| Research background and ideasPancreatic cancer(PC)is a digestive tract tumor with high malignancy.Its mortality rate ranks the fourth in the world and the ninth in China.Pancreatic cancer is known as the "king of cancer" because of its hidden incidence,rapid progression,poor prognosis and high mortality.At present,surgical resection and chemotherapy are important ways to treat pancreatic cancer,while targeted treatment is still rarely reported in pancreatic cancer.Therefore,it is particularly important to explore the pathogenesis of pancreatic cancer and find the molecular target of pancreatic cancer.Previous studies showed that serine protease 3(PRSS3),as an oncogene,was highly expressed in pancreatic cancer.In order to explore the potential mechanism of PRSS3 imbalance in pancreatic cancer,first of all,we analyzed the database of Targetscan and Miranda online and found that the 3’-UTR sequence of PRSS3 was complementary to miR-217.Then,quantitative PCR analysis of PC tissues and cell lines showed that miR-217 expression in pancreatic cancer tissues and cells was lower than that in paracancerous tissues;By analyzing the clinical data of patients,we found that patients with low expression of miR-217 had shorter survival time than those with high expression of miR-217,and it was related to lymph node metastasis.Inhibition of miR-217 increased the malignant phenotype of pancreatic cancer cells.As we all know,circular RNA is a new kind of star molecule.Unlike linear RNA,circular RNA is a covalent closed loop molecule without 5 ’cap structure and 3’ ploy A tail.It has been reported that most of circRNAs are located in the cytoplasm of eukaryotic cells and stably expressed.It was also found that circRNA has many functions in tumor,such as participating in transcription regulation and splicing,interacting with RNA binding proteins,participating in translation,competing with mRNA to adsorb microRNA.In order to find the circRNA related to miR-217,we first predicted on-line the Starbasev2.0 database and verified it by RNA pull-down assay,and found that there was a binding site between circ-ADAM9 and miR-217.Then,we conducted quantitative detection of circ-ADAM9 in pancreatic cancer tissues and cell lines,and analysis on its clinical prognosis and cell function experiments,all of which confirmed that circ-ADAM9 was involved in the development of pancreatic cancer.Further mechanism studies showed that circ-ADAM9 enhanced the expression of PRSS3 by sponge adsorption of miR-217,weakened the inhibition of miR-217 on PRSS3,and activated ERK/VEGF signaling pathway.In short,the ceRNA regulatory network of circ-ADAM9/miR-217/PRSS3 plays a key role in the development of PC by regulating ERK/VEGF signal pathway,which provides a new idea and direction for targeted treatment of pancreatic cancer.Materials and Method1 Collection of tissue samples and clinical data of pancreatic cancer patientsWe collected fresh frozen tissue samples from pancreatic cancer patients,extracted RNA by Trizol method,and then reversely transcribed the RNA into cDNA,which was stored in refrigerator at-80℃.Pancreatic cancer cell lines and normal pancreatic ductal epithelial cell lines were purchased from the Shanghai,Chinese Academy of Sciences.The total RNA in the cell lines was extracted by the Trizol method.The total RNA was reverse transcribed into cDNA and stored in a refrigerator at-80℃ until use.Clinical information of pancreatic cancer patients was collected,including hospitalization number,gender,age,smoking history,drinking history,TNM stage,lymph node metastasis,survival time,etc.2 Expression level and clinical characteristics of miR-217/circ-ADAM9 in pancreatic cancer tissue samplesWith the tissue samples cDNA,the expression of miR-217/circ-ADAM9 in pancreatic cancer patients with different TNM stage and lymph node metastasis was detected by qRT-PCR.Using cell line sample cDNA as a template,qRT-PCR technology was used to detect the expression levels of miR-217 and circ-ADAM9 in different cell lines.3 The effect of miR-217 expression on the malignant phenotype of pancreatic cancer cell lineMimics and inhibitors of miR-217 which were purchased from Shanghai Jima Company were transfected into the pancreatic cancer cells MiaPaca2 and Capanl respectively by Lip2000 liposome transfection method.After 48 hours of transfection,the transfection efficiency was detected by qRT-PCR.CCK8,migration and invasion test were conducted to detect the effect of miR-217 on the malignant phenotype of pancreatic cancer cell line.Western blot was used to detect the effect of miR-217 on the expression of PRSS3,pERK,ERK,VEGF and other proteins in pancreatic cancer cell lines.4 The effect of circ-ADAM9 expression level on the malignant phenotype of pancreatic cancer cell linespcD-ciR-ADAM9 was constructed and transfected into the pancreatic cancer cells MiaPaca2 and Capanl by lipofection 2000.After 48 hours,the transfection efficiency was detected by qRT-PCR;CCK8,migration and invasion tests were used to detect the effect of miR-217 on the malignant phenotype of pancreatic cancer cell lines,and Western blot was used to detect the effect of circ-ADAM9 on the expression of PRSS3,pERK,ERK,VEGF and other proteins in pancreatic cancer cell lines.5 Interaction between miR-217 and PRSS3 and circ-ADAM9 in pancreatic cancer cell lineTwo groups of double fluorescein reporter plasmids were constructed,one was pmirglo-PRSS3-3’-UTR-WT and pmirglo-PRSS3-3’-UTR-MUT,the other was pmirglo-circ-ADAM9-WT/pmirglo-circ-ADAM9-MUT.The above two plasmids were co-transfected with miR-217 and control simulant respectively into the pancreatic cancer cells MiaPaca2 and Capanl by Lip-2000 method.48 hours later,the cells were collected and the relative luciferase activity was detected by double luciferin report test.The biotin labeled miR-217 and control simulant purchased from Shanghai Jima Company and were transfected into the pancreatic cancer cells MiaPaca2 and Capanl by Lip2000.After 48 hours,the enrichment of circ-ADAM9,circ-ATAD1,circ-SYPL1,circ-SNF8,circ-ZCCHC14 and miR-217 were detected by RNA pull-down assay.6 Statistical analysisStatistical software SPSS22.0 was used to analyze the results of the experiments.The data was represented by mean±standard deviation,the difference between groups was represented by t test or chi square test,the correlation coefficient was tested by Pearson correlation coefficient,the survival curve was analyzed by Kaplan Meier chart and long rank test,and the graph was made by GraphPad Prism 7.0 software.*p<0.05,**p<0.01 and***p<0.001.Results1 miR-217 is a potential upstream negative regulatory gene of PRSS3 in pancreatic cancerThe results of online prediction and double Luciferase Report showed that the 3’UTR region of PRSS3 was complementary to miR-217.Compared with the paracancerous tissues,the expression of miR-217 was lower in pancreatic cancer tissues.Combined with clinical data analysis,it was found that the higher TNM stage,the lower expression level of miR-217 in patients,as the patients with lymph node metastasis.Survival analysis showed that the survival rate of patients with low expression of miR-217 was lower with the prolongation of survival time.2 miR-217 suppresses the malignant phenotype of pancreatic cancer cells by targeting PRSS3The results of fluorescence quantitative PCR and Western blot showed that the mRNA and protein expression levels of miR-217 and PRSS3 were increased,while the mRNA and protein expression levels of miR-217 and PRSS3 were decreased.Inhibition of miR-217 and cell function test showed that the proliferation,migration and invasion of pancreatic cancer cells increased.However,inhibition of PRSS3 can save the malignant phenotype of pancreatic cancer cells;on the contrary,overexpression of miR-217 and PRSS3 can also inhibit the malignant phenotype of pancreatic cancer cells.3 In pancreatic cancer cell line,circ-ADAM9 acts as sponge to adsorb miR-217The results of online prediction and double Luciferase Report showed that miR-217 and circ-ADAM9 were complementary pairing.The results of on-line prediction and RNA pull-down assay showed that circ-ADAM9 could enrich miR-217.4 Expression level and clinical characteristics of circ-ADAM9 in pancreatic cancer tissue samplesCompared with the paracancerous tissues,the expression of circ-ADAM9 was higher in patients with TNM stage and lymph node metastasis.Survival analysis showed that the survival rate of patients with high expression of circ-ADAM9 got lower with the prolongation of survival time.5 Mechanism of circ-ADAM9/miR-217/PRSS3 axis in pancreatic cancer cell lineOverexpression of circ-ADAM9,quantitative PCR and Western blot showed that the mRNA and protein expression levels of PRSS3 were increased,and the protein expression levels of ERK and VEGF were also increased.Inhibition of circ-ADAM9,decreased the expression of mRNA and protein of PRSS3,as well as the protein expression of ERK and VEGF.Cell function experiment show that,with the overexpression of circ-ADAM9 and inhibition of PRSS3,the proliferation,migration and invasion of pancreatic cancer cells were also inhibited.Conclusion1.PRSS3 is regulated by circ-ADAM9/miR-217 axis.2.The ceRNA regulatory network of circ-ADAM9/miR-217/PRSS3 plays a key role in the development of PC by regulating the ERK/VEGF signal pathway. |
