The Screening Study Of Serum MicroRNA Biomarkers With Transgenic Kras^G12D Mouse Model Of Pancreatic Pre-malignant Lesions

BackgroundPancreatic ductal adenocarcinoma is a deadly disease, which is difficult for early diagnosis. Only a small percentage of patients are suitable for surgical interventions at the time of diagnosis. Recent studies showed that it might take approximately ten years from initial mutation to pancreatic cancer. The disease progression starts from initial genetic mutation, pre-malignant lesions, eventually to more clinically overt symptoms; therefore, there should be an opportunity for early diagnosis of pre-malignant pancreatic lesions, therefore improving the therapeutic efficacy. However, the clinical available biomarkers lack enough sensitive to diagnose pre-malignant diseases. MicroRNA is a 22-nucleotide RNA, which is stable in serum and serves as potential molecule for pre-malignant disease diagnosis. This study used transgenic mouse model to recapitulate disease progression from normal pancreas to pre-malignant disease. The transgenic mouse bears a KrasG12D mutation, which is activated only in pancreas due to pancreas specific promoter named Pdx-1. A cohort of Kras^G12D mutant mice and Kras wildtype （WT） control mice was raised. This study used a microRNA array chip technology to screen for potential biomarkers, and validated by real-time PCR reactions.Objective1. Breed Kras^G12D mice, and raised them without drug treatment to observe natural course of disease progression.2. Use caerulein to induce pancreatitis and to accelerate the disease progression.3. Use microRNA array to screening for up-regulated serum microRNA for pancreas pre-malignant lesion with pancreatitis.4. Use bioinformatics to screen for pancreas-specific microRNA.5. Use real-time PCR method to validate those microRNA after screening.Methods1. The mouse tails were collected enzymatically digested to yield DNA. Then, specific primers confirmed the presence or absence of Kras^G12D and Pdxl-cre genes for each mouse.2. The mouse was subjected to intra-abdominally injection of caerulein to induce pancreatitis. The severity of pancreatitis was controlled by the length of caerulein-injection time. The mouse was sacrificed, and the pancreas tissue was collected, embedded, and subjected to hematoxylin and eosin （H&E） stain, and immunostaining3. The severity of pancreatic pre-malignant lesions was quantified by randomly selected field of H&E stains. Immunocells were quantified by immunostaining.4. The peripheral blood was collected by retro-orbital bleeding, and separated by centrifugation to yield serum.5. The microRNA quantification was conducted by microRNA array. The up-regulated microRNA was selected based on stringent bioinformatics calculations.6. The up-regulated microRNAs were further analyzed and compared with previous published literature. Those with more abundance in pre-malignant or malignant pancreatic cancer tissues were further validated by real-time PCR analysis. This step was to select those microRNAs with diagnostic potential in pancreatic pre-malignant lesion.Results1. We inbred a number of 143 mice, in which 15 mice were Kras^G12D/Cre with spontaneous pancreatic pre-malignancy tendency.2. Six Kras^G12D/Cre mice were injected with caerulein to induce pancreatitis. Other nine Kras^G12D/Cre mice were without caerulein injection as a natural progression group.3. All mice had pancreatic pre-malignant lesion, which was confirmed by H&E stain. Immunocells were quantified by immunostaining of the pancreas.4. MicroRNA microarray showed a panel of 54 microRNAs with up-regulation in pre-malignant group. Ten out of 54 were previously reported to be more abundant in pancreatic pre-malignant and malignant lesion. Real-time PCR validation of the peripheral serum samples for the natural progression group identified miR-210-3p as more abundant in pre-malignancy group.ConclusionsKras^G12D/Cre recapitulates human pancreatic malignancy progression.This mouse model is suitable for screening studies for serum biomarker of pre-malignant diseases. MiR-210-3p could serve as potential biomarkers for pancreatic pre-malignant disease.