to the damage and functional abnormalities of podocytes.microRNA?miRNA?is a class of endogenous single-stranded,non-coding RNA molecules including about 22 nucleotides in length.It is thought to modulate gene transcription and expression by translational inhibition and destabilization of messenger RNAs?mRNAs?by binding to its target gene to form a complete or incomplete complementary pairing.miRNAs are expressed in many species,including plants,animals and even some algae.In fact,some studies showed that knocking out some special enzymes such as Dicer or Drosha that play an important role in the production of miRNAs could lead to proteinuria and glomerulosclerosis in podocytes^[1].It is indicated that miRNAs play an important role in the physiological and pathological process of podocytes.miR-200b is a member of the miR-200 family and miR-200b-3p is obtained from the 3'arm of pre-miR-200b.Many studies have shown that miR-200b-3p plays an important role in the regulation of epithelial-mesenchymal transformation?EMT?.It suggested that overexpression of miR-200b-3p could inhibit the transfer of epithelial cells to mesenchymal cells in bladder cancer^[2].Most of studies on miR-200b-3p focused on cancer,and there were few studies on podocytes and kidney.Raman Agrawal and other studies have showed that miR-200b family is abundant expression in kidney^[3];Li and other studies have showed that miR-200b-3p can promote podocyte differentiation by the repression of RSAD2^[4].Therefore,miR-200b-3p plays a vital role in regulating the physiological function of podocytes.Transient Receptor Cation Channel 6?TRPC6?is nonselective Ca²⁺-permeable cation channels.It is reported that the mutation of TRPC6 gene leads to focal segmental glomerulosclerosis?FSGS?^[5];The activation of TRPC6 could activate calcineurin,and then decrease the expression of the cytoskeletal protein F-actin,which can lead to the cytoskeletal rearrangement of podocytes,and cause the damage of podocyte structure and the production of proteinuria^[6].Therefore,the function of TRPC6 has a great effect on the physiological process of podocytes.The large conductance Ca²⁺-activated potassium channel(BK_Ca)is activated by the increase of local cytosolic Ca²⁺and the depolarization of the membrane potential.It is reported that BK_Ca and TRPC6 channels are co-expressed in podocytes,and TRPC6 may provide a Ca²⁺source for BK_Ca activation.Moreover,BK_Ca plays an important role in regulating the self-balance of Ca²⁺in podocytes.Therefore,this study studies the effects of miR-200b-3p inhibition on TRPC6 and BKCa in podocytes.The conditionally immortalized mouse podocyte cell line?MPC5?was used in our study.The differentiated mature podocytes were used to record the whole-cell currents of TRPC6 and BK_Ca in the CON group,miR-200b-3p inhibitor group and siRNA CON group.The levels of protein and mRNA were also detected in our study.The results were shown as follows:1.The down-regulation of mi R-200b-3p inhibited the currents of TRPC6 channel.The results also showed that down-regulation of miR-200b-3p decreased the levels of mRNA transcription and protein expression of TRPC6in podocytes.2.The down-regulation of miR-200b-3p inhibited the amplitude of BK_Ca currents.The results also showed that down-regulation of mmu-miR-200b-3p reduced the levels of mRNA transcription and protein expression of BK_Ca in podocytes and the I-V curve moved towards to right.Moreover,the activation curve shifted to the depolarization.3.Effects of miR-200b-3p inhibition on the intracellular Ca²⁺concentration:the results of Ca image showed that down-regulation of miR-200b-3p resulted in the decrease of the intracellular Ca²⁺concentration.4.Effects of TRPC6 inhibition on the BK_Ca channel:the results showed that TRPC6 inhibition decreased the amplitude of BK_Ca currents in podocytes,and the I-V curve moved towards to right.Moreover,the activation curve shifted to the depolarization.In summary,the down-regulation of miR-200b-3p inhibited the currents of TRPC6 and BK_Ca channels.Moreover,the transcription level and protein expression of TRPC6 and BK_Ca channels were both reduced in podocytes,and the intracellular Ca²⁺concentration was also decreased.It was also found that the inhibition of TRPC6channel resulted in the decrease of the BK_Ca channel currents.This suggests that the down-regulation of mmu-miR-200b-3p inhibits currents of TRPC6,and the inhibition of TRPC6 currents led to the decrease of intracellular Ca²⁺concentration in podocytes,which may reduce the Ca²⁺source required for BK_Ca activation,resulting in the inhibition of BK_Ca currents.