The Molecular Mechanism Study On USP11 Promotes Cell Proliferation Of Melanoma By Regulating NONO

USP11,which belongs Ubiquitin-Specific Proteases(USP)family of deubiquitinase,is a cysteine protease,and widely distributes in various tissues of the human body.The human USP11 protein has two isoforms,one is 963 Aa in length,the most common one is 920 Aa.USP11 contains a N-terminus DU domain,which consists of a DUSP(domain present in USPs)domain and a UBL(ubiquitin-like)domain,and a second UBL domain in the catalytic domain.C275(Cys,cysteine)and C283 are two key deubiquitinase active sites.There are a variety of proteins interacted with USP11,such as HPV-16E7,PML,E2F1,XIAP and p21.The ubiquitin chains on those interactors can be removed by USP11,thereby the interactors are more stable than before.USP11 plays a vital role in the occurrence and development of many diseases,including a variety of tumors.NONO protein is a multifunctional protein,which locates in the nucleus usually.It participates in a variety of cellular physiological activities,including but not limited to: activation of internal immune response,RNA nuclear retention,RNA shearing,transcription regulation,DNA uncoiling,DNA repair,cell proliferation regulation,circadian rhythm maintenance,paraspeckle formation of nuclear substructure,etc.NONO protein is abnormally expressed in various of tumors,such as renal cell carcinoma,lung cancer,breast cancer,leukemia,prostate cancer,colorectal cancer,etc.,and it plays important roles in tumor cell proliferation,metastasis,invasion and other biological processes.In melanoma,NONO protein is often highly expressed,which speeds up the progression of melanoma.In vitro studies showed that cell proliferation slowed down after the NONO gene was knockdowned,meanwhile,cell apoptosis increased,connexin-43 expression increased,and cell-to-cell communication was enhanced.In addition,in DNA damage repair in melanoma cells after exposure to UV,NONO/PSF complex plays an important role,and knockout of NONO will affect this process.Although Melanoma inhibitory activity(MIA)can regulate the NONO protein level at the transcription level,the m RNA and protein levels of NONO in melanoma cells are not correlated,which suggesting that there are regulatory mechanisms beyond the transcription level in the regulation of NONO.In order to further explore the function of USP11 and identify more USP11 substrates,we performed TAP/MS(tandem affinity purification/mass spectrometry)and found 70 proteins that may interact with USP11 in the USP11 precipitate,including NONO.Through literature mining,both USP11 and NONO can promote proliferation in melanoma.In order to confirm the interaction between NONO and USP11,and their relationship in promoting melanoma proliferation,we first used Co-IP,GST-Pulldown,and immunofluorescence to confirm solidly the direct interaction between USP11 and NONO in the nucleus of melanoma cells,and used a series of truncated USP11 proteins to explore the approximate segments of USP11 involved in the binding of USP11-NONO.Since NONO's m RNA and protein levels are not correlated,there should be other mechanisms to regulate NONO.Some studies have shown that NONO can be degraded through the ubiquitin-proteasome pathway,we then investigated the effect of USP11 on NONO protein in melanoma cells.The results show that overexpression of USP11 can increase the level of NONO protein in a dose-dependent manner,knocking down USP11 causes a decrease of NONO protein;Q-PCR results show that USP11's changes didn't affect NONO m RNA levels,suggesting that USP11 regulated NONO beyond the transcription level;USP11 regulates NONO via the ubiquitin-proteasome system,ubiquitination assay confirmed that USP11 can remove ubiquitin chains on NONO protein.In the end,we investigated the ubiquitination type of NONO,and the results showed that USP11 is mainly involved in K48-deubiquitination.Both USP11 and NONO have proliferation-promoting functions in melanoma,so,is there any connection between them? We first investigated the proliferation activity of USP11 in melanoma cells,the results showed that USP11 promoted proliferation.Next,we generated stable transgenic melanoma cell lines with USP11 knockdown and NONO re-introduction.The cell viabilities were down-regulated after the USP11's knockdown,up-regulated after the introduction of NONO.The CCK-8,colony formation assay,and In Vivo Tumorigenesis Study proved USP11 promote melanoma proliferation in a partially NONO-dependent way.Compared with normal skin tissues,the protein levels of both USP11 and NONO in clinical melanoma tissues were significantly up-regulated.Moreover,the level of USP11 protein and NONO protein in melanoma tissue showed a significant positive correlation,and a survival curve analysis showed that elevated USP11 protein levels were associated with poor prognosis.In summary,this project found that NONO protein is a new important substrate molecule of USP11 from melanoma cells,meanwhile,USP11 is the first deubiquitinase NONO protein;USP11 up-regulates the protein level of NONO via ubiquitin-proteasome system,thereby mediates the cell proliferation function of melanoma.Compared with normal skin tissue,the protein levels of USP11 and NONO are both significantly upregulated in clinical melanoma tissue,and they are positively correlated.The findings will deepen the view on mechanism of melanoma occurrence and development,and could provide new ideas for the diagnosis of melanoma and the development of new targeted drugs.