| BackgroundPancreatic cancer is a relatively common highly malignant digestive system tumor,of which the patient's five-year survival rate is less than 5%,the median survival time is less than 6 months.Pancreatic ductal adenocarcinoma(PDAC)is the most common histological type of pancreatic cancer,accounting for about 85%-90% of all pancreatic cancer cases.The high mortality and poor prognosis of pancreatic cancer is mainly due to the lack of specific clinical symptoms of the disease at the early stage and the associated diagnosis and prognosis molecular biomarkers.Thus,the majority of patients have been diagnosed at a locally advanced or metastasis that have lost surgery opportunities,and the tumor is poorly response to Chemotherapy and easy to recurrence.In recent years,with the development of medical technology,the survival time of most cancers has been stably increased,while advances have been slow for pancreatic cancer.Therefore,the early diagnosis of pancreatic cancer is an urgent problem to be solved.Most of the pancreatic ductal adenocarcinoma(PDAC)is developed by three precursor lesions,pancreatic intraepithelial neoplasia(PanIN),intraductal papillary mucinous neoplasm(IPMN),and mucinous cystic neoplasm(MCN).PanIN,especially the high-level of PanIN(PanIN-3),is the most common non-invasive precursor lesion of PDAC.Early detection and surgical resection can improve PDAC 5-year survival rate from 6% for stage IV to about 50% for stageI.If we can find specific molecular biomarkers of pancreatic cancer,it can help us diagnose the disease in itsprecursor stage or screen high-risk groups.The survival rate of patients with pancreatic cancer will be greatly improved.Pancreatic cancer is considered to be a complex genetic disease.Besides,the progression and development of pancreatic cancer involves a variety of oncogenes,tumor suppressor genes,miRNAs and epigenetic changes.Therefore,to make clear the mechanisms under lies the pathogenesis of PDAC are of vital importance.MicroRNAs(miRNAs)are a class of short,endogenous non-coding single-stranded RNAs,length 19-24 nucleotides,that regulate gene expression mainly by base-pairing with its target mRNA 3'UTR region,leading to mRNA degradation or translation repression and 30% of the protein-encoding gene expression was regulated in the post-transcriptional level.The abnormal expression of miRNAs is closely related to the development of various tumors,such as pancreatic cancer,liver cancer,lung cancer,breast cancer,colorectal cancer and so on.Recent studies have shown that miRNAs such as miR-21,miR-155,miR-196 a and miR-210 are important in the development of pancreatic cancer and can serve as potential molecular biomarkers for this deadly tumor.Therefore,to discover the specific miRNAs of pancreatic cancer,especially in PanIN-3 and explore its underlying mechanism,will be conducive to the screening,prevention,diagnosis,treatment and prognosis of pancreatic cancer ObjectiveThis study aims to find differential expression miRNAs by screening PanIN-3 and PDAC tissues and then verify them in different kinds of samples.Investigate the correlation between miR-1290 in human pancreatic cancer andclinic-pathological characteristics.Investigate the effect of miR-1290 on the biological behavior of pancreatic cancer.Use bioinformatics analysis to predict itspossible downstream target gene and try to find out the possiblepathways and related mechanisms.Methods 1.We had previously profiled the expression of microRNAs in PanIN-3 and pancreatic cancer tissues VS normal pancreas tissues using miRNA microarray.2.These miRNAs were further verified using the in-situ hybridization(ISH)and real-time qPCR was performed to test the differential expression of miRNAs in plasma ingroups of patients with PDAC,non-PDAC and normal andpaired PDAC cancer and adjacent non-cancer normal tissues.3.The correlation between aberrant expression levels of candidate miRNAs and their clinico-pathological data was also analyzed.4.Through a series of experiments in vitro,the effects of miR-1290 on the growth,proliferation,migration,invasion and apoptosis of pancreatic cancer cell lines were observed.5.We also further studied the biological behaviors of miR-1290 by subcutaneous tumor-implanting in nude mices.6.Use bioinformatics(MiRDB,Targetscan,MiRanda)analysis to predict its possible downstream target gene and then verify the relationship by double luciferase reporter gene system.Further study its effects on the biological behavior of pancreatic cancer cell lines 7.Study the expression ofIKKa,the target gene of miR-1290,in pancreatic cancer and adjacent normal tissues via real-timeqPCR,Western Blot and immunohistochemistry.Explore the possible signaling pathways.Results 1.Tissue miRNA microarray showed more than 30 kinds of miRNAs were significantly up-regulated or down-regulated(more than 5-fold)in PanIN-3 and pancreatic cancer tissuescompared with normal pancreatic ductal epithelium and low-degree PanIN tissues.19 of them were selected as candidate miRNAs.miR-31-5p,miR-29a-5p,miR-21-5p,miR-200b-3p,miR-192-5p,miR-146b-5p,miR-1290,miR-101-3p,let-7a-5p,miR-196a-3p,miR-29a-3p,miR-34a-3p and miR-155 were up-regulated while miR-105-3p,miR-216a-5p,miR-218-2-3p,miR-34a-5p,miR-513c-3p and miR-887 were down-regulated.2.In situ hybridization showed 6 miRNAs(miR-31-5p,miR-101-3p,miR-1290,miR-34a-3p,miR-21-5p and miR-155)were significantly higher expressed in PanIN-3 and cancer tissues than in low grades PanIN lesions.3.Real-time PCR showed the expression levels of miR-21-5p,miR-31-5p,miR-155 and miR-1290 were higher in the plasm of patients with pancreatic cancer than that in non-cancerous tumor patients and healthy volunteers.Differences were statistically significant.The ROC curve indicated that miR-1290 and miR-31-5p had better potential diagnostic value for screening pancreatic cancer.AUC was respectively 0.848 and 0.829.4.Real-time PCR of paired pancreatic cancer and adjacent normal tissues showed that the relative expression of miR-1290 and miR-31 in pancreatic cancer were significantly higher than those in normal tissues.Differences were statistically significant.For little researches were on miR-1290,we chose miR-1290 as our study object and found that miR-1290 expression level was statistically associated with the degree of pancreatic cancer differentiation and lymph node metastasis.5.Expereiments in vitro:(1)miR-1290 has the highest expression in pancreatic cancer cell line PANC-1 and has the lowest expression in pancreatic cancer cell line AsPC-1.AsPC-1 was selected as miR-1290 overexpressing cell line,while PANC-1 was used as miR-1290 inhibitory cell line.(2)Transfection experiments showed that miR-1290 was successfully overexpressed in AsPC-1 and was successfully inhibited in PANC-1.(3)The colony formation cell assay showed that overexpression of miR-1290 can significantly promote cell proliferation and colony formation,whereas inhibition of miR-1290 expression,cell proliferation and colony-forming abilities decreased.(4)CCK8 assay showed that the ability of proliferation was improved by overexpressing miR-1290.Tthe number of viable cells in miR-1290 overexpression group was significantly larger than that in control groupafter 24 hours,while after 24 hours the number of viable cells in miR-1290 inhibition group was significantly less than that in control group.(5)Overexpressing miR-1290,the ability of migration and invasion was significantly increased.Inhibiting miR-1290 can make the cell migration and invasion capacity decreased.(6)Flow cytometry assay showed overexpressing or inhibiting miR-1290 had no effect on cell apoptosis or cell cycle.6.The subcutaneous tumor implant experiment in nude mice showed that overexpressing miR-1290 can increase the ability of tumor formation and the volume of tumors.On the contrary,inhibition of miR-1290 can reduce the tumorigenic ability of pancreatic cancer cells and the tumor volume decreased as well.7.IKKa might be one of miR-1290 target genes through cross-analyzed by bioinformatics analysis in 3 databases(MiRDB,Targetscan and MiRanda).8.The relative expression of IKKa mRNA and protein were significantly repressed by miR-1290.While the expression of CHUK protein was significantly influenced by miR-1290 expression levels and the lower expression of IKKa was statistically associated with the degree of pancreatic cancer differentiation and lymph node metastasis.Dual-luciferase reporter assay,in addition,showed the site of interaction between miR-1290 and IKKa was located at 843-849 sites.9.Inhibiting the expression of IKKain pancreatic cancer cell lines can promote the ability of proliferation,migrationand invasion.Overexpressing IKKa can reverse miR-1290's effect on pancreatic cancer cell lines.Western blot showed no significant change in the expression of NF-kB signaling pathway realtive proteins IKBa(11)p65 and p50 along with the change of miR-1290 and IKKa in pancreatic cancer cell lines.Conclusions 1.Through microRNA microarray,in situ hybridization and real-time qPCR in tissue and plasma validation,6 miRNAs was thought to be pancreatic cancer-related miRNAs: miR-31-5p,miR-101-3p,miR-1290,miR-34a-3p,miR-21-5p and miR-155.2.The expression of miRNAs in pancreatic cancer tissues was partly consistent with that in plasma of pancreatic cancer patients,suggesting that miRNAs in plasma expected to be potential biomarkers for pancreatic cancer diagnosis,treatment and prognosis.3.miR-1290 was highly expressed in both pancreatic cancer tissue and pancreatic cancer patients' peripheral plasma,and the level of miR-1290 was correlated with tumor differentiation and lymph node metastasis,indicating miR-1290 might be a potential biomarker of pancreatic cancer.4.miR-1290 can promote proliferation,migration,invasion and tumorigenic ability of pancreatic cancer cells to participate in the biological behavior of pancreatic cancer regulation,indicating that miR-1290 played an important role of oncogene in pancreatic cancer development.5.miR-1290 promotes proliferation,migration and invasion of pancreatic cancer cells via directly targeting IKKa(binding site 3'UTR 843-849).miR-1290/IKKa could be novel diagnostic and therapeutic target of pancreatic cancer.6.IKKa is probably one of miR-1290's target gene.miR-1290's targeted inhibition of IKKa is probably through NF-kB-independent pathway. |
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