| Objective: Pancreatic cancer is a kind of digestive tract cancer with high malignancy,which is difficult to screen,diagnose and treat in early stage.The poor prognosis of pancreatic cancer is closely related to its special anatomimic location,occult disease,lack of special symptoms in the early stage and lack of effective early diagnosis and treatment methods.More than 80% of the patients have metastasis or are in the advanced stage when they are diagnosed with pancreatic cancer and cannot receive surgical resection,so they can only take comprehensive treatment including chemotherapy,radiotherapy and chemotherapy and nutritional support.However,the overall 5-year survival rate is only about 5%.Even in patients treated with surgery,the 5-year survival rate was less than20%.In order to improve the poor prognosis of patients with pancreatic cancer,finding new tumor markers and finding molecular therapeutic targets have become a hot topic.Previous studies have shown that long non coding RNA(lncrna)can regulate gene expression at epigenetic level and play a key role in regulating cancer cell behavior(including proliferation,apoptosis,invasion and metastasis).A large number of studies have confirmed that lncrna can activate target gene expression by binding specific mimicroRNA(miRNA),and affect the malignant biological behavior of tumor.AGR2 is related to metastasis,invasion,proliferation and drug resistance of many kinds of tumors,which is considered as a promising marker for early detection and treatment.Therefore,the function of AGR2 in pancreatic cancer and its role in signal transduction need further study.In recent years,studies have shown that there is interaction between RNAs of different lengths,especially the formation of the lncrna miRNA target gene regulatory network.It is very important to clarify the interaction and function of RNA molecules.Therefore,the purpose of this study is to find the lncrna and mi RNA interacting with AGR2,and to explore how this regulatory network plays a role in the malignant biological behavior of pancreatic cancer,analyze its mechanism in pancreatic cancer,and provide new potential biomarkers for the diagnosis and treatment of pancreatic cancer.Methods: The study used 36 cases of ductal cell carcinoma of the pancreas and its paracancer tissues were collected from the Shengjing Hospital of China MedicalUniversity from January 2014 to June 2017.All the patients had no other malignant diseases.Through bioinformatics analysis,differentially expressed mRNA and lncrna in pancreatic cancer were screened out in geo database.The expression of AGR2 in pancreatic cancer was detected by immunohistochemistry.The expression of LINC01207,miR-143-5p and AGR2 in 36 pairs of pancreatic cancer and adjacent tissues were detected by qRT-PCR.The binding sites of LINC01207 and miR-143-5p,miR-143-5p and AGR2 were confirmed by double luciferase reporter gene.RNA pull down and RNA binding protein immunoprecipitation experiments were carried out to further verify the binding relationship.The expression of LINC01207,miR-143-5p and AGR2 were detected by qRT-PCR.In the pancreatic cancer cell line,we constructed the cells which were silent LINC01207 and over expressed miR-143-5p by transfection of lentivirus plasmid.The proliferation activity of cells was detected by MTT method,apoptosis was detected by TUNEL method and flow cytometry,autophagy acid vesicles were detected by acridine orange staining,autophagy related protein LC3? was detected by immunofluorescence.Western blot was used to detect the expression of AGR2,LC3 ?,p62,beclin-1,Bcl-2 and Bax at protein level.To elucidate the molecular mechanism of LINC01207/miR-143-5p/AGR2 pathway involved in apoptosis and autophagy of pancreatic cancer cells.Results: 1.By retrieving the geo database,downloading the expression data of pancreatic cancer chip(gse22780,gse32676,gse71989,gse91035 and gse16515)and annotation probe files,we found that AGR2 was highly expressed in pancreatic cancer.Through the data analysis of gse16515 chip,we found that LINC01207 and AGR2 were highly expressed in pancreatic cancer,and there was a positive correlation.Through the prediction and analysis of rna22 online website and the verification of the targeting relationship by double luciferase reporter gene,it was found that there was a binding site between LINC01207 and miR-143-5p,and there was a binding site between AGR2 and miR-143-5p.The results of RNA pull-down and rip showed that miR-143-5p and LINC01207 could bind directly,and miR-143-5p could target AGR2.2.The expression of LINC01207,miR-143-5p and AGR2 in pancreatic cancer and adjacent tissues was detected by qRT-PCR.The results showed that the expression of LINC01207 and AGR2 increased(P<0.05)and the expression of miR-143-5p decreased(P<0.05)compared with normal pancreatic tissues.3.qRT-PCR was used to detect the expression of LINC01207,miR-143-5p and AGR2.Compared with the control group,the expression of LINC01207,miR-143-5p and siRNA-LINC01207+miR-143-5p mimic decreased,and the expression of miR-143-5p increased(P<0.05).Compared with the control group,the expression of LINC01207 and AGR2 in mi R-143-5p inhibitor group increased,and the expression of miR-143-5p decreased.4.The results of MTT showed that the cell growth of siRNA-LINC01207,miR-143-5p mimic and siRNA-LINC01207+miR-143-5p mimic group was inhibited,and the cell growth of siRNA-LINC01207+miR-143-5p mimic group was more inhibited(P<0.05),and the cell growth of miR-143-5p inhibitor group was increased(P<0.05).5.Compared with the control group,the apoptosis percentage of siRNA-LINC01207,miR-143-5p mimic and siRNA-LINC01207+miR-143-5p mimic group increased significantly,and the apoptosis percentage of siRNA-LINC01207+miR-143-5p mimic group increased more significantly(P<0.05),and the apoptosis percentage of miR-143-5p inhibitor group decreased significantly(P<0.05).6.Compared with the control group,the percentage of autophagic acid vesicles in siRNA-LINC01207,miR-143-5p mimic and siRNA-LINC01207+miR-143-5p mimic group increased,and the autophagy in siRNA-LINC01207+miR-143-5p mimic group increased more significantly(P<0.05,P<0.01).In miR-143-5p inhibitor group,the percentage of autophagic acid vesicles decreased(P<0.05).The results of immunofluorescence staining showed that the expression of membrane type autophagy related protein LC3 ? in siRNA-LINC01207,miR-143-5p mimic and siRNA-LINC01207+miR-143-5p mimic group increased,and the expression of membrane type LC3? in siRNA-LINC01207+miR-143-5p mimic group increased more significantly(P<0.05,the expression of membrane type LC3? in miR-143-5p inhibitor group decreased(P<0.05).7.Western blot was used to detect the expression of autophagy and apoptotic protein.Compared with the control group,siRNA-LINC01207,miR-143-5p and siRNA-LINC01207+miR-143-5p mimic,the expression of AGR2 and p62 decreased in si-agr2 group,the expression of LC3? and beclin-1 increased,but the level of Bcl-2/Baxdecreased,and the indicators of si RNA-LINC01207+miR-143-5p mimic group changed more significantly(P<0.05).In miR-143-5p inhibitor group,the expression of AGR2 and p62 increased,the expression of LC3 ? and beclin-1 decreased,but the level of Bcl-2/Bax increased(P<0.05).Conclusion: 1.In pancreatic cancer,there are different expressions of LINC01207,miR-143-5p and AGR2.The expression level of LINC01207 and AGR2 is up-regulated,while that of miR-143-5p is down regulated.2.LINC01207 as a ceRNA,can up regulate the expression of AGR2 by binding with mir-143-5p,forming the lncRNA-miRNA-target gene regulatory network.3.LINC01207 is highly expressed in pancreatic cancer and plays a role in promoting cancer.Silencing LINC01207 or enhancing miR-143-5p can promote the proliferation,autophagy and apoptosis of pancreatic cancer cells.It provides a new basis for studying the malignant biological behavior of pancreatic cancer. |
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