Studies On Mitosis Regulation Of Ubqln4 And The Role Of Long Non-coding RNA SNHG6 In Gastric Cancer

Mitosis is a very complicated and orderly regulated process,through which the parental cells deliver the genetic information carried by parental cells to the daughter cells.There are a variety of protein post-translational modifications in eukaryotic cells,including ubiquitination,phosphorylation and dephosphorylation,glycosylation and deglycosylation.Post-translational modifications can be involved in almost every aspect of cellular activities by causing changes in the structure,activity,and cellular localization of the certain proteins.Among the many post-translational modifications,phosphorylation is one of the most widely studied.A series of phosphorylation and dephosphorylation events on cell cycle-related proteins constitute a phosphorylation network for cell cycle regulation.Ubqln4,also known as A1Up,is a member of the Ubiquilins(Ubqlns)protein family,which harbors two important domains:The Ubiquitin-like domain(UbL)can bind to the proteasome,and the ubiquitin-associate domain(UBA)can interact with mono-or polyubiquitin chains.By these two domains,the Ubqlns act as shuttle factors to transport specific ubiquitinated substrate proteins to the proteasome.Recent studies have revealed that Ubqln4 can be phosphorylated by ATM and remove the ubiquitinated MRE11 protein from damaged chromosomes,thereby participating in the early steps of homologous recombination-mediated DNA damage repair.At present,there are few studies on Ubqln4 gene function.In our previous studies,we have confirmed that Ubqln4 played an anti-cancer effect on gastric cancer(GC)cells by inhibiting proliferation,inducing cellular senescence and inducing cell cycle arrest.To clarify the mechanism of Ubqln4.we identified the potential interacting proteins of Ubqln4 in cells by immunoprecipitation-mass spectrometry.Functional annotation analysis of 329 proteins identified by mass spectrometry revealed that the potential interacting proteins of Ubqln4 were enriched in the ribosome pathway,systemic lupus erythematosus signaling pathway,proteasome pathway and endoplasmic reticulum protein processing signaling pathway.we further verified that Ubqln4 could negatively regulate RNF114,one of the E3 ligases of p21,and promoted p21 protein acceleration,which mediated the anti-cancer effect of Ubqln4 in GC cells.We found that Ubqln4 had effect on cell cycle of GC cells,but the specific mechanism has not been explored.Based on this,we separated the cells of each cycle phase by cell cycle synchronization and detected the change of Ubqln4 expression level.We found that the electrophoretic mobility of Ubqln4 during mitosis is lower than that of other cell cycle phases.We then purified the mitotic Ubqln4 protein by immunoprecipitation and detected the expression of total phosphorylated level of Ser/Thr residues using Western blot,the results showed that the decrease in electrophoretic mobility of Ubqln4 during mitosis was caused by its phosphorylation modification.Combining synchronization of cell cycle with Western blot,we confirmed that the phosphorylation of Ubqln4 appeared with the onset of mitosis and disappeared with the exit of the mitosis.Results of the kinase inhibitor screening and in vitro kinase assays demonstrated that phosphorylation of Ubqln4 during mitosis is dependent on the kinase CDK1-Cyclin B complex.We also found that Ubqln4 interacted with PLK1,one of the cell cycle kinases.Using protein half-life detection and ubiquitination experiments we revealed that Ubqln4 promoted the binding of PLK1 to ubiquitin,which was degraded by the proteasome pathway and the interaction of Ubqln4 with PLK1 was dependent on its UBA domain and STI1-2 domain.In order to find the phosphorylation site of Ubqln4,we utilized the cell cycle synchronization immunoprecipitation mass spectrometry process and verified that Ubqln4 was phosphorylated at multiple Ser/Thr sites between 133-160 amino residues.In order to clarify the effect of Ubqln4 on the cell cycle,we constructed the RFP-H2B-HeLa cell line as a tool to monitor the cell cycle progression using Time-Lapse live cells imaging assay,which suggested that Ubqln4 can prolong the mitosis phase and obstacle the exit of mitosis of HeLa cells.In summary,we first discovered the phosphorylation of Ubqln4 during mitosis and confirmed that the phosphorylation of Ubqln4 was dependent on CDK1 kinase.We also revealed the effect of Ubqln4 on the proteasome degradation pathway of PLK1.Our research lays a good foundation for revealing the biological role of Ubqln4 in cell cycle and has a certain guiding significance for the further mechanism study of Ubqln4.Gastric cancer is one of the most common cancers.In 2018,there are an estimated 1,030,000 new cases and 780,000 deaths of GC worldwide,accounting for 5.7%of new cancer cases and 8.2%of cancer deaths,respectively.The incidence rate and mortality rate rank sixth and fifth among all cancers,respectively.Nearly half of new cases and more than half of deaths occurred in Asia.Increasingly LncRNAs-related studies show that LncRNAs play an important role in the gene regulatory network of GC development,and LncRNA can participate in many processes such as tumor proliferation,invasion and metastasis.SNHG6,also known as U87HG,was first sequenced by leichang in hepatocellular cancer(HCC)tissues and found to be expressed higher in hepatocarcinoma tissues than that in adjacent normal tissues.The expression level of SNHG6 was closely related to clinical stages and portal vein tumor thrombosis of HCC patients.SNHG6 played an oncogenic role in the proliferation and drug resistance in HCC cells.However,the mechanism of SNHG6 in other tumors is not definitely clear.In this study,we first analyzed the data of the public database Oncomine and found that the expression level of SNHG6 in GC tissues is higher than that in normal gastric tissues.Furthermore,we used the clinical serum samples of GC patients to verify the expression level of SNHG6 and found that the expression level of SNHG6 in serum samples of GC patients was significantly higher than that in serum samples of normal volunteers.We examined the expression levels of SNHG6 in five GC cell lines and the gastric mucosal immortalized cell line GES-1.The results showed that SNHG6 also expressed higher in GC cells compared with the immortalized gastric mucosal cells.We selected GC cell lines(MGC-803 and MKN45)with higher expression of SNHG6 and GC cell line(SGC-7901)with lower expression of SNHG6 to separately establish the SNHG6-knockdown and SNHG6-overexpression GC cell lines in vitro by lentiviral infection.The effects of knockdown/overexpression of SNHG6 on proliferation and the colony formation compacity of GC cells were detected by CCK-8 and colony formation assays.The results showed that knockdown of SNHG6 significantly inhibited the proliferation and colony formation ability of GC cells,whereas the overexpression of SNHG6 promoted the proliferation and colony formation of GC cells.We further tested the tumorigenic ability of GC cells in vivo after knockdown of SNHG6 using subcutaneous tumor formation in nude mice.The results revealed that the tumorigenic ability of GC cells in nude mice was significantly inhibited after SNHG6 knockdown.SA β-gal staining showed that knockdown of SNHG6 induced senescence in GC cells.The results of qPCR and Western blot experiments confirmed that knockdown of SNHG6 promoted the expression of p21 both in mRNA and protein levels.We further found that knockdown of SNHG6 activated ERK1/2,JNK and p38 signaling pathways and inhibited EZH2 expression.EZH2 shows an inhibitory effect on the transcription of p21.Using inhibitors of ERK1/2,JNK and p38 pathway to treat GC cells,we found that the inhibition of JNK pathway almost blocked the accumulation of p21 protein induced by SNHG6 knockdown,the inhibition of p38 pathway had no effect on the accumulation of p21 triggered by SNHG6 knockdown,and blockade of the ERK1/2 pathway showed an opposite effect with the inhibition of the JNK pathway.We speculated that the activation of the JNK pathway triggered by SNHG6 may play the dominant role in the regulation of p21.We also confirmed that the inhibition of cell proliferation and induction of cellular senescence of GC cells caused by SNHG6 knockdown was largely dependent on the increase in p21 expression through rescue experiments,which means that p21 may be a key effector of SNHG6 in GC regulation.In conclusion,our study suggested that SNHG6 exerted a significant effect on the regulation of GC cells by affecting the proliferation and senescence.The mechanical studies demonstrated that the regulation of SNHG6 on p21 is to a large extent dependent on the activation of JNK pathway and EZH2 expression.