| PDAC(pancreatic ductal adenocarcinoma)is one of the most malignant types of cancer,the incidence of PDAC is increasing,however,the mechanism behind it is still unclear.ncRNA(Non coding RNA)is a class of RNAs without protein coding ability,nonetheless,more and more studies have found that ncRNA plays pivotal role in tumorigenesis.NcRNA could be divided into miRNA(microRNA)and lncRNA(long nocoding RNA)in terms of nucleotide size.In this study,we chose miR 212,as well as H19 to explore the role of them played in pancreatic cancer and mechanism.By using qRT PCR,Western Blot,Dual luciferase assay,CCK 8 assay,migration assay,and Transwell assay,we found that both miR 212 and H19 functioned as oncogene in PDAC.miR 212 is correlated with the TNM stage of PDAC,additionally,miR 212 can promote pancreatic cancer cell migration and invasion by regulating its target gene PTCH1 H19 is found to be related to metastasis of PDAC,furthermore,we demonstrated that H19 could promote pancreatic cancer cell migration and invasion at least partially by antagonizing let 7’s inhibition on its target gene HMGA2(High Mobility Group AT hook2)which mediated EMT(Epithelial Mesenchymal transition).These findings suggest that miR 212 and H19,as members of ncRNA play important role in PDAC and may serve as a prognostic biomarker as well as providing an attractive therapeutic target for PDAC.Section1: miR 212 promotes pancreatic cancer cell growth and invasion by targeting the Hedgehog signaling pathway receptor patched 1【Objective】 To explore the expression of miR 212 in PDAC(pancreatic ductal adenocarcinoma),and its role on pancreatic cancer cell function as well as the possible mechanism).【Methods】 Quantitative real time PCR(qRT PCR)was performed to detect the expression of miR 212 in PDAC tissues and pancreatic cancer cell lines.Next,pancreatic cancer cells were divided into three groups,miR 212 mimic、miR 212 inhibitor and negative control were transfected into each group,then CCK 8 assay was used to test the proliferation effect of miR 212 on pancreatic cancer cell,colony formation assay was performed to detect the effect of miR 212 on colony formation of pancreatic cancer cell.In addition,the wound healing assay as well as transwell assay were used to examine the influence of miR 212 on migration and invasion ability of pancreatic cancer cell.Furthermore,bioinformatic analysis tool was used to predict the potential target of miR 212,then dual luciferase activity assay,qRT PCR and Western Blot were performed to validate the predicted target gene.In addition,qRT PCR as well as Immunohistochemistry staining were used to explore its expression in PDAC tissues.Then we used TCGA data to analyse the effect of predicted target gene of miR 212 on prognosis of PDAC patients.Besides,we performed statistical method to speculate the correlation between miR 212 expression and its predicted target gene expression in PDAC tissues.Furthermore,miR 212 and predicted target gene were co tranfected into pancreatic cell to demonstrate whether miR 212 influence pancreatic cancer cell function by regulate its target gene.【Results】 miR 212 was up regulated in PDAC and pancreatic cancer cells compared with adjacent tissues and normal ductal cells.Using both gain of function and loss of function experiments,miR 212 was demonstrated to promote pancreatic cancer proliferation,colony formation,migration and invasion.Patched 1(PTCH1)was predicted as an candidate target gene of miR 212 by bioinformatic analysis tool,after that,the results of dual luciferase activity assay,qRT PCR and Western Blot validated that PTCH1 was a target gene of miR 212 in pancreatic cancer.In addition,PTCH1 was down regulated in PDAC and low expression level of PTCH1 is related with poor survival.Furthermore,the expression of miR 212 was negatively correlated with expression of PTCH1.At lastly,co transfetcion of miR 212 and PTCH1 into pancreatic cancer cell showed that PTCH1 can partly override miR 212’s effect on pancreatic cancer which means that miR 212 influence on pancreatic cancer at least partly by regulating its target gene PTCH1.【 Conclusions】 miR 212 was up regulated in PDAC,miR 212 can promote pancreatic cancer cell growth and invasion by targeting the patched 1.Section2: lnc RNA H19 promotes pancreatic cancer metastasis by derepressing let 7 ’s suppression on its target HMGA2 mediated EMT【Objective】 To explore the expression of H19 in PDAC,and its impact on the invasion and metastasis of pancreatic cancer【Methods】 qRT PCR was performed to detect the expression of H19 in PDAC tissues and pancreatic cancer cell lines compared with adjacent tissues and normal pancreatic ductal cells.PDAC tissues were divided into two groups based on whether metastasis happened.RTCA was used to identify the ability of migration and invasion of the three pancreatic cell lines,PANC 1,SW1990 and Bx PC 3,trying to figure out is there any relationship between the ability of migration as well as invasion and the expression of H19 in the three pancreatic cancer cell lines.In vitro study,we transfected pancreatic cells with H19 small interfering RNA(H19 siRNA)to knock down the expression of H19 in pancreatic cancer cells,and try to find out is there any difference in terms of migration and invasion by The wounding healing assay and Transwell assay.In addition,qRT PCR and Western Blot were performed to explore the effect of knock down of H19 expression on let 7,and its target gene HMGA2(High Mobility Group AT hook2)as well as well as EMT(epithelial mesenchymal transition)marked gene E cadherin,N cadherin and Vimenin. Furthermore,we co transfected pancreatic cancer cells with H19 siRNA and let 7 inhibitor to see whether H19 functioned on pancreatic cancer by antagonize let 7.【Results】 H19 was up regulated in PDAC tissues and pancreatic cancer cell lines,compared with those PDAC tissues without metastasis,the expression of H19 was even higher in those PDAC tissues with metastasis.RTCA showed that PANC 1 has the most ability of migration and invasion,Bx PC 3 has the least ability,and SW1990 between those two cell lines,interestingly,this trend was the same in terms of the expression of H19 in these three pancreatic cancer cell lines,which implicated that there is some correlation between the expression of H19 and the ability of migration and invasion of pancreatic cancer cells.Subsequently,the efficacy of knockdown of H19 by H19 siRNA was evaluated in vitro,and we found that down regulation of H19 impaired pancreatic cancer cell invasion and migration.Furthermore,Western Blot demonstrated that H19 siRNA induced downregulation of HMGA2 and impaired EMT,However,qRT PCR showed that the expression level of let 7 didn’t change,which means that H19 act as a sponge to antagonize let 7’s ability to inhibit its target gene.At lastly,co tranfection assay showed that H19 promoted PDAC cell invasion and migration at least partially by increasing HMGA2 mediated EMT through antagonizing let 7.【 Conclusions 】 H19 was up regulated in PDAC,and H19 could promote pancreatic cancer metastasis by derepressing let 7 ’s suppression on its target HMGA2 mediated EMT... |
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