Study Of Targeted Therapy With Double MiRNAs Expressed By Oncolytic Adenovirus In Pancreatic Cancer

?Background & Objectives? Pancreatic ductal adenocarcinoma(PDAC)is one of the most fatal cancers in our country,due to its high degree of malignancy,increased incidence,high mortality and unsatisfactory treatment efficacy.In spite of enormous progress in medical image,new chemotherapy and immunotherapy,the 5-year survival rate of pancreatic cancer is still below 5%.Mi RNAs are small noncoding RNAs that regulate gene expression by affecting translation of m RNAs.A large number of evidence suggests that miRNAs are dysregulated in various types of tumors and play an important role as oncogenes or tumor suppressor genes.The expression profiling of miRNA in pancreatic ductal adenocarcinoma is significantly changed and can regulate the expression of corresponding target gene.Studies have shown that miRNAs play a big role in the occurrence and development of cancer and likely regulate diverse biological processes,including cell cycle,repairing of damaged DNA,cell apoptosis and tumor metastasis.Nowadays,plasmid and virus were used to carry miRNAs so as to change its expression in PDAC and achieve the treatment of tumor.For example,studies have shown that low expression of miR-34 a and miR-let7 in human pancreatic cancer cells act as tumor suppressor gene.Nanoparticles and CC9 peptides were successfully used to carry miR-34 a for the treatment of PDAC and to inhibit the growth of subcutaneous transplanted pancreatic tumor.Meanwhile,Torrisani et.al improved the expression of miR-let7 carried by plasmid or lentiviral vector in order to inhibit the proliferation of PDAC cells.However,the intervention therapy of single miRNA on pancreatic cancer was limited.Thus,miR-34 a and miR-let7 were simultaneously cloned to oncolytic adenovirus vector and obtain recombinant adenovirus therapy system(Ad CEAp-miR34a-let7)in our study.This system has high levels of expression and release of miR-34 a and miR-let7 which plays a role of tumor suppressor in pancreatic cancer by the regulation of CEA promoter.And then achieve the synergistic antitumor effect and targeted intervention therapy to pancreatic cancer cells.?Method? 1.Construction and identification of adenoviral vector: The adenovirus therapy system Ad CEAp-miR34a-let7,positive control group Ad CEAp-miR34 a and Ad CEAp-miRlet7 were constructed using the tumor suppressor sequences miRNA-34 a,miRNA-let7 and CEA promoter-regulated hybrid oncolytic adenovirus Ad5F35 which was generated in our previous study.Meanwhile,Ad CEAp-EGFP and Ad5-EGFP carrying enhanced green fluorescent protein(EGFP)were constructed.Then,the expression of virus genome was identified.2.Identification of specific proliferation activity of adenovirus: Human pancreatic cancer cell lines Panc-1,Capa-2,SW1990,Bx PC3 and normal human fibroblast cells BJ,MRC-5 were infected with Ad CEAp-EGFP and Ad5-EGFP.Then,the percentage and strength of EGFP-positive cells were observed and counted under a fluorescence microscope.After infected with Ad CEAp-miR34a-let7,Ad CEAp-miR34 a,Ad CEAp-miRlet7 and Ad5-miR34a-let7,virus titre was measured by tissue culture infectious dose 50(TCID50)methods while gene expression of adenovirus E1 a gene was detected by Western Blotting.3.Gene expression of miRNAs mediated by adenovirus: The pancreatic cancer cell lines Panc-1,Capa-2,SW1990,Bx PC3 and normal fibroblast cells BJ,MRC-5 were infected with Ad CEAp-miR34a-let7 and Ad5-miR34a-let7 at an MOI of 1pfu/cell.The expression of miR-34 a and miR-let7 were measured by q RT-PCR after extraction of total miRNAs.4.The influence of biological behavior of pancreatic cancer cells treated by double miRNAs mediated by adenovirus: The pancreatic cancer cells and normal fibroblast cells were infected with Ad CEAp-miR34 a,Ad CEAp-miRlet7,Ad CEAp-miR34a-let7 and Ad5-miR34a-let7 at an MOI of 1pfu/cell.Meanwhile,the parental cells without adenovirus were cultured synchronously as blank control.Cell viability of pancreatic cancer cells and normal fibroblast cells were tested by MTT assay after treated with double miRNAs mediated by adenovirus.The influence of cell migration and invasion of pancreatic cancer cells were detected by transwell chamber assay.Abilities of apoptosis of pancreatic cancer cells after treated by this therapy system were tested by flow cytometry.The expression of corresponding target proteins and signal molecules of miR-34 a and miR-let7 were detected by Western Blotting.5.Animal model experiments: The subcutaneous pancreatic xenograft in nude mice were established and treated with intra-tumor injection of experimental adenoviruses mentioned above.The growth variation of tumor was observed and recorded.Then,we draw the growth curve in order to evaluate its inhibiting ability to cancer xenograft models.?Results? 1.Identification of specific proliferation activity of adenovirus: The proliferative oncolytic adenovirus carrying EGFP reporter gene(Ad CEAp-EGFP)had a significant proliferation in pancreatic cancer cells(Panc-1,Capa-2,SW1990 and Bx PC3)and did not proliferate in normal fibroblast cells(BJ and MRC-5).Non-proliferative oncolytic adenovirus(Ad5-EGFP)did not proliferate in all cell lines.Ad CEAp-miR34a-let7,Ad CEAp-miR34 a and Ad CEAp-miRlet7 had significant proliferation in four pancreatic cancer cell lines except in normal fibroblast cells.Ad5-miR34a-let7 did not have significant proliferation in all cell lines.Pancreatic cancer cells Panc-1,Capa-2,SW1990 and Bx PC3 infected with Ad CEAp-miR34a-let7 were found to express E1 a gene by Western Blotting test,while normal fibroblast cells BJ and MRC-5 did not express E1 a gene.2.Expression of miRNAs mediated by proliferated Adenovirus: We found that the expression of miR-34 a and miR-let7 were significantly increased in pancreatic cancer cells infected with Ad CEAp-miR34a-let7 and the expression levels in normal fibroblast cells were low by q RT-PCR.The expression levels of miR-34 a and miR-let7 were low in all cells treated by Ad5-miR34a-let7.3.Expression of double miRNAs mediated by proliferated oncolytic adenovirus could decrease the cell viability of pancreatic cancer cells: The cell viability of pancreatic cancer cells Panc-1 and SW1990 in Ad CEAp-miR34a-let7 group were found to be higher than in Ad CEAp-miRlet7 group(P<0.01,P<0.05)and negative control group(P<0.001,P<0.01).The difference of cell viability between Ad CEAp-miR34a-let7 group and Ad CEAp-miR34 a group was not significant.4.Expression of double miRNAs mediated by proliferated oncolytic adenovirus could inhibit migration and invasion ability of pancreatic cancer cells: We found that the migration and invasion rate of pancreatic cancer cells in Ad CEAp-miR34a-let7 group were lower than in Ad CEAp-miR34 a group,Ad CEAp-miRlet7 group,negative control group and blank control group by Transwell testing.The migration and invasion ability of pancreatic cancer cells in Ad CEAp-miR34a-let7 group were significantly lower than in Ad CEAp-miRlet7 group and negative control group(P<0.001).There were no significant difference between Ad CEAp-miR34a-let7 group and Ad CEAp-miR34 a group.5.Expression of double miRNAs mediated by proliferated oncolytic adenovirus could improve apoptosis of cancer cells: After infected with miRNAs mediated by oncolytic adenovirus,the apoptotic rate of Panc-1 cells increased dramatically.The difference was statistically significant between Ad CEAp-miR34a-let7 group and negative control group or Ad CEAp-miRlet7 group(P<0.05).But there was no significant difference between Ad CEAp-miR34a-let7 group and Ad CEAp-miR34 a group(P=0.054).6.Expression of double miRNAs mediated by proliferated oncolytic adenovirus could affect the gene expression profiling: The expression of target gene of miR-34a(cyclins D1,E2F1,Bcl-2)and miR-let7(HMGA2,CRD-BP)were found to be down-regulated in Ad CEAp-miR34a-let7 group and in Ad CEAp-miR34 a group by Western Blotting.The difference were statistically significant(P<0.05).7.Double miRNAs expression mediated by oncolytic adenovirus suppressed the growth of xenograft tumor in nude mice: The pancreatic cancer xenograft in nude mice was successfully established.After intra-tumor injection of adenovirus mentioned above,the speed of tumor growth in Ad CEAp-miR34a-let7 group was found to be slower than in any other groups.The tumor volume in Ad CEAp-miR34a-let7 group did not increase after treatment of 21 days and even have a trend of decreasing.The antitumor effect among Ad CEAp-miR34 a group,Ad CEAp-miRlet7 group and Ad5-miR34a-let7 group do not have significant difference.?Conclusion? In our previous research,we constructed a tumor-specific proliferated adenovirus mediated by CEA promotor and identified its effectiveness.In this study,a therapy system with two tumor suppressive miRNAs was constructed on the basis of the adenovirus vector.Consequently,we not only exert the synergistic antitumor effects of two miRNAs,but also make it specificly acted on pancreatic cancer cells.Thus,we provide a new weapon for the therapy of pancreatic cancer.Here are the main conclusions: 1.A therapeutic system(Ad CEAp-miR34a-let7)carrying double miRNAs by the vector of tumor-specific proliferated adenovirus was successfully constructed.This therapeutic system could proliferate in pancreatic cancer cells specifically.Then,this system was found to express E1 a gene and express high levels of miR-34 a and miR-let7 in pancreatic cancer cells.So we conclude that it has pancreatic cancer specificity and does not have effect on normal cells.2.Expression of double miRNAs mediated by tumor-specific proliferated oncolytic adenovirus could inhibit the cell viability,migration and invasion ability of pancreatic cancer cells.Meanwhile,the apoptosis rate was improved and gene expression profiling of miR-34a(cyclins D1,E2F1,Bcl-2)and miRNA-let7(HMGA2,CRD-BP)were down-regulated after treated by this therapeutic system.3.The expression of double miRNAs mediated by oncolytic adenovirus could suppress the growth of xenograft tumor in nude mice after intra-tumor injection of this therapeutic system.