Studies On CDK1/Cyclin B1-mediated UBQLN4 Phosphorylation In Mitosis And The Role Of LncRNA PRR34-AS1 In Gastric Cancer

The core of cell life cycle is that a single parent cell completes the genome replication and even distribution,and finally produces two identical progeny cells.The cell cycle is a cycle that successfully completes the events required for cell reproduction.CDK1 and PLK1,as classic cell cycle kinases,occupy the core position in the entire cell cycle regulation process.Protein phosphorylation and ubiquitination modification are two common types of post-translational covalent modifications(PTMs)in eukaryotic organisms,and run through the cell cycle.The Ubiquitin-proteasome system(UPS)is a multi-step reaction process that degrades unwanted proteins in a highly specific manner in eukaryotic organisms,and regulates the protein levels and activities,as well as cell cycle and cell proliferation.UBQLN4,also known as AlUp,is a member of the family of ubiquitin-binding proteins(Ubqlns)family.Current studies have found that UBQLN4 can act as shuttle transporters to transfer failed proteins from mitochondria and endoplasmic reticulum to the proteasome for degradation in time,and maintain protein homeostasis in the cytoplasm.UBQLN4 is highly expressed in many aggressive tumors,and its overexpression is positively correlated with the disease-free survival rate of HCC patients.UBQLN4 functions as a tumor suppressor by p53-dependent and p53-independent manner in gastric cancer.At present,there are few studies on UBQLN4 in cell cycle regulation.Bioinformatics analysis showed that UBQLN4 was differentially expressed in a variety of human tumors,and there was copy number amplification.Its DNA amplification status in a variety of tumor tissues was significantly positively correlated with the increase in mRN A levels,and had different prognostic values in different tumors.In addition,it was found that UBQLN4 and multiple cell cycle regulatory proteins showed a highly correlated expression in a variety of tumors.Subsequently,we observed that the cellular localization of exogenous UBQLN4 was cycle-related through cellular immunofluorescence combined with laser confocal.The localization between the endogenous and exogenous UBQLN4 protein was not completely consistent.The endogenous UBQLN4 was mainly located in the nucleus,and the cytoplasmic localization signal of exogenous UBQLN4 was significantly enhanced.The exogenous UBQLN4 and PLK1 could co-localize in the centrosome at the metamitosis;at the end of mitosis,they no longer co-localized.Further studies on UBQLN4 verified that the phosphorylation of UBQLN4 at the serine/threonine sites during mitosis(M phase)was not tumor-specific.By constructing a series of site-directed mutants,we have clarified that the phosphorylation sites of UBQLN4 in M phase were 12 serine and threonine residues in the 133-313 amino acid region.The phosphorylation of UBQLN4 by the CDK1/Cyclin B1 complex depended on the presence of these sites.The mutant mutated all these sites were called phosphorylation sites mutant,namely PSM.Co-IP assay showed that the interactions of PSM with CDK1 and PLK1 were not completely dependent on the phosphorylation of UBQLN4.We used cell cycle synchronization to arrest cells in different phases and found that UBQLN4 phosphorylation can inhibit the expression level of PLK1 protein during mitosis.The protein half-life detection and ubiquitination experiment revealed that PSM inhibited ubiquitination and degradation of PLK1.Hence,UBQLN4 regulated the ubiquitination and degradation of PLK1 in M phase depending on its phosphorylation.Moreover,we combined flow cytometry,laser confocal microscopy and time-lapse dynamic live cell monitoring system to observe the changes in cell division and mitosis of UBQLN4 and PSM.The results indicated that UBQLN4 phosphorylation significantly prolonged the duration of mitosis,especially metaphase.Its phosphorylation can also reduce abnormal nuclear morphology during cell division,and leaded to a significant reduction in abnormal mitotic phase of cells.Meanwhile,some of our studies showed that UBQLN4 phosphorylation was also involved in tumorigenesis and development.Endogenous knockout of UBQLN4 may activate different protein regulatory pathways in different cells.In short,we have elucidated the mechanism by which CDK1/Cyclin B1-mediated UBQLN4 phosphorylation in mitosis regulates PLK1 degradation and the mitotic process.We are the first to prove that UBQLN4 phosphorylation can promote the degradation of PLK1 through the ubiquitin-proteasome pathway,prolong the duration of mitosis and maintain genome stability.These studies reveal that UBQLN4 is a key factor regulating the cell cycle,and clarify the functional positioning of UBQLN4 and its phosphorylation in the cell cycle regulatory network,which is of great significance for the subsequent functional research of UBQLN4.At present,gastric cancer(GC)is still the major cancer burden in China.LncRNA PRR34-AS1 belongs to the antisense LncRNA.There are few studies on it at present,and its expression and biological function in gastric cancer are not yet known.This study aimed to explore the role of PRR34-AS1 in GC.We detected the expression level of PRR34-AS1 in multiple GC serum samples and multiple pairs of GC tissues by qPCR,and found that its expression was low.Subsequent chi-square analysis showed that the expression of PRR34-AS1 in GC serum samples was not significantly correlated with multiple clinical characteristics of tumor patients,but was only significantly correlated with the clinical stage,while there was no significant correlation in GC tissues.In addition,ROC curve analysis showed that the AUC of PRR34-AS1 was 0.709,with a 95%confidence interval of 0.611-0.807.These results indicate that PRR34-AS1 has certain diagnostic efficacy in gastric cancer and can be used as a potential serum diagnostic marker for GC.Next,we verified that PRR34-AS1 was low expressed in a variety of GC cells.The full-length sequence of PRR34-AS1 was amplified by RACE technology,and GC cell lines overexpressing PRR34-AS1 were constructed.Subsequent studies found that PRR34-AS1 showed obvious anti-tumor effect in GC cells,and its overexpression can significantly inhibit the proliferation,plate colony formation,cell migration and tumorigenesis in nude mice.In addition,we also explored the relationship between PRR34-AS1 and the drug resistance of GC cells,and found that after overexpression of PRR34-AS1,the survival rate of AGS cells treated with cisplatin for 48 h was significantly increased,and the ability of the cells to resist cisplatin was enhanced.In HGC-27 cells treated with 5-Fu for 24 h and 48 h,the survival rate of PRR34-AS1 overexpression group was significantly lower than that of control group,and its drug sensitivity was significantly improved.The subcellular of LncRNA is crucial to its function.We have confirmed by RNA-FISH and nuclear and cytoplasmic component separation that PRR34-AS1 was mainly located in the nucleus in GC cells,partly concentrated in the nuclear membrane and rarely distributed in the cytoplasm.We performed transcriptome sequencing on the control group and overexpressing PRR34-AS1 group of GC cells AGS and HGC-27.GO analysis showed that the differential genes of the two groups were involved in biological processes such as angiogenesis and extracellular matrix organization.In terms of biological function,they all showed ATP binding.KEGG pathway analysis showed that the differential genes in the two groups of cells were clustered in the PI3K-Akt signaling pathway.Through the prediction analysis of transcription factors and their target genes,it was found that the main differential transcription factors in the two groups of cells were distributed in the ETS and TF-bZIP families.In brief,this study suggests that PRR34-AS1 is low expressed in GC serum and tissues and can be used as a potential serum diagnostic marker for GC.PRR34-AS1 overexpression can inhibit the malignant proliferation of gastric cancer,which is related to the drug resistance of GC cells.The above research provides a certain foundation and idea for the study of the role and mechanism of PRR34-AS1 in GC,and provides a new direction for the precise treatment and personalized treatment of gastric cancer.