Researches On Molecular Mechanisms Of Autophagy Dysfunction Induced By Silica Nanoparticles

Autophagy is a highly conserved,selective process of biodegradation in plants,animals and microbes(e.g.,epiphyte).Autophagy plays an important role in many physiological and pathological processes,such as nutritional deprivation,environmental stress,cancer and neurodegenerative diseases.Its main process starts from the autophagosome which have double membrane structure.Subsequently,autophagosomes and lysosomes fuse to autolysosomes.The cytoplasm contents are swallowed,and they are digested by lysosomal enzymes.The degradation products are eventually recovered and reused in the cytoplasm.Nanomaterials,it is a material which composed of basic components by nanoparticles or those with one-dimensional dimensions between 1 and 100 nm.Recent studies have shown that a variety of nanomaterials could induce autophagy in cells,such as quantum dots,gold nanoparticles,carbon nanotubes,metal oxide nanoparticles,nano-fullerenes,and so on.The mainstream viewpoint holds that the autophagy induced by nanoparticles is one of the main cytotoxic mechanism of nanomaterials.It limits the application of nanomaterials in biomedicine.At present,the research mainly focuses on the discovery of nanomaterials which could induce autophagy activation,the design and optimization of nanomaterials which could regulate autophagy.It is very important to study the deep molecular mechanism of nanoparticles on autophagy activation.Protein kinases are extensively involved in the process of autophagy by phosphorylation of ATG and autophagy-related regulatory proteins.Whether protein kinases play an important role in nanoparticle induced autophagy is unclear.In this research,the study selected Si NPs-induced autophagy as the research object.The NRK cell lines were treated with the Si NPs.Combining the next-generation sequencing technology,the identification technology of proteome and phosphoproteome,bioinformatics prediction method,molecular cell experiment and biochemical experiment.This study quantified transcriptomes,proteomes and phosphoproteomes,design and establish the prediction method for kinase which are involved in the regulation of autophagy activation.The study predicted and verified two new kinases.The molecular mechanism of the kinases which were involved in the regulation of Si NPs-induced autophagy were revealed.Our study provides valuable reference information for the application and optimization of nanomaterials.(1)This study explored the physicochemical properties of three kinds of Si NPs,and found that all sizes of Si NPs have good dispersion in the aqueous phase,and showed good stability in the near physiological environment.This result reveals the actual state of nanoparticles when they contacted with NRK cells.It is important to explain the mechanism of Si NPs induced autophagy.Then the NRK-GFP-LC3 cells were treated with different concentrations of different size of Si NPs.With the help of confocal microscopy,the study observed that three kinds of Si NPs could increase the numbers of GFP-LC3puncta,and the higher concentration treatment had a stronger inductive effect.After NRK cells were treated with different size of Si NPs,the study detected the protein level of LC3-II by western blot.The study found that 16 nm Si NPs could significantly increase the protein level of LC3-II.By means of TEM and the images of co-location of r Si NPs with NRK-GFP-LC3 cells,the study found that Si NPs were swallowed by autophagosomes,then autophagosomes fused with lysosomes.Therefore,the study has a conclusion that Si NPs could induce autophagy and promote the formation of autophagosomes,and the effect of autophagy by Si NPs is characterized by size and concentration dependence.(2)NRK cells were treated with Si NPs,the protein level of SQSTM1 was measured by western blot.The study found that the protein level of SQSTM1 was significantly increase,it means that Si NPs could block the autophagy flux.This result was further verified by the experiment of m RFP-GFP-LC3 fluorescence indicator.The study added autophagy inhibitors 3-MA and CQ to the experiment of Si NPs induced autophagy,detected the protein levels of LC3-II and SQSTM1,and discuss the effect of 3-MA and CQ on autophagy flux.Consider all the results,the study found that the high concentration of Si NPs could effectively block autophagy flux and promote autophagosomes accumulation by inhibit lysosomes degradation.(3)In our opinion,if a protein kinase could regulate autophagy,the level of transcriptional expression,protein expression,kinase phosphorylation and kinase activity may have a change.So,NRK cells were treated with the 16 nm of Si NPs by time gradient,and identified gene transcription level,protein expression and phosphorylation sites by the next-generation sequencing technology,the identification technology of proteome and phosphoproteome,respectively.Then,based on the integration of multiple omics data,the study developed a new algorithm of computational prediction of master autophagy-regulating kinases.This study considered the change of kinase activity in the process of autophagy and the biological function of known kinase,predict 21 potential kinase which could regulate Si NPs-induced autophagy.(4)Experimental verification was carried out on these predicted kinases.The study used si RNA library knock down 21 potential kinase gene,detected the protein levels of LC3-II by western blot.The study found the protein and phosphorylation level of CDK4and CDK7 are significantly changed in Si NPs-induced autophagy,CDK4 and CDK7 may participate in the regulation of Si NPs induced autophagy.Knock down CDK4 and CDK7kinases in NRK-GFP-LC3 cells,then cells were treated with Si NPs and r Si NPs,the study found that the numbers of GFP-LC3 puncta in cells were significantly reduced.The study also further verified the above results in He La and Hep G2 human cancer cell lines.The autophagy inhibitor CQ was added into the knockdown experiment,the results showed that the functions of CDK4 and CDK7 mainly occurred in the autophagy activation rather than autophagy flux.Moreover,the CDK4 specific inhibitor ON123300,the CDK7 specific inhibitor THZ1 and the autophagy inhibitor CQ were added into Si NPs-induced autophagy experiment,the results showed that the inhibition of CDK4 and CDK7 activity can inhibit the expression level of LC3-II and the accumulation of GFP-LC3 puncta.These results suggest that CDK4 and CDK7 are important kinases which involved in autophagy activation induced by Si NPs.Taking all the result into consideration,the study concludes that the protein kinases CDK4 and CDK7 are important kinases involved in regulate Si NPs-induced autophagy activation.(5)In addition,by exploring the effects of autophagy activators etoposide,H₂O₂ and tunicamycin on autophagy,the study found that various stress pressures which caused by Si NPs may contribute to the activation of CDK4 and CDK7.In general,this study selected Si NPs-induced autophagy as the research object.The NRK cell lines were selected as the cell model.Combining the next-generation sequencing technology,the identification technology of proteome and phosphoproteome,bioinformatics methods,molecular cell experiment and biochemical experiment,the study identified and quantified transcriptomes,proteomes and phosphoproteomes,designed a new algorithm of computational prediction of master autophagy-regulating kinases,predicted and identified important kinases in the process of Si NPs-induced autophagy activation,and revealed the molecular mechanism of phosphorylation in this process.Our study provides an important reference for the application and optimization of nanomaterials.