Research On The Effects And Mechanisms Of Doramectin Inducing Necroptosis And Uchl1 In Glioma Cells

Glioma is an extremely aggressive primary central nervous system tumor.Due to it’s invasiveness and drug resistance,traditional treatment methods and drugs cannot kill the tumor,causing great pressure and burden to patients,families and society.Therefore,it is of great significance to create new therapeutic means or develop new effective chemotherapy drugs for the treatment of glioma.Doramectin,a derivative of avermectin,belongs to macrolide class of antiparasitic drugs,which is widely used in animal husbandry to inhibit parasites in vivo and in vitro.Compared with other macrolide antibiotics,it has the advantages of longer half-life and faster absorption.In previous studies,we have demonstrated that Doramectin can promote apoptosis,autophagy,inhibition,migration and invasion of glioma cells both in vitro and in vivo.Therefore,doramectin may be a potential new anticancer drug.With the deepening of research,researchers have found that the anti-apoptosis of glioma has become a major problem hindering the treatment of glioma,and necroptosis,as a new programmed death,can well solve this problem.However,the effect of doramectin on necroptosis of glioma cells has not been reported.Therefore,this study first explored the influence of Doramectin on the necroptosis of glioma,then searched for potential interacting proteins that affect the necroptosis process,and finally explored the function of this protein in glioma.The results of this experiment are as follows:(1)Doramectin induced mitochondrial damage of glioma C6 cells to produce ROS,leading to necroptosisThrough the analysis of GO and KEGG enrichment in the RNA sequencing results of glioma C6 cells treated with doramectin,it was found that differential genes were enriched in ubiquitin proteolysis,necroptosis and other aspects.In addition,the signal pathway map showed that doramectin could promote necroptosis induced by RIPK1/RIPK3/MLKL pathway.According to the observation under inverted microscope,glioma C6 cells showed obvious and rapid death with the increase of drug concentration.Second,the lactate dehydrogenase test showed that this mode of death caused the leakage of cell contents.Flow cytometry showed that 40 μM doramectin induced necrosis.Subsequently,transmission electron microscopy,scanning electron microscopy and fluorescence microscopy demonstrated that the death of glioma cells induced by doramectin had the morphological characteristics of early apoptotic chromatin marginalization and necrotic cell membrane swelling and rupture.The results of Western Blot and RT-q PCR further proved that doramectin induced cell necrosis was necroptosis.However,at low concentrations,the proteins and indicators related to necroptosis were not significant,so the necroptosis may not be directly induced.Secondly,according to the addition of necroptosis inhibitor Nec-1 by flow cytometry and Western Blot,the results further proved that necroptosis was not directly induced by drugs.Combined with the existing literature and sequencing results,it is speculated that mitochondria may be the "culprit" of necroptosis.According to the results of DCFH-DA probe detection and JC-1 staining,doramectin induces mitochondrial membrane potential decline and production of reactive oxygen species in a dose-dependent manner,and the experimental results after the addition of reactive oxygen scavenger NAC showed that the proportion of necroptosis decreased significantly.In conclusion,ROS plays a major role in the necroptosis induced by doramectin.(2)RIPK3 and Uchl1 interact with each other at the protein levelFirstly,RIPK3 protein was enriched by immunocoprecipitation.Mass spectrometry showed the presence of Uchl1.Subsequent laser confocal microscopy showed that Uchl1 was mainly expressed in the nucleus,while RIPK3 was mainly expressed in the nuclear membrane and cytoplasm.In addition,the interaction between RIPK3 and Uchl1 in cells was demonstrated by proximity linking assay,and Western Blot assay confirmed that Uchl1 could remove single ubiquitin molecules on RIPK3.(3)Silencing Uchl1 inhibited tumor development both in vitro and in vivoUchl1 was silenced using Sh RNA silencing vectors.Subsequently,MTT assay and plate cloning assay showed that the proliferation ability of cells decreased significantly after silencing Uchl1.The cell adhesion experiment showed that the adhesion ability of cells was inhibited after silencing Uchl1.Scratch healing assay and Transwell chamber assay showed that silencing Uchl1 inhibited C6 cell migration and invasion.Flow cytometry showed that silencing Uchl1 result G2/M phase cycle arrest in cells.In addition,the silencing of Uchl1 reduced the proportion of necroptosis induced by TBZ.In vivo experiments showed that silencing Uchl1 was beneficial to control the development of intracranial tumors.In summary,we found that doramectin can significantly induce mitochondrial damage and promote necroptosis in vitro.Meanwhile,RIPK3 and Uchl1 interact with each other.In addition,silencing Uchl1 can significantly inhibit the occurrence and development of tumors both in vitro and in vivo.In conclusion,doramectin may become the next potentially effective chemotherapy drug in the treatment of glioma,and Uchl1 may become a potential target for the treatment of glioma.