DDR2 Expression In Alcoholic Liver Fibrosis Rats And Construction Of RNAi Lentiviral Vectors Targeting DDR2 Gene

Background and objective Discoidin domain receptors(DDRs) belong to a novel receptor tyrosine kinase subfamily which was found during the study of tyrosine kinase proteins expressed in human malignant tumors.The main function of DDRs is to recept changes of collagen content and conformation in microenvironment,and to adjust cellular reactions in the manners of adhension,migration,differentiation,growth and proliferation. They can be divided into DDR1 and DDR2 in mammals.It has been indicated that DDR2 plays important roles in many pathologic processes include liver fibrosis,etc.Our previous work of animal experiments indicated that DDR2 expressions in alcoholic liver fibrosis (ALF) rat models increased in a time-dependent manner,accompanying with alcoholic intragastric administration and liver fibrosis progreeion.And DDR2 expressions were positively correlated with collagen typeâ… ,â…¢,â…£as well as serum hyaluronic acid(HA) and laminin(LN).Furthermore,DDR2 expressions were down-regulated accompanying with alleviation of liver fibrosis after treatment.All of these studies show that DDR2 plays an important role in ALF formation and progression.In this study,we analysis the role of discoidin domain receptor 2(DDR2) and matrix metalloproteinase 2(MMP2) in development of rat alcoholic liver fibrosis.And based on the above-mentioned studies,it is designed to construct RNA interference(RNAi) lentivirus vectors targeting rat DDR2 gene,package RNAi lentivirus particles and screen the efficient RNAi letiviruses,in order to provide a sufficient RNAi tool to be used in further study of action mechanism of DDR2 and clinical treatment of liver fibrosis and other related diseases.Methods1.Immunohistochemical detection of DDR2 and MMP2 in ALF ratsAfter fed normally for a week,16 adult male Wistar rats were divided into two groups randomly,10 for model group(M group) and 6 for normal control group(N group).The rats of the model group were given olive oil diet plus the intragastric administration of alcohol and pyrazol mixture.The alcohol was administrated to rat in distillate spirit form, the quantity and concentration of which increased every two weeks from 4 g/kg·d,30%to 9.6g/kg·d,60%with pyrazol(25mg/kg·d) dissolved.The rats in N group were given equal normal sodium.To get liver samples,the animals in M group and N group were all sacrificed at the end of 16th week.One part of the liver samples taken were used to observe the pathologic changes of liver with the methods of HE stains and Masson stains; another part were used to detect the expressions of DDR2 and MMP2 by immunohistochemical staining.2.Construction and identification of DDR2 RNAi lentiviral vectors(1) Preparation of RNAi lentivirus vectors targeting rat DDR2 gene.According to the sequence of rat DDR2 gene available in GenBank,three pairs of some interference RNAs(siRNA) targeting DDR2 were designed,named siRNA1, siRNA2 and siRNA3.Then three double-stranded DNA(dsDNA) oligonucleotides containing corresponding short hairpin RNA(shRNA) and cohesive ends of Age I and EcoRI were designed and synthesized.Each of the dsDNA oligonucleotides was ligated with lentivirus vector pGCSIL-GFP digested by enzyme Age I and EcoRI respectively.In order to obtain vshRNA recombinant lentiviral vectors DDR2/LV-shRNA,ligation products were transformed into competent Escherichia coli cells freshly prepared with CaCl₂ method and the recombinant positive clones selected by ampincillin were identified by PCR and sequencing.(2) Package and titer determination of recombinant vshRNA lentiviral particlesThe DDR2/LV-shRNA,pHelper 1.0 and pHelper 2.0 plasmids were co-tranfectd into 293T cells by lipofectamine 2000 to package and amplify the recombinant vshRNA lentiviral particles,named DDR2/Lenti-shRNA1,DDR2/Lenti-shRNA2 and DDR2/Lenti-shRNA3 respectively.Virus titers were determined by serial dilution assay in 293T cells.(3) Screening of effective DDR2 vshRNA lentivirusNRK52E cells were infected by DDR2 vshRNA lentiviral particles in vitro.72 hours later,infection efficiencies were measured by green fluorescence protein(GFP) expression in cells viewed under a fluorescence microscope.Five days later,cells were collected and mRNA expression levels were examined by real time PCR.The expression ratios were calculated by using 2^{-ΔΔCt} methed and gene silencing effects of DDR2 in the cells infected by lentiviral particles of each group were compared.The most effective RNAi target was selected simultaneously. Results1.After 16 weeks of alcoholic intragastric administration for model establishment, histopathology of the liver in M group showed typical liver fibrosis,and the deposition of collagen fibers in liver tissue increased significantly compared with that of N group (P＜0.01).Immunohistochemical examinations showed that expressions of DDR2 and MMP2 in liver tissue of M group increased compared with those of N group(P＜0.01),and the distrabution of DDR2 was consistent with that of collagen fibers.2.As expected,the result of PCR indicated that products of recombinant positive clones ligated with vshRNA fragment were 343 bp(24bp was cut off from each vector), while those of control empty vector clone were 306 bp.The sequencing result indicated that the DDR2 vshRNA nucleotide sequences of all groups wre inserted correctly.Recombinant vshRNA lentiviral particles were packaged and amplified in 293T cells by lentiviral vector system,with viral titers reaching to 2×10⁸ TU/mL.The infection efficiency of recombinant vshRNA lentiviral particles each group was above 80%.Real time PCR displayed that inhibition effect of DDR2/Lenti-shRNA1 and DDR/Lenti-shRNA3 on DDR2-mRNA expression was above 80%in NRK52E cells.Conclusions1.The rat alcoholic liver fibrosis models could be successfully established through olive oil diet plus the intragastric administration of alcohol and pyrazol mixture.DDR2 contributed to the development of ALF,which was likely related to the interaction of collagen and hepatic stellate cell mediated by MMP2.2.Three RNAi lentivirus vectors targeting rat DDR2 gene were successfully constructed.Recombinant vshRNA lentiviral particles of high titers could be obtained by RNAi lentivirus vectors packaged in 293T.DDR2/Lenti-shRNA1 and Lenti-shRNA3/DDR were identified as effective DDR2 RNAi-lentivirus due to efficient and specific inhibition of DDR2 gene expression in NRK52E cells.