| 1.Backgroud:Bladder cancer(BC)is the tenth most common malignancy worldwide.90%of bladder cancers are urothelial carcinoma,also known as transitional cell carcinoma.Squamous and adenocarcinoma,although less common(5%and 2%respectively),are found to be more advanced and have a higher mortality rate.There were an estimated 573,275 new cases and 212,536 deaths globally in 2020.There were an estimated 81,400 new cases of bladder cancer and 17,980 bladder cancer deaths in the United States in 2020.In China,the incidence and mortality of bladder cancer have also increased significantly in the past decade.Bladder cancer is more common in men and is thought to be related to smoking and occupational exposure.Bladder cancer can be classified as either nonmuscle-invasive bladder cancer(NMIBC)or muscle-invasive bladder cancer(MIBC).NMIBC is found in 70%of patients who are initially diagnosed with bladder cancer.Patients with NMIBC were treated with bladdersparing strategies,such as transurethral resection of bladder tumor(TURBT),followed by intravesical immunotherapy or chemotherapy;Patients with MIBC received neoadjuvant chemotherapy and surgical resection of the bladder.Immunotherapy based on immune checkpoint has also achieved good results.With standard treatment,the 5-year survival rate of NMIBC is 90%.However,about 15-20%of NMIBC,especially those with Tis or advanced T1,can progress to MIBC.About 10-34%of patients diagnosed with stage T1 bladder cancer die within 5 years.However,the 5year survival rate of MIBC is only about 60-70%,the 5-year survival rate of pT2N0 patients is only 60-70%,and that of PT3-4AN0 patients is only 40-50%.Even with radical cystectomy and pelvic lymph node dissection for MIBC,50%of patients still end up with distant metastasis.In recent years,a large number of biomarkers have been developed for the diagnosis,monitoring and prognosis prediction of bladder cancer,but up to now,no biomarker similar to the role of PSA in prostate diagnosis and monitoring can be applied to bladder cancer.Glycosylation is an important form of post-translation modification(PTM)of protein,which is the process of glycan linking to peptide chain under the action of glycosyltransferase.It can occur in all organisms,and has a great influence on the function of protein,and plays an important role in the malignant transformation and metastasis of tumor.The two main types of protein glycosylation are N-glycosylation and O-glycosylation.N-glycosylation connects nitrogen atoms on the side chain of asparagine to N-acetyl glucosamine(GlcNAc)in the endoplasmic reticulum.N-glycan is further processed and differentiated in golgi apparatus.O-glycosylation can link the oxygen atom on the side arm of threonine or serine to N-acetyl glucosamine(GalNAc).O-glycans can be synthesized in the endoplasmic reticulum,golgi apparatus,and even in the cytoplasm.Abnormal glycosylation is a hallmark of cancer.Many tumor-associated glycans,such as CA19-9,AFP,CEA and PSA,are abnormally expressed in tumors and play an important role in tumor progression and metastasis.They can be used as markers for diagnosis,monitoring and prognosis.Compared with normal tissue,tumor cells often show abnormal glycation patterns and new tumorspecific antigens.Many highly glycosylated proteins,such as adhesive proteins or proteases,play important roles in cancer metastasis.Hypolycosylation can lead to protein misfolding and endoplasmic reticulum stress(ERS),which is also closely related to the development and prognosis of cancer.Ribophorin II(RPN2)is a highly conserved glycoprotein that exists only in the rough endoplasmic reticulum and is a subunit of the Oligo-Saccharyl-transferase complex(OST).It can promote the binding of oligosaccharides to the common n-X-S/T motif of asparagine residues of polypeptide chain and participate in the Nglycosylation process of proteins.As a subunit of OST,abnormal expression of RPN2 may lead to abnormal glycosylation of proteins,thus affecting tumor progression.Nglycosylation has been shown to enhance the stability of EGFR and PD-L1,inhibit their degradation,promote tumor cell proliferation and mediate immune escape.RPN2 has been found to be involved in glycosylation of tumor cell surface receptors or immune checkpoints.For example,RPN2 can participate in the glycosylation of CD63 and Pglycoprotein on the surface of breast cancer,promote the progression of breast cancer and enhance its drug resistance.In addition to participating in protein glycosylation,RPN2 has been reported to promote tumor progression through other mechanisms.RPN2 can directly bind gSK-3β in glioma,inhibit the degradation of βcatenin and other substrates by GSK-3β in Wnt signaling pathway,promote the progression of glioma and enhance its drug resistance.RPN2 also inhibits autophagy,promotes MMP-9 expression,and promotes hepatocellular carcinoma proliferation,migration,invasion and epithelial mesenchymal transformation(EMT)through phosphorylation of STAT3 and NF-κB pathways.In this study,bioinformatic analysis revealed that RPN2 was highly expressed in bladder cancer tissues and differently expressed in different subgroups of bladder cancer,and predicted unfavorable outcome of bladder cancer.The different expression of RPN2 in different subgroups was confirmed by immunohistochemistry in clinical tissue samples.We also explored the effects of RPN2 on the biological functions of bladder cancer cells and the mechanism through which RPN2 was regulated and facilited the progression of bladder cancer,providing a new potential target for the diagnosis,monitoring and treatment of bladder cancer.2.Objective1)To investigate the expression level of RPN2 in bladder cancer tissues,the expression differences in various subgroups of bladder cancer and the effects on the prognosis of bladder cancer patients.2)To investigate the effects of RPN2 on biological functions such as proliferation,migration,invasion and EMT of bladder cancer cells.3)To investigate the effects of RPN2 on EGFR and PD-L1 glycosylation and immune cell function in bladder cancer cells.4)To explore the mechanisms by which RPN2 expression is regulated by IncRNA LUC AT1/miR-181 c-5p axis to promote the progression of bladder cancer,providing potential targets for the diagnosis and treatment of bladder cancer.3.Methods3.1 The expression of RPN2 in bladder cancer tissues and its prognostic value1)GSE3167 and GSE7476 datasets were downloaded from GEO database,and differential expression genes between bladder cancer and normal tissues were analyzed using the function limma in R.Next,the intersection of the differential genes obtained from the two datasets was obtained.Then,we performed Cox univariate and multivariate regression analysis to confirm RPN2 to be further investigated.2)Five bladder cancer datassets(GSE3167,GSE7476,GSE13507,GSE83586 and GSE120736)were obtained from GEO database and BLCA dataset from TCGA database to compare the expression differences of RPN2 between bladder cancer,normal tissue and bladder cancer subgroups.3)71 paraffin-embedded bladder cancer specimens,19 of which had matched normal para-cancer tissues,were obtained.We cut formalin-fixed paraffin-embedded tumor tissue blocks into 4μm sections and performed immunohistochemistry.The immunoreactivity of RPN2 in the mitochondria was scored according to the intensity and proportion of positively stained cells.We compared the immunoreactivity of RPN2 to evaluate the RPN2 expression in different subgroups.4)TCGA-BLCA bladder cancer patients were divided into RPN2 high expression group and low expression group according to the median expression level of RPN2.Age,gender,T stage,N stage,M stage,AJCC stage and grade were included in statistical analysis.Survival analysis,Cox univariate and multivariate analysis were performed for OS and DFS.3.2 RPN2 regulates the proliferation,migration,invasion and EMT of bladder cancer1)Four bladder cancer cell lines(5637,T24,RT4 and J82)were used to extract proteins,and Western blottingting was used to evaluate the expression of RPN2 in the four bladder cancer cell lines.2)siRPN2 was designed,synthesized and transfected into bladder cancer cells T24 and 5637.Western blottingting was used to detect the efficiency of siRNA interference with RPN2 in bladder cancer cells.3)After knockdown of RPN2 in bladder cancer cells T24 and 5637,ccK-8 assay,clone formation assay,wound healing assay and transwell assay were used to evaluate the proliferation,migration and invasion of bladder cancer cells.4)After knockdown of RPN2 in bladder cancer cells T24 and 5637,Western blotting was used to evaluate the expression changes of EMT-related markers in bladder cancer cells3.3 RPN2 can promote the glycosylation of EGFR and PD-L1 of bladder cancer cells and inhibit T cell functions1)After knocking down RPN2 in bladder cancer cells T24 and 5637,Western blotting was used to evaluate the changes of EGFR and PD-L1 in bladder cancer cells.2)After knocking down RPN2 in bladder cancer cells T24 and 5637,Western blotting was used to evaluate the changes of PI3K,Akt,P-PI3K and P-Akt in bladder cancer cells.3)CD8+T cells were co-cultured with bladder cancer cells.After knocking down RPN2 in bladder cancer cells T24 and 5637,CD8+ T cells were co-cultured with bladder cancer cells,respectively.The concentrations of IL-2 and INF-y in the culture medium were tested using ELISA.3.4 RPN2 is regulated by lncRNA LUCATl/miR-181c-5p axis1)Design and synthesise miR-181c-5p inhibitors(anti-miR-181c-5p)and mimics(miR-181c-5p mimic).After transfection with anti-miR-181c-5p and miR-181c5p mimic,the expression level of miR-181c-5p was evaluated by RT-qPCR.2)After anti-miR-181c-5p and miR-181c-5p mimic were transfected into bladder cancer cells,the expression level of RPN2 mRNA in bladder cancer cells was evaluated by RT-qPCR.3)After anti-miR-181c-5p and miR-181c-5p mimic were transfected into bladder cancer cells,Western blotting was used to evaluate the expression level of RPN2 protein in bladder cancer cells.4)After knockdown of lncRNA LUCAT1 in bladder cancer cells,the expression level of miR-181c-5p was evaluated by RT-qPCR.5)After lncRNA LUCAT1 was knocked down in bladder cancer cells,the mRNA expression level of RPN2 was evaluated by RT-qPCR,and the protein expression level of RPN2 was detected by Western blotting.6)A rescue experiment was conducted.After lncRNA LUCAT1 was knocked down in bladder cancer cells,miR-181c-5p was also knocked down,and the protein expression of RPN2 in bladder cancer cells was detected by Western blotting.7)To verify the direct interaction of miR-181c-5p and lncRNA LUCAT1,lncRNA LUC AT1 wild-type and mutant plasmids(mutant 1 and mutant 2)were constructed.Bladder cancer cells were transfected with miR-181c-5p mimic and lncRNA LUCAT1 wild-type or mutant plasmids,then the luciferase activity was tested.8)To verify the direct interaction between miR-181c-5p and RPN2 mRNA,RPN2 mRNA plasmids containing 3’-UTR wild-type and mutants(mutants 1 and 2)were constructed.Bladder cancer cells were transfected with miR-181c-5p mimic and RPN2 mRNA plasmids of 3 ’-UTR wild-type or mutant(mutants 1 and 2),then the luciferase activity was tested.4.Results4.1 The expression of RPN2 in bladder cancer and its prognostic value1)Differentially high and low expression genes in bladder cancer were obtained from GSE3167 and GSE7476,respectively.Next,we intersect the high expression genes and low expression genes respectively.A total of 110 high expression genes and 213 low expression genes were obtained.Then,the bladder cancer patients in the TCGA-BLCA dataset were divided into the high expression group and the low expression group according to the median expression level of each gene.Combined with the clinical data,Cox univariate and multivariate regression analysis were performed to obtain 8 different genes with independent prognostic significance for OS.The survival analysis of OS and DFS between the RPN2 high and low expression groups were performed.and it was found that there were statistical differences in OS and DFS between the RPN2 high and low expression groups.RPN2 was identified as the gene to be further investigated.2)Subgroup analysis in GSE3167,GSE7476,GSE13507,GSE83586,GSE120736 and TCGA-BLCA showed that RPN2 was highly expressed in bladder cancer tissues compared with normal or para-cancer tissues.It is highly expressed in MIBC,G3 grade,high grade,AJCC Ⅲ/Ⅳ stage and recurrent bladder cancer tissues.3)In the clinical specimens of bladder cancer tissues,the expression of RPN2 protein was significantly higher than that in para-cancer tissues and paired para-cancer tissues,higher in MIBC than that in NMIBC,and higher in high grade cancer tissues than that in low grade cancer tissues.4)Spearman correlation analysis showed that the expression level of RPN2 was significantly positively correlated with the infiltration degree of B cells,CD8+T cells,macrophages,neutrophils and dendritic cells,and the expression levels of EGFR,PD-L1,AKT and mTOR.5)There were significant differences in OS and DFS of bladder cancer patients between high and low RPN2 expression.Cox univariate and multivariate regression analysis reveal that RPN2 was an independent prognostic factor for OS.4.2 RPN2 regulates the proliferation,migration,invasion and EMT of bladder cancer1)Among all bladder cancer cell lines,the expression level of RPN2 protein was higher in T24 and 5637 cell lines.2)siRNA of RPN2 was designed and synthesized.Compared with the control group,siRPN2 significantly reduced the protein expression level of RPN2 in bladder cancer cells T24 and 5637.3)RPN2 can promote the proliferation and clone formation of bladder cancer cells.CCK-8 showed that knockdown of RPN2 could significantly inhibit the proliferation of bladder cancer cells.Clone formation test indicated that knockdown of RPN2 significantly inhibit the clone formation of bladder cancer cells.4)RPN2 can promote the migration and invasion ability of bladder cancer cells.Wound healing test showed that knockdown of RPN2 could significantly inhibit the migration ability of bladder cancer cells.Transwell experiment showed that knockdown RPN2 significantly inhibited the migration and invasion ability of bladder cancer cells.5)RPN2 could affect the expression of EMT-related proteins.WB experiment showed that knockdown of RPN2 significantly down-regulated the expression of N-cadherin and Vimentin,and up-regulated the expression of E-cadherin.4.3 RPN2 can promote the glycosylation of EGFR and PD-L1 of bladder cancer cells and inhibit T cell functions1)After knockdown of RPN2,EGFR molecular weight decreased,electrophoresis speed increased in bladder cancer cells.2)After knockdown of RPN2,PI3KS Akt,p-PI3K and p-Akt were decreased,the values of p-PI3K/PI3K and p-Akt/Akt were significantly decreased in bladder cancer cells.3)After knockdown of RPN2,IL-2 and INF-γ concentrations in the medium of CD8+T cells co-cultured with bladder cancer cells were significantly increased.4.4 RPN2 is regulated by lncRNA LUCAT1/miR-181c-5p axis1)After transfection of anti-miR-181 c-5p in bladder cancer cells,the expression level of miR-181c-5p was significantly reduced.In contrast,miR-181c-5p expression was significantly increased after transfection with miR-181c-5p mimics in bladder cancer cells T24 and 5637.2)After knockdown of miR-181c-5p in bladder cancer cells,the expression level of RPN2 mRNA was significantly increased.After overexpression of miR-181c-5p in bladder cancer cells,the expression level of RPN2 mRNA was significantly decreased.3)After knockdown of miR-181c-5p in bladder cancer cells,the expression level of RPN2 protein was significantly increased.In contrast,overexpression of miR181 c-5p in bladder cancer cells significantly reduced the expression level of RPN2 protein.4)After knockdown of lncRNA LUCAT1 in bladder cancer cells,the expression level of miR-181 c-5p was significantly increased.5)After knockdown of IncRNA LUCAT1 in bladder cancer cells,the mRNA and protein expression levels of RPN2 were significantly increased.6)Rescue experiment found that after knockdown of IncRNA LUC AT1 in bladder cancer cells,the expression level of RPN2 protein was significantly decreased,and followed by knockdown of miR-181 c-5p,the expression level of RPN2 protein was significantly increased.7)The luciferase activity was significantly decreased in bladder cancer cells transfected with miR-181c-5p mimic and wild-type IncRNA LUC AT1.The luciferase activity was also decreased in T24 and 5637 bladder cancer cells transfected with miR-181c-5p mimic and mutant IncRNA LUCAT1,but the decrease degree was smaller than that in bladder cancer cells transfected with miR181 c-5p mimic and wild lncRNA LUCAT1.It was proved that Mir-181 C-5p could directly bind to IncRNA LUCAT1.8)Luciferase activity was significantly decreased in bladder cancer cells transfected with miR-181c-5p mimic and 3 ’-UTR wild-type RPN2 mRNA.The luciferase activity was also decreased in T24 and 5637 bladder cancer cells transfected with miR-181c-5p mimic and 3 ’-UTR mutant RPN2 mRNA,but the decrease degree was smaller than that in bladder cancer cells transfected with miR-181c-5p mimic and 3’-UTR wild-type RPN2 mRNA.It was proved that miR-181c-5p could directly bind to the 3’-UTR end of RPN2 mRNA..5.Conclusion1)RPN2,as an oncogene,is highly expressed in bladder cancer tissues,and its expression level is higher in MIBC,high stage,high grade,metastatic and recurrent tissues.The OS and DFS of patients between high and low expression groups are significantly different.RPN2 can be used as an independent prognostic factor of OS.2)Functional studies confirmed that RPN2 could promote the proliferation,migration and invasion of bladder cancer cells.3)RPN2 can promote the glycosylation of EGFR and PD-L1 in bladder cancer cells,activate PI3K/Akt pathway,and inhibit CD8+T cell functions.4)The expression level of RPN2 in bladder cancer can be regulated by lncRNA LUCATl/miR-181c-5p axis. |
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