Ubiquitin Specific Protease USP39 Is Indispensable In The Regulation Of Inflammatory Factors In NF-?B/STAT3 Signaling Pathway

Ubiquitin Specific Peptidase 39(USP39)is a member of the ubiquitinase family.However,unlike other members,USP39 lost the ubiquitin proteasome activity.The reason is its catalytic role of three key amino acid residues(cysteine,histidine,aspartic acid/asparagine)respectively replaced by aspartic acid,serine,glutamic acid.Study found that USP39 mainly reflected the splicing of pre-mRNA.Besides,it also closely related to some biological functions in breast cancer,liver cancer and prostate cancer,especially in tumor cell proliferation,apoptosis,migration,and invasion,but researches about the molecular mechanism of such function were not deep enough,and there were no study reported the function of USP39 in inflammatory disease.In our study,we firstly used activators(TNF-?,LPS)and inhibitors(BAY11-7082)of NF-Kappa B signaling pathway to stimulate the cells,in order to activate or inhibit the NF-Kappa B pathway.After that,we found the NF-Kappa B signaling pathway had no effect on the spatial orientation of USP39,besides,the pathway had a contrary effect on the transcriptional expression and protein expression of USP39,so we guessed that there existed a negative feedback between USP39 and NF-Kappa B pathway to maintain the steady expression level of both USP39 and NF-Kappa B,or the protein degradation of USP39 was accelerated under the condition of activating NF-Kappa B pathway,or there were other signaling pathways could regulate the expression of USP39.In order to study whether there existed a negative feedback between USP39 and NF-Kappa B pathway to maintain the steady expression level of both USP39 and NF-Kappa B,we respectively overexpressed and knocked down the expression of USP39 in different cells to test the condition of NF-Kappa B signaling pathway.The results showed that USP39 could not activate the pathway.However,knockdown of USP39 could significantly raised the transcriptional level of STAT3.According to the above results,we further studied whether the knockdown and rescue of USP39 had consistent regulation on the expression of STAT3.And according to the structural features of USP39,we build different plasmids,which contains different domains of USP39,to study the regulation of different domains on the expression of STAT3 signaling pathway.After the research,it is found that USP39 can inhibit the transcriptional level and protein level of STAT3,and the USP domain of USP39 is crucial in the process of the transcriptional regulation.It is reported that STAT3 plays an important role in inducing and maintaining inflammatory microenvironment,and USP3 9 could regulate the expression of various inflammatory factors.Therefore,we overexpressed STAT3 in colon cancer cell line HCT116 and HCT8,and detected the expression of some inflammatory factors.The results showed that STAT3 played a pro-inflammatory role in colon cancer cell lines.Combine the fact that USP39 could negatively regulate the expression of STAT3,we further study whether USP39 can regulate the expression of inflammatory factors.The results showed that knockdown of USP39 in HCT116 cells could promote the mRNA expression of proinflammatory factors IL-18 and IL-32,at the same time significantly inhibit the transcription of anti-inflammatory factors IL-15.In addition,knockdown of USP39 in HT29 cells could promote the mRNA expression of proinflammatory factors IL-1??IL-6?IL-18 and IL-32,at the same time significantly inhibit the transcription of anti-inflammatory factors IL-15.To sum up,this paper has preliminarily confirmed that the NF-kappa B signaling pathway has no influence on the spatial orientation of USP39,besides,the pathway had a contrary effect on the transcriptional expression and protein expression of USP39.In addition,the results showed that USP39 could not activate the NF-kappa B signaling pathway,but USP39 could inhibit the transcriptional level and protein level of STAT3,and the USP domain of USP39 is crucial in the process of the transcriptional regulation.This study also found that STAT3 played a role in promoting inflammation in intestinal cancer cells,and it is preliminarily determined that USP39 played an anti-inflammatory role in colorectal cancer cell line HCT116 and HT29.