MiR-30b-5p And MiR-148a-3p Suppress The Autophagy-lysosome Pathway Via Different Mechanisms

Macroautophagy(hereafter referred to as autophagy)is a fundamental physiological process for maintaining intracellular homeostasis.By degrading misfolded proteins and aging or damaged organelles,autophagy allows cells to resist external stimuli such as starvation and drug damage,which is closely related to the development of neurodegenerative diseases,cardiovascular diseases and tumors.Fusion between autophagosomes and lysosomes is an important process in the autophagy pathway.Autophagic cargoes are ultimately degraded by acid hydrolases in the lysosomes,so the lysosomal biogenesis is essential for the autophagy process.microRNA(miRNA)is a class of endogenous small non-coding RNA with a length of about 22 nucleotides.It is able to participate in the regulation of more than 60% of genes in the body through multiple mechanisms.In recent years,many studies have showed that miRNA could regulate the autophagy process by targeting different autophagy-related genes.However,in these studies miRNA all play a role in the cytoplasm through the classical molecular mechanism and their roles in the nucleus are hardly studied.In this study,we aim to explore the regulation of lysosomal biogenesis and autophagy process by different ways in the nucleus and cytoplasm of miR-30b-5p and miR-148a-3p,respectively,which provides new ideas for expounding the novel mechanisms of miRNA and treating autophagy-related diseases.1.The inhibition of TFEB-mediated lysosomal biogenesis and autophagy by miR-30b-5p in the nucleus:(1)miR-30b-5p binds to the CLEAR element directly: Firstly,we found that miR-30b-5p could bind to the CLEAR element directly through the pull-down assays and sRNA-seq.Subsequent luciferase assays showed that miR-30b-5p was able to inhibit the luciferase activity of the CLEAR element.Next,through EMSA experiments,miR-30b-5p was found to inhibit the binding of the CLEAR element to the nuclear proteins.(2)miR-30b-5p binds to the promoters of lysosomal genes and suppress their transcription through the CLEAR elements: Luciferase assays showed that miR-30b-5p could inhibit the luciferase activity of lysosomal promoters such as CTSA,CTSD,MCOLN1 and etc.While,it lost its inhibitory effect when the putative binding site on miR-30b-5p was mutated.(3)miR-30b-5p inhibits the transcriptional activity of TFEB in the nucleus: The results of FISH and qPCR experiments showed that miR-30b-5p was distributed in the nucleus and the amount of translocation into the nucleus increased under starvation.Subsequent ChIP assays showed that miR-30b-5p was able to reduce the aggregation of TFEB and RNA polymerase II in the promoters of lysosomal genes.(4)miR-30b-5p suppresses the expression of lysosomal genes: Mass spectrometry,qPCR and Western blot experiments showed that miR-30b-5p was able to inhibit the expression of most lysosomal genes.The results of Immunofluorescence assays indicated that miR-30b-5p could inhibit the expression of LAMP1,a lysosomal marker.In addition,we explored the effect of miR-30b-5p on the lysotracker and the results were consistent with the Immunofluorescence experiment.After knocking down Importin8,a nuclear transporter that mediated the mature miRNA into the nucleus,miR-30b-5p lost its inhibitory effect on lysosomal genes,which provides a key evidence for the function of miR-30b-5p in the nucleus.(5)Inhibition of miR-30b-5p on the biogenesis of autophagosomes and autophagic flow: The results of Western blot experiments suggested that miR-30b-5p could reduce the expression of LC3 II,a marker of autophagosome and the degradation of SQSTM1,an autophagic substrate.To further explore the effect of miR-30b-5p on autophagic flow,we transfected it and the tandem EGFP-mCherry-LC3 plasmids into HEK293 cells to detect the autophagy process.The results showed that miR-30b-5p inhibited the biogenesis of autophagosomes and the fusion of autophagosomes and lysosomes.After knockout of miR-30b-5p by CRISPR/CAS9,the mRNA level,protein level and luciferase activity of lysosomal genes were all up-regulated.(6)The regulatory effect of miR-30b-5p on autophagy-lysosome process in the mouse liver: After starvation treatment in mice for 48 hours,we found that liver was the most affected organ with elevated autophagy levels.Therefore,we injected the recombinant adeno-associated viruses containing pri-miR-30b-5p into mice at caudal vein.After 9 weeks,the mice were fasted for 48 h and then their viscera were removed for analysis.The results showed that the lysosomal biogenesis and autophagy levels decreased in the livers of mice treated with miR-30b-5p.2.miR-148a-3p inhibits autophagy by binding to CTSA directly:(1)miR-148a-3p binds to 3'UTR of CTSA directly and inhibits its luciferase activity: By predicting from the TargetScan database,we found that the 5'seed sequence of miR-148a-3p had a match of 8 bases with the 3?UTR of CTSA.We subsequently cloned the 3' UTR of miR-148a-3p into the psiCHECK-2 plasmid to construct a luciferase system.The results of luciferase assays showed that miR-148a-3p could inhibit the luciferase activity of 3'UTR of CTSA mRNA.(2)miR-148a-3p inhibits the protein and mRNA levels of CTSA: Through qPCR and Western blot experiments,we found that miR-148a-3p could significantly inhibit the mRNA and protein levels of CTSA.Notably,the inhibition of CTSA protein by miR-148a-3p will be significantly enhanced after being treated with EBSS for 2 hours or 3 hours.In addition,the inhibition of CTSA mRNA and protein by miR-148a-3p is time-and concentration-dependent.Afterwards,we further verified the effect of miR-148a-3p by Immunofluorescence experiments.(3)miR-148a-3p suppresses lysosomal biogenesis as well as autophagy: Firstly,we analyzed by Mass Spectrometry and found that miR-148a-3p was able to reduce the protein levels of most lysosomal genes.Subsequently,we confirmed the results of Mass Spectrometry analysis by Western blot experiments.After that,LC3 was used as a marker of autophagosome to explore the regulatory effect of miR-148a-3p on autophagy process through western blot experiments and the results showed that miR-148a-3p significantly reduced the conversion of LC3 I to LC3 II,indicating a lower level of autophagy.Whereas under the treatment of bafilomycin A1,the lysosomal biogenesis decreased and the degradation of autophagic substrates dropped.Thus,the inhibition on the autophagy process by miR-148a-3p was performed by inhibiting the lysosomal biogenesis process.(4)miR-148a-3p does not affect the transcriptional activity of TFEB: The results of Chromatin Immunoprecipitation(ChIP)assays showed that the overexpression of miR-148-3p had no significant effect on the aggregation of TFEB or RNA polymerase II at the promoters of lysosomal genes,which indicating that miR-148a-3p does not affect the transcriptional activity of TFEB.(5)miR-148a-3p does not affect the expression of TFEB: Firstly,we predicted the sequence matching between the miR-148a-3p and the mRNA of TFEB through the TargetScan database and found that the seed sequence of miR-148a-3p was complementary to the 3'UTR of TFEB mRNA.Subsequently,luciferase assays showed that the overexpression of miR-148a-3p did not significantly affect the luciferase activity of the 3'UTR of TFEB mRNA.Whereas the database prediction results showed that there was no strong base complementarity between the promoter of the TFEB and miR-148a-3p either.Subsequent qPCR and western blot showed no significant effect of miR-148a-3p on TFEB mRNA as well as protein levels.To sum up,through database prediction and the experimental exploration of function and mechanism in vivo and in vitro,this study showed that miR-30b-5p was able to inhibit the transcriptional activity of TFEB in the nucleus by occupying its binding site and thus inhibit the expression of autophagy-lysosome related genes;whereas miR-148a-3p could inhibit the expression of CTSA a lysosomal gene at the posttranscriptional level by binding to its 3'UTR the seed sequence and thereby inhibit the lysosomal biogenesis and autophagy process.In the former,miRNA functions through a novel mechanism,while the latter is through the classical mechanism of miRNA.This study suggested that miRNA could regulate the autophagy-lysosome pathway through multiple mechanisms,providing new ideas for elaborating novel mechanisms of miRNA and the treatment of autophagy related diseases.