The Study On Pancreatic Cancer Metastasis By The Regulation Of ZFAS1

Background: pancreatic cancer(PC)is a kind of digestive system tumor with a high degree of malignancy.The incidence of PC is concealed and the early symptoms are not obvious.Most of the patients are diagnosed as advanced stage.The prognosis of the patients is very poor,and the five-year survival rate is less than 8%.There are many limitations in the traditional treatment of pancreatic cancer.Early pancreatic cancer can benefit from surgery resection,but the recurrence rate and distant metastasis rate are very high.Most patients with advanced pancreatic cancer are not sensitive to radiotherapy and chemotherapy.In recent years,the new targeted therapy is becoming a research hotspot,and the metastasis and invasion of pancreatic cancer is an important factor affecting the prognosis of patients with pancreatic cancer.Therefore,it is of great significance to study the mechanism of metastasis of pancreatic cancer and explore the key target genes for targeted treatment of pancreatic cancer.In recent years,long non-coding RNA has been a research hotspot.It does not encode protein itself,but can regulate gene expression at multiple levels and plays an important role in the development of the body and cell differentiation.long non-coding RNA has many mechanisms,which can combine with specific proteins and change the activity and cell location of proteins;it can reconstruct chromatin or affect the upstream transcription of coding genes to regulate the downstream gene expression;some molecules,including long non-coding RNAs,have binding sites with micro RNA and play a role as mi RNA sponge in cells.The mechanism is called the competitive endogenous RNA(ce RNA)mechanism.The expression of ZFAS1(ZNFX1 antisense 1)is abnormal in various diseases including cardiovascular disease,inflammation and tumor,but there are few reports in pancreatic cancer.The expression level of ZFAS1 in pancreatic cancer,whether there is regulation of biological function of pancreatic cancer and its underlying mechanism are also not clear,which needs further study.Objective: predicted and verified the expression level of ZFAS1 in pancreatic cancer tissues and cells,analyzed the clinical correlation of ZFAS1,evaluated the value of ZFAS1 as a diagnostic index of pancreatic cancer,predicted and verified the biological function and potential mechanism of ZFAS1 regulating metastasis of pancreatic cancer.Methods: This study explored the role of ZFAS1 in pancreatic cancer from three levels:phenomenon,function and mechanism through human pancreatic cancer tissue chip,in vitro cell assay and in vivo animal model.In clinical samples,we first predicted the expression difference of ZFAS1 in pancreatic cancer through bioinformatics database,and then divided the whole data into two groups according to the expression value of ZFAS1.We carried out GSEA to predict the tumor-related biological function related to the high expression of ZFAS1.Then,the expression of ZFAS1 in human pancreatic cancer and adjacent tissues was detected by in situ hybridization,and the subcellular localization of ZFAS1 was determined,and the clinical correlation between ZFAS1 and pancreatic cancer was analyzed with related data.In vitro,we used transient transfection to knock down the expression of ZFAS1,RT-PCR was used to detect the transfection efficiency,and wound healing assay and transwell migration assay were used to detect whether ZFAS1 had influence on the metastasis of pancreatic cancer.Bioinformatics predicted the binding site of mi R-3924 binding to ZFAS1,by constructing stable ZFAS1-silenced pancreatic cancer cell line,the potential mechanism of regulating pancreatic cancer metastasis were validated through the corresponding dual luciferase report assay and the corresponding rescue assay of wound healing and transwell migration assay.After ZFAS1 silencing or mi R-3924 overexpression,the expression levels of FAK,RHOA and ROCK2,which is the direct targets of mi R-3924,were detected by western blot.In vivo,the tail vein metastasis model of nude mice was established.After killing of nude mice,the influence of ZFAS1 silencing on pancreatic cancer SW1990 cell line hepatopulmonary metastasis was verified by observing the number of hepatopulmonary metastasis colonies with HE stain.Results: included clinical tissue validation,in vitro validation and in vivo validation.Clinical tissue related validation results1.The results of differential expression analysis of standardized data sets in GEO database showed that the expression level of ZFAS1 in pancreatic cancer was significantly higher than that in adjacent pancreatic cancer;TCGA and ONCOMINE database showed the same results;TCGA database analysis showed that the high expression of ZFAS1 was related to drinking habits,sex and tumor stage.2.The results of in situ hybridization showed that the expression level of ZFAS1 in human pancreatic cancer was significantly higher than that in human pancreatic cancer,and ZFAS1 was distributed both in the cytoplasm and nucleus.3.The KM curve showed that the prognosis of patients with high expression of ZFAS1 was obviously poor;the high expression of ZFAS1 was not related to tumor grade(P=0.576),TNM stage(P=0.431),sex(P=0.794),age(P=0.798),tumor size(P=0.279),perineural invasion(P=1)and lymph node metastasis(P=0.285);the AUC of ROC curve was 0.747.4.The results of GSEA showed that the high expression of ZFAS1 in pancreatic cancer was related to focal adhesion pathway,extracellular matrix receptor interaction and m TOR pathway,focal adhesion pathway is the most relevant pathway.In vitro results1.The expression level of ZFAS1 in pancreatic cancer cell lines was also higher than that in normal pancreatic ductal epithelial cells.In transient transfection,si-ZFAS1 significantly reduced the expression level of ZFAS1 in pancreatic cancer cell lines BXPC-3 and SW1990 compared with si-NC.Transwell migration assay showed that ZFAS1 knockdown significantly reduced the number of cells moving from the upper chamber to the lower chamber in pancreatic cancer cell lines BXPC-3 and SW1990.Wound healing assay results showed that ZFAS1 knockdown significantly reduced the migration rate of BXPC-3 and SW1990 cell lines.2.Bioinformatics database prediction showed that mi R-3924 has binding site with ZFAS1;dual luciferase report assay showed that the ratio of firefly luciferase to renilla luciferase activity in wild-type luciferase plasmid group is significantly higher than in mutant luciferase plasmid group,suggesting the direct binding of mi R-3924 and ZFAS1;transwell migration assay showed that the over expression of mi R-3924 significantly reduced the size of pancreatic cancer cell lines BXPC-3 and SW1990.Wound healing assay showed that over expression of mi R-3924 significantly reduced the migration rate of pancreatic cancer cell lines BXPC-3 and SW1990.3.BXPC-3 and SW1990 cell lines with stable silencing of ZFAS1 were constructed by lentivirus and screened by puromycin.RT-PCR showed that ZFAS1 was stable silenced.And mi R-3924 inhibitor was transfected in BXPC-3 and SW1990 cell lines with stable silencing of ZFAS1,Transwell migration assay showed that the inhibition of mi R-3924 reversed the effect of ZFAS1 silence on the number of cells migrating from the upper chamber to the lower chamber;wound healing assay showed that the inhibition of mi R-3924 reversed the effect of ZFAS1 silence on cell mobility.4.Bioinformatics database predicted the binding sites of ROCK2 with mi R-3924.The dual luciferase report showed that the ratio of firefly luciferase to renilla luciferase activity in the wild-type luciferase plasmid group was significantly higher than that in the mutant luciferase plasmid group,suggesting the direct binding of mi R-3924 and ROCK2.Western blot showed that ZFAS1 silencing and mi R-3924 over-expression could both reduce the expression level of ROCK2,RHOA and FAK,which are the upstream genes of ROCK2 in focal adhesion pathway.In vivo results The results of HE staining showed that the stable silence of ZFAS1 significantly inhibited the number of liver metastasis colonies of pancreatic cancer in nude mice.Conclusion: 1.The expression of ZFAS1 in pancreatic cancer was significantly higher than that in adjacent pancreatic cancer.2.The high expression of ZFAS1 is related to the poor prognosis of pancreatic cancer.3.ZFAS1 promotes pancreatic cancer metastasis in vivo and in vitro.4.Mi R-3924 inhibited metastasis of pancreatic cancer in vitro.5.ZFAS1 regulates the metastasis of pancreatic cancer by competitive endogenous binding mi R-3924 and further affect the expression of focal adhesion pathway.