The Role And Mechanism Of Long Non-coding RNA CASC19 In Gastric Cancer

Background and purpose:Gastric cancer(GC)is one of the most common malignant tumors of the digestive tract in the world,and its morbidity and mortality are ranked fourth and fifth in global cancers,respectively.The morbidity and mortality of GC in China account for more than half of the world.In general,the occurrence of GC is the result of the interaction of genetic and environmental factors.At present,surgical resection is still the main method of GC treatment.In China,more than 80% of patients are usually diagnosed with advanced GC at the initial diagnosis,and the 5-year survival rate is less than 30%.Although great progress has been made in the treatment of GC,the survival rate of GC patients is still very low.Therefore,there is an urgent need to find new molecular biomarkers and specific therapeutic targets for GC.With the development of high-throughput sequencing technology,long non-coding RNA(lncRNA),which was previously considered to be "noise" and "junk" of transcription,has attracted the attention of many scientists.Studies have found that lncRNA is highly tissue-specific and is involved in a variety of biological process in tumor cells.In addition,lncRNAs can participate in the development and progression of tumors through various methods such as epigenetic modification,transcriptional regulation,and post-transcriptional regulation.Although lncRNA is still in the exploration stage in the study of GC,some lncRNA may play a key role in the pathological process of GC,which also provides new ideas for the diagnostic and treatment of GC.Therefore,lncRNA has great potential as a diagnosis biomarker and therapeutic target for GC.In this study,lncRNA cancer susceptibility candidate 19(CASC19)was selected for analysis.CASC19 is located in the 8q24.21 region of the chromosome known as the genetic desert.Currently,the role of CASC19 in GC has not been reported.We found that CASC19 was up-regulated in GC,and was significantly associated with infiltration depth,TNM staging,distant metastasis,and overall survival(OS)based on the Cancer Genome Atlas(TCGA)database.Then,we obtained the 717 bp full-length sequence of CASC19 in GC cells using the Rapid Amplification of cDNA ends(RACE)experiment,and confirmed that this is a new transcript by UCSC.We performed a series of experimental verifications on the role and regulatory mechanism of CASC19 in clinical samples,cell models and animal models,in order to provide reference for the diagnosis and treatment of GC.Materials and Methods:1.The RNA sequencing and clinical trait data of 375 GC patients were obtained through the TCGA database.We performed weighted gene co-expression network analysis(WGCNA)and differential gene expression analysis to screen lncRNAs related to GC progression and prognosis.Then,the relationship between target lncRNA expression and clinicopathological characteristics and prognosis was analyzed.Finally,Gene Set Enrichment Analysis(GSEA)was performed to explore lncRNA-related signaling pathways.2.We collected 80 pairs of fresh GC tissues and adjacent control tissues that were surgically resected from The 940 th Hospital of Joint Logistics Support Force of Chinese People’s Liberation Army and Lanzhou University First Hospital from September 1,2019 to January 30,2020.Real-time Quantitative PCR(qRT-PCR)was used to detect the expression level of CASC19 in GC tissues and adjacent control tissues,and the relationship between the expression of CASC19 and the clinicopathological parameters of GC patients was analyzed.The correlation between CASC19 and the downstream target gene NPM1 was analyzed.Receiver operator characteristic(ROC)curve was used to evaluate the significance of CASC19 in the diagnosis of GC.3.QRT-PCR was used to detect the expression level of CASC19 in human GC cell lines(AGS,BGC-823,MGC-803,HGC-27 and MKN-45)and normal gastric mucosal epithelial cell lines(GES-1).RACE experiment was used to identify the full-length sequence of CASC19 in GC cells.CASC19 shRNA and over-expressing lentivirus were prepared.The shRNA lentivirus infected MKN-45 and BGC-823 cells,and the over-expressing lentivirus infected HGC-27 and AGS cells.QRT-PCR was used to detect knockdown and overexpression efficiency.The effects of CASC19 on the proliferation of GC cells were examined using CCK-8 experiments,cell clone formation experiments,and EdU cell proliferation experiments.Cell scratch test,Transwell migration and invasion experiments were used to detect the effect of CASC19 on the migration and invasion ability of GC cells.Flow cytometry and Western Blot(WB)experiments were used to detect the effect of CASC19 on GC cell apoptosis.The effects of CASC19 on the expression of epithelial-mesenchymal transition(EMT)-related proteins E-cadherin,Vimentin and N-cadherin were measured by WB experiments.4.We constructed a stable transfected cell line by using Luciferase-tagged CASC19 shRNA lentivirus and MKN-45 cells.We selected 4-5 week old BALB/c-nu male nude mice for animal experiments.Ten nude mice were randomly divided into two groups: sh-CASC19 group and sh-NC group,5 in each group.A subcutaneous xenograft tumor model of nude mice was constructed by injecting stable transfected cell lines into the scapula of nude mice.The tumor volume was measured daily,and the tumor growth status was monitored using a small animal live imager.Nude mice were sacrificed for 4 weeks.Half of the tumor was fixed with 10% neutral formalin solution,and the other half was stored in a liquid nitrogen tank.QRT-PCR was used to detect the expression of HDAC1 and NPM1 mRNA in transplanted tumors.The expressions of Ki-67,PCNA,HDAC1 and NPM1 proteins in transplanted tumors were detected by IHC and WB experiments.5.We used gene chip technology to detect the expression of downstream functional genes after CASC19 knockdown and identify possible downstream target genes for CASC19.We perform classical pathway analysis,upstream regulatory analysis,disease and function analysis,regulatory effect analysis,and interaction network analysis through Ingenuity pathway analysis(IPA)software.Finally,30 differentially expressed genes(DEG)were selected for qRT-PCR verification.6.The nuclear cell separation experiment was used to determine the subcellular localization of CASC19 in GC cells.RNA pull-down experiment was used to detect proteins that interact with CASC19.Chromatin Isolation by RNA Purification(ChIRP)experiments were used to detect proteins and genes acting simultaneously with CASC19,and downstream proteins and genes were identified by mass spectrometry(MS)and sequencing(Seq),respectively.WB experiment was used to detect HDAC1 protein expression in RNA pull-down elution products.RNA-binding protein immunoprecipitation(RIP)experiment was used to reverse verify the interaction between HDAC1 protein and CASC19.Chromatin immunoprecipitation(ChIP)experiment of HDAC1 was used to detect genes interacting with HDAC1.ChIP-qPCR experiment was used to verify the binding of HDAC1 to the NPM1 promoter.The double luciferase reporter gene experiment was used to verify the binding of CASC19 and HDAC1 to the NPM1 promoter simultaneously.ChIP-qPCR experiment was used to verify the binding of MYC to the CASC19 promoter.Research results:1.CASC19 was identified as the core lncRNA by WGCNA method for subsequent analysis and verification.CASC19 was up-regulated in GC samples from the TCGA database,and its overexpression was significantly associated with T stage(P = 0.034),TNM stage(P = 0.022),and lymph node metastasis(P <0.001)in GC patients.Compared with GC patients with low CASC19 expression,GC patients with CASC19 overexpression had shorter OS(P = 0.015).Cox analysis revealed that CASC19 overexpression was an independent risk factor for OS in GC patients(HR = 1.524,95% CI = 1.003-2.316,P = 0.049).GSEA analysis showed that most of the genes downstream of changes caused by CASC19 overexpression were enriched in cancer-related and classical signaling pathways.2.CASC19 was significantly up-regulated in GC tissues,and its expression level was 4.2 times than that in adjacent control tissues.CASC19 expression was positively correlated with GC tumor size(P <0.001),depth of invasion(P = 0.011),TNM stage(P <0.001),and lymph node metastasis(P = 0.003).CASC19 binding protein HDAC1 was up-regulated in GC tissues,while CASC19’s downstream target gene NPM1 was down-regulated in GC tissues,and the expression of NPM1 was negatively correlated with CASC19(R =-0.361,P = 0.001).Compared with adjacent control tissues,HDAC1 protein was overexpressed in GC tissues,while NPM1 protein was underexpressed in GC tissues.The AUC of CASC19 in the ROC curve is 0.952(95% CI: 0.920-0.984,P?<?0.0001),suggesting that CASC19 has a better diagnostic effect on GC.3.CASC19 is upregulated in the human GC cell lines,which is present in GC cells at a length of 717 bp.CASC19 is considered not to have protein encoding capabilities through the UCSC website,CPAT tool,and the ORFfinder website.The expression of CASC19 in MKN-45 and BGC-823 cells was successfully down-regulated by shRNA lentivirus,and the expression of CASC19 in AGS and HGC-27 cells was successfully up-regulated by overexpression of lentivirus.CASC19 knockdown inhibited the proliferation,migration,and invasion of MKN-45 and BGC-823 cells,and promoted the apoptosis of MKN-45 and BGC-823 cells.CASC19 overexpression promoted the proliferation,migration and invasion of HGC-27 and AGS cells,and inhibited the apoptosis of HGC-27 and AGS cells.In addition,CASC19 knockdown inhibited the expression of anti-apoptotic proteins Bcl-2 and interstitial proteins Vimentin and N-cadherin in MKN-45 and BGC-823 cells,and promoted the expression of the apoptosis proteins Casepase-3,Casepase-7 and Casepase-9 and epithelial protein E-cadherin.CASC19 overexpression promoted the expression of Bcl-2,Vimentin and N-cadherin proteins in HGC-27 and AGS cells,and inhibited the expression of Casepase-3,Casepase-7,Casepase-9 and E-cadherin proteins.4.The volume and weight of subcutaneously transplanted tumors in the sh-CASC19 group were significantly smaller than those in the sh-NC group.HE staining revealed that the tumor cells in the sh-NC group showed increased nuclear volume,increased mitotic figures,and giant cell proliferation.The expression levels of Ki-67 and PCNA protein in sh-CASC19 group were significantly lower than those in sh-NC group.The expression levels of HDAC1 mRNA and protein in the transplanted tumors of sh-CASC19 group were not significantly different from those of sh-NC group.Compared with the sh-NC group,the expression levels of NPM1 mRNA and protein in the transplanted tumors of the sh-CASC19 group were significantly increased.5.CASC19 knockdown resulted in an increase of 1,433 DEGs and a decrease of 369 DEGs.IPA analysis showed that CASC19 may interact with THBS1 and TGFBR2,and then affect the proliferation and apoptosis of GC cells.Thirty DEGs were selected for qRT-PCR verification,which was consistent with the results of chip experiments.6.CASC19 is mainly located in the nucleus of GC cells,suggesting that CASC19 may play a role through epigenetic regulation.RNA pull-down and ChIRP-MS experiments identified 96 proteins that CASC19 might bind.Bioinformatics predicted a binding region between CASC19 and the histone modified protein HDAC1.WB and RIP experiments showed that HDAC1 can directly bind to CASC19,and there was no significant difference in HDAC1 mRNA and protein expression after up-or-down CASC19,which proved that HDAC1 is not regulated by CASC19.ChIRP-seq and ChDAC-seq experiments of HDAC1 identified 281 genes that HDAC1 and CASC19 may co-regulate on the genome.Bioinformatics analysis showed a peak-calling peak in the promoter region of the NPM1 gene,suggesting that CASC19 might bind to the NPM1 promoter.ChIP-qPCR experiment showed that CASC19 knockdown reduced the binding of HDAC1 to the NPM1 promoter.QRT-PCR and WB experiments demonstrated that NPM1 is regulated by CASC19 and HDAC1.The double luciferase reporter experiment confirmed that CASC19 inhibited NPM1 expression by recruiting HDAC1 to bind to the NPM1 promoter.In addition,ChIP-qPCR experiment showed that MYC can bind to the CASC19 promoter,and qRT-PCR experiment proved that MYC can increase the expression of CASC19.Conclusions:In this study,we found that lncRNA CASC19 located in the 8q24.21 region of chromosome is upregulated in GC and related to the progress and prognosis of GC by the TCGA database.CASC19 was found to have a length of 717 bp in GC cells by RACE.We performed a series of in vitro and in vivo validations of CASC19 in GC cell lines,GC clinical samples,and animal models,and demonstrated that CASC19 is an oncogene in GC.CASC19 is mainly distributed in the nucleus of GC cells.Mechanistically,CASC19 recruits HDAC1 to the promoter region of NPM1 to deacetylate the promoter region of NPM1,which inhibits the transcriptional activity of NPM1 and reduces the expression of NPM1.In addition,we also found that CASC19 is regulated upstream by the transcription factor MYC.Our study demonstrates a new mechanism by which CASC19 inhibits NPM1 expression through epigenetic regulation,and adds new evidence for a better understanding of the role and mechanism of lncRNA in GC.CASC19 is expected to become a potential diagnostic marker and therapeutic target for GC.