A Study On The Mechanism Of Development Of A Novel Modified MiR-15a Mimic With Enhanced Therapeutic Potential For Treatment Of Pancreatic Ductal Adenocarcinoma

Background:Pancreatic ductal adenocarcinoma（PDAC）is one of the most fatal malignancies leading to an estimated 300,000 deaths each year worldwide,with an overall 5-year survival rate of less than 6%and less than 20%of patients undergoing surgical resection^[1].Late clinical presentation,early metastasis and recurrence are mainly responsible fo r poor prognosis^[2].Currently,the main therapeutic approaches are curative resection,adjuvant chemotherapy（FOLFIRINOX and nab-paclitaxel-gemcitabine）and radiotherapy^[3-8].Despite extensive research and development efforts over several decades enhancing the clinical management of PDAC,the outcomes remain unsatisfactory^[9-11].Various strategies focusing on targeting protein coding oncogenes such as KRAS,Myc,PI3K/AKT/mTOR have failed to yield any clinically useful inhibitors^[12-14].Hence,there is an urgent need to discover and develop novel diagnostic and therapeutic strategies for PDAC.Emerging evidence demonstrates that epigenetic regulation（methylation,non-coding RNAs）is one of the major mechanisms contributing to cancer resistance.microRNAs（miRNAs）are a class of small non-coding RNAs（18-22 nucleotides in length）that play an important role in post-transcriptional regulation by binding to the 3’-UTR of target mRNA,resulting in translation suppression or mRNA degradation^[15-17].The dysregulation of miRNAs is tightly correlated with proliferation,apoptosis,invasion,metastasis and resistance^[18,19].Recently,many studies report that a range of miRNAs function as tumor suppressors or oncogenes in the pathogenesis and progression of PDAC,modulating diverse key targets and pathways such as KRAS,TP53,PI3K/AKT,NF-κB and Hedgehog signaling^[20-24].miR-15a is one of the miRNAs that has been shown to be a major contributor in PDAC^[22,24].It was first reported by Calin et al.that deletion of miR-15a/miR-16 is important in the development of chronic lymphocytic leukemia（CLL）^[25].Recent studies from our group and others have demonstrated that miR-15a acts as tumor suppressor by regulating a number of key targets（such as BMI-1,BCL-2,YAP-1,DCLK-1 etc.）in gastric cancer,colorectal cancer and PDAC^{[21,22,26,27]}.However,it was not clear at present that if it was possible to modify miR-15a to improve the efficiency in suppressing PDAC and it needed to be further studied.Based on these evidences,we focused on the chemotherapeutic medicine for PDAC.As the classic chemotherapeutic drug,5-fluorouracil（5-FU）is a cornerstone chemotherapeutic agent for treating PDAC and many other solid tumors.5-FU is an analogue of uracil with a fluorine atom at the C-5 position in place of hydrogen.The 5-FU metabolite,FdUMP forms a suicide ternary complex with the enzyme thymidylate synthase（TS）and Ch2THF,thereby block the binding of normal substrate dUMP and inhibiting dTMP synthesis to cause DNA damage.The clinical reports demonstrated that 5-FU could enhance the therapeutic efficiency and prolong the patients’overall survival time^[28,29].However,5-FU is easy to form chemoresistance,and loses the inhibitory effect on pancreatic cancer cell proliferation.Patients are prone to early recurrence and metastasis,which eventually leads to treatment failure.Increasing evidence indicated that there were multiple uracil（U）residues of the guide stand of miR-15a,and theoretically it’s possible to replace the uracil（U）residues of the guide stand of miR-15a with 5-FU^[26,30].Therefore,we suppose that it’s possible to synthesize a chemical compound using miR-15a and 5-FU,and apply it to PDAC research.This compound has both the anti-cancer effect of miR-15a and the therapeutic effect of 5-FU on cancer cells.In this study,we attempt to synthesize the novel compound using miR-15a and 5-FU,and further explore the function of this compound and the potential molecular mechanism in PDAC.Objectives:To Investigate the expression,function and clinical significance of miR-15a in PDAC;explore the function,molecular mechanism and the therapeutic potential of novel compound（5-FU-miR-15a）,and provide new ideas for developing new diagnostic and therapeutic strategies for PDAC.Methods:1.We detected the expressions of miR-15a and miR-16 between PDAC tissue and normal pancreas tissue by real-time qRT-PCR;downloaded TCGA data and analyzed the correlation between miR-15a and PDAC patients’prognosis using Kaplan-Meier survival analysis;explored the reason leading to the different expression of miR-15a and miR-16 in pancreatic cancer using real-time qRT-PCR,the separated technology of nucleus and cytoplasm and western blotting;investigated the effect of miR-15a on proliferation,cell cycle and the downstream targets in pancreatic cancer by cell transfection,WST-1proliferation array,flow cytometry and western blotting.2.We constructed the novel compound（5-FU-miR-15a）;investigated the effect of5-FU-miR-15a on proliferation and cell cycle by WST-1 proliferation array,flow cytometry;identified the specific targets of 5-FU-miR-15a and measured the expressions of these targets in pancreatic cancer by cell cycle PCR,TargetScan&miRDB predictive software,western blotting,luciferase report gene system and real-time qRT-PCR.3.We tested the expression of the downstream target（CDC2）of Wee1 in pancreatic cancer tissue by IHC,and assessed the relationship between CDC2 and PDAC patients’prognosis via Kaplan-Meier survival analysis;miR-15a regulated the activity of Wee1/Chk1/CDC2 signaling pathway was investigated by DNA damage induced experiment,western blotting and real-time qRT-PCR.4.The efficiency of 5-FU-miR-15a in enhancing the chemosensitivity of pancreatic cancer cell to gemcitabine was detected by cell transfection,cytotoxicity assay,and synergistic effect experiment.5.The animal model of metastatic pancreatic cancer was established to demonstrate if the 5-FU-miR-15a could inhibit the formation of metastatic tumors of pancreatic cancer,overcame the chemoresistance,enhanced the therapeutic effect of gemcitabine on pancreatic cancer,and improved the prognosis.6.The expression profile of circRNAs and some significant differential expression of circRNAs in PDAC tissue were detected and identified using arraystar human circRNA array analysis and bioinformatic analysis.Multiple binding sits between circRNAs and miR-15a or miR-506 were identified and the relevance was analyzed by real-time PCR and bioinformatic analysis.Results:1.The expression of miR-15a was significantly reduced in PDAC compared with normal pancreas（P=0.0014）;There was no expression difference of miR-16 between normal pancreas and PDAC（P=0.4118）;The expression level of miR-15a was tightly associated with PDAC patients’prognosis,and the median survival time was 1502 days（high expression）and 511 days（low expression）respectively（P<0.0001）.2.Mature miR-15a was significantly reduced compared with miR-16（P<0.0001）,while there were no differences between primary and precursor of miR-15a and miR-16 in PDAC clinical samples（P>0.05）.In pancreatic cancer cell lines,there were no differences between pri-miR-15a and pri-miR-16 expression,as well as pre-miR-15a and pre-miR-16.However,mature miR-15a was significantly reduced compared with mature miR-16（P<0.05）.3.miR-15a significantly inhibited pancreatic cancer cell proliferation,and triggered cell cycle arrest at G1 phase with a significant reduction in G2 phase.Additionally,there was a visible reduction of cyclin A expression along with increased levels of p27 in pancreatic cancer cells treated with miR-15a.（P<0.05）4.The inhibitory effectof miR-15a on pancreatic cancer cell proliferation was further enhanced by 5-FU-miR-15a;5-FU-miR-15a inducedthe increased S phase besides G2checkpoint reduction;miR-15a and 5-FU-miR-15a significantly reduced the expression levels of Wee1 and Chk1,while 5-FU-miR-15a visibly decreased the expression levels of the specific targets（BMI-1 and Yap-1）of miR-15a.（P<0.05）5.CDC2 was significantly over-expressed in PDAC tissue compared with normal pancreas tissue;the overall survival time of patients with Cdc2 high-expression was shorter than that of patients with Cdc2 low-expression;miR-15a significantly reduced CDC2expression;miR-15a could inhibit the activity of Wee1/Chk1/CDC2 signaling pathway.（P<0.05）6.5-FU-miR-15a had a synergistic effect when combined with gemcitabine,as well as sensitized pancreatic cancer cells to gemcitabine and enhanced the therapeutic effect of gemcitabine on pancreatic cancer cells;5-FU-miR-15a significantly inhibited metastatic tumor growth and the inhibitory effect was enhanced in combination with gemcitabine,as well as 5-FU-miR-15a overcomed chemoresistance and prolonged the survival time.（P<0.05）7.Multiple circRNAs dysregulated in pancreatic cancer;diverse circRNAs had different binding sites of miR-15a or miR-506;circRNAs could regulate miR-15a or miR-506 by“sponge effect”.（P<0.05）Conclusion:1.miR-15a expression was significantly reduced in pancreatic cancer tissue and cell,and was closely related with patients’prognosis.2.The reason leading to the different expression of miR-15a and miR-16 in pancreatic cancer was that reduction of mature miR-15a occurred during processing of pre-miR-15a to mature miR-15a.3.miR-15a inhibited pancreatic cancer cell proliferation and triggered cell cycle arrest.4.5-FU-miR-15a inhibited pancreatic cancer cell proliferation and metastatic tumor growth;affected cell cycle progression;sensitized pancreatic cancer cells to gemcitabine,overcomed chemoresistance and prolonged the survival time;down-regulated Wee1,Chk1,BMI-1,Yap-1,and maintained the target specificity of miR-15a.5.CDC2 expression was up-regulated in pancreatic cancer,and was tightly correlated with patients’prognosis;miR-15a inhibited the activity of Wee1/Chk1/CDC2 signaling pathway by down-regulating them their expressions.6.Multiple circRNAs dysregulated in pancreatic cancer;diverse circRNAs had different binding sites of miR-15a or miR-506;circRNAs could regulate miR-15a or miR-506 by“sponge effect”.