Locked Nucleic Acid In Situ Hybridization Analysis Of Hsa-miR-96and Hsa-miR-217Expression During Pancreatic Ductal Adenocarcinoma Development

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly invasive tumor which is difficult to be early detected because the clinical symptoms usually appear too late. Most cases are diagnosed at an advanced stage, and the prognosis is the worst among all solid tumors. If effective interventions could be provided during non-invasive precancerous period, the survival rate of pancreatic cancer would be greatly improved. Pancreatic intraepithelial neoplasia (PanIN) is the most common non-invasive precursor lesion of PDAC.MicroRNAs (miRNAs) is an endogenous non-coding single-stranded small RNA of19to24nucleotides(nt) in length that function as a post-transcriptional regulating molecule. Mature miRNAs can function as oncogenes or tumor suppressor genes. In recent years, many studies have confirmed that many miRNAs are up-regulated or down-regulated in PDAC. Previous studies in this group have both indicated that hsa-miR-96and hsa-miR-217are down-regulated in PDAC using microarray technology and quantitative PCR. hsa-miR-96and hsa-miR-217may function as tumor suppressor genes. Whether hsa-miR-96and hsa-miR-217have been down-regulated in the most important non-invasive precursor lesion(PanIN) of PDAC, and play a potential role in carcinogenesis of PDAC, which has not yet been reported in the literatur. Based on the previouse study in our group, in this study the expression of hsa-miR-96and hsa-miR-217in PanIN and PDAC were analyzed using the method of locked nucleic acid in situ hybridization (LNA-ISH), with chronic pancreatitis and normal pancreatic tissue as reference control tissues.Objective:To study the role of hsa-miR-96and hsa-miR-217in pancreatic ductal adenocarcinoma development and potential role in diagnosis of PDAC.Methods:(1)191cases of paraffin-embedded pancreatic specimens, including119cases of PDAC,22cases of chronic pancreatitis and47cases of other pancreatic tumors were collected from the archives of the Department of Pathology, Peking Union Medical College Hospital from the year2008-2010. Follow up was made, and the time of follow-up was17-50months. All the clinical data were reviewed. And all available slides were evaluated for the presence, grade of PanIN lesions using the four-tier classification.(2)Paraffin-embedded pancreatic specimens contain with PanIN, including45cases of PDAC,10cases of chronic pancreatitis and cases of other pancreatic tumors were selected in this study. The miRNAs hsa-miR-96and hsa-miR-217were analyzed using locked nucleic acid in situ hybridization (LNA-ISH). The differences of miRNA expression among sample sets were analyzed with the Chi-squared test.Results:(1) There was a progressive increase from other types of pancreatic tumors to pancreatitis and to ductal adenocarcinoma in the frequency of overall pancreatic intraepithelial neoplasia lesions (24.0%,12/50;54.5%,12/22; and70.6%,84/119; respectively). The frequency of higher grade pancreatic intraepithelial neoplasia lesions (2and3) in ductal adenocarcinoma patients was31.1%(37/119), which was significantly higher than the one in chronic pancreatitis(0%,0/22) or other types pancreatic tumors(2%,1/50)(P<0.05). The frequency of PanIN was significant difference among the different age groups (P<0.05), and was increased with the age. There was no significant difference of the frequency of the PanIN between male and female gender.(2) There was a progressive reduce from PanIN-1, PanIN-2, to PanIN-3in the expression of hsa-miR-96(91.3%,22/23;70.6%,12/17; and22.2%,4/18; respectively) and hsa-miR-217(95.7%,22/23;70.6%,12/17;27.8%,5/18; respectively). The expressions of hsa-miR-96and hsa-miR-217in PanIN-2/3lesions were significantly different from normal pancreatic tissue and PanIN-1(P<0.01).(3) In PDAC,31.1%(14/45) and42.2%(19/45) of samples showed positive expression of hsa-miR-96and hsa-miR-217in tumor cells, respectively, whereas all corresponding adjacent normal pancreatic tissue and chronic pancreatitis samples were positive or weakly positive (P<0.05).(4)Positive expression of hsa-miR-96and hsa-miR-217was observed in the fibroblasts of22.2%(10/45) and24.4%(11/45) of PDAC, respectively, which was significantly lower than chronic pancreatitis (70%,7/10and60%,6/10). These differences were statistically significant (P=0.003).(5)There was no statistical difference in survival between the negative group and positive group of hsa-miR-96or hsa-miR-217.Conclusion:(1) The frequency of overall PanIN lesions and higher grade PanIN lesions (2and3) in PDAC were significantly higher than the one in chronic pancreatitisor other types pancreatic tumors.(2)hsa-miR-96and of hsa-miR-217abnormal expression was observed in early stage (PanIN-2/3) of PDAC.(2)These microRNAs(hsa-miR-96and hsa-miR-217) may play an important role in the development of PDAC and may serve as potential biomarker.(3)There was different expression of hsa-miR-96and hsa-miR-217between PDAC and chronic pancreatitis.(4) Down-regulation of hsa-miR-96and hsa-miR-217in PDAC exists not only in cancer cells, but also in the stromal fibroblasts, which maybe provide new ideas about the microenvironment of PDAC.