| Objectives:Gastric cancer(GC)is one of the most common malignancies with unfavourable prognosis,and there is still no ideal therapeutic target.Therefore,it is urgent to study the mechanism of the occurrence and development of GC.As one of the most common RNA modifications in nature,alternative polyadenylation(APA)is involved in a variety of biological processes.Numerous studies have shown that APA plays a crucial role in the progression of malignancies such as liver cancer,glioblastoma,cervical cancer,bladder cancer,breast cancer,and prostate cancer.Cleavage and polyadenylation specific factor 1(CPSF1),as a critical APA-related factor,is involved in the occurrence and development of many cancers.Nevertheless,the roles and underlying mechanisms of CPSF1 remain unclear in GC.Consequently,we performed the study to explore the biological role of CPSF1 and CPSF1-mediated mechanistic models in GC,in order to find therapeutic targets for GC and provide a theoretical basis for the clinical diagnosis and treatment of GC.Methods:1.Analyze the data in the Cancer Genome Atlas(TCGA)database and Gene Expression Omnibus(GEO)database by bioinformatics methods,and explore the expression of CPSF1 in GC and the relationship between the expression level of CPSF1 and the prognosis of GC patients;2.60 pairs of GC tissues and matched paracancer tissues were collected to verify the above bioinformatics conclusions by immunohistochemical assay;3.Clinical information and postoperative follow-up status were collected to explore the relationship between CPSF1 expression and clinicopathological parameters and prognosis;4.Western blot assay was performed to explore the expression of CPSF1 in GC cell lines,and appropriate cell lines were selected for functional experiments;5.Lentivirus was used to construct stable GC cell lines with low CPSF1 expression,and these stable GC cell lines with low CPSF1 expression were used for functional experiments in vitro(cell counting assay,CCK8 assay,clone formation assay,Transwell assay,wound healing assay,cell cycle assay and cell apoptosis assay)to explore the effects of CPSF1 knockdown on proliferation,metastasis,apoptosis and cycle of GC cells in vitro;6.CPSF1-overexpression plasmid was used to construct GC cell lines with CPSF1 overexpression,and these GC cell lines with CPSF1 overexpression were used for functional experiments in vitro(cell counting assay,CCK8 assay,clone formation assay and Transwell assay)to explore the effects of CPSF1 overexpression on proliferation and metastasis of GC cells in vitro;7.Stable GC cell lines with low CPSF1 expression were used for mouse xenograft assay(subcutaneous tumorigenicity assay,lung metastasis constructed by tail vein injection and in vivo imaging assay)to explore the effects of CPSF1 knockdown on the proliferation and metastasis of GC cells in vivo;8.Stable GC cell lines with low CPSF1 expression and control GC cells were sent to the company for RNA-seq,and the sequencing data were analyzed to screen out the downstream genes with both 3’UTR lengthening and down-regulated expression after CPSF1 knockdown;9.Western blot assay was performed to verify the protein level of NAD(P)dependent steroid dehydrogenase-like(NSDHL)after CPSF1 knockdown;10.The effect of CPSF1 knockdown on the 3’UTR length of NSDHL was verified by using the Integrative Genomics Viewer(IGV);11.Western blot assay was performed to explore the expression of NSDHL in GC cell lines,and appropriate cell lines were selected for functional experiments;12.Lentivirus was used to construct stable GC cell lines with low NSDHL expression,and these stable GC cell lines with low NSDHL expression were used for functional experiments in vitro(cell counting assay,CCK8 assay,clone formation assay and Transwell assay)to explore the effects of NSDHL knockdown on proliferation,metastasis of GC cells in vitro;13.Stable GC cell lines with low NSDHL expression were used for mouse xenograft assay(subcutaneous tumorigenicity assay,lung metastasis constructed by tail vein injection)to explore the effects of NSDHL knockdown on the proliferation and metastasis of GC cells in vivo;14.Rescue assay:Overexpressing NSDHL in stable GC cell lines with low NSDHL expression,and performing functional experiments in vitro(cell counting experiments,CCK8 assay,clone formation assay and Transwel assay)to explore whether NSDHL could reverse the phenotype induced by the CPSF 1 knockdown on GC cells.Results:1.Both bioinformatics analysis and immunohistochemical assay suggested that the expression level of CPSF 1 was higher in GC tissues compared to normal gastric mucosal tissues or paired adjacent nontumor tissues;2.Both bioinformatics analysis and the follow-up information of GC clinical samples all suggested that patients with higher CPSF1 expression had a worse prognosis;3.CPSF1 expression levels were closely associated with tumour size,TNM stage and lymph node metastasis,and patients with higher CPSF1 expression have worse clinicopathological parameters;4.Western blot assay suggested that the expression level of CPSF1 in GC cell lines was significantly higher than that in normal gastric cells,and the expression level of CPSF1 was the highest in AGS and MGC-803 cells;5.Functional experiments in vitro showed that CPSF1 knockdown dramatically inhibited GC cell proliferation,clonal formation,wound healing,migration and invasion,promoted apoptosis of GC cells,and inhibited cells into the division phase;6.Functional experiments in vitro showed that overexpression of CPSF1 promoted GC cell proliferation,clonal formation,migration and invasion;7.Xenograft assays showed that CPSF1 knockdown significantly inhibited tumorigenic ability and lung metastasis ability of GC cells in vivo;8.The results of RNA sequencing indicated that CPSF1 knockdown resulted in significant changes in the expression level and 3’UTR length of numerous downstream genes,including 12 downstream genes with both 3’UTR lengthening and down-regulated expression;9.Western blot assay suggested that the knockdown of CPSF1 resulted in the decrease of NSDHL protein expression level;10.Using IGV,we found that CPSF1 knockdown resulted in a significant reduction in the proximal poly(A)site usage in the 3’UTR region of NSDHL gene,while the usage proportion of distal APA was significantly higher than that of proximal APA,namely,the 3’UTR elongation of NSDHL;11.Western blot assay suggested that the expression level of NSDHL in GC cell lines was significantly higher than that in normal gastric cells,and the expression level of CPSF1 was the highest in AGS and MGC-803 cells;12.Functional experiments in vitro showed that NSDHL knockdown dramatically inhibited GC cell proliferation,clonal formation,migration and invasion;13.Xenograft assays showed that NSDHL knockdown significantly inhibited tumorigenic ability and lung metastasis ability of GC cells in vivo;14.Rescue assays showed that the overexpression of NSDHL could abrogate the decreases in the proliferation,clone formation,migration and invasion capacity of GC cells caused by CPSF1 knockdown.Conclusions:This study showed that both CPSF1 and NSDHL played a role in promoting the progression of GC,and CPSF1 was at least partly due to enhancing the expression of NSDHL through APA mechanism,thus promoting the progression of GC.In general,this study offered new insights for targeted therapy of GC. |
