Experimental Study Of Lentivirus-mediated RECK Gene Therapy On Pancreatic Carcinoma

Objective To investigate the expression of reversion-inducing-cysteine-rich protein with Kazal motifs(RECK) gene in human pancreatic carcinoma tissues and pancreatic carcinoma cell lines and to explore the relationship between RECK expression and the clinicopathological characteristics.Methods Immunohistochemical SP method was used to detect the expression of RECK protein in 42 cases of resected specimens of pancreatic carcinoma and their corresponding normal pancreatic tissues.Western Blotting was used to examine the expression of RECK protein in human pancreatic carcinoma cell lines(PANC-1,MIAPaCa-2,AsPC-1).All the statistical analysis were performed by using SPSS 13.0 statistical software to determine the relationship between RECK expression and the clinicopathological characteristics.Results All of the three pancreatic carcinoma cell lines did not express RECK protein.The overall positive rate of RECK expression was 45.2%(19/42) in pancreatic carcinoma,88.1%(37/42) in normal pancreatic tissue,the expression level of RECK was significantly lower in tumor tissues than in the normal tissues(P<0.01).The positive rate of RECK expression in the cases of TNM I+II stage(58.3%,14/24) was significantly higher than that in the cases of TNM III+IV stage(27.8%,5/18,P<0.05).The positive rate of RECK expression in cases without lymph node metastasis(58.6%,17/29) was significantly higher than that in cases with lymph node metastasis(15.4%,2/13,P<0.01).The positive rate of RECK expression in cases without local infiltration(71.4%,10/14) was significantly higher than that in cases with local infiltration(32.1%,9/28,P<0.05).Conclusions RECK expression was reduced in pancreatic carcinoma tissues and undetected in pancreatic carcinoma cell lines.RECK expression was closely related to the invasion and metastasis of pancreatic carcinoma and might become a new prognostic marker for pancreatic carcinoma.Part II Construction of recombinant lentiviral vector expressing the RECK geneObjective To construct and package a recombinant lentiviral vector carrying RECK gene and to provide the basis for further experiments in vivo and in vitro.Methods RECK gene was amplified from plasmid pINCY-RECK by PCR technique and cloned into the expression plasmid of lentiviral vector,pGC-FU,to generate the lentiviral expression vector,pGC-FU-RECK.The correct RECK gene was confirmed by PCR identification and sequencing.pGC-FU-RECK was then used to transfect 293T cells.RECK protein expression in 293T cells was dectected by Western Blotting analysis.Recombinant lentiviruses carrying RECK gene(LV-RECK) were produced by 293T cells following the co-transfection of pGC-FU-RECK and packaging plasmids-pHelper1.0 and pHelper2.0.The virus titer was measured.Results (1)Plasmid pGC-FU-RECK carried the correct RECK gene and could express RECK protein in 293T cells.(2)The recombinant lentiviruses LV-RECK could be produced by co-transfection of pGC-FU-RECK and packaging plasmid to 293T cells.The titer of virus was 2×108 TU/ml.Conclusions The recombinant lentiviral vector carrying RECK gene was produced successfully,which will provide the basis for the further study on function of RECK gene in pancreatic carcinoma and gene therapy. Part III Experimental study of human pancreatic carcinoma cell line PANC-1 infected with LV-RECK in vitroObjective To investigate the effects of RECK gene over-expression on the biological characteristics of human pancreatic carcinoma cell line PANC-1.Methods LV-RECK was used to infect human pancreatic carcinoma cell line PANC-1,and the efficiency of infection was detected.The RECK gene expression was measured by Real-time polymerase chain reaction(PCR) and Western Blotting.Cell proliferation and apoptosis were examined by 3-(4,5-dimethylthiazole-2-yl) -2,5- diphenyltetrazolium bromide (MTT) assay and flow cytometry.The ability of invasion was detected using Transwell chambers.The expression and activity of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) were measured by Real-time PCR,Western Blotting,and gelatin zymography.Results PANC-1 cells were successfully infected with LV-RECK,and the infection efficiency was above 85%.RECK mRNA and protein expressions were significantly increased in PANC-1 cells of experimental group compared with negative control and blank control groups(P<0.05).Meanwhile,the invasion ability of PANC-1 cells of experimental group was potently suppressed(P<0.05).The activity of MMP-2 and MMP-9 in PANC-1 cells of experimental group were significantly decreased(P<0.05).However,no significant difference was observed in the cell proliferation and apoptosis among the three groups(P>0.05).There was also no significant difference in the expression of MMP-2 and MMP-9 among the three groups(P>0.05).Conclusions LV-RECK could infect PANC-1 cells at high levels of efficiency and mediate RECK gene expression in PANC-1 cells.RECK gene over-expression could inhibit MMP-2 and MMP-9 posttranslationally without affecting proliferation and apoptosis of PANC-1 cells,without affecting the expression of MMP-2 and MMP-9.Therefore,RECK gene over-expression could potently suppress the invasion ability of Panc-1 cells in vitro. Part IV Experimental study of recombinant RECK gene therapy on pancreatic carcinoma in vivoObjective To investigate the effects of LV-RECK therapy on human pancreatic carcinoma xenograft in nude mice.Methods Subcutaneous xenograft tumor models of human pancreatic carcinoma were established in nude mice firstly,then divided into experimental group,negative control group and blank control group randomly.The three groups of nude mice were respectively intratumorally injected with LV-RECK,LV-EGFP and normal saline(NS).Antitumor effect and liver and lung metastasis were investigated.Immunohistochemical SP method was used to detect the expression of RECK protein and microvessel density(MVD).Terminal deoxynucleotidyl transferase mediated dUTP-DIG nick end labeling(TUNEL) was used to detect the apoptosis of tumor cells and survival analysis was performed.Results Compared with negative control and blank control groups,the volume of subcutaneous xenograft tumor in experimental group was significantly decreased(P<0.05).The tumor growth inhibition rate was 52.68%.RECK protein in experimental group was reexpressed.MVD of experimental group was significantly less than those of control groups(P<0.05).Apoptotic index(AI) of experimental group was significantly higher than those of control groups(P<0.05).The rate of liver and lung metastasis of nude mice in experimental group was significantly less than those in control groups(P<0.05).The survival time of nude mice in experimental group was significantly longer than those in control groups(P<0.05).Conclusions LV-RECK could mediate RECK gene expression in human pancreatic carcinoma subcutaneous xenografts of nude mice.RECK gene over-expression could inhibit neovascularization of pancreatic carcinoma,induce apoptosis of tumor cells,inhibit the growth and metastasis of tumor xenograft and improve the prognosis of tumor-bearing mice.This would offer a new way for pancreatic carcinoma treatment.