| Objective: pancreatic cancer is a highly malignant tumor with very poor prognosis in the digestive system.With the change of people's living and eating habits,such as the intake of high-fat highenergy food and heavy drinking and smoking,the incidence rate and incidence of pancreatic cancer are increasing year by year,coupled with environmental pollution and food safety problems.In China,pancreatic cancer ranks fifth in the mortality rate of malignant tumors.Because the early symptoms of pancreatic cancer are hidden and symptoms are obvious,it is already in the middle and late stage.There are local infiltration,invasion and distant metastasis of adjacent organs,resulting in low resectable rate of surgery,and the effect of chemotherapy and radiotherapy is not satisfactory.Therefore,how to solve the early detection,early diagnosis and early treatment of pancreatic cancer has become one of the focuses of public health in China and around the world.With the deepening of basic research on tumor,it is found that there are a large number of non coding RNA in cancer cells,which is related to the occurrence and development of tumor.These noncoding include lncrna,mi RNA,si RNA,pi RNA,circrna,etc.,which do not have the function of encoding proteins.At present,long non coding RNA(lncrna)has attracted more and more attention.Lncrna is a kind of noncoding RNA with base chain length of more than 200 bp.In mammalian genome sequences,about 4%-9% of the transcripts produced by sequences are lncrna,while the proportion of RNA encoding proteins is only 1%.Initially,these lncrnas were considered as the "noise" of gene transcriptome and did not have biological functions,so they were not paid attention to.At present,more and more studies show that lnc RNA is closely related to the occurrence and development of tumors.It can participate in the proliferation and invasion and metastasis of malignant tumors through chromatin modification,genomic imprinting,transcriptional activation,post transcriptional regulation and protein function regulation.Lnc RNA CERS6 antisense RNA 1(CERS6-AS1)plays a key regulatory role in the progression of breast cancer.However,the characteristics of cers6-as1 involvement in PDAC carcinogenicity have not been determined.Here,we tried to measure the expression of cers6-as1 in PDAC and further explore the role of cers6-as1 in PDAC carcinogenesis.The molecular mechanism of CERS6-AS1 regulating PDAC is also elucidated.It is confirmed that cers6-as1 / mir-15a-5p / FGFR1 pathway can provide new ideas for PDAC treatment.Results:(1)the expression profile of lncrnas in PDAC was evaluated by gene expression profile interaction analysis(http://gepia.cancer-pku.cn/)? Among these lncrnas,cers6-as1 is one of the most significantly highly expressed lncrnas.Compared with adjacent non tumor tissues,the expression of cers6-as1 in PDAC tissues was higher.In addition,overexpression of cers6-as1 was also verified in all four tested PDAC cell lines.PDAC patients were divided into two groups according to the median expression of cers6-as1 in this patient cohort,and it was found that patients with high cers6-as1 showed significantly shorter overall survival than patients with low cers6-as1.To analyze whether cers6-as1 is related to PDAC disease progression,specific si RNA targeting cers6-as1 was used to inhibit the expression of endogenous cers6-as1.All three si RNAs reduced the expression of cers6-as1 in PANC-1 and SW1990 cells.Si-cers6-as1#2 showed the highest silencing efficiency and was selected as the loss of function test.Cers6-as1 silencing significantly reduced the proliferation of PANC-1 and SW1990 cells.Flow cytometry analysis showed that the apoptosis rate in the two PDAC cell lines increased significantly after cers6-as1 deletion.In addition,the migration and invasion ability of PANC-1 and SW1990 cells decreased after cers6-as1 gene knockout.In conclusion,cers6-as1 may play a carcinogenic role in PDAC.(2)To elucidate the molecular events in which cers6-as1 plays a role,lnclocator was used(http://www.csbio.sjtu.edu.cn /Bioinfo / lnclocator /)predicts the distribution of cers6-as1.Cers6-as1 is expected to be mainly located in the cytoplasm,which is further confirmed by cell cytoplasmic / nuclear separation analysis.The putative mi RNA complementary to cers6-as1 was predicted using the bioinformatics tool Starbase 2.0.Seven mi RNAs were predicted as potential targets of cers6-as1.Then,the expression of these candidate genes was measured in PDAC cells with cers6-as1 deletion.After cers6-as1 silencing,mir-15a-5p increased significantly,while the expression of other candidate genes did not change.In addition,mir-15a-5p was weakly expressed in PDAC tissues,which was negatively correlated with the expression of cers6-as1.Luciferase reporter analysis was performed to determine whether cers6-as1 directly binds to mir-15a-5p in PDAC cells.In PANC-1 and SW1990 cells,the expression of exogenous mir-15a-5p significantly decreased the luciferase activity of cers6-as1-wt in PANC-1 and SW1990 cells,while the luciferase activity of cers6-as1 mut did not change after the up regulation of mir-15a-5p.In addition,cers6-as1 and mir-15a-5p were co immunoprecipitated by anti-ago2 antibody in PANC-1 and SW1990 cells.Overall,cers6-as1 acts as Cerna in PDAC and adsorbs mir-15a-5p directly.(3)To evaluate the effect of mir-15a-5p overexpression on PDAC cells.The increase of mir-15a-5p in PDAC cells was achieved by transfection of mir-15a-5p simulant.Ectopic mir-15a-5p expression inhibits the proliferation of PANC-1 and SW1990 cells and promotes apoptosis.In addition,mir-15a-5p simulant transfection resulted in significantly impaired cell migration and invasion of PANC-1 and SW1990 cells.We then identified the downstream target of mir-15a-5p in PDAC cells,and two mir-15a-5p binding sites were observed in the 3 '-UTR region of FGFR1.Because this gene has a well-known contribution to the tumorigenesis and progression of PDAC,this gene was selected for further experimental confirmation.Luciferase report analysis confirmed that mir-15a-5p simulant reduced the luciferase activity of FGFR1 wt;However,when the binding site is mutated,this inhibition is eliminated.The up regulation of mir-15a-5p decreased the expression of FGFR1 in PANC-1 and SW1990 cells.In addition,the level of FGFR1 m RNA in PDAC tissue was high and negatively correlated with the level of mir-15a-5p.In general,these targets in fgmir-15a-15 a are directly determined.(4)After confirming that cers6-as1 acts as mir-15a-5p sponge,we next studied whether cers6-as1 controls the expression of FGFR1 in PDAC cells.The deletion of cers6-as1 resulted in a significant decrease in FGFR1 expression in PANC-1 and SW1990 cells.Inhibition of mir-15a-5p can eliminate the inhibitory effect of cers6-as1 gene knockout on FGFR1 expression in PANC-1and SW1990 cells.In addition,a positive expression relationship between FGFR1 m RNA and cers6-as1 was confirmed in PDAC tissues.In conclusion,cers6-as1 actively regulates the expression of FGFR1 in PDAC cells by isolating mir-15a-5p.(5)To evaluate the tumor promoting effect of mir-15a-5p / FGFR1 on cers6-as1 in PDAC cells.PANC-1 and SW1990 cells were transfected with si-cers6-as1 and mir-15a-5p inhibitors alone or in combination.The downregulation of cers6-as1 decreased cell proliferation and promoted apoptosis;However,CO transfection with mir-15a-5p inhibitor reversed both effects.In addition,mir-15a-5p inhibition eliminated the inhibitory effect of si-cers6-as1 on the migration and invasion of PANC-1 and SW1990 cells.FGFR1 overexpression plasmid pc DNA3,1-fgfr1 was used to increase the expression of FGFR1 protein in PDAC cells.pc DNA3.1-fgfr1 or pc DNA3 1 was introduced into PANC-1 and SW1990 cells together with si-cers6-as1.The up regulation of cers6-as1 effectively reversed the effects of si-cers6-as1 on the proliferation,apoptosis,migration and invasion of PANC-1 and SW1990 cells.In conclusion,cers6-as1 promotes the carcinogenicity of PDAC cells by controlling the mir-15a-5p / FGFR1 axis.(6)The subcutaneous xenograft model was established by injecting SW1990 cells stably overexpressing sh-cers6-as1 or sh NC into mice.The results showed that the tumor growth and weight of sh-cers6-as1 group were significantly inhibited compared with SH NC group.In addition,RT q PCR analysis showed that cers6-as1 expression was down regulated in tumors derived from SW1990 cells stably expressing sh-cers6-as1.In addition,in cers6-as1 silenced xenotumor grafts,the level of mir-15a-5p increased and FGFR1 protein decreased.Therefore,these findings confirm that cers6-as1 gene knockout can impair the growth of PDAC tumors in vivo.Conclusion: the expression of cers6-as1 is up-regulated in PDAC,and cers6-as1 gene knockout significantly inhibits the progression of PDAC.Cers6-as1 acts as mir-15a-5p sponge in PDAC cells,thereby increasing the expression of FGFR1,thus playing a key role in the tumorigenesis of PDAC.In conclusion,our data provide new insights into the pathogenesis of PDAC and suggest that cers6-as1 / mir-15a-5p / FGFR1 axis may be a feasible therapeutic target in PDAC treatment. |
PDF Full Text Request