The Expression Of MiR-506 In Pancreatic Ductal Adenocarcinoma And Normal Pancreatic Tissue

Objective:Pancreatic ductal adenocarcinoma(PDAC)is a kind of malignant tumor with high mortality,and the trend of morbidity and mortality has been rising in recent years.MicroRNAs(miRNAs)are small non-coding RNAs that function as regulators of gene expression at the post-transcription level.MicroRNA-506 is located in X chromosome and influences various processes,such as cell proliferation,apoptosis,senescence,cell cycle,cell differentiation,epithelial-to-mesenchymal transition,angiogenesis,invasion and metastasis by post-transcriptional regulation to its targets.There are few literatures about the expression and significance of miR-506 in pancreatic tissue and PDAC,and they compared PDAC with the whole of pancreatic ducts,acinus and islands in relative literature.Most of the PDACs were originated from ductal epithelial cells,it is more accurate to compare the expression level of miR-506 in PDACs and normal pancreatic ductal epithelial cells to evaluate the role of miR-506 in PDAC.So we intend to detect the expression level of miR-506 in paraffin embedded PDAC tissues by two methods,and analyze the relationship between miR-506 expression and clinicopathological characteristics in order to understand the role of miR-506 in the development of PDAC.Moreover,we intend to detect the expression level of miR-506 in pancreatic ducts and acinus/islands respectively.Then we will compare the differences between the components of normal pancreatic tissues and PDAC in miR-506 expression to understand the expression changes in PDAC tumorigenesis.Methods:1.We used tissue microarray and miRNA in situ gybridization(ISH)combined with morphology to detect the expression level of miR-506 in PDAC,normal pancreatic ducts and acinus/island.And we analyzed the associations between miR-506 expression and the clinicopathologic characteristics of PDAC patients.2.We used laser capture microdissection(LCM)and hand-cutting to separate normal pancreatic ducts,acinus/island and PDAC cells from paraffin embedded tissue,extracted RNA respectively,reversed transcription and analyzed the expression level of miR-506 by quantitative Real-Time Polymerase Chain Reaction(qRT-PCR).Then we analyzed the associations between miR-506 expression and the clinicopathologic characteristics of PDAC patients.3.We compared detection results between miRNA and qRT-PCR and identified features of miR-506 expression in normal pancreatic tissues and PDAC.We analyzed the advantages and disadvantages of the two methods to detect the expression of miRNA in paraffin embedded tissues.Results:1.MiR-506 ISH based on TMA was completed in 110 cases of PDAC.The results showed that miR-506 was up-regulated and down-regulated in 72(65.5%)and 38(34.5%)PDAC cases respectively.Moreover,the expression of miR-506 in T3-T4,poor differentiated and lymph node metastasis PDAC cases was lower than in T2(P=0.004),well and moderately differentiated(P=0.023)and no lymph node metastasis(P=0.033)PDAC cases.We found no association between miR-506 expression and age(P=0.139),sex(P=0.127),tumor site(P=0.213),size(P=0.638),vascular invasion(P=0.595)and stage(P=0.063).The qRT-PCR detection of miR-506 was completed in 100 cases of PDAC paraffin embedded tissues.Statistical analysis of the clinicopathological characteristics in 100 PDAC cases were done.The results showed that the expression level in tumor diameter>4cm and T3-T4 PDAC cases were lower than in tumor diameter?4cm(P=0.04)and T1-T2(P=0.023)PDAC cases.We found no association between miR-506 expression and age(P=0.229),sex(P=0.417),tumor site(P=0.21),degree of differentiation(P=0.305),vascular invasion(P=0.295),lymph node metastasis(P=0.401)and stage(P=0.389).2.87 cases of normal pancreatic tissue on TMA were detected by miR-506 ISH.The results showed that miR-506 was up-regulated and down-regulated in 9.6%(7/73)and 90.4%(66/73)in normal pancreatic ducts epithelia cases respectively,and up-regulated and down-regulated in 68.3%(45/66)and 32.7%(21/66)in normal pancreatic acinus/islands cases respectively.Compared with the test results in the first part in PDAC,the expression of miR-506 was up-regulated in normal pancreatic ducts epithelia(P<0.001)but down-regulated in normal pancreatic acinus/islands(P<0.001).In 100 cases of PDAC and 17 cases of normal pancreatic tissue,the acinus / islets and ducts were cut by LCM and hand-cutting.The results showed that miR-506 was up-regulated and down-regulated in 8%(8/100)and 92%(92/100)in normal pancreatic ducts epithelia cases respectively,and up-regulated and down-regulated in 6%(6/100)and 94%(94/100)in normal pancreatic acinus/islands cases respectively.A further analysis of 10 paired PDAC and its normal pancreatic tissue was made.The results showed miR-506 was down-regulated in PDAC compared with paired normal pancreatic ducts and acinus/islands(P<0.0001).3.In PDAC,the results of miR-506 ISH were basically the same as that of qRT-PCR,and the high expression of miR-506 was often found in early T stage.In the comparison of different components between PDAC and normal pancreatic tissue,the results of miR-506 ISH and qRT-PCR were different.The reasons for the difference might as follows.First,sample size was small.Secondly,miR-506 ISH was done on TMA,and pancreatic ducts in normal tissues was very few.Thirdly,the miRNA ISH results were evaluated by a semi-quantitative method,based on the product of the staining intensity and the proportion of positive cells,which were different from the quantitative analysis of qRT-PCR.Fourth,the most important possibility was that miR-506 is located in cytoplasm.Compared with normal pancreatic ducts,PDAC has larger nuclear / cytoplasma ratio.Less cytoplasm made the relative concentration of miRNA larger,staining deeper and stronger expression in visual.The results was more subjective.Lastly,miRNA ISH kit recommended reference for U6 which were expressed in the nucleus,and miR-506 were expressed in the cytoplasma.There were questions about matching.Although qRT-PCR can not directly combine with histomorphology,it was more reliable to get specific tissue components by with LCM/hand-cutting,and the results obtained by quantitative detection were more reliable.Conclusion:1.It is feasible to detect the expression of miRNA by qRT-PCR in formalin fixed and paraffin embedded tissues,in view of the little effects of miRNA on the tissue degradation.Used qRT-PCR for specific tissue components by LCM/hand-cutting can compensate for the disadvantages that qRT-PCR cannot take into account histological.2.Although miRNA ISH can be combined with morphology,the semi-quantitative evaluation by staining intensity results in a lower precision than qRT-PCR.Choosing the right research object,finding and using more reasonable internal parameters,and increasing the sample size may increase the detection efficiency of miRNA ISH.3.The qRT-PCR results based on LCM and hand-cutting confirmed that the expression of miR-506 in PDAC was significantly lower than that of normal pancreatic ductal epithelium,and its expression level was negatively correlated with the poor prognosis of PDAC.This suggests that down-regulated miR-506 may be related to the tumorigenesis and development of PDAC,but it still needs large sample studies,in vitro experiments and animal experiments to confirm.